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Tio2 has been investigated intensely since the 1970s when it was discovered that it is an active photocatalyst . Although most surface science studies focus on the most thermodynamically stable rutile tio2(110) face, there is a growing interest in other rutile terminations as well as anatase tio2 surfaces . The rutile tio2(011) surface has received particular interest because of a reportedly enhanced photoactivity . Most studies of tio2(011) report a (2 1) reconstruction, the structure of which was initially unclear, with two proposed models: a titanyl model and a microfacet model . However, three independent diffraction studies have clarified the surface structure, all pointing to the diffraction model shown in figure 1a . Theoretical calculations also find this to be the most stable of the proposed models . Both the beanlike and zigzag motifs that appear in scanning tunneling microscopy (stm) images recorded close to and far from the surface, respectively, could also be reproduced by stm images simulated from the diffraction model . Ball and stick models of (a) the diffraction model for the tio2(011)-(2 1) phase, (b) the model proposed for tio2(011)-(4 1)-, and (c) the model proposed for tio2(011)-(4 1)-. Red balls are ti and blue balls are o. o atoms that form the (2n 1) rows are shaded lighter . Surface unit cells are indicated in yellow and the green ellipse indicates the region between the added (2 1)-like rows where our nc - afm and stm images do not show much detail . In addition to the (2 1) phase, kubo et al . Also report noncontact atomic force microscopy (nc - afm) and stm images of a coexisting (4 1) phase . Ahmed et al . Also report a (4 1) reconstruction following a wet preparation, but this phase does not survive a uhv anneal . Here, we report on a series of (2n 1) reconstructions that are revealed by nc - afm and stm images . Two types of (4 1) reconstruction were observed, which we refer to as (4 1)- and (4 1)-. The (4 1)- surface has the same structural elements as the widely reported (2 1) reconstruction . In an analogous fashion, it is also possible to have an array of such (2n 1) reconstructions; indeed, we observe a series of (2n 1)- reconstructions where n = 25 . In contrast, the (4 1)- reconstruction seems to be a unique structure without higher order analogues . The proposed structure for (4 1)- has the same structural elements as the (2 1) phase but with the addition of {111} microfacets . The experiments were performed in osaka using a custom - built nc - afm housed in an ultrahigh - vacuum chamber (with a base pressure of 5 10 torr) and operated at room temperature . The tio2(011) crystal (pi - kem) was prepared using repeated cycles of ar - ion bombardment (2 kev) for approximately 5 min and annealing between 1073 and 1273 k for 1025 min . Preparation of a different tio2(011) crystal (mateck gmbh) with a lower annealing temperature of 943953 k led to the more commonly observed (2 1) termination . However, further systematic study is required to establish a definitive recipe for preparation of (2n 1) terminations . Nc - afm images were obtained using the frequency modulation detection method, with the cantilever oscillation amplitude kept constant (peak - to - peak amplitudes 176278). The data presented here were taken with two silicon cantilevers which had resonant frequencies in the range 155156 khz . A dc voltage (vcpd) is added between the tip and sample that minimizes the average tip sample contact potential difference . Stm images were obtained using the same cantilevers, biased with a voltage (vs), with the oscillation still active such that the current is time - averaged (it). In some cases, the tips were treated by electrical pulses or nanoindentation procedures to ensure sufficient conductivity for stm measurements . Figure 2a shows a large - area nc - afm image of the tio2(011) surface . The image is characterized by a number of bright rows aligned to the [011] direction . There are several domains present, which are shaded with different colors in figure 2b . On the right - hand side, the line profile in figure 2c is obtained from the green line shown in figure 2b, which crosses two (4 1) domains . It is clearly evident from the line profile that two types of (4 1) reconstruction are present: on the left - hand side, the corrugation of the rows is about 1.5, whereas on the right - hand side, it is about twice this: 3 . We refer to these as the (4 1)- and (4 1)- reconstructions, respectively . Apart from the greater corrugation of the (4 1)- phase compared with (4 1)-, the phase can also be distinguished by its smoother appearance due to a lower density of defects, fixed (4 1) periodicity, and a broader appearance of the rows . (a) nc - afm image (350 226, f = 1.5 hz, vcpd = 0.5 v) of the tio2(011)-(2n 1) surface . (c) line profile taken along the green line in (b) that shows a low corrugation for (4 1)- and a greater corrugation for (4 1)-. In figure 2b, the (4 1)- regions are unshaded, whereas the (4 1)- region is shaded light - blue . The center of the image contains rows mostly with a (6 1) periodicity and a very narrow (2 1) domain that is shaded yellow . The (6 1) region is further separated into three domains, shaded light - red and dark - red . The unit cells of the two light - red regions contain two adjacent bright rows, whereas the unit cell of the dark - red region contains only one bright row . It is also apparent that the two light - red regions are out - of - phase with respect to each other, as highlighted by the white guideline in figure 2b . This suggests that the bright rows in the regions shaded light- and dark - red are the same . In contrast to the (4 1)- regions, it can also be seen that the other (2n 1) regions merge with each other without discernible barriers . For instance, the row highlighted by the black guideline in figure 2b going from top to bottom straddles (6 1), (2 1), and (4 1)- regions . On the basis that the rows from the (2n 1) phases [apart from (4 1)-] can simultaneously form part of the (2 1) and the higher order (2n 1) phases, we propose that with the exception of (4 1)-, all the bright rows from the (2n 1) reconstructions have the same structure as that of the (2 1) reconstruction . As such, these phases will be referred to collectively as (2n 1)-. A series of schematic models for these (2n 1)- structures are shown in figure 3a . By definition, if the added (2 1) rows are packed with saturation density, then the frequently reported (2 1) reconstruction will be formed . When the spacing of the added rows is doubled, the (4 1)- reconstruction is formed as shown in figure 3a . Consistent with the nc - afm image in figure 2, where two types of (6 1)- reconstruction were observed depending on how many bright rows are present (labeled (6 1)- i and (6 1)-ii in figure 2b), two (6 1)- models are shown in figure 3a using either one or two added rows per unit cell . The models are constructed from (2 1) units shown light blue and unreconstructed (1 1) units in dark blue . In (a), the (2 1) units are on a flat (1 1) platform, whereas in (b), the second layer also contains (2 1) units . The high - resolution nc - afm image in figure 4a reveals further substructure within the added rows . The rows have a zigzag motif reminiscent of that reported previously in stm images of the (2 1) surface . The zigzag can be simply defined by a triangle as shown in figure 4a and highlighted in figure 4b . Measurements of the dimensions show that the triangle is isosceles in nature, the equal sides being 3.8 0.4 and the long side being 5.45 . The latter side is in line with the unit cell along the [011] direction and used to calibrate the measurements . The dimensions of this triangle are remarkably close to those measured from stm images of the tio2(011)-(2 1) phase with zigzag contrast . (a) nc - afm image (100 35, f = 38.9 hz, vcpd = 0.9 v) of tio2(011)-(2n 1)-. The light blue lines indicate a 2 spacing with double - ended arrows indicating 2, 4, and 6 periodities . A zigzag motif is observed that can be described by the isosceles triangle drawn red . A number of dark defects can be observed, some of which are circled in white . (b) magnified part of the image shown in the white square in (a) with the measured dimensions . (c) line profile taken from the green line in (a) that shows high peaks for the bright rows and lower peaks for the darker rows . One of the darker rows is marked with a red line . The same 2, 4, and 6 periodicities are marked as in (a). Empty - state stm images taken from a similar area of the surface are shown in figure 5 . The same zigzag motif is discernible, and again, it can be described by an isosceles triangle with a long side of 5.45 and equal sides of 3.9 0.3 . This gives strong evidence to support our model where the added rows are composed of the same rows that form the (2 1) phase . Stm images of tio2(011)-(2n 1)- with image parameters of (a) 200 100, vs = 3.5 v, it = 0.025 na and (b) 100 62, vs = 3.5 v, it = 0.027 na . The green rectangle in (a) marks the approximate area of the image in (b). Some point defects are marked with green crosses, and the light - blue lines mark some rows with (2 1) periodicity . The zigzag motif of the rows is highlighted by red triangles and the area marked by the white square is magnified in the inset . The image in the inset has an fft filter applied to minimize the periodic noise and accentuate the zigzag motif . In empty - state stm, when the tip is relatively close to the sample, a beanlike contrast is found that is dominated by tunneling into o 2p states because the o atoms protrude further out of the surface . On the other hand, when the tip is further from the surface, the zigzag contrast is found . The zigzag contrast is dominated by tunneling into ti 3d states because of its longer decay length compared to the o 2p states . Given that the zigzag contrast is electronic in nature, it does not necessarily follow that a similar contrast should be seen in nc - afm . However, a similar interplay between the decay of the tip sample potential and the surface geometry could be at play, and theoretical calculations would shed more light on this . Note that at this stage, it is also not clear if the zigzag motif in nc - afm arises from ti, and this could be established by simultaneous measurement of nc - afm and stm . While evidence has been presented for the structure of the added rows of the (2n 1)- reconstructions, the platform on which the added rows sit has not yet been discussed . The models in figure 3a show these added rows on a (1 1) platform simply to highlight the periodicity of the added rows . Two (6 1) units are marked in figure 4a: one on the left - hand side and one in the center of the image . That on the left - hand side has a darker row (marked with a red line) as well as a bright row in the unit cell . The line profile in figure 4c also shows this extra darker row clearly, the peak being about 1 lower than those of the bright rows . On the other hand, there does not appear to be an extra darker row between the added rows of the (4 1) part of the (6 1) structure in the center of the image . Figure 6a shows an nc - afm image with a (4 1)- region adjacent to a (6 1)- region . The (4 1)- region is clearly composed of alternating brighter and darker rows, also highlighted in the line profile in figure 6b . Figure 6c shows a higher resolution image of the (4 1)- region . The rows have an almost identical appearance, except the upper added rows are slightly broader . This is because they lie topographically higher and therefore part of their side structure is resolved . Likewise, in the nc - afm image of the (6 1)- region shown in figure 6e, the unit cell consists of one bright row and two dark rows each with a similar appearance . Nc - afm images and line profiles of tio2(011)-(2n 1)-. (a) image parameters are 150 75, f = 7.5 hz, vcpd = 0.6 v. (b) line profile along the green line marked in (a). (c) image parameters are 62.5 30, f = 9.1 hz, vcpd = 0.6 v. (d) line profile along the green line marked in (c). (e) image parameters are 70 35, f = 7.5 hz, vcpd = 0.6 v. (f) line profile along the green line marked in (e). In (a), (c), and (e), the light - blue lines indicate a 2 spacing with double - ended arrows indicating 4 and 6 periodicities and green crosses marking the position of point defects . For easy comparison, the line profiles are drawn with the same x - axes scales as their corresponding images . We therefore propose that the platform on which the added (2n 1)- rows stand can be either the unreconstructed (1 1) surface or rows with the (2 1) structure, as shown in the schematic models of figure 3a, b, respectively . When n> 2, there are several configurations in which the (2 1)-like rows can be arranged to make the (2n 1)- structures . For instance, two types of (6 1) structure are shown in figure 3b: (i) a structure that would appear in nc - afm as one bright row and one darker row, as seen in figure 4a, and (ii) a structure with one bright row and two darker rows, as observed in figure 6e . A ball and stick model of the (4 1)- structure (including a darker row) is shown in figure 1b that corresponds to the schematic in figure 3b . All other higher order (2n 1)- phases can be visualized using this model and arranging the units as shown in the schematics of figure 3a, b . We note that while the scanning probe images give good evidence for the general structure proposed, the detailed structure between the rows is unknown (i.e., the region circled in green in the model of figure 1b) and can probably be best addressed by computer modeling given that the structure does not have the long - range order required for quantitative diffraction studies . Figure 7 shows a high - resolution image of the (4 1)- phase . As with the images presented of the (2n 1)- phases the zigzag can be described by an isosceles triangle similar to those in figures 4b and 5b: the long side is 5.45, and the shorter equal sides are 4.3 0.3, similar to that found for the (2n 1)- phases here and in stm images of the (2 1) termination . The model we propose tentatively is therefore again based on elements of the diffraction model for the (2 1) phase . However, in this case, we remove every other row of the (2 1) model to create a microfaceted structure somewhat similar to that proposed by kubo et al . And illustrated in figure 1c . Nc - afm image (40 40, f = 9.5 hz, vcpd = 0.6 v) of tio2(011)-(4 1)-. The red triangle highlights the zigzag structure . The proposed model would account for the greater corrugation observed in the nc - afm images for this phase compared to the (2n 1)- phases . Such a microfaceted structure exposes the less stable {111} faces and this could explain why the (4 1)- structure does not develop further higher order structures like (6 1) and (10 1) because the proportion of the less stable {111} facets increases with the size of the microfacet . For the same reason, the (4 1)- phase is likely to have a higher energy than the (2n 1)- phases . This could explain why when starting from an as - purchased crystal, the (4 1)- phase was only observed during the first 21 sputter / anneal cycles, whereas the (2n 1)- phases were still observed after 64 cycles . In this scenario, the more stable (2n 1)- phases would tend to dominate upon repeated annealing . Note that while the (4 1)- model can be created by removing (2 1) units from the (2 1) phase, this does not carry any implication on how the phase is formed . It may be that the reconstructions grow out from the surface as has been shown for the rutile tio2(110) surface . In the high - resolution stm and nc - afm images shown in figures 46, several defects (or agglomerations of defects) can be seen, and some of these are marked with crosses and circles . Figure 8a, b show nc - afm and stm images, respectively, that are taken from the same area of the surface in the vicinity of the images in figures 4,5 . Specifically, this region mainly has a (6 1)- configuration where two bright rows make up the unit cell . (a) nc - afm image (350 350, f = 8.4 hz, vcpd = 0.9 v) of tio2(011)-(2n 1)-. The light - blue lines mark a 2 spacing, and the double - ended arrow marks the 6 periodicity; (b) stm image (350 350, vs = 2 v, it = 0.027 na) of the same area as (a); (c) and (d) are duplicates of (a) and (b), respectively . Green crosses mark defects present in both images, blue crosses mark defects visible in the nc - afm image but not the stm image, red crosses mark defects visible in the stm image but not the nc - afm image, and the yellow cross marks a defect that appears dark in the nc - afm image but bright in the stm image . The green crosses in figure 8 highlight coincident defects in the stm and nc - afm image . In one case, a bright defect in the stm appears dark in the nc - afm image and this defect is marked yellow . Red crosses indicate defects only seen in the stm image and blue crosses mark those that appear only in the nc - afm image . The majority of crosses are green, indicating that most of the defects are detectable in both images . As the nc - afm image was recorded 7 min after the stm image, at least some of the defects that cannot be matched between the images may arise from diffusion . Although dark defects that appear in stm images of tio2(011)-(2 1) with zigzag contrast have been assigned to oxygen vacancies, bright defects have been assigned to adsorbed hydrogen . In sequential stm images and sequential nc - afm images taken from the same area of the tio2(011)-(2n 1)- phase (not shown), several of the bright defects change their positions, indicating that at least some of the defects can diffuse rather easily even at room temperature . On rutile tio2(110) at room temperature, adsorbed hydrogen is known to diffuse either intrinsically or facilitated by molecular water . On the other hand, given that the (2n 1)- phase shares the same basic structure as tio2(011)-(2 1), the easy diffusion of the defects supports the assignment of the bright defects observed by tao et al . To adsorbed hydrogen . As with images in stm, it is well - known that the contrast obtained by nc - afm can change depending on the nature of the tip apex . For instance, a subtle difference in contrast can be seen between the images in figure 6a, c . Both nc - afm images contain (4 1)- regions with bright and darker rows . In the image in figure 6a, the bright rows have a height of 0.6, and the darker rows have a height of 0.2, so that the height difference between them is 0.4 . On the other hand, in the image in figure 6c, the bright rows have a height of 0.5, and the darker rows have a height of 0.3, with the height difference being only 0.2 . As such, the 4 1 periodicity in figure 6a is clear, whereas the (4 1) periodicity in figure 6c is only just discernible . More drastic tip changes can be seen in the series of five nc - afm images shown in figure 9 . These were recorded sequentially, and each image has a different contrast . In figure 9a, the bright defects can be seen together with the bright (2n 1)- rows . In figure 9b, the contrast is similar, but the defects and rows appear more smeared out ., there is a drastic contrast change: the (2n 1)- rows still appear bright, but the contrast is only dark between rows with (2 1) periodicity . Between rows with a greater periodicity, there is a bright band . The defects are visible as very well - resolved bright spots, but the (2n 1)- rows themselves are not resolved . In figure 9d, the contrast is similar to that in figure 9b, but the (2n 1)- rows dominate, and the defects are invisible . Finally, in figure 9e, there is another drastic tip change: the defects are again very well - resolved, but in contrast to the image in figure 9c, the (2n 1)- rows are also well resolved . Sequential (200 60, vcpd = 0.9 v) nc - afm images of tio2(011)-(2n 1)- with f = (a) 6.1 hz, (b) 6.1 hz, (c) 6.7 hz, (d) 6.0 hz, and (e) 5.8 hz . The light - blue lines indicate a 2 spacing and in (a)(d), the red guidelines mark equivalent rows . The green crosses highlight some defects that are visible in (a)(c). In (d), despite the image being more or less in the same area as (a)(c), the defects are invisible, whereas the image in (e) was taken from a slightly different position . The images in figure 9 show that the point defects themselves can alter in appearance from very well - resolved to invisible . However, the contrast of these defects can also invert completely: although the defects presented in the images of figures 6, 8 and 9 almost all appear bright, the defects in the image of figure 4 appear as dark depressions . It is clear that point defects on tio2(011)-(2n 1)-, and by extension tio2(011)-(2 at least some of the defects are adsorbed hydrogen, but it is not yet clear if that is the only defect present . Furthermore, it is evident that like on the rutile tio2(110) surface, several types of contrast are possible in nc - afm . Both the origin of the different contrasts and the unambiguous assignment of defects could likely be resolved with further work combining stm and nc - afm with theoretical simulation . In conclusion, we have used nc - afm and stm to study the rutile tio2(011) surface . A series of (2n 1) reconstructions were observed, including two types of (4 1) reconstruction: (4 1)- and (4 1)-. High - resolution nc - afm and stm images suggest that the (4 1)- phase has the same structural elements as the more widely reported (2 1) termination . Closely related higher - order (2n 1)- phases where n = 35 were also observed . The (4 1)- reconstruction also has a structure based on the (2 1) reconstruction but with additional microfacets of {111} character . Higher - order analogues were not observed for the (4 1)- phase . Although not definitively assigned, the same point defects were observed in both nc - afm and stm images . In sequentially imaged areas, the nc - afm contrast was subject to changes, and the point defects appeared clearer in some cases and invisible in others . Further study combining theoretical simulations with stm and nc - afm may be able to explain such tip changes as well as identifying the point defects.
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Heart diseases, as the most common cause of death in iran and worldwide, have always been considered by the public and healthcare policy makers . Consequently, increasing the number of heart surgery centers has become an indicator of healthcare extension . Almost all of healthcare managers place the expansion of heart centers at the top of their career priorities . This issue is usually supported by national politicians, e.g. Members of parliament and city council, beyond the people s concern . Therefore, purchasing and launching highly developed technologies has become a touchstone to assess politician s capacities . It varies from an ordinary clinic to the most advanced open - heart surgery and imaging facilities, such as catheterization labs . Despite this progress, waiting time is regarded as an appropriate determinant in evaluating healthcare quality, heart surgery prognosis (1) and patient satisfaction (2, 3), and all research efforts have been centered around measuring clinics or emergency department s waiting time (4, 5). Therefore, doing a research about estimating heart surgery waiting time seemed necessary . In countries with general health insurance coverage, waiting time limits healthcare access and decreases inpatient payment . Consequently, they will have more sources for providing state surgical or hospital services . Of course, it can harm people s health and cause inadequate use of elective services . Besides that, according to siciliani et al . (6), less than 10 days waiting time can reduce total costs and it is better that managers use other methods for rationing, instead of waiting time . Besides being an important factor influencing patient satisfaction, waiting time is a quality measure for outpatient services . (7) assumed that waiting time, staff motivation and patient education are more effective on patient satisfaction than specialized team provision . Several scholars believe the time assigned by physicians to patients visit is more satisfying than waiting time for receiving services (8). Waiting time is an expression of hospital services accessibility and a measure for hospital performance . (9) mentioned about an increasing pressure for the implementation of an equitable and patient needs - based system to rationing surgery . They also assumed that national official data on waiting lists for spain and catalonia do not allow conclusive lessons to be learned, regarding the impact that the austerity measures are having on waiting times for patients . The same has happened for 132 thousands and 785 thousands of people in nederland and great britain, respectively . Accompanied by efforts for providing healthcare, prioritization of people in waiting lists can ensure the assignment of services to neediest ones . A small number of clinicians work on priority setting and, among them, fewer specialists engage with ranking of surgical heart services . Ontario practitioners decision is an example of waiting time application in reducing the impact of resource constrains . This deprivation is in contrast to american policy, which is based on low income or lack of insurance (10). Eisenberg (11) highlighted that sociological factors, such as age, gender, race and social level, influence waiting time average . In 2005, the cardio - start charity group conducted several heart surgeries in golestan province, iran . Afterwards, golestan university of medical sciences, golestan, iran, decided to establish a heart center in amir al - momenin hospital of kordkooy district . The main goal was reducing referral of golestan and adjoining provinces patients to tehran and other large cities . One of the most important reasons for the urgent establishment of this action was the long waiting list of patients in the aforementioned regions . The aim of this research is to study waiting time and its influencing factors, which helps to assess center s effectiveness after 9 years . We expect that this study may fill the current deficit in domestic literature about cardiac surgery waiting time and re - introduce it as an important measure for assessing the effectiveness of high technology purchasing and establishment at end - users level . This cross - sectional study performed in 2013 involved 156 patients referred to amir al - momenin hospital of kordkooy district, kordkooy, iran, for different kinds of heart surgeries . The patients were evaluated about demographic characteristics, accompanying disorders, economic status, education and type of surgery . Based on expert opinions (heart surgeons), heart performance index was the most important factor affecting our response variable (waiting time). Due to this opinion and using the new york heart association classification, which classifies heart performance in four classes, sample size was identified as 39 patients for each class (156 patients), which formed the study patients groups . Our four patient groups were as follows: the first group comprises patients whose activity is not compromised anyway . In contrast to this, the fourth one cannot do any activity without trouble (12). Participants were selected from patients who were nominated by heart surgeons for elective surgery . Because waiting time does not apply to urgent patients, they were excluded from study . All of the 156 cases were selected from patients spending recovery period in open - heart intensive care unit (icu). They took notes about the date of admission, place of residence, date of referral from surgeon, accompanying diseases, impression, preoperative heart performance etc . Afterwards, more information about economic status, occupation, and other demographic data were collected through interviews with patients or relatives . Data about heart performance or left main coronary artery disease were derived by observing results of coronary angiographies and consultation with the physician . Before any action, patient satisfaction to join in the study and attendance of researchers to their medical records were required . Therefore, we constructed a variable based on patient s annual income and asset scales, such as having personal automobile, computer, washing machine, and similar devices . For income scoring, living conditions were valued by two - thirds of asset score and one - third of income score . Based on this scoring we had: - poor group: monthly income <167 usd or 5000000 rials (iran currency) and having two assets (question number 10 of checklist - maximum score of 10). - good group: monthly income 334 usd plus four points or more assets - minimum score 21 . - moderate economic condition: scores from 10.1 to 21 . In data analysis, kolmogorov - smirnov and shapiro tests were conducted first, to determine the normality of data . Because waiting time, as the main variable, was not normal in any subgroups, non - parametric tests like man - whitney and kruskal - wallis substituted parametric tests, like the t test and anova . Quantitative variables, described by average and standard deviation were reported through presenting p value, by using mean rank report . For ethical consideration, interviews with patients and relatives and accessing patients profile details have been done with patients permission and hospital coordination . Inhabitants of urban areas were 69.8% and while 30.2% lived in rural areas . In terms of marital status, 1.9% were single, 94.9% married, 0.6% divorced and 2.6% widows or widowed men . Therefore, it was predictable that our sample would be composed of different ethnicities . In this sense, we had 82.7% persian or fars (as an ethnicity), 6.4% turkmen (a large minority who have formed, with fars ethnicity, the indigenous population of golestan province), 10.3% sistani (other minority who emigrated from southeast of iran to the north) and 0.6% others, which are composed of baluch, cossacks and turks . The literacy rate was also calculated in our study, revealing 36.5% illiterate, 27.6% at elementary or literacy movement organization (a governmental agency which was established by decree of the islamic republic of iran, in 1980, to teach reading and writing to adults and children who are deprived of education), 13.5% junior high school, 14.7% high school diploma, 0.6% associate degree and 7.1% bachelor or more (table 1). Regarding to occupational level, 4% were unemployed, 5.8% government employee, 4.5% full - time self - employed (5-day in a week, 8 hours a day), 26.6% part - time self - employed, 25.3% housekeeper, 26% retired or disabled and 3.2% were farmer (table 2). Review of patients medical records revealed that, in terms of preoperative heart performance index, 19.1% were class i, 44.7% class ii, 27% class iii and 9.2% class iv . Out of the respondents, 20% had left main coronary artery involvement versus 21.2% left main equivalent disease . Provision of surgical equipment (13.5%), specialist absence (12.8%), equipment failure (0.6%), financial problems (28.2%), personal problems (18.6%), patient s health condition (1.3%), specialist recommendation (12.8%), specific drug consumption (3.8%), hospital admission delay (1.9%) were the most important causes of delay in surgical procedure . In 2.6% of cases, 87.2% had coronary artery bypass grafting (cabg), 6.4% heart valve surgery, 4.5% both of the previous surgeries and 1.9% other kind of heart surgery (table 3). Concerning to outcome of surgery, most patients had complete remission (89.1%), while several had partial remission (20%) and fewer died (0.6%). The current survey showed that 21.1% of participants had poor, 50.3% moderate and 28.6% good economic condition . There was no significant relation between waiting time and marriage status (p = 0.85 and mean rank was 93.33, 78, 56, 50, 72.25 for singles, married, divorced and widows respectively). There was no significant relation between waiting time and gender (p = 0.371). Based on the findings (table 4), waiting time was significantly influenced by economic status, as kruskal - wallis test shows (p = 0.037). There was no significance between waiting time and ethnicity, place of residence and religion (p value was 0.664, 0.08 and 0.429, respectively). Findings of relationship between waiting time and type of surgery, type of disease, accompanying disease, and preoperative heart performance are shown in tables 3 and 5 - 7 . There was no relation between waiting time and presence of hypertension (htn) and history of myocardial infarction (p = 0.983 and 0.274, respectively). The current study shows no relation between waiting time and demographic characteristics, while many investigations around the world have revealed this fact . A study conducted by ray and his colleagues reveals that long waiting time in women is due to more sensitivity they have about their health and not because of gender . Therefore, their health problem is discovered in early stages and enough time is spent to prepare for the surgical intervention . In a study conducted by ray et al . (13), p values were 0.518 and 0.481 for men and women, respectively, in comparison with ours, which was 0.371 . (14) assumed that waiting time and gender have no association with each other . 15), in their study titled queuing for coronary surgery also concluded the same . According to the study by arnesen et al . (14), age group 70 years had the shortest waiting time among other age groups . In the current study, this age group had longer waiting time than the age group <70 (28.6 versus 21.5 days, respectively, p = 0.068). (13) showed that surgery outcome is affected by several risk factors, such as history of heart surgery and myocardial infarction, and there was no relation with waiting time . Ajami and ketabi (16), studied bottlenecks in the discharge process, concluded that patient s financial problem influences waiting time, as an important factor . (17) studied the effects of socioeconomic status on access to invasive cardiac procedures . Pell et al . (18) concluded that socioeconomically deprived patients, besides their worse medical condition and less access to investigations, are further disadvantaged, because should wait longer for surgery because of lower priority . Kee et al . (19) found no significant relation between occupational condition and waiting time, which is consistent with our study (p = 0.201). Naylor et al . (15) concluded that there is an inverse relation between patient s neighborhood income quartile and waiting time . The current study, similarly to previous studies about economic status, is exposed to the risk of under reporting the financial status of the participants . This is especially due to costly services of heart surgery and fear of failure to commute . Therefore, using a combined variable, formed from patients self - reporting, and having living facilities reduce this concern . Hurst and sisiliani (20), in their article, titled tackling excessive waiting times for elective surgery in 12 oecd countries, reported that average waiting time is about 3 months, which could be increased to 1 year . In denmark, prolonged waiting time for cabg surgeries was reduced permanently through increasing practices and supporting capacity buildings (20). Hurst and sisiliani (20) also reported that the proportion of studied people who waited for more than 12 weeks for surgery was 58.1% in portugal, 41.7% in switzerland and 36.3% in italy . Other findings of similar studies have revealed 28% in norway, 19.4% in germany, 18.5% in spain, 16.1% switzerland, 15.2% netherlands and 13.3% hungary (20). Waiting time average was about 30 days in england, in 1991, which increased to about 190 days, in 2001 (21). One of the proposed solutions for decreasing the need to elective surgeries is the substitution of public financing by private methods . When the public services are in poor condition, or private services are strong and choosing of the providers could be done by patients, this is useful . We should consider that increasing private insurance coverage can cause more demand and give the opposite result . Private insurance coverage can give the patient a shift from public services to private sector . However, this decrease in waiting time can come at the cost of undermining incentives to buy voluntary private insurance . In fact, waiting lists with other factors such as income, age and political preferences have a major role in inducing private insurance purchasing . On the other hand, a patient who has been confronted with paying increased co finally, if the private sector confronts with limiting marketing legislation or staff shortage, accessing to shorter waiting lists may be postponed . Based on our research, patients with just basic insurance had no difference regarding waiting time compared with more expensively insured patients . (14) considered socioeconomic characteristics as important waiting time influencing factors and especially focused on urology, gynecology and orthopedic elective surgeries . They estimated waiting time between 6 and 846 days (61, as average). In their study, patients with malignancy or serious complications had shorter waiting time and the factors affecting waiting time are patient s conditions, physician s situation and type of disease . Our research did not found a significant relation between waiting time and disease type . In other words, patients with left main coronary artery involvement had an average waiting time of about 18.2 days, whereas for patients without the aforementioned involvement it was 23.6 days . (p = 0.128). In naylor et al . Research (15), symptoms and anatomy of lesion were the most important waiting time determinants . (13) showed that most patients (86%) had diabetes mellitus (dm), involvement of three vessels, stenosis of left main or both vessels . We found that 50% of our participants had dm (table 5) and 20% stenosis of left main artery . There was no association between waiting time for patients who just had dm (22.3 days) and patients with both dm and hypertension (21.7 days, p = 0.662). No comparison was found between different ethnic group waiting times, in literature review . As mentioned earlier, although golestan province population is composed of various ethnic groups, our research did not show any significant difference between waiting time of ethnic groups (p = 0.82). Patients usually do not talk about asking extra - payment by physician or referring them to private sector, for more charge . Although the reality pointed above is a limitation of our study, several general statements, such as financial problem or specialist recommendation, as the first and third factors influencing waiting time, reflect this concern . Launching heart surgery wards has always been one of politicians favorites, whether they are directly related to the health sector or not . Patients who decide to undergo surgery, on one hand, are worried that their decision will not necessarily lead to cure or even result in death . On the other hand, they are worried that their intention may impose catastrophic costs . Therefore, it is acceptable that the proximity of heart surgery wards to people s accommodation can be an important factor for establishment . This strategy, in addition to diminishing waiting time, can reduce insensible costs, such as commuting costs . It should be noted that, due to the lack of specific studies for the waiting time for elective cardiac surgery, its standard are selected according to the researcher s opinion . Therefore, we recommend that other studies about heart surgery waiting time should consider different types of heart diseases . Except the economic condition, this suggests that the studied heart center, regardless of gender, settlement, education and occupation, considers fairness in determining waiting time . However, if waiting time for several conditions is considered, this should make managers aware of falling into other kinds of unfairness or in better expression, mismanagement . For example, we found that women in all age groups had longer waiting time compared to men . By opinion of ray et al . (13) this translates that women are more sensitive to their health, although the difference probably has other points that should be considered . Although more attention should be given to patients over 70 years, the waiting list in this group is longer than young people . Consulting with specialists did not show any cost - effective analysis regarding qaly league tables, to justify this difference . Surprisingly, patients with poor heart condition deserve to undergo surgery earlier, even if our study shows no significant association . Currently, specialist opinion is the third factor in waiting time determination (12.8%). It seems that managers should review their waiting time standards and change them to more important determinants . (22), in new zealand, today we witness a move to more explicit decisions . As noted, patient s economic condition is the only variable which significantly associates with waiting time . On the other hand, financial and personal problems are also important factors that cause waiting time elongation (28.2 and 18.6%, respectively). These facts highlight the role of the ministry of health, insurance and other supporting organizations in protecting privileged groups . Increasing health insurance makes the load of hospital costs and physician s informal payments more bearable . In addition to increasing health insurance coverage, waiting time for heart surgery can be further reduced by providing the cost of surgery . Implementing strategies, like in the for costs of patients injured in road accidents, considering psychological consultation and introducing technical and professional abilities, besides providing comfortable atmosphere, could be definitely useful . Freeman and denham (25) suggest three important principles for nurses to cope with the anxiety of patients and their relatives . Second, clear communication at the admission about the expected surgical time and potential problems that might lead to delays . Third, empowerment or use of a contract that establishes the nurses intent to see the patient goes to surgery at the scheduled time (25). (26) stressed that long waiting time is considered an independent and exclusive risk factor for patients dissatisfaction . They recommend that giving information about delay and its causes act as a right strategy in patient satisfaction . As noted earlier, reporting the economic condition by the patient is not reliable . Because of that, we used a summary measure composed of reported income and living amenities . Another limitation is concerns the standard of heart surgery waiting time, that is derived subjectively from expert opinions . Participatory management, with application of total quality management principles, could improve waiting time . In consistence with mossadegh - rad (27) opinion, it could be appropriate if the area of participative management, such as staff training, creating culture of participation by directors and employees, proper incentives and commitment of senior management is available . They believe that correcting patients perception about waiting time, through improving patient - physician interactions, greater understanding of patients about their care, and improving their waiting experience, are key elements in promoting patients satisfaction . Finally, we should not be unaware about the role of dm in heart surgery success rate . This finding underlines that we should, as soon as possible, determine the role of dm or controlling it in the prognosis of heart surgery . It is important that not all the diabetic patients mentioned previous history of myocardial infarction . Therefore, the fact that dm directly affect the prognosis, regardless of its role in predisposing to atherosclerosis, could be considered by clinical researchers (29). (30) assumed that the risk of coronary artery disease increases with the severity, rather than duration of diabetes mellitus . For a given level of severity of dm, the risk of cad did not differ by anatomic location, within the coronary tree . Launching heart surgery wards has always been one of politicians favorites, whether they are directly related to the health sector or not . Patients who decide to undergo surgery, on one hand, are worried that their decision will not necessarily lead to cure or even result in death . On the other hand therefore, it is acceptable that the proximity of heart surgery wards to people s accommodation can be an important factor for establishment . This strategy, in addition to diminishing waiting time, can reduce insensible costs, such as commuting costs . It should be noted that, due to the lack of specific studies for the waiting time for elective cardiac surgery, its standard are selected according to the researcher s opinion . Because of that, our study fairly has inherent weaknesses . Therefore, we recommend that other studies about heart surgery waiting time should consider different types of heart diseases . Except the economic condition, this suggests that the studied heart center, regardless of gender, settlement, education and occupation, considers fairness in determining waiting time . However, if waiting time for several conditions is considered, this should make managers aware of falling into other kinds of unfairness or in better expression, mismanagement . For example, we found that women in all age groups had longer waiting time compared to men . By opinion of ray et al . (13) this translates that women are more sensitive to their health, although the difference probably has other points that should be considered . Although more attention should be given to patients over 70 years, the waiting list in this group is longer than young people . Consulting with specialists did not show any cost - effective analysis regarding qaly league tables, to justify this difference . Surprisingly, patients with poor heart condition deserve to undergo surgery earlier, even if our study shows no significant association . Currently, specialist opinion is the third factor in waiting time determination (12.8%). It seems that managers should review their waiting time standards and change them to more important determinants . (22), in new zealand, today we witness a move to more explicit decisions . As noted, patient s economic condition is the only variable which significantly associates with waiting time . On the other hand, financial and personal problems are also important factors that cause waiting time elongation (28.2 and 18.6%, respectively). These facts highlight the role of the ministry of health, insurance and other supporting organizations in protecting privileged groups . Increasing health insurance makes the load of hospital costs and physician s informal payments more bearable . In addition to increasing health insurance coverage, waiting time for heart surgery can be further reduced by providing the cost of surgery . Implementing strategies, like in the for costs of patients injured in road accidents, considering psychological consultation and introducing technical and professional abilities, besides providing comfortable atmosphere, could be definitely useful . Freeman and denham (25) suggest three important principles for nurses to cope with the anxiety of patients and their relatives . Second, clear communication at the admission about the expected surgical time and potential problems that might lead to delays . Third, empowerment or use of a contract that establishes the nurses intent to see the patient goes to surgery at the scheduled time (25). (26) stressed that long waiting time is considered an independent and exclusive risk factor for patients dissatisfaction . They recommend that giving information about delay and its causes act as a right strategy in patient satisfaction . Reporting the economic condition by the patient is not reliable . Because of that, we used a summary measure composed of reported income and living amenities . Another limitation is concerns the standard of heart surgery waiting time, that is derived subjectively from expert opinions . Participatory management, with application of total quality management principles, could improve waiting time . In consistence with mossadegh - rad (27) opinion, it could be appropriate if the area of participative management, such as staff training, creating culture of participation by directors and employees, proper incentives and commitment of senior management is available . They believe that correcting patients perception about waiting time, through improving patient - physician interactions, greater understanding of patients about their care, and improving their waiting experience, are key elements in promoting patients satisfaction . Finally, we should not be unaware about the role of dm in heart surgery success rate . This finding underlines that we should, as soon as possible, determine the role of dm or controlling it in the prognosis of heart surgery . It is important that not all the diabetic patients mentioned previous history of myocardial infarction . Therefore, the fact that dm directly affect the prognosis, regardless of its role in predisposing to atherosclerosis, could be considered by clinical researchers (29). (30) assumed that the risk of coronary artery disease increases with the severity, rather than duration of diabetes mellitus . For a given level of severity of dm, the risk of cad did not differ by anatomic location, within the coronary tree.
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Civil unrest is not new to the united states or the world . In the period between september 11, 2001, and the anaheim police shooting in 2012, there were 21 nationally recognized incidents of civil unrest.4,5 since the police shooting of 16-year - old kimani gray in 2013 in baltimore, maryland,6 there have been 12 incidents of civil unrest in the united states, some lasting days or weeks . What may surprise many is that the incidence of civil unrest is far less than what was seen in the 1960s and early 1970s, a time when the country was stirred by what many viewed as an unjust war (the vietnam war).4,5 these decades were also a time when the nation was facing the reality that we lived in an unjust society that needed to address civil rights . The figure demonstrates that as a country, we have consistently experienced some level of civil unrest . The good news is that we historically have come out the other side as a more just and stronger country . The incidents range from relatively minor or major and from a few hours to multiday events.4,5 timeline of civil unrest in the united states . One author of this article has spoken to many people in ferguson since the death of michael brown . The 2 most striking lessons learned were the need to train street medics and the need to address the role of security in hospitals . During the time of civil unrest in ferguson, the deaconess faith community nurse ministries recognized that, unlike what often occurs in a natural disaster, there were not a large number of spontaneous unaffiliated or affiliated health care volunteers asking to participate in providing care . Consequently, they worked with the community to organize and host a street medic training for interested lay leaders and health care professionals in october 2014 . This was in preparation for a moral monday demonstration being held in the st louis region . The training session focused on preparing participants with the following information and skills: providing basic first aid for protestors, handling of contact with riot agents, dealing with emergent issues of people with preexisting health conditions (ie, asthma, hypertension, etc), and counseling of those impacted by the psychological trauma of being on the front line during protests . The deaconess faith community nurse ministries, under the leadership of its executive director, the reverend donna smith - pupillo, rn, is dedicated to improving and promoting the health of body, mind, spirit, and community throughout the st louis region.7 one of its guiding principles is to be a care advocate for the voiceless and marginalized in the health care system . Rev smith - pupillo shared with the article authors that it is a special call for a nurse to be able to use your skills to help people concerned about justice to further the greater good . However, she quickly pointed out that this type of response is not for everyone . Just as we see in almost every disaster, there are those who want to help, but when they get to the field where care is needed, they are not emotionally prepared for what they encounter . Whether a nurse is working in the acute care setting of a hospital or providing care in the community, personal security is an issue during civil unrest . R. bartram, eastern regional director of security of the mercy health care system, noted that during the civil unrest in ferguson, the safety of home health care workers needed to be addressed.8 one suggestion to help ensure the safety of these workers was to make all visits before noon because much of the unrest occurred during the evening hours . Other suggestions included rescheduling visits and, when appropriate, conducting a visit via telephone . The safety concerns of staff working in the acute care setting were different but equally significant . Leaders in the mercy security department stayed in contact with administrators throughout the hospital to communicate measures taken to ensure safety for the workers in the acute care setting.8 hospital nurse leaders and other administrators were acutely aware of the varying views of protestors . Because it was likely that, at some point, protestors, police officers, or both might present to the hospital for care, the administration and staff needed to know how to respond and to ensure provision of nursing care that demonstrated a respect for human dignity.9 for example, during the unrest in ferguson, 2 protestors presented to one of the local hospitals for care . The other was brought in by the st louis county police department to determine whether the person could be taken to jail without jeopardizing the individual's health.8 although the presence of security personnel at the hospital was increased during august and november 2014, staff had to be reminded of the purpose of the security personnel, as well as the rights of those protesting and picketing . It was not the role of the local police department to respond to protestors who engaged in legal activities, but they would respond if protestors engaged in illegal activities, such as blocking patients' access to the facility.8 it is important to remember that in such situations, the presence of police in the hospital can be viewed by protesters as adversarial, so their presence may make the situation more difficult to manage . These situations point out the similarities and differences between natural disaster response and actions best suited to the civil unrest . The 3 primary differences are the role of security, the availability of volunteers, and the importance of being the trusted patient advocate . In a disaster, likewise, the role of trusted advocate can help diffuse any tension that may exist within the health care setting between those who identify with the protesters and those who identify with the police . Trust is a key quality that nurses bring to the table during civil unrest . In the missouri event, nurse leaders in the local county health department worked with staff to ensure they were able to meet the community's needs . The main location of the saint louis county department of public health is approximately 2 miles from ferguson and serves many clients who live in that town or in neighboring communities . During the early days of civil unrest, these included delivering medications to residents when they were unable to leave their homes because of safety concerns . However, personnel did not go into ferguson or neighboring communities to conduct case finding in the immediate aftermath of rioting and violence.10 many families in baltimore have been impacted by decades of racial injustice, poverty, and multiple adversities . This has resulted in poor access to basic human needs such as employment, quality education, and healthy foods . These conditions are associated with an accumulation of exposure to traumatic events, compounded by circumstances that result in physical and mental health disparities . Ultimately, these lead to poor health outcomes.8,11 in april 2015, the death of an african american man injured while in police custody spurred several days of angry protest and civil unrest in baltimore city.12 this event sparked a strong response across the nation and forced sentiments to the surface that have been brewing among baltimore's citizens for decades . Fires were set in the city; assaults were perpetrated against police officers; and 7 community pharmacies were looted by thieves seeking opioid medications . The resulting burden upon the city hospitals and local health department was significant and long lasting . Between april 27 and may 8, 2015, the baltimore city health department was a lead agency in the unrest response and recovery activities . Emergency physicians and nurses participated in many facets of the disaster response . Just as occurs in an emergency medical situation, a public health code was eventually proposed as a model for centralizing, reacting to, and debriefing after situations of civil unrest.13 as nurses, we must all decide what role we will play during a crisis . To do this, we must each ask the following: can you respond?do you have family responsibilities that must take priority (dependent elder, young children)?will your job allow you time off?are you physically able to respond?are you psychologically prepared?are you trained?are you prepared for the possibility of being arrested? Do you have family responsibilities that must take priority (dependent elder, young children)? One nurse shared with the authors that she could not risk being arrested, or losing her job, and as a result not be able to feed her children . This was not an unusual response from nurses who wanted to take part in the protest because they felt the need to stand for justice . I invested this money in school so i'm not just going to throw that away because i want justice . I can be heard in a way that i can't get locked up . Another nurse leader, the reverend tracy blackmon, rn, was able to take vacation time because her place of employment was supportive of her work . On the contrary, a student nurse leader reported that she was told by angry community members she would never get a job . She stated some even tried to get her school to denounce her work rather than support her choice . Nurses must keep the focus on the humanity of the situation.3 we must ask ourselves if we will support our nurse leaders who choose to be active participants in protests . Disaster preparedness and response are a cycle that begins with planning and continues through recovery . In disaster response as the recovery drags on, the enthusiasm of the larger community and health care professionals diminishes long before the recovery is complete . During civil unrest, there are many who want to join the protest and/or support the protesters . As nurses, we must remember that long after the celebrities and the cameras have gone, the community and the nurses who live in the impacted communities need a constant presence and assurance that others in their profession are there to support them . Based on the lived experience of those writing this article, the people we have met along the way, the literature available, and the leaders who emerged during community crises, there are 10 musts in preparing for civil unrest . These include the following: training of hospital security in crowd control during riots.identification of local leaders to be trained for emergency management and as street medics.inclusion of local leaders and students in drills that teach how to stay safe during a riot.establishment of a communication plan for local leaders, street medics, and hospitals.2modeling of behavior by nurse leaders, who manage the largest workforce in the hospital and are trusted members of the community . (it is essential that the nurse leader model what they expect from other organizations.2)recognition of the intersection of complex issues by nurse managers who can then intervene with staff when there is a lack of understanding.planning of an orderly triage by enlisting the support of local and prominent community leaders.2planning for the use of mobile clinics or provision of home visits during the crisis.designing of hospital and community partnerships to help heal young people impacted by violence with case management, mentorship, and evidence - based trauma interventions.14training of all nurses in trauma informed care . Inclusion of local leaders and students in drills that teach how to stay safe during a riot . Establishment of a communication plan for local leaders, street medics, and hospitals.2 modeling of behavior by nurse leaders, who manage the largest workforce in the hospital and are trusted members of the community . (it is essential that the nurse leader model what they expect from other organizations.2) recognition of the intersection of complex issues by nurse managers who can then intervene with staff when there is a lack of understanding . Planning of an orderly triage by enlisting the support of local and prominent community leaders.2 planning for the use of mobile clinics or provision of home visits during the crisis . Designing of hospital and community partnerships to help heal young people impacted by violence with case management, mentorship, and evidence - based trauma interventions.14 training of all nurses in trauma informed care . Preparedness and training for civil unrest are essential to not only patient outcomes and emotional well - being but also organizational and community outcomes . Nurse leaders must recognize that managing patients and personnel requires being aware of and addressing myriad emotions and prospective on social issues that lead to civil unrest, which is above and beyond the issues of security, crisis communication, and alterations in practice necessary during a disaster . Nurses are members of a profession who can lead the way for humane, competent care even during times of civil unrest.
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They include sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming of energy metabolism and evading immune destruction 1 . The most common system for classifying the extent of spread of cancer is the american joint committee on cancer / union internationale contre le cancer (ajcc / uicc) tnm classification 24 . The tumour staging gives an estimation of the degree of tumour progression at the time of the surgical resection . The higher the degree of tumour progression, the greater the chance that the tumour will have undergone clonal evolution and acquired a set of unfavourable characteristics, such as the ability to invade lymphatic or blood vessels or to metastasize to distant sites . Furthermore, multiple tumour - cell parameters are an indication of the intrinsic biology of the tumour . The tnm classification has been used for over 80 years and is valuable in estimating the outcome of patients for a variety of cancers 24 . The union for international cancer control issued the seventh edition of the tnm classification guidelines . This static measurement integrating the tumour grade and stage is correlated with a dynamic process, such as the time to occurrence or death . The system is used in clinical trials to select patients who are eligible for inclusion, and in cancer registries to compare outcomes between different series, across different countries and over different time periods 5 . An accurate, stable internationally agreed staging system is essential to global progress in this disease . Its main aim should be to provide prognostic information and, based on this information, individual treatment decisions can then be made . In daily practice and in guidelines, the tnm category is directly linked to treatment strategies and, as such, changes in the tnm staging system have a considerable and direct impact on the cancer care that patients receive . This tnm staging system has stood the test of time but provides incomplete prognostic information . Clinical outcome can significantly vary among patients within the same histological tumour stage 5 . In some patients, advanced - stage cancer can remain stable for years, and although rare, partial or full regression of metastatic tumours can occur spontaneously 6 . In contrast, relapse, rapid tumour progression and patient death is associated with approximately 25% of tnm i / ii stage colorectal cancer (crc) patients, despite complete surgical resection and no evidence of residual tumour burden or distant metastasis 6 . Unfortunately, the predictive accuracy of the traditional staging system relies on the assumption that disease progression is a tumour cell - autonomous process . The focus of this classification is solely on the tumour cells and fails to incorporate the effects of the host immune response 7 . The phenotype of a tumour is not governed only by the epithelial component but also by the tumour environment, that is, other cells in contact with the tumour, the mesenchyme and the inflammatory infiltrate . These components determine the net inputs to the cell, which include ligands, cell cell adhesion molecules, metabolites, oxygen and multiple soluble factors 1 . Relatively little information is available on the spatial organization of key proteins and cells, although new imaging techniques offer the potential for high - resolution measurements of the spatio - temporal dynamics of large numbers of proteins 8 . The tnm classification was never surpassed in multivariate analysis by alternative methods such as immunohistochemistry for tumour biomarkers, flow - cytometry for dna content, molecular signatures or genetic features . However, it was shown that the analysis of a specific type of intratumoural immune response was indeed surpassing the tnm classification in multivariate analysis 9,10 . Thus, tumour progression has now to be considered as the result of a balance between an invasive tumour process and a defence system whose major component is constituted by the host immune response . These rely on tumour cell characteristics, including morphology, molecular pathways, mutation status, cell origin and gene expression - based methods, and allow the distinction of multiple, often overlapping, subtypes . Adeno nos, adenocarcinoma not otherwise specified; transit - amplifying - r, transit - amplifying - resistant; transit - amplifying - s, transit - amplifying - sensitive; cin, chromosomal unstable; msi, microsatellite unstable; cimp, cpg island methylator phenotype . (bottom) classification based on the host immune response using the immunoscore . A morphology - based classification a number of histological variants are described: mucinous, signet ring cell, medullary, micropapillary, serrated, cribriform comedo - type, adenosquamous, spindle cell and undifferentiated . Crc is molecularly defined into three groups: chromosomal unstable (cin), microsatellite unstable (msi) and cpg island methylator phenotype (cimp). Most cases arise through the cin pathway, with imbalances in chromosome number and loss of heterozygosity . These cancers have accumulation of mutations in specific tumour suppressor genes and oncogenes that activate pathways critical for crc initiation and progression . A high degree of msi (msi - high) is present in 15% of crcs and represents a specific type of genomic instability characterized by frequent microsatellite length mutations . Frameshift mutations in microsatellite instability high (msi - high) colorectal cancers are a potential source of targetable neo - antigens 11 . Most msi - high cancers are caused by epigenetic silencing of a mismatch - repair gene (mlh1). This silencing typically occurs in tumours of the cpg island methylator phenotype (cimp - high) 12,13 . Cimp - high represents a specific type of epigenomic instability that is characterized by widespread promoter cpg island methylation and epigenetic gene silencing 15 . Nonetheless, different from the msi subgroup, cimp - high patients have similar characteristics to msi, and have been associated with old age, female gender, proximal tumour location, poor tumour differentiation, braf mutation, wild - type tp53, high - level of global dna methylation and stable chromosomes (cin) 12,13 . Msi tumours have a more favourable prognosis and are less prone to lymph node or distant metastasis 16 . Cimp - high might be a prognostic marker independent of the presence of msi and braf mutation 17 . A third method to classify crcs is based on mutation analysis . Following the discoveries from bert vogelstein et al that tumours result from the sequential accumulation of alterations in oncogenes and tumour suppressor genes and the appearance of driver mutations, several mutations are now regularly tested in crcs . These include the apc, kras, tp53, braf, nras, pi3kca and ctnnb1 genes . Somatic mutations in codons 12, 13 and 61 of kras, and more recently nras, predict innate resistance to mabs targeting epidermal growth factor receptor 1820 . The braf v600e mutation is an adverse prognostic factor in stage iv colorectal cancer but does not have clinical utility at present 21 . The fourth and fifth methods, assessing the cell of origin and gene expression, respectively, to classify crcs are molecular based techniques . Using this approach, six groups of crcs were recently defined, based on similarities with distinct cell types within the normal colon crypt and the response to classic chemotherapy and targeted therapies 22 . Other gene expression - based analysis described three groups of crc patients 23, which correlated with two of the known molecularly defined groups, namely the msi (renamed ccs2) and the cin (ccs1) groups, whereas the third group corresponded partly with the cimp group (ccs3). Further analysis of precursor lesions (serrated polyps) suggested that this latter group could be derived from the serrated pathway . Some partial overlap exists between both proposed gene expression - based classifications, especially with respect to the group that is defined by the msi - high phenotype . Other gene signatures were also reported and, strikingly, most of the genes were unique to each signature . Interestingly, both profiles emphasize the importance of zeb1, a transcription factor regulating the epithelial however, emt in crc is generally not a feature of the whole tumour but is seen at the invasive margin (i m). The stroma of human colorectal tumours was shown to contain twist1-positive cancer cells with mesenchymal phenotypes 25 . Emt is characterized by tumour cell budding, nuclear expression of -catenin, loss of cdh1 (e - cadherin), gain of cdh2 (n - cadherin) and alteration of other epithelial cell adhesion molecules 26 . These features are often not molecularly defined but the result of interplay between the tumour and the microenvironment . Thus, gene expression classification may be prone to bias, due to different percentages of tumour cells in the sample, but also due to lack or uncontrolled presence of the i m in the sample used for rna extraction . These issues were not addressed in either study 22,23 . Because of the limitation of gene expression signatures, and to facilitate implementation and testing in large patient series unfortunately, half of the patients turned out to be unclassifiable 22 and the absence of cdx2 positivity in all ccs3 samples (25% of crcs) 23 does not correspond with extensive literature data (98% positivity in crcs) 27 . The carcinogenic process that gives rise to an individual tumour is unique . It is postulated that tumours with similar characteristics share common pathogenic mechanisms and progression patterns . However, other major parameters have to be taken into consideration, in particular the tumour microenvironment . Importantly, neoplastic cells interact with host non - neoplastic cells (including immune cells) and extracellular matrix in the tumour microenvironment, and those components influence each other and modify an integral phenotype of any given tumour 28 . Many markers, signatures and methods have been described to evaluate the prognosis of cancer patients, yet very few such markers and laboratory assays translate into clinical practice or reach the statistical power of the tnm classification . Modern classification of tumours is based on the recognition of disease entities that are characterized by morphological, phenotypical and genetic markers . Each classification system needs to be reliable, reproducible, clinically relevant and biologically meaningful . First, the inevitable presence of non - neoplastic cells, including immune cells, in tumour areas means that dna (or rna) from the tumour area is not contaminants, often> 50% of non - tumour cells, may in fact have a profound biological meaning . Thus, the degree of immune cell infiltration may correlate with tumour molecular changes or may mask a correlation between a tumour marker and the outcome, because contaminating non - neoplastic cells can influence the results of a tumour molecular assay . For sensitive mutation detection, sequencing technologies allow the detection of approximately 5% of mutant alleles . For quantitative dna methylation assays, this is even more problematic with gene expression signatures, where a different degree of non - neoplastic cells is observed among the assays . Second, a precise characterization of specific tumour - infiltrating cells requires the use of an immunohistochemical technique to detect in the tissue the presence and localization of specific antigens expressed by subsets of immune cells . The evaluation of immune cells in haematoxylin and eosin (h&e)-stained sections can be done at a low cost compared to an immunohistochemical technique . However, an evaluation of a specific subset of immune cells is generally not possible with h&e - stained sections . For example, lymphocytes with opposite functions, such as cd4 t cells with th1 orientation versus th2 orientation versus immune cells with regulatory functions (treg cells), or nk cells, nkt cells, b cells or cytotoxic cd8 t cells, are indistinguishable without proper marker evaluation and require antibody labelling by immunohistochemistry . Nowadays this is simple, as specific antibodies with high affinity and a high signal: noise ratio allow the detection of these immune cell subpopulations with a single staining . Third, it is known that immune cells are scattered in the core of the tumour (ct) within the tumour stroma and the tumour glands, in the invasive margin (i m) and in organized lymphoid follicles distant from the tumour . A statistically significant correlation between immune cell density in each tumour region (ct and i m) and patient outcome has been shown in colorectal cancer 9 . Further, the combined analysis of tumour regions (ct plus i m) improved the accuracy of prediction of survival for the different patient groups compared with single - region analysis 9 . Given the major clinical importance of distinct tumour regions, it is appropriate to conduct immune cell infiltration evaluation systematically in the two separate areas, the core of the tumour and the invasive margin 9,10 . For routine practice, this requires immune cell evaluation on whole - tissue sections, taking into account their location . Fourth, objective ways of counting immune cells need to be achieved in order to remove the subjectivity of field selection and imprecise semi - quantitative evaluation . Most of the tumour markers are generally more complicated to quantify than immune cells, since only a fraction of the tumour cells express the antigen and the staining intensity has often been taken into consideration . Standardized and reproducible measurement of the intensity of staining, and hence quantitation of protein expression, is intrinsically difficult using immunohistochemistry . In contrast, immune cell types are easier to quantify because well - characterized markers exist, giving complete membrane staining for immune cell subpopulations (eg cd3 for t cells), allowing counting cells as individual cells . Nowadays, digital pathology and image analysis software can detect stained immune cells and determine their densities (n cells / mm) in histological sections . Validation studies demonstrated the high concordance of these automated systems in comparison to optical counts 9,29 . This is particularly important to facilitate routine pathology and to speed up the process of quantification . In particular, given the huge number of infiltrating immune cells within a tumour (eg a mean of 75 000 cd3 cells present on a 4 m - thick section of tumour slide from a crc stage i / ii patient), it would be unrealistic to ask pathologists to count them all . In addition, immune cells such as cd3 lymphocytes are often aggregated in complex cell clusters . Algorithms for segmenting clusters of densely packed cells permit a precise counting of cells . Given the major importance of the determination of the density of immune cells 9,30 to predict a patient's outcome, it is now required to take advantage of the digital pathology to determine the exact count of stained cells and the surface of the tissue analysed . Fifth, the heterogeneity of a tumour applies to tumour cells but to the microenvironment as well . Upon evaluation of whole - tissue sections the selection of the tissue areas to study may depend on subjective interpretation, and the evaluation needs to be validated by a second independent observer blinded for the other results . The use of computer - assisted image analysis provides important advantages, as all the fields are analysed in the whole tumour section 32 . For example, the determination of the mean density of stained cells in a tumour region is supported by an objective computer - based cell - counting method, leading to a good level of reproducibility between users . A growing body of literature 9,10,30,3336 supports the hypothesis that cancer development is influenced by the host immune system . This may offer powerful prognostic information and facilitate clinical decision making regarding the need for systemic therapy 7 . Accumulat - ing evidence suggests that cd3 9,10,30,34,3743, cd8 9,10,34,38,4352 and cd45ro 9,10,34,40,47,49,53,54 cells have roles in antitumour immune responses . Collected from large cohorts of human crcs (with sample sizes n = 415, 599 and 602, respectively) demonstrated that the number, type and location of tumour immune infiltrates in primary tumours are prognostic for disease - free survival (dfs) and overall survival (os) 9,10,30 . Notably, several large studies of crcs (with sample sizes n = 843 and n = 768, respectively) have shown that tumour lymphocytic reaction and t cell subpopulations (cd8, cd45ro, foxp3) are significant prognostic biomarkers, even after adjusting for stage, lymph node count and well - established prognostic tumour molecular biomarkers, including microsatellite instability (msi), braf mutation and line-1 hypomethylation 54,56 . A possible msi tumours often contain intra - epithelial t cells in response to the expression of neo - antigens on the cell surface 42, in particular those that do not undergo non - sense medicated decay 11 . Furthermore, crcs from a large cohort (n = 1197) and external validation (n = 209) confirmed the prognostic value associated with cd3, cd8 and cd45ro (ptprc) t cell infiltration in crcs 43 . A recent meta - analysis 55 summarized the impact of immune cells, including b cells, nk cells, mdsc, macrophages and all subsets of t cells on clinical outcome from more than 120 published articles . Importantly, the beneficial impact of the immune infiltrate with cytotoxic and memory t cell phenotype has been demonstrated in cancers from diverse anatomical sites; including colorectal cancer but also malignant melanoma, lung, gastric, oesophageal, head and neck, breast, bladder, urothelial, ovarian, cervical, prostatic and pancreatic cancer, hepatocellular carcinoma, medulloblastoma and merkel cell carcinoma 55 . It is interesting to note that the implications of this immune phenotype apply not only to various organs of cancer origin, but also to various cancer cell types, ie adenocarcinoma, squamous cell carcinoma, large cell cancer and melanoma . Thus, general characteristics emerge in which cytotoxic t cells, memory t cells and th1 cells are associated with prolonged survival 35,57,58 . The prognostic impact of other immune cells, such as b cells, nk cells, mdscs, macrophages and a subset of t - helper populations (th2, th17, treg cells), differ depending on the type of cancer and on the cancer stage 55 . Altogether, the publications indicate that a precise analysis of the immune component of the tumour microenvironment by computer - based analysis may be essential to managing patients better . Thus, precise analysis of the tumour microenvironment by pathologists may be essential for future clinical implementation and better patient management . An expertise in this new emerging field is now warranted to translate it into the clinical practice . A potential clinical translation of these observations is the establishment of a scoring system designated immunoscore (figure 2), derived from the immune contexture (figure 3) 33,35,55, and based on the numeration of two lymphocyte populations (cd3/cd45ro, cd3/cd8 or cd8/cd45ro), both in the core of the tumour (ct) and in the invasive margin (i m) of tumours, as a clinically useful prognostic marker in colorectal cancer 34 . Detailed description of the immune contexture in comparison to the immunoscore has been described previously 35,36 . The immunoscore provides a score ranging from immunoscore 0 (i0) when low densities of both cell types are found in both regions, to immunoscore 4 (i4) when high densities are found in both regions . This test has a dual advantage: first, it appears to be the strongest prognostic factor for dfs, disease - specific (dss) and os, including at early - stage colorectal cancers; and second, it has biological meaning (adaptive immune response to tumours) and provides a tool or a target for novel therapeutic approaches, including immunotherapy (as recently illustrated in clinical trials boosting t cell responses with anti - ctla4, anti - pdcd1 (pd-1) and anti - cd274 (pd - l1) 5962 . Thus, considering the probable universal character of the immune control of tumours, it is essential for patients to take into account the immune parameter as a prognostic factor and to introduce the immunoscore as a component of cancer classification 6,7,35,63,64 . (bottom) immunohistochemistry of a colorectal tumour stained for cd3 t cells (brown). The immunoscore was shown to be very powerful, for instance, in crc patients with clinically localized colorectal cancer, with no detectable tumour spreading to lymph nodes or distant organs . These patients are usually treated only by a surgical removal of the tumour; however, approximately 25% of these patients will have recurrence of their disease, indicating that occult metastases were already present at the time of curative surgery . No tumour - associated marker predicts the recurrence of this subgroup of patients that could have a benefit for an adjuvant therapy . The immunoscore (i) approach (with an enumeration of cd8 and cd45ro cells in the ct and the i m) was applied to two large independent cohorts (n = 602). Only 4.8% of patients with a high i4 relapsed after 5 years and 86.2% were still alive . In contrast, 72% of patients with a low score (i0 and i1) experience tumour recurrence and only 27.5% were alive at 5 years . These i0 and i1 patients could potentially have benefited from adjuvant therapy if the immunoscore had been incorporated into the tumour staging 34 . The immunoscore classification, demonstrating the prevalence of immune infiltrates, was shown to have a prognostic significance superior to that of the ajcc / uicc tnm classification system . For all patients with crc stages i / ii / iii, multivariate cox analysis revealed that the immune criteria remained highly significantly associated with prognosis (dfs, dss, os). In contrast, the histopathological staging system (t stage, n stage and tumour differentiation) was no longer significant 10 . Indeed, the immune pattern remained the only significant criterion over the classical ajcc / uicc tnm classification for dfs and os, and led to an editorial entitled tnm staging in colorectal cancer: t is for t cell and m is for memory accompanying the publication by mlecnik et al 10,65 . It has thus been suggested that the prevalence of tumour immune infiltrates, more than the tumour status, could be a key indicator for recurrence, metastasis and therefore clinical outcome (figure 4). These results suggest that once human cancer becomes clinically detectable, the adaptive immune response plays a critical role in preventing tumour recurrence . The ability of effector - memory t cells to recall previously encountered antigens leads to a protective response . Following primary exposure to antigen, memory t cells disseminate and are maintained for long periods of time 66 . The trafficking properties and the long - lasting antitumour capacity of memory t cells could result in long - term immunity in human cancer . Over the past few years, the area of immune regulation at the level of the tumour microenvironment has gained a forefront position in cancer research, in crc 9,10,30,34,55, melanoma 67 and all other cancer types 7 . The immunoscore, initially described several years ago 9 as a prognostic factor 10,34, could also play a role as a marker to predict the response to biotherapies targeting the immune checkpoints 63,64, schematic representation of cancer classification based on tumour - microenvironment - related parameters (immunoscore), based on tumour extension and invasion (t, n, m stages), and based on five alternative tumour - related methods . The inherent complexity of immunohistochemistry, in conjunction with protocol variability, contributes to the variability of the results obtained large - scale assay harmonization efforts have already been witnessed, conducted for commonly used immunological assays of peripheral blood immune cell populations 68,69 . A clinical validation of the immunoscore with standardized procedures is necessary to reach clinical applicability for individual patients . We performed multiple immunoscore quality controls to test the accuracy and repeatability of the method . We first observed that the automated cell - counting method achieved a very good level of correlation with the optical counting for cd3 and cd8 immunostainings and an excellent reproducibility of the count of stained cells . In addition, the variablity between users for the determination with the software of the immune cell densities in tumour regions was 5.2% and 2.5% for cd3 and cd8, respectively (unpublished data). All the tests indicated a reliable assessment of the immunoscore . To be used globally in a routine manner, evaluation of a novel marker should have the following characteristics: do - able in routine, feasible, simple, inexpensive, rapid, robust, reproducible, quantitative, standardized, pathology - based and powerful . Importantly, for patients with rectal cancer treated by chemoradiotherapy (pcrt) before surgery, preoperative chemoradiation therapy induces histological reactions precluding the realization of an immunoscore, since the delineation of the analysed tumour regions (ct and i m) is often no longer practicable . Also, assessment of anti - tumour immunity by the immunoscore is inappropriate in biopsies, since the limitation of the material precludes a precise delimitation of the tumour and the invasive margin . Characteristics of a good biomarker and of the immunoscore . To assess the immunoscore in clinical practice and measure its prognostic value, we are conducting a prospective multicentre study (french phrc programme) for 600 patients from seven hospitals bearing a colorectal cancer treated by primary surgery . In an effort to promote the utilization of the immunoscore in routine clinical settings, we initiated a worldwide immunoscore consortium, with the support of the society for immunotherapy of cancer (sitc) 64 . Several thousands of tumours from patients will be immunoscore - tested by the 23 centres from the worldwide consortium . The worldwide immunoscore consortium, composed of international expert pathologists and immunologists, identified a strategy for the organization of worldwide retrospective study for the validation of the immunoscore in colon cancer . Because of background staining and loss of antigenicity in stored sections (cd45ro) and granular staining (gzmb), it was decided to employ the two easiest membrane stains, cd3 and cd8 . Thus, the combination of two markers (cd3 and cd8) in two regions (ct and i m) was agreed for validation in standard clinical practice . Precise quantification is currently performed on whole slide sections (figure 2), following the recommended initial guidelines . The purpose of the ongoing immunoscore worldwide consortium is to validate the following points: first, to demonstrate the feasibility and reproducibility of the immunoscore; second, to validate the major prognostic power of the immunoscore in routine for patients with colon cancer stages i / ii / iii; and third, to demonstrate the utility of the immunoscore to predict stage ii colon cancer patients with high risk of recurrence . These participants represent 23 centres from 17 countries, including asia, europe, north america, australia and the middle east (australia, austria, canada, china, czech republic, france, germany, india, italy, japan, the netherlands, qatar, spain, sweden, switzerland, the uk and the usa). It is hoped that this initiative will result in the implementation of the immunoscore as a new component for the classification of cancer, tnm - i (immune). The immunoscore should better define the prognosis of cancer patients, better identify patients at high risk of tumour recurrence 70, help to predict and stratify patients who will benefit from therapies 35 and, ultimately, help save the lives of patients with cancer . Jg is coordinating the worldwide immunoscore initiative, conceived the study and wrote the manuscript; jg and fp initiated the immunoscore project; bm, gb, fp, cl, ab and jg performed the initial experiments related to the immunoscore; fp and hka participated in the drafting of the manuscript . Al, cb, gb, ft, pd, ah, ma, ll, mm, fg, fp, fmm, baf and jg were experts involved in the design of the immunoscore study and expert pathologists participating to the inaugural immunoscore workshop . All authors, jg, bm, gb, hka, ab, cl, al, iz, ah, cb, idn, rp, gvm, gb, ft, pd, mm, ll, fg, ma, cda, fvv, ez, lc, pso, shb, bgw, mr, ln, yk, sh, ko, so, pg, pw, ns, tt, ky, psp, sns, yw, sk, fas, paa, fmm, baf, vs, fp, are participants of the initial worldwide immunoscore task force study and have read and approved the manuscript.
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Genetic testing is a tool that reads the dna sequence of genes to identify variations . Not all variations have a noticeable effect on health or other traits, and a great deal of research is devoted towards identifying those that do . Over the last decade, the price of sequencing has dramatically decreased while the number of known genetic variants with human health associations has grown . As a result today, genetic tests can be used to screen a patient's risk of developing cancer or genetic diseases, for non - invasive early prenatal testing or to identify the likelihood of rare genetic diseases . At the time of writing, clinvar, a user - generated database of genetic variations hosted by the national institutes of health, contains over 100,000 variations in more than 6000 genes . As genetic testing becomes more integrated into everyday clinical practice, more attention has been placed on the regulatory structure overseeing this field . The fda has traditionally regulated clinical genetic testing in a patchwork approach depending on the type of test offered . Tests in which a physician sends a sample to a laboratory for analysis by scientists are classified as laboratory - developed tests (ldts). The fda claims jurisdiction over ldts, but historically has exercised enforcement discretion, choosing not to regulate them . However, if a non - physician orders an equivalent test, the fda classifies it as a direct to consumer test and as such is subject to pre - market review, even if the test is otherwise identical to an ldt . Finally, the fda considers kits that enable testing to be done by non - experts in vitro diagnostics (ivds), subject to pre - market review and approval . These classifications have created an inconsistent regulatory landscape where oversight of genetic testing is determined not by the risk of the test, but by the customer and method of testing . The fda has recently issued draft guidance on new regulations for ldts, intending to subject them to pre - market review and approval . These guidelines call for measures including but not limited to pre - market approval, test registration, adverse event reporting, and establishment of quality control systems . Ldts will be stratified into three tiers based on risk, with the level of pre - market review varying by tier . Risk will not be based on the complexity of the test, but on its use and thus the harm to the patient if the test should give either a false positive or negative result . This pre - market review is intended to evaluate the analytical and clinical validity of ldts, which are not assured by current oversight . A test is analytically valid if it correctly identifies the sequence, copy number, or other marker being examined . Clinical validity describes the ability of that marker to correctly predict or detect the clinical condition for which the test is intended to screen . In some sense, these proposed regulations bring regulatory coherence to genetic testing: all forms of genetic tests will be subject to pre - market approval by the fda . This harmony is part of the motivation behind these regulations, as the fda has expressed concern that manufacturers of ivds will attempt to classify themselves as ldts to avoid fda regulatory oversight . However, whether the fda even has the regulatory authority to regulate ldts has stirred controversy . Since ldts are not physical items for sale but a process performed by researchers in a laboratory, proponents claim that ldt testing methods should qualify as a practice of medicine not subject to fda authority; this interpretation may also have support in legislative intent . Despite these claims, this analysis will assume fda authority over ldts and focus on the degree to which the fda should regulate them . This will involve examining existing oversight of these tests, their risks, and the extent to which the fda's guidelines address these risks . This paper will close by examining alternatives that may be more suitable for regulating this industry . Currently, the primary regulatory body for laboratories that perform genetic testing is the center for medicare and medicaid services (cms). However, cms does not examine the safety of the test nor does it require demonstration of the clinical validity of the test . In other words, cms certifies the qualifications of the laboratory and the testing methods, but it does not make assurances about its applications to the clinical setting . Clia certification then can be seen as an indication of laboratory quality . Certifying the methods in a laboratory means a lab does not have to seek approval each time it develops a new test or improves existing methods . So long as these changes are validated and properly documented, clia standards are satisfied . This regulatory focus on a laboratory's analytical methods instead of specific review of each test is an approach to managing risk that eliminates a layer of oversight between innovation and the patient . The national institute of standards and technology, the cdc, and commercial vendors have developed quality control standards that laboratories may use to validate the accuracy of their methods . Additionally professional societies, such as the american college of medical genetics and the clinical laboratory standards institute play an important role in drafting guidelines for clinical use of genetic tests . Finally, the college of american pathologists runs a laboratory accreditation program, which requires member laboratories to undergo proficiency testing to certify their performance . While these guidelines and standards are beneficial for participating laboratories in verifying their quality and methods above clia standards, there is no requirement to follow them or receive accreditation before listing a test . Beyond the voluntary nature of accreditation and self - regulation, in addition to safety and efficacy concerns, cms does not require (1) registration of available ldts, (2) demonstration of clinical validity, (3) adverse event reporting, (4) any mechanism or quality system assurance to demonstrate safe manufacture of tests, or (5) post - market tracking of ldts . The fda believes that these problems lack of assurance around safety and clinical validity plus the lack of adverse event reporting create a regulatory gap that necessitates fda intervention . As a rationale for increasing regulatory scrutiny of ldts, the fda cites the changing market landscape for these tests . The fda argues that enforcement discretion was a viable model when the tests were simple and more locally applied, but the growth in scale, complexity, and importance of these tests necessitate increased oversight . To continue with minimal fda oversight, the fda argument goes, would leave regulatory gaps that could lead to patient harm . An inaccurate result could very well shape a physician's diagnosis and treatment recommendations, and so the fda is rightfully concerned that these results should be analytically accurate . Beyond just ensuring that tests can report variants accurately, the fda also wants to ensure the clinical validity of its tests . These tests can shape medical, personal, and lifestyle decisions due to their ability to screen for or predict genetic diseases . Genetic tests currently inform decisions such as whether to terminate a pregnancy, undergo a preventative mastectomy, or adjust drug dosages based on genetic sensitivity . It is important to make sure the implications of a genetic variant are known and understood to avoid misapplication or misinterpretation of a finding . There is an additional risk from uncertainty . The impact of a genetic variant on a condition (its effect size) can often be quite small because of interactions with other genes and the environment . The probabilistic nature of some genetic tests can make demonstrating the utility of these tests difficult . It may be difficult for physicians to make diagnoses or inform clinical decisions based on measures with small predictive value . The potential for mitigating effects of genetic background, gender, or lifestyle make it difficult to know how the effects of genetic variants reported in the literature apply to other patient groups . These are risks of clinical utility, or how useful the results of genetic tests will be to physicians . While the fda is not addressing clinical utility in its guidance, it is an important consideration . Because most of these risks deal with the inherent uncertainty of genetic testing, they also carry an additional risk of potentially unnecessary economic costs to the healthcare system . Traditionally, the fda's regulatory philosophy is to operate under the precautionary principle, which mandates the provider of a product to first prove that the product is not harmful before being marketed and sold . In most cases, this requires that manufacturers demonstrate both the safety and efficacy of drug and device products before they can be used in patients . This perspective errs on the side of rejection or delay of beneficial treatments in order to prevent approval of an unsafe or ineffective treatment . In other words, there may be delays in new treatments or fewer treatments overall, but we can be confident that the treatments that do exist are safe and efficacious . The precautionary principle is highly appropriate for traditional drugs and devices for several reasons: (1) there are large potential risks to patients from untested products, (2) the products are usually for general application and so the risks can be examined in a small number of targeted studies, (3) the market delay imposed by regulation is proportional to the lengthy time a product is in development, (4) the path towards demonstrating safety and effectiveness is achievable and often clear, and (5) the product is not expected to change once it is in the market . Each of these qualities contributes to a large but knowable risk to patient safety that merits caution . Overall, this approach works well in fields like small molecule therapies or implantable devices where moderate innovation disruption is offset by prevention of large and real risks . Genetic testing differs from products regulated using the precautionary principle . Differences include: (1) high analytical accuracy of genetic tests used to inform / screen rather than provide diagnosis; (2) clinical findings are often probabilistic and interpretation is individualized; (3) effect sizes from tests are often extremely low and interpretation is subject to change over time; (4) genetic testing is more rapidly changing with short development times new variants are continuously discovered and testing methods can be quickly improved; and (5) genetic tests involve manual procedures and thus methods will vary across each laboratory . Each of these differences either lowers the risk of a genetic test or makes proof of efficacy more difficult and burdensome relative to development time . In a patent - protected industry that rewards compliance with a legal monopoly and pricing power, the fda approach is an appropriate fit . Existing players have sufficient resources to meet fda criteria or find investors who can expect high margins if a product is approved . Genetic testing by contrast is marked by competition and in most cases tests are performed in in - house laboratories with limited ability to scale . Overall, the fda's approach is mismatched with the current state of genetic testing, and it risks undermining the entire ldt industry . The fda's proposed ldt regulations could have myriad effects on the genetic testing industry . Thousands of laboratories compete to develop new tests or apply new techniques allowing for rapid innovation . For example, clinical laboratories developed an hiv screening test two years before an fda - approved kit became available . As mentioned above, our knowledge of clinically relevant genetic variants and methods for testing is continuously expanding . Patients directly benefit from this rapid transition from discovery to the clinic . By compelling a pre - market review, the fda will slow down laboratories ability to provide physicians with tests based on genetic research . While true of any form of pre - market regulation, the effects are amplified with genetic testing due to the pace of discovery and improvement in the field . The human genome has tens of thousands of tests that can potentially be offered, if one were to develop one sequencing test for each gene . Rather than requiring a general ability to sequence genes correctly, the fda's draft regulations ask for validation of individual tests . By current estimates, there are more than 11,000 laboratories authorized to perform ldts, and genetic tests for more than 1200 conditions are available today . Some genetic tests examine multiple loci at once, and a requirement to validate each site increases the regulatory burden commensurately; this will discourage the use of new, powerful parallel sequencing technologies that allow for multisite testing . If every modification of a testing method or substitution of a gene panel requires a new submission, patients and physicians may have to wait months or longer for new tests . In a field where science updates continuously, there is a risk of a widening lag between the latest science and latest medicine . Worse still, this regulation could create disincentives for laboratories to improve or update their methods because scarce resources will only allow submission and resubmission for a limited number of tests . As an example of this, the fda - approved hiv test mentioned above has changed little since initial approval because of regulatory cost of updating the test . Ultimately, such a system could reshape the industry into one more closely resembling pharma with fewer players and high costs, thus negating much of the upside of the ldt model . Further adding to the worry about disruption, the fda has not yet declared whether it will expand its staff to accommodate the more than 11,000 genetic tests that will need to be reviewed and approved (to say nothing for the other forms of ldts). The fda will have a herculean task once it assumes this responsibility, increasing its workload significantly . This expansion would come when other efforts at the fda are underway to expand its reach into mobile health applications and clinical decision software . It is difficult to see how the fda can ensure speedy review of ldt tests while not extending the time frame of other 510(k) reviews . The length of 510(k) reviews has already increased by 25% from 2008 to 2012, and review times for pathology ivds, a division that may be the best fit for absorbing ldt review, took an average of 324 days to review in 2012, more than twice the length of the average device . Altogether, it is difficult to believe that the fda will be able to review these tests in a timely manner . An additional risk of increased fda regulation is the effect it will have on the genetic testing industry as a whole . Currently, many genetic tests are provided by institutional laboratories charging for testing as a service . Because of the uncertain clinical utility of many tests, it is currently difficult for many genetic tests to be reimbursed by payers, further eroding margins . It is likely that the financial burden imposed by submitting these tests for approval will cause many of these tests to be pulled from the market and may even force some labs to be shut down . These regulations may have the unintended consequence of reducing competition that drives the market, further slowing innovation and raising prices for patients . Genetic testing informs serious medical decisions and this has prompted the fda to act in an attempt to guarantee their analytical and clinical efficacy . However, reports about harms from genetic testing often do not focus on the accuracy or marketing of a test, but the misuse or misinterpretation of these tests by physicians and patients . Many genetic tests may invariably produce ambiguous or uncertain results, which may have serious consequences for the patient . The fda hopes to protect the patient by assuring the analytical and clinical efficacy of genetic testing, but this focuses attention on the wrong part of problem . Genetic counselors from arup laboratories reviewed physician - ordered genetic tests and found that around 30% of these were incorrectly ordered . Inappropriate clinical application is one of the biggest risks of genetic testing, and it cannot be solved by fda action . This is a problem with the practice of medicine, and can only be addressed through better education and guidelines for physicians on how to apply these tests to clinical care . Attention to the validity of the tests, typically quite high, is a distraction and will divert attention and resources away from the larger issue of better incorporating these tests into care . Not only will the fda's actions reduce the number of overall tests and providers and slow the pace of innovation, but it may also shift the focus of regulators and professional associations towards compliance and away from solving this larger problem . Even with clia oversight and self - regulation, it is clear that regulatory gaps remain, and the fda should be commended for attempting to fill these gaps using the tools it has available . Weighing the clinical risks from untested analytical and clinical validity and the practical risks from the proposed regulations, a strong argument can be made that the proposed ldt regulations will do more harm than good . These regulations also fail to address some of the larger problems in genetic testing around improving clinical utility, physician education, and how to most appropriately incorporate these tests into the practice of medicine . Regulation itself imposes systemic risks and consideration of its use should attempt to strike a balance between options . As a regulatory body, the fda's actions directly shape patient and physician options, and so the fda's regulations should be tailored to ensure safe and effective products without overly burdening test providers . A more appropriate regulatory structure for genetic testing would address the larger risks of inappropriate testing or interpretation while preserving the nimbleness and competition that make it a beneficial market for consumers . The fda should leave in place its proposal to register and list all genetic tests, an effort that was already underway, led by the national institutes of health . An alternative to requiring pre - market approval for each genetic test would be to implement a genetics licensing program that qualifies laboratories offering genetic tests generally . The fda should work with cms to expand laboratory clia certification beyond analytical methods to include regular analytical validity audits of a representative sample of tests, and require laboratories to enroll in a proficiency - testing program to retain their license . This alone should do much to ensure analytical validity while imposing a lesser burden; a cdc study found validity of tests highly correlated with whether a lab participated in proficiency testing . Cms can incentivize participation by making medicare and medicaid reimbursement for tests contingent on whether it was performed in a licensed lab . Under this model, once a lab is cleared to offer genetic tests, they only need to register a new or updated test when it is offered . Overall this would ensure analytical quality from laboratories without spreading their resources too thin, protecting smaller providers and thereby maintaining the competitive nature of the industry . Further proficiency testing standards must be developed, but regulators could leverage existing tests and partner with nist and professional organizations to expand existing programs rather than creating new ones de novo . Without pre - market approval for each individual test, clinical concerns would need to be addressed in some other manner . Scientific literature is currently one source for demonstrating clinical validity, and the fda has indicated receptiveness to using it in lieu of trials for pre - market review . Thus, the only new data in an fda submission may be proof analytical validity . If clinical validity can be demonstrated with existing data, and a licensing model is enough to ensure high analytical validity with lower burdens, why is pre - market approval necessary in the first place? Licensed laboratories could continue to market products much as they are today and test registration can publicly reference scientific literature to demonstrate clinical validity . The fda can review these claims at its leisure in collaboration with the ftc and monitor for fraudulent marketing . Regulators would not be alone; the current competitive environment of ldts creates incentives for the industry to monitor itself and report its bad actors . Overall, addressing regulatory gaps through registration, laboratory licensing, and expansion of clia harnesses existing industry dynamics to reinforce regulations with a small regulatory burden . Proposed guidelines drastically alter the industry landscape to achieve compliance, which risks undermining many of the industry's benefits . The final problem that still remains unaddressed is that of clinical utility . In many cases, the clinical utility of a test many tests are incorrectly ordered by physicians and not properly integrated into standards of care . However, these are problems with the practice of medicine and will not be solved by increased regulatory oversight . In fact, the fda's increased oversight may only distract from improvement in this area by forcing resources to be allocated towards fda compliance . Genetic testing is in an area with substantial knowledge gaps; both regulators and providers have acknowledged this issue . While cms is in the best position to address these gaps using the existing clia framework, it has shown a reluctance to do so and the fda has stepped in . Nonetheless, it is hard to believe that the tools available to the fda are a better fit for the task . While the fda's goals are admirable in trying to bring regulatory coherence to the genetic testing industry, the fda is imposing an undue regulatory burden that will change the industry in a way that harms providers and patients while not addressing the greatest problems . These problems are best solved by better clinical guidelines from medical associations, increased proficiency testing from laboratories (through cms and self - regulation), and reliance on the latest peer - reviewed research which will allow this industry to stay innovative, agile, and open.
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The prevalence of obesity in early childhood has shown some signs of decreasing; however, for the hispanic population, it continues to remain high, estimated at 16.7% for 25-year - old hispanic children in the united states, double that of the general population . There are few effective interventions that have enrolled obese, hispanic children from this age group . There are also very few prevention studies shown to be effective in hispanic families [3, 4]. Of the intensive, clinical interventions shown to be effective, a major limitation is the intensity required as this limits the intervention's potential public health scope and brings about challenges of sustainability [58]. Peer or parent mentors are an alternative to professional counseling for delivering information and assisting with behavioral change . Parent mentors have been used as an effective intervention model in coaching other parents on their child's diabetes management and in childhood asthma . This model may also be an attractive option for the treatment of early childhood obesity because it requires a relatively low amount of resources to initiate a parent mentoring program, and there is potential for empowerment and sustainability within the community . Head start is a government - funded program for low - income families that includes early learning and school readiness, health and development screenings as well as daily meals, and family well - being to strengthen parent - child relationships . One of head start's core values is the empowerment of families, and they serve a population at high risk for obesity; therefore, the parent mentor model of intervention meshes well with their structure and operational values . The data on obesity interventions in head start centers in other locations have yielded variable results . The positive deviance approach was used as the premise for building the intervention as it is well suited for high - risk populations and family empowerment . Positive deviance is the idea that, even among those most at risk for an adverse outcome, there are some individuals in the community who find a way to succeed, and, by identifying how those individuals or families succeed, one can identify successful strategies and behaviors that utilize local resources and knowledge . This approach was historically used in malnutrition though it has recently been explored in obesity [1417]. This study was designed to evaluate the feasibility of a clinical trial to test the hypothesis that parent mentors using positive deviance - derived education could be an effective intervention for early childhood obesity in the context of a head start program serving a low - income, hispanic community . This was a pilot, randomized clinical trial designed to evaluate the feasibility of an intervention using parent mentors trained in positive deviance behaviors to reduce adiposity among obese 25-year - old children . All of the parent - child dyads recruited for the study were actively enrolled in a head start program, neighbors in need of services (ninos inc . ). The full protocol for this trial is described in greater detail elsewhere and is also available by contacting the corresponding author . Staff identified 139 families with at least one 25-year - old obese child from the 16 centers in the geographic area of this study, cameron county, south texas . Identified families were contacted by letter and phone to explain the study and determine interest in participating . Eligibility was determined as having a body mass index (bmi) 95th percentile for age and gender . Exclusion criteria were intellectual disability, severe development delay, seizure disorder, diabetes, cerebral palsy, any genetic problem, and inability to communicate in either english or spanish . Sixty parents provided written consent, and they and their child were enrolled in the study . Parent - child dyads were randomized 1: 1 to intervention or comparison in blocks of six using redcap . The randomization allocation table was generated by a research assistant and was concealed from the principal investigator and staff . Enrollment and assignment of participants to intervention and comparison arms were carried out by clinical research unit staff . Parent mentors were recruited from cameron county head start centers and trained as described previously . Briefly, these individuals were required to have a 25-year - old child at a healthy weight who was enrolled in head start at the time; the parents themselves could be of any weight . Four parents received a one - day intensive training on the content of the parent mentor manual and on reflective listening, with three parents selected for participation based on their engagement in the training . The parent mentor manual was developed using the american academy of pediatrics guidelines on obesity prevention and the previous study on identified positive deviance practices done in cameron county . There were five main foci that the parent mentors were instructed on as being potentially effective strategies to share with their parent mentees: dealing with behavior problems without using food, identifying internal motivators for healthy habits, organizational strategies for feeding, accurate perceptions of weight, and effective snacking strategies . They completed worksheets monthly for each encounter with their mentee and reported what topics were discussed . Parent - child dyads randomized to the intervention arm were assigned to one of three parent mentors . Home assessments with the parent and child were conducted by each parent mentor at baseline and three months after enrollment . A standardized approach to the visit included asking about five main areas (positive deviance behaviors listed above), and at least one phone call per month was made by each parent mentor in order to reinforce those behaviors . Meetings were conducted on a monthly basis by parent mentors, and each mentor was allowed to implement her own curriculum in accordance with the goals discussed with the participating group . Parent - child dyads randomized to the community health worker comparison arm had the opportunity to attend one of three monthly community meetings held at head start centers . These meetings were conducted by a local promotora or community health care worker . In contrast to the intervention arm meetings, the comparison arm meetings followed a structured setting outlined by the eatplaygrow curriculum . Topics discussed during the meetings included health benefits of fruits and vegetables, limiting unhealthy foods, physical activity, portion control, and sleep, using an interactive format via songs, exercise, story time, and snacks . During the hour - long meeting, the first ten minutes were reserved for signing in and serving fruits and vegetables, the following forty - five minutes were for content delivery, and the final five minutes were used to remind parents of the following meeting and discuss any questions . Children were allowed to attend the meetings with their parents but it was not a requirement . In contrast to the intervention arm, parent - child dyads did not receive any home visits or follow - up phone calls throughout the study . The only contact time between the community health worker and parents was during the hour - long community meeting once a month . Therefore, a control group was identified with no intervention other than usual care in head start which consists of providing healthy meals and messaging on healthy habits in newsletters . The control group children were identified as obese (95th bmi percentile for age and gender), were enrolled in head start over the same period of time, attended the same local head start centers, and were matched to each study participant by sex and on their initial bmi z - score (within 0.3 units). The average baseline bmi z - score difference between participants in the trial and community control group children was 0.03 units . All measures described below were administered in either english or spanish depending on the preference of the individual participant . The primary outcome was bmi z - score change at the end of the six - month intervention; bmi z - scores were calculated using centers for disease control standards . A postintervention follow - up children were measured and weighed without shoes and in light clothes by trained research staff . Secondary outcome measures assessed at baseline, at the end of intervention (six months), and six months after intervention (12 months from baseline) included health - related quality of life using the pediatric quality of life inventory (pedsql 4.0, mapi research trust, lyon, france), feeding behaviors measured by the comprehensive feeding practices questionnaire (cfpq), dietary intake assessed using the block kids food screener (bkfs) developed by nutritionquest (berkeley, ca, usa), screen time, sleep, and outside play using standardized questions previously described [18, 2628]. Growth (height) velocity was calculated over a twelve - month period, and each individual was plotted on a sex specific growth velocity chart . Recruitment was evaluated by assessing how many parent - child dyads were screened and contacted to achieve one dyad enrolled . Participation was calculated using a point system for each potential interaction in each arm of the study with fourteen possible points in the intervention arm and six points possible for the comparison arm . These points were categorized into high participation 65%, some participation 1% and <65%, and no participation = 0% . The primary clinical outcome of bmi z - score was analyzed under the intention - to - treat principle . Linear mixed models with a random intercept were used to evaluate the primary outcome using randomization group, time (0, 6, and 12 months coded as intervals rather than a continuous variable to evaluate period effects), and their interaction (group time) as main effects adjusted for baseline measurements; an interaction term between baseline measurement and time was included if significant to account for the intercept variation . This modeling approach was also used for the secondary outcomes of the pedsql 4.0 scales, cfpq scales, and diet and activity measures . Data were analyzed using spss (version 23; ibm spss statistics, ibm corporation, chicago, il). Weight maintenance in this age group with continued growth in height leads to a decrease in bmi z - score among obese children of about 0.5 units over 6 months; this approximates a moderate effect size . With an expected mean bmi z - score of 2.5 at baseline, a standard deviation of 0.5, and a two - sided of 0.05, 30 participants in each group provided 48% power to detect a difference of moderate effect size, 86% power to detect a large effect size (reduction in bmi z - score of 0.8), and 12% power to detect a small effect size (reduction in bmi z - score of at least 0.2). This study was approved by the university of texas health science center at san antonio institutional review board . The total program enrollment for the cameron county head start includes more than 2,700 students . From this cohort of students, 139 families were assessed for eligibility to participate in the randomized clinical trial . Ultimately, 79 families were excluded and 60 were enrolled in the trial . Of the sixty parent - child dyads initially enrolled in the study, forty - eight completed the six - month follow - up visit (end of intervention) (80%) and forty - one completed the twelve - month visit (six months after intervention) (68.3%) (figure 1). Participation in both the intervention and the comparison groups overall was high, with 76% of all participants meeting the benchmark of 65% of possible interactions to qualify as having high participation . Only two of the parent - child dyads had no participation recorded while still completing all study visits for measurements, both in the comparison arm of the study . In assessing adherence, the parent mentors followed a standardized approach of discussing five main areas (positive deviance behaviors) when conducting home visits and phone calls throughout the trial . Their discussion of each area by proportion of all documented interactions was perceptions of weight, 84.3% (95% ci: 75.9, 92.7); snacking strategies, 95.3% (95% ci: 91.6, 99.0); dealing with behavior problems and emotions, 69.2% (95% ci: 56.2, 82.2); organization and taking control, 77.1% (95% ci: 67.8, 86.5); and figuring out why healthy habits are important, 78.6% (95% ci: 69.3, 87.7). Comparing the baseline demographics and anthropometrics, there were no significant differences between the randomized groups (table 1). There was no difference in baseline demographics between completers (n = 48) and noncompleters (n = 12) at six months . Those completing the twelve - month visit were less likely to be employed at baseline (44%) compared to noncompleters (78%), p = 0.05, and otherwise there were no differences . For the outcome of bmi z - score, there was no difference in the mean change between the parent mentor and community health worker groups (mean difference: 0.02 (95% ci: 0.26, 0.22)). Both had a significant reduction in mean bmi z - score by time (p <0.001, table 2). Using estimated marginal means adjusted for baseline values, the estimated change in bmi z - score from baseline to end of intervention at six months was 0.24 (95% ci: 0.34, 0.15), and that between the end of intervention and the twelve - month time point was 0.00 (95% ci: 0.10, 0.11). When separated by baseline bmi z - score quartiles, participants starting at a higher z - score intercept had the largest overall z - score change with a mean of 0.68 (95% ci: 1.1, 0.24). There was no effect on systolic blood pressure; diastolic blood pressure percentiles decreased overall by a mean of 5.53 (95% ci: 9.74, 1.31) at six months, with no effect by group (p = 0.96) (table 2). For sleep, no differences by group were observed; the mean baseline for the parent mentor group was 10.7 hours (sd = 1.42) and was 10.7 (sd = 1.40) for the community health worker group . In the analysis adjusting for baseline and multiple measures, there was a significant increase from baseline to six months (p = 0.04) but then there was a decrease from six months to twelve months (p = 0.002) resulting in overall no difference between baseline and twelve months (p = 0.16) (table 2). Weekday screen time had a trend toward significance in the interaction of randomization group time (p = 0.08) with the intervention group decreasing screen time from a mean of 3.3 (95% ci: 2.3, 4.2) at six months to 2.1 (95% ci: 1.5, 2.7) at twelve - month follow - up . Out of the forty - one participants who completed the six - month postintervention, 12.2% (5) had a growth (height) velocity <50th percentile, 29.3% (7) were between the 50th and 90th percentile, and 70.7% (29) had a growth velocity> 90th percentile . For the nonrandomized control group comparison, their baseline bmi z - score was 2.65 (95% ci: 2.38, 2.93) (table 3) and their mean age at baseline was 45.6 months (95% ci: 44.0, 47.2). The difference between their baseline and six - month bmi z - score was not significant (p = 0.08, paired t - test). As a group over a twelve - month period, they did have a significant decrease in bmi z - score of 0.32 (95% ci: 0.53, 0.10), with a mean z - score at twelve months of 2.33 (95% ci: 2.02, 2.66). Using the block ffq as the primary assessment of dietary intake, there was a significant reduction in sugary beverage intake overall with a mean change of 0.14 servings (95% ci: 0.23, 0.04) at six months but not by group (p = 0.96), with the significance occurring between baseline and end of intervention at six months (p = 0.001) and sustained with no change at twelve months (six months after intervention) (p = 0.83 for six versus twelve months) (table 4). There was also a significant decrease in sugar added to food or drink over time of 1.22 tsp (95% ci: 2.12, 0.32), and no changes were seen by time or group for vegetable intake (p = 0.36 for time) or whole grain intake (p = 0.12). The overall caloric intake decreased between baseline and six months with a mean difference of 119.99 (95% ci: 252.23, 12.25 p = 0.07) and was then stable between end of intervention and twelve months (p = 0.51). The quality of life scales assessed using the pedsql 4.0 showed no significant changes from baseline to end of intervention or six months after intervention in either group, except in emotional functioning, with a significant increase occurring between baseline, 81.3 (95% ci: 76.9, 85.8), and end of intervention, 86.8 (95% ci: 83.1, 90.6) (p = 0.003). The following are mean overall scores for the intervention and comparison group throughout the trial: baseline, 84.4 (95% ci: 81.3, 87.5); end of intervention, 83.6 (95% ci: 79.2, 88.1); and six months after intervention, 85.7 (95% ci: 81.5, 90.0). Finally, all of the cfpq scales showed significance by time except the monitoring measure (p = 0.24) (table 5). The encourage balance and variety in diet scale was the only measure to show significance by group (p = 0.009) with a higher score reported at the end of intervention and six months after intervention for the intervention group versus the comparison group, with mean score of 4.5 (95% ci: 4.2, 4.8), 4.8 (95% ci: 4.6, 5.0) for the intervention arm and 4.3 (95% ci: 4.0, 4.5), 4.5 (95% ci: 4.2, 4.7) for the comparison arm, respectively, over time . The scales that are most important and related to the intervention are environment, emotion regulation, restriction for health, restriction for weight control, and modeling . These scales were more heavily discussed during the parent mentor training before the beginning of the study . This is the first clinical trial to our knowledge that uses parent mentors as an intervention mechanism for childhood obesity . An important aspect of this trial was that it was conducted in a low - income, hispanic population who, in the united states, are at higher risk of obesity compared with white children . The data on recruitment, participation, and retention suggest that a full - scale clinical trial would be feasible in this setting . The high participation rates for both intervention and comparison groups in the present study indicate that our approach was successful in terms of engaging and motivating parents of obese children . Moreover, our findings provide evidence that parent mentors are effective at facilitating this type of engagement in their peers . It has been suggested that interventions sponsored or supported by government agencies and/or big organizations (i.e., top - down approaches) are more sustainable compared to bottom - up approaches like interventions driven by community - based organizations . Our parent mentor intervention was driven by an independent social network, which is different compared to standard bottom - up approaches that are dependent on formal institutional support . This is significant because it suggests that a positive deviance approach, with peer mentors serving as agents of change, may hold the key to improving sustainability . At the same time, the relative success of these mentors may be dependent on their own capacities, and the variation between mentors in efficacy will need further testing in full - scale trial . Both the parent - child dyads randomized to receive a parent mentor and those randomized to receive health education from a community health worker experienced a decrease in their adiposity as measured by bmi z - score . The diet and activity changes that were measured were consistent with this decrease in adiposity, and the plateauing of those diet and activity changes between the end of the intervention at six months and the twelve - month follow - up is consistent with their weight stabilization . A recent study comparing childhood obesity interventions with and without a true control group suggests that careful interpretation of results in those without a true control should be considered due to possible regression to the mean and biases favoring resolution . For the control group that we did employ, the reduction in the control group's bmi z - scores over twelve months makes it impossible to attribute the reduction seen in both of our intervention groups to the intervention . However, the lack of a significant effect in the first six months for the control children in contrast to the stronger reduction in the first six months of the two intervention groups suggests there is some additional benefit to receiving the intervention that should be evaluated in a larger clinical trial . Other trials demonstrating success in reducing adiposity in this age group have utilized multidisciplinary teams including a dietician, psychologist, or behavioral counselor and some physical activity coaching [7, 8]. One trial specifically tested a mentoring component in addition to multidisciplinary education and found a significant benefit of health coaching by a paraprofessional mentor compared with the educational intervention alone [6, 32]. In a multidisciplinary intervention study with a usual care control, a larger effect size on bmi z - score was seen (mean difference 0.77, 95% ci: 0.27, 1.26) than in our study; other studies using a degree of intensity more similar to that described in this study (using motivational interviewing or health coaches) have seen a similar, more modest effect . One important aspect of this study was its conduct in partnership with a head start program . While the head start program staff did not provide any direct education for the parents involved in the study, they did encourage their involvement . The potential for a scalable and effective program for reducing obesity has significant potential impact given the scope of head start and the population the programs serve . Prior studies targeting obesity in head start have been mixed with one study showing no reduction in adiposity and even increases in some groups, whereas others have found decreases associated with usual head start participation . Another series of trials found no significant effect with a weight - focused intervention but then after including a parent - focused component found significant decreases from baseline in both the intervention and the control groups, suggesting a possible role for usual head start participation . Using the cfpq, we observed an increase in parental report of more child self - control of their own eating behaviors, a decrease in regulation of child's emotional states through use of food, an increase in parent promotion of well - balanced food intake, an increase in availability of healthy foods in the home, a decrease in use of food as a reward for child behavior, an increase in encouragement of child's own involvement in meal planning and preparation, an increase in parent demonstration of healthy eating, a decrease in parent pressure of child eating more food at meals, an increase in parent control of child's food intake to limit less healthy foods and maintain child's weight, and an increase in techniques to encourage consumption of healthy foods . One challenge of using the positive deviance method to develop the intervention is that there were no specific behavioral mapping tools for the targeted behaviors . However, given that one area of the positive deviance - based intervention was on how to address behaviors without food, the cfpq findings are encouraging that the intervention had a causal relationship with the behavior changes reported . Interestingly, the only difference between the intervention and comparison group was an increase in parent reported encouragement of a balanced and varied diet in the intervention group . Prior observational studies have shown a correlation between restrictive feeding practices and increased risk for overweight [3638]; however, some studies have suggested a role for restriction at least in early childhood, and the role of restrictive practices in the context of an intervention for obesity where a parent is trying to change patterns is less clear and should be explored in future interventional studies . Finally, reporting bias is certainly a possible influence on the cfpq results, particularly after intervention . The change in the health - related quality of life measure of emotional functioning may be a result of the interventions or relating to the natural maturation process . Also, the initial scale ratings for quality of life are lower than previously reported means of healthy children and similar to that of another study of obese preschoolers . However, these could also be unrelated to the child's weight status and be more related to their socioeconomic status or another confounding variable . The limitations of this study include the secondary outcome measures of screen time, activity and sleep being by parental report, though any recall bias would probably have affected both groups equally . The limitation of no true control that was randomized is discussed above, though this study does add to the literature on describing the potential effect of usual head start participation . The study was underpowered to detect a difference between groups for a small effect size on adiposity, which is what we observed . However, the focus of this study was on feasibility of a novel intervention and study design . Finally, we cannot separate the effect of the content delivery versus the health coaching or support by the promotora with this study design . A significant challenge in addressing the childhood obesity epidemic, particularly the disparities seen in minority populations, requires scalable and culturally acceptable interventions . The data presented suggest that the method of using parent mentors with minimal training is a potentially feasible intervention with a small effect size that warrants further exploration . Positive deviance as a method of deriving solutions from a high - risk community also warrants further testing in clinical studies . Finally, the role of early education centers, particularly head start, in addressing early childhood obesity is promising from both this and other recent studies.
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Superficial infections such as burn, wound and post - surgical site infections are important causes of emergency health care - associated problems all around the world . Superficial infections cause longer hospital stays, more expensive hospitalizations and increased mortality (1). The annual superficial infection care products market is projected to reach $15.3 billion by 2010 (1). Pseudomonas aeruginosa are non - fermentative, aerobic, gram - negative rod shape bacteria, which substantially contribute to wound - related morbidity and mortality worldwide . They are widely distributed, mostly in hospital environments and are one of the most important agents of hospital - acquired superficial infections, ecthyma gangrenosum and black necrotic lesions (2, 3). Superficial infections caused by p. aeruginosa are one of the most prevalent causes of hospitalization and emergency health care references all around the world (2 - 6). Treatment of superficial infections caused by p. aeruginosa often requires antibiotic therapy yet the levels of antibiotic resistance in the rough strains of these bacteria have increased over time (7 - 11). Therefore, it is essential to study the levels of antibiotic resistance in the p. aeruginosa isolates of each region and even each hospital . In the recent years, the growing incidence of p. aeruginosa has been of particular concern . The incidence of p. aeruginosa in superficial and wound infections is becoming more serious in developing countries like iran (12, 13). This issue is of higher importance for females and elders, due to their relatively lower levels of immune system . The present study was carried out in order to study the antibiotic resistance pattern of p. aeruginosa isolated from cases of superficial infections referred to the emergency health care units of iranian hospitals . Ethical committees of the educational hospitals approved the general principles and framework of the present investigation . Personal information of all patients remained confidential . From june 2014 to october 2015, a total of 300 swab samples were taken from patients with superficial infections referred to the emergency health care units of iranian hospitals . Swab samples were taken from various types of superficial infections including wound (n = 110), burn (n = 90) and post - surgical site (n = 100) infections . Personal information like age and gender were recorded for each sample and all samples were transferred to the laboratory in a cooler with an ice pack . Swab samples were inoculated on blood, macconkey (merck, germany) and nutrient agar (merck, germany) and incubated at 37c for 24 hours . Colonies that produced pyoverdin, pyocyanin and pyorubin pigments were transferred to nutrient agar and subcultured more than one time to obtain pure cultures . The isolates were identified using conventional biochemical tests such as motility, oxidase, catalase, citrate utilization, gelatinase liquefaction, urease production, nitrate reduction, alkaline protease production, triple sugar iron agar, oxidative - fermentative, indole, lecithinase production and hemolysin production . The mueller - hinton agar (merck, germany) medium was used for this purpose . Antibiotic resistance of p. aeruginosa strains against 12 commonly used antibiotics, including norfloxacin (30 g / disk), ampicillin (10 u / disk), imipenem (30 u / disk), gentamycin (10 g / disk), ciprofloxacin (5 g / disk), cefepime (30 g / disk), cotrimoxazole (30 g / disk), polymyxin b (300 u / disk), meropenem (10 g / disk), amikacin (30 u / disk), ceftazidime (30 g / disk) and aztreonam (30 g / disk) antibiotic agents (oxoid, uk) was analyzed using the clinical laboratory standard institute protocol (clsi) (14). A single colony was inoculated on 5 ml of brain heart infusion broth and incubated over night at 37c . Then 1.5 ml of a saturated culture was harvested with centrifugation for five minutes at 14,000 rpm . The cell pellet was resuspended and lysed in 200 l of lysis buffer (40 mm tris - acetate ph 7.8, 20 mm sodium - acetate, 1 mm edta, 1% sds) by vigorous pipetting . To remove most proteins and cell debris, 66 l of 5 m nacl solution was added and mixed well, and then the viscous mixture was centrifuged at 12,000 rpm for 10 minutes at 4c . After transferring the clear supernatant to a new eppendorf tube, an equal volume of chloroform was added, and the tube was gently inverted at least 50 times when a milky solution was completely formed . Following centrifugation at 14,000 rpm for five minutes, the supernatant was then removed to another eppendorf tube and double volume of 100% ethanol was added . The tubes were gently inverted five to six times, then centrifuged at 10000 rpm for five minutes . The supernatant was discarded and 1 ml of ethanol (70%) was added to the pellet, and tubes were centrifuged at 10000 rpm for five minutes . Finally, the supernatant was discarded and the pellet was dried for 10 minutes at room temperature and was then resuspended in 100 l h2o . The dna concentration was determined by measuring absorbance of the sample at 260 nm, using a spectrophotometer (15). Genomic dna extracted from the bacterial colonies was confirmed to be p. aeruginosa using the pcr technique . The pcr mixture contained 200 m of each dntp (fermentas, germany), pcr buffer (10 mm tris / hcl, 50 mm kcl, 1.5 mm mgcl2, ph 8.3), dmso at a final concentration of 4%, 12.5 pmol of each primer (f: 5- gggggatcttcggacctca -3 and r: 5- tccttagagtgcccacccg -3, 956 bp) (16), 1 u taq dna polymerase (fermentas, germany) and 25 ng dna template . The dna was amplified in a programmable thermal cycler (eppendorf, mastercycler 5330, eppendorf - netheler - hinz gmbh, hamburg, germany) pcr device using the following protocol: 94c for one minute, 30 cycles of 94c for 35 seconds, 58c for 60 seconds, 72c for 60 seconds, and 72c for five minutes . P. aeruginosa atcc 27853 were used as positive controls and distilled water (d. w, merck, germany) was used as a negative control in all pcr reactions . Fifteen microliters of pcr products were resolved on a 1.5% agarose gel containing 0.5 mg / ml of sybr green in trisborate edta buffer at 90 v for 40 minutes, also using suitable molecular weight markers . The results were transferred to a microsoft excel spreadsheet (microsoft corp ., redmond, wa) for analysis . Chicago, il) for significant relationships between incidences of antibiotic resistance of p. aeruginosa isolated from the samples of superficial infections . The chi - square test and fisher s exact 2-tailed test analysis were performed in this study . Ethical committees of the educational hospitals approved the general principles and framework of the present investigation . From june 2014 to october 2015, a total of 300 swab samples were taken from patients with superficial infections referred to the emergency health care units of iranian hospitals . Swab samples were taken from various types of superficial infections including wound (n = 110), burn (n = 90) and post - surgical site (n = 100) infections . Personal information like age and gender were recorded for each sample and all samples were transferred to the laboratory in a cooler with an ice pack . Swab samples were inoculated on blood, macconkey (merck, germany) and nutrient agar (merck, germany) and incubated at 37c for 24 hours . Colonies that produced pyoverdin, pyocyanin and pyorubin pigments were transferred to nutrient agar and subcultured more than one time to obtain pure cultures . The isolates were identified using conventional biochemical tests such as motility, oxidase, catalase, citrate utilization, gelatinase liquefaction, urease production, nitrate reduction, alkaline protease production, triple sugar iron agar, oxidative - fermentative, indole, lecithinase production and hemolysin production . The mueller - hinton agar (merck, germany) medium was used for this purpose . Antibiotic resistance of p. aeruginosa strains against 12 commonly used antibiotics, including norfloxacin (30 g / disk), ampicillin (10 u / disk), imipenem (30 u / disk), gentamycin (10 g / disk), ciprofloxacin (5 g / disk), cefepime (30 g / disk), cotrimoxazole (30 g / disk), polymyxin b (300 u / disk), meropenem (10 g / disk), amikacin (30 u / disk), ceftazidime (30 g / disk) and aztreonam (30 g / disk) antibiotic agents (oxoid, uk) was analyzed using the clinical laboratory standard institute protocol (clsi) (14). A single colony was inoculated on 5 ml of brain heart infusion broth and incubated over night at 37c . Then 1.5 ml of a saturated culture was harvested with centrifugation for five minutes at 14,000 rpm . The cell pellet was resuspended and lysed in 200 l of lysis buffer (40 mm tris - acetate ph 7.8, 20 mm sodium - acetate, 1 mm edta, 1% sds) by vigorous pipetting . To remove most proteins and cell debris, 66 l of 5 m nacl solution was added and mixed well, and then the viscous mixture was centrifuged at 12,000 rpm for 10 minutes at 4c . After transferring the clear supernatant to a new eppendorf tube, an equal volume of chloroform was added, and the tube was gently inverted at least 50 times when a milky solution was completely formed . Following centrifugation at 14,000 rpm for five minutes, the supernatant was then removed to another eppendorf tube and double volume of 100% ethanol was added . The tubes were gently inverted five to six times, then centrifuged at 10000 rpm for five minutes . The supernatant was discarded and 1 ml of ethanol (70%) was added to the pellet, and tubes were centrifuged at 10000 rpm for five minutes . Finally, the supernatant was discarded and the pellet was dried for 10 minutes at room temperature and was then resuspended in 100 l h2o . The dna concentration was determined by measuring absorbance of the sample at 260 nm, using a spectrophotometer (15). Genomic dna extracted from the bacterial colonies was confirmed to be p. aeruginosa using the pcr technique . The pcr mixture contained 200 m of each dntp (fermentas, germany), pcr buffer (10 mm tris / hcl, 50 mm kcl, 1.5 mm mgcl2, ph 8.3), dmso at a final concentration of 4%, 12.5 pmol of each primer (f: 5- gggggatcttcggacctca -3 and r: 5- tccttagagtgcccacccg -3, 956 bp) (16), 1 u taq dna polymerase (fermentas, germany) and 25 ng dna template . The dna was amplified in a programmable thermal cycler (eppendorf, mastercycler 5330, eppendorf - netheler - hinz gmbh, hamburg, germany) pcr device using the following protocol: 94c for one minute, 30 cycles of 94c for 35 seconds, 58c for 60 seconds, 72c for 60 seconds, and 72c for five minutes . P. aeruginosa atcc 27853 were used as positive controls and distilled water (d. w, merck, germany) was used as a negative control in all pcr reactions . Fifteen microliters of pcr products were resolved on a 1.5% agarose gel containing 0.5 mg / ml of sybr green in trisborate edta buffer at 90 v for 40 minutes, also using suitable molecular weight markers . The results were transferred to a microsoft excel spreadsheet (microsoft corp ., redmond, wa) for analysis . Chicago, il) for significant relationships between incidences of antibiotic resistance of p. aeruginosa isolated from the samples of superficial infections . The chi - square test and fisher s exact 2-tailed test analysis were performed in this study . The present investigation was carried out to study the prevalence of antibiotic resistance of p. aeruginosa isolated from various types of superficial infections . Table 1 shows the total distribution of p. aeruginosa in the swab samples taken from various types of superficial infections . Of the 300 studied swabs, 172 (57.3) samples were found to be contaminated with p. aeruginosa . The results of the culture technique were also confirmed using the pcr method (figure 1). Swab samples, which were taken from female cases (64.2%), patients older than 70 years (68.5%) and cases of burn infections (66.6%), had the highest prevalence of p. aeruginosa . Statistically significant differences were seen in the prevalence of p. aeruginosa between male and female cases (p = 0.039), younger than 10-years - old and older than 70-years - old patients (p = 0.016) and cases of burn infections and wound infections (p = 0.041). M, 100 bp ladder; 1, positive samples (956 bp); 2, positive control (p. aeruginosa atcc 27853); 3, negative control (distilled water (d.w, merck, germany)). Table 2 shows the antibiotic resistance pattern of p. aeruginosa isolated from various types of superficial infections . We found that the p. aeruginosa strains of superficial infections harbored the highest levels of resistance against ampicillin (93%), gentamycin (89.5%), ciprofloxacin (82.5%) and amikacin (77.3%), and also the lowest levels of resistance against meropenem (2.3%), imipenem (2.9%), polymyxin b (21.5%) and cotrimoxazole (31.9%). P. aeruginosa strains of males had a higher prevalence of antibiotic resistance than females (p = 0.026). Statistically significant differences were seen between the type of infection and prevalence of antibiotic resistance (p = 0.044), and also between the age of patients and prevalence of antibiotic resistance (p = 0.032). The results of the present study showed that p. aeruginosa has a higher prevalence in various types of superficial infections . To the best of our knowledge, this finding is the highest prevalence of p. aeruginosa in swab samples of superficial infections . Lower prevalence rate of p. aeruginosa in human superficial infections have been reported previously by ranjan et al . (2006) (32%) (4), oguntibeju and nwobu (2004) (33.2%) (5), masaadeh and jaran (2009) (27.78%) (6) and siguan et al . High prevalence of p. aeruginosa in the clinical samples of our study maybe due to the fact that the type of samples (swabs samples of the site of infection) and health care managements is different with those of other investigations . In fact, the presence of environmental pollution, especially in the hospital environment as well as contaminated and lack of optimal disinfection of instruments and equipment of hospitals are the main reasons for the high prevalence of p. aeruginosa (62%) in post - surgical site infections of our study . Low levels of healthcare management in iranian healthcare units and hospitals have been recognized from the results of our study and the results of various previous iranian investigations (13, 18, 19). Higher sensitivities of female skin are a reason for the higher prevalence of p. aeruginosa in their superficial infections . Al - hasan et al . (2008) (22) and khan et al . (2008) (7) reported a higher prevalence of p. aeruginosa clinical infections in males than females, which were different to our results . Their reason for the high prevalence of bacteria in males is that they are more in contact with the polluted outside home environment . Therefore, they are more prone to get superficial infections . Aging, decrease in the levels of keratin skin cells and reduction in the level of immunity are reasonable factors for the higher prevalence of p. aeruginosa in older than 70-years - old patients . High prevalence of p. aeruginosa in old patients has been reported previously (23 - 25). In despite of the results of a previous investigation, which showed a high prevalence of p. aeruginosa in children (26), the results of our study showed that less than ten years old patients had a lower prevalence of bacteria . One possible explanation for this finding is that the age range of younger than ten - year - old patients of our study was eight to ten years . On the other hand, there were no younger than eight - year - old pediatrics in our study population . Our study also focused on the prevalence of antibiotic resistance in p. aeruginosa strains of superficial infections . We found that p. aeruginosa isolates had the highest levels of resistance against ampicillin (93%), gentamycin (89.5%), ciprofloxacin (82.5%) and amikacin (77.3%). In a study conducted in nepal (27), there was no resistance against ampicillin, gentamicin, norfloxacin and ofloxacin . The prevalence of resistance against ceftriaxone, cephalexin, ciprofloxacin and cotrimoxazole were 50%, 100%, 50% and 100%, respectively . An indian investigation revealed that the p. aeruginosa isolates of wound swab samples harbored the highest levels of resistance against tobramycin (66.3%), ciprofloxacin (87.93%), ceftazidime (73.27%), cefixime (84.48%), gentamycin (78.44%), amikacin (21.55%) and ofloxacin (87.93%), which was similar to our findings . In a study, which was conducted in ethiopia (28), 40% of p. aeruginosa isolates were resistant to seven antibiotics including amoxicillin, ampicillin, ciprofloxacin, norfloxacin and gentamicin . In the cases of burn infections (29), 70% of p. aeruginosa isolates were positive for metallo - beta - lactamase, with high prevalence of antibiotic resistance against ceftazidime (70%), chloramphenicol (68%) and gentamicin (62.5%). A recent iranian investigation (30) revealed that the p. aeruginosa strains isolated from the site of burn infections were resistant to cloxacillin (91.8%), cotrimoxazole (86%), cefazolin (83.7%), carbenicillin (74.4%), piperacillin (69.9%), ceftazidime (68.8%), ciprofloxacin (66.3%), tobramycin (58.2%), amikacin (48.8%) and gentamicin (37.2%), while the most effective antibiotic was imipenem with a resistance rate of 23.3%, which was similar to our results . Irregular and unethical antibiotic prescription and even self - treatment by strong antibiotics cause such high levels of resistance in the p. aeruginosa strains of our investigation . Differences in the idea of medical practitioners in antibiotic prescription cause variations in the levels of antibiotic resistance against different antibiotics . In addition, the differences in the bactericidal activities of antibiotics and also difference in difficulty in developing resistance against various antibiotics are two other reasons for differences in the levels of antibiotic resistance . The present study was one of the most extensive prevalence reports of p. aeruginosa and its antibiotic resistance pattern in the burn, post - surgical site and wound infection samples of emergency health care units of iranian hospitals . Our results showed that resistant strains of p. aeruginosa had a high prevalence in patients older than 70-years - old and especially in the samples taken from the site of burn infections . In keeping with this, the prevalence of these bacteria in cases of wound and post - surgical infections and also other studied groups were considerable . Also, because of the variation of resistance pattern in each hospital, it is important for each region and even hospital to formulate their own antibiotic policy, according to their local resistance pattern . We recommend the initial prescription of meropenem, imipenem and polymyxin b antibiotics for treatment of the cases of superficial infections in iran.
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Elucidation of the role played by small rna molecules in the context of viral life cycles has led to the identification of new drug targets that fall outside the realm of traditional drug design . In particular, the transactivation response element (tar) of hiv is critical in regulating transcriptional elongation of the hiv genome . Tar is a highly conserved stem - loop rna located at the 5-end of viral transcripts . For elongation to occur, the hiv transactivator protein (tat) the ensuing tat - tar complex is then responsible for recruitment of the positive transcription elongation factor complex, ptefb, resulting in processive elongation . The tat - tar complex is a particularly attractive drug target because it is highly conserved and difficult for the virus to develop resistance, which is a problem that hinders the effectiveness of conventional hiv therapies . The most effective strategies to inhibit tat - tar formation rely upon mimicking the binding region of the tat protein . Initial attempts to design tar - binding molecules focused on dissecting the tat peptide and synthesizing peptides corresponding to the highly basic, arginine rich region of tat that binds to the trinucleotide bulge of tar . The truncated tat peptides are able to compete with tat in vitro for binding to tar, however peptides are typically not viable drug candidates due to low bioavailability and stability . For instance, a nonamer peptoid was developed that had significant potency against the tat - tar interaction [8, 9]. Alternatively, cyclic peptides, and peptides comprised of d - amino acids had similar effects [1012]. All of these molecules share a common feature in that they display highly cationic sidechains . We felt that the high degree of cationic charge associated with these molecules would lead to promiscuous rna binders . Thus, we sought to create molecules that could retain similar binding properties while minimizing cationic charge about the periphery of the scaffold . Initial results from our lab showed that highly functionalized polyamines were able to serve as tar - binding molecules specific for the trinucleotide bulge . Our design strategy served to relegate cationic charge to the interior of the polyamines to decrease nonspecific charge - charge interactions, while projecting sidechains out from the backbone to direct specificity . The first generation of polyamines showed promising results, yielding a polyamine that bound tar specifically and with a kd of ~6 m at the bulge . These polyamines, however, suffered from an inefficient oligomer synthesis that was not possible to develop into a combinatorial library . Due to the limited structural information available on tar - binding molecules, we felt that expanding our strategy to allow for combinatorial library synthesis and subsequent screening was more likely to yield positive results . As such, we designed a new class of molecules, polyamino - amido oligomers (paas), that could be synthesized through modified solid phase peptide synthesis . The secondary amines in the backbone, which should be protonated at physiological ph, are spaced at intervals corresponding to the spacing of the phosphate groups of the rna backbone in order to form ammonium - phosphate salt bridges . From this general backbone scheme, while molecular modeling is often used as a tool for drug discovery, we felt that diversity - oriented approaches were more likely to yield positive results due to the dynamic nature of tar rna and the limited rational - based information available for design of small - molecule rna binders . Furthermore, we wanted to reduce the number of highly exposed cationic charges that are prevalent in most rna - binding small molecules which convey high binding affinity but also afford low specificity . In this paper, we describe the synthesis of the monomeric building blocks for paas as well as a parallel library synthesis . A quantum dot - based screen was implemented for an on - bead screening of the library for tar binding . An fmoc - protected amino acid (10 mmol) and diea (1.6 ml) were dissolved in dry ch2cl2 (50 ml) and cooled to 0c . Edc (2.4 g, 12 mmol) and hobt (1.8 g, 12 mmol) were added to the reaction . The reaction mixture was stirred at 0c for 10 minutes to pre - activate the carboxylic acid . After 10 minutes, n, o - dimethylhydroxylamine hydrochloride (1.2 g, 12 mmol) and diea (2.0 ml) were added to the reaction mixture . The reaction was allowed to slowly warm to room temperature and was stirred for a total of 16 hours . The reaction was then transferred to a separatory funnel with ch2cl2 (100 ml) and washed with 2 m hcl (3x, 50 ml), saturated aqueous nahco3 (2x, 50 ml), and brine (2x, 50 ml). The organic layer was dried over sodium sulfate and solvent was removed under reduced pressure to yield pure weinreb amides as solid white foams . Dry thf (50 ml) and weinreb amide (5 mmol) were added to an oven dried 250 ml round bottom flask, placed under n2, and cooled to 0c in an ice bath . Lithium aluminum hydride (250 mg, 6.25 mmol) was slowly added to the solution over a period of approximately 30 seconds . The reaction was stirred vigorously at 0c for 60 minutes and then slowly quenched with a 1 m solution of nahso4 (50 ml). The biphasic mixture continued to stir at 0c for 10 minutes at which point it was transferred to a separatory funnel using etoac (50 ml) and brine (50 ml). The aqueous layer was extracted with etoac (75 ml) and the combined organic layers were washed with 1.5 m hcl (2x, 50 ml), saturated aqueous nahco3 (2x, 50 ml), brine (2x, 50 ml). The organic layer was dried over sodium sulfate and concentrated under reduced pressure . An fmoc - protected -amino aldehyde (4.5 mmol) was dissolved in dry ch2cl2 (30 ml) at room temperature . To the stirring solution was added a benzyl protected amino acid hydrochloride (4.9 mmol) and diea (0.85 ml). Nabh(oac)3 (6.3 mmol) was immediately added to the reaction mixture and stirred vigorously for 75 minutes . The reaction was quenched with a mixture of saturated aqueous k2co3 (10 ml) and saturated aqueous nahco3 (30 ml) and allowed to stir for an additional 10 minutes . The biphasic mixture was transferred to a separatory funnel and extracted with ch2cl2 (3x, 30 ml). The organic layers were combined and dried over sodium sulfate, filtered, and concentrated under reduced pressure . The crude product was purified using a biotage flash chromatography system (40+m column, 60: 40 hexanes: etoac). The paa secondary amine backbone (2.0 mmol) was dissolved in dry ch2cl2 (40 ml) and stirred at room temperature . To the solution was added di - tert - butyl dicarbonate (0.9 g, 4 mmol) and diea (0.6 ml, 2 mmol). The reaction was allowed to stir for 48 hours and was then transferred to a separatory funnel with ch2cl2 (50 ml). The reaction mixture was washed with 1 m hcl (2x, 50 ml), saturated aqueous nahco3 (2x, 50 ml), and brine (2x, 50 ml). The crude product was purified using a biotage flash chromatography system (40+m column, 80: 20 hexanes: etoac). A parr flask was purged with n2 and then charged with 10% palladium on carbon . The palladium catalyst was wetted with a minimum amount of methanol (~5 ml) while under an n2 atmosphere . The paa - monomer ester (1.5 mmol) was dissolved in a minimal amount of methanol (typically ~20 ml) and added to the parr flask . The flask was placed under an h2 atmosphere (40 psi) and shaken on a parr shaker for 2 hours . The reaction mixture was then filtered through a bed of celite to remove the palladium from the mixture . The resulting solution was concentrated under reduced pressure to yield an off - white solid as the crude product . The crude product was purified using a biotage flash chromatography system (40+m column, 0%5% meoh gradient in ch2cl2). Several beads from each well of the paa library were transferred to a 384-well filter plate, keeping their spatial separation and orientation intact . The beads were first washed with water (5x, 50 l), then 1x tk buffer (50 mm tris, 20 mm kcl, 0.1% triton x-100, ph 7.4; 4x50 l). To each well, bsa (0.1 mg / ml) was added in 1x tk buffer (20 l) and agitated with mechanical shaking for 60 minutes at room temperature . The microplate was drained under vacuum and washed with 1x tk buffer (3x, 50 l). Following bsa blocking, bulge mutant tar in 1x tk buffer (2.5 m, the library was incubated with bulge mutant tar for 24 hours at 4c before being drained under vacuum . Immediately following solvent removal, a mixture of bulge mutant tar (2.5 m) and 5-biotin labeled tar (dharmacon, 250 nm) in 1x tk buffer (20 l / well) were introduced to the library . (note: rna was snap - cooled by heating at 95c for 5 minutes followed by an immediate transfer to dry ice for 5 minutes to promote hairpin formation .) The library was incubated with this solution for 2.5 days at 4c, drained, and washed with water . To each well a solution of qdot605 (50 nm, 15 l / well) in 1x tk buffer was added and agitated at room temperature for 3 hours . The solution was drained and each well was washed with 1x tk (3x50 l), followed by a 2 hour wash with 1x tk buffer and drainage under vacuum . The library was then visualized using a fluorescent microscope equipped with a triple bandpass filter . Beads that appeared red or orange under the microscope were selected for further characterization while those that were green were disregarded . Polyamine monomers were synthesized starting from commercially available fmoc - protected amino acids utilizing a solution - phase reductive amination strategy . Initially we sought to synthesize five polyamine monomers starting from orthogonally protected serine, tryptophan, tyrosine, 4-amino - phenylalanine, and phenylalanine (scheme 1). The monomers were selected based on their likelihood to interact with folded rna, thus amino acids capable of -stacking, hydrophobic interactions, and hydrogen bonding were selected . We chose to exclude certain high - affinity moieties, such as guanidinium groups, due to their strongly cationic nature and tendency to interact non - specifically . This choice aligns with our design strategy, in which positive charge is sequestered to the backbone to reduce non - specific charge - charge interactions with the rna backbone . The fmoc - protected amino acid was first converted to the weinreb amide (1) in high yield under edc - mediated amide bond forming conditions . Subsequently, 1 was reduced to the corresponding aldehyde (2) using lithium aluminum hydride . The aldehyde product was taken forward without additional purification to the reductive amination step where 2 was condensed with the hydrochloride salt of benzyl glycinate . Following chromatographic purification, the secondary amine was protected with a boc group to afford the paa monomer ester . Hydrogenolysis of the benzyl ester yielded the paa monomer (5). From these five monomers, a library of 125 paa trimers the synthesis was designed to be compatible with the development of an on - bead screen for rna binding . In order to create a library suitable for aqueous screening conditions, tentagel - nh2 resin was chosen as a synthetic platform due to its unique ability to swell in both aqueous and organic solvents . In addition, the library synthesis was performed in 96-well filter plates to provide a physical separation between distinct paas . The strategy outlined provided an accessible synthetic platform that negated the need for molecular deconvolution during the screening process . The spacer was deprotected and paa trimers were synthesized through hatu - mediated solid phase peptide synthesis (see supplementary material available online at doi:10.1155/2012/971581). Upon completion of the trimers, a global deprotection of the backbone and sidechain protecting groups afforded a 125-member library of resin - bound paa trimers . Initially we aimed to develop a simple fluorescent screening procedure for assessing tar binding to our resin - bound paas . To this end, we adapted protocols developed by the rana and kodadek laboratories to our system [12, 1416]. Since on - bead binding and high activity in solution are not necessarily correlated, our assay design was carefully chosen based on validated literature results . The incubation conditions mimicked those reported by rana, whose bead - based assays showed strong positive correlation to appropriate off - bead activity measurements . The paa - containing beads were first incubated with a solution of bsa to block any nonspecific binding to the bead surface . Subsequently, the library was incubated with a bulge mutant tar construct containing a single base bulge, rather than the wildtype trinucleotide bulge . The bulge mutant acted as a competitive inhibitor to exclude compounds that were not specific for the bulge region of tar . Next, 5-biotinylated wildtype tar was added to the resin in the presence of an excess of bulge mutant tar . Binding events were visualized using a fluorescent microscope equipped with a triple bandpass filter after the addition of streptavidin - coated quantum dots (qdot605). This method allowed for the visualization of positive beads as red in color where beads containing nonbinding polyamines were seen as green under the microscope (figure 2). From this initial screen the six brightest beads were selected, as determined by visual inspection and verified independently by a second researcher, and resynthesized on a larger scale to allow determination of binding constants . To describe the paas, the standard one - letter amino acid abbreviations are used to describe the sequence of sidechains with the understanding that in this paper the backbone is represented by the chemical structure in figure 1 for a paa backbone . The six paas selected based on the screen were sff, yff, fff, sys, ysf, and fyy . Previous work in our lab found rna footprinting studies using terbium (iii) ions as an rna cleavage agent to be a reliable method for quantification of polyamine binding to tar rna [13, 17]. The six selected paas were synthesized, purified by reversed phase hplc, and quantified by uv absorbance . The paas were then titrated into buffered solutions containing tar up to 1 mm concentrations and effects on rna cleavage patterns were assessed as a function of paa concentration via denaturing gel electrophoresis . The results of these experiments identified the sequence xff as a binding motif specific for the bulge region of tar . Three of the six ligands selected showed appreciable binding affinity for the bulge region, with sff, yff, and fff exhibiting binding constants in the low micromolar range (figure 3). The best ligand derived from our initial screen was sff (figure 4), exhibiting a kd of 14 m for the bulge region . Additionally, no binding was observed for the loop region of the tar rna, suggesting that our screen effectively selected for molecules that specifically interacted with the desired bulge region rather than nonspecific rna binders (see supplementary material). We next sought to probe the importance of sidechain interactions in an effort to define a minimal binding motif for the bulge region . The most direct way for us to probe this question was to substitute each sidechain in our best ligand (sff) with a moiety deemed to be unlikely to interact with the rna . In analogy to alanine scans in peptides, we synthesized the alanine - derived paa monomer and iteratively replaced each monomer in the sff paa with one bearing a methyl sidechain . We found that all three sidechain replacements yielded compounds with very low affinity for the tar target, thus leading us to conclude that all three sidechains were critical to maintain binding affinity . The paa scaffold was intentionally designed such that modifications to the backbone could be easily installed in order to determine structural activity relationships (sars). Therefore, modification to the backbone of the sff core motif could lead to enhanced binding affinity for tar . In this vein, we aimed to synthesize a library of compounds based on the sff core but bearing sidechains in place of the glycine subunit of the paa monomers . Six new monomers were synthesized, where the key step involved condensation between serine or phenylalanine aldehydes and the hydrochloride salts of either tryptophan, tyrosine, or lysine (figure 5). The new monomers and the original serine and phenylalanine monomers were incorporated into a 64-member paa library, where all library members contained the previously identified sff core . The library was again subjected to the qdot based screen and the six brightest hits were chosen for further characterization . Unfortunately, all paa derived from the second screen showed either very weak affinity for tar or no affinity at all in the footprinting assays (see supplementary material). As biological knowledge has advanced, rna has emerged as a viable pharmaceutical target . To date, there are few de novo designed molecules that are capable of specifically interacting with folded rna . In this paper, we have presented a class of readily synthesized oligomers that can specifically bind to the bulge region of folded tar rna . In developing these molecules, we implemented a quantum dot based fluorescent screening protocol that readily identified ligands for rna . Our screening protocol allowed for the determination of a minimal binding motif from the initial paa library . Quantification of binding affinity via footprinting analysis yielded a low micromolar binder of tar rna specific for the bulge region . We anticipate that further understanding of rna - mediated biological pathways will serve to validate rna as a viable drug target . Consequently, the ability to rapidly develop drug candidates that are able to distinguish between subtle differences in rna secondary structure will be of the utmost importance . We feel that the methods and results presented in this work represent a step toward developing a general class of rna binding molecules that can suit this purpose (see supplementary material).
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The selection of outcome measures to be applied is one of the most important factors in designing a clinical trial . The outcome measures present a major factor influencing the duration of a trial, likelihood of detecting a therapeutic effect, size of the sample and acceptance of study results . Selection of outcome measures is particularly difficult for multiple sclerosis (ms) trials because ms affects patients in many different ways . Among many difficulties, walking problems are very common and important for ms patients; about three - quarters of them experience mobility problems and they consider walking as the most valuable bodily function . Also, new developments in the pharmacotherapy to improve walking in ms patients have occurred . Therefore, the assessment of walking ability in ms patients is of a great importance for clinical research and practice . Assessment of walking ability in ms patients began in 1955 with the first measure of disease severity, the kurtzke disability status scale . Since that time, there has been a proliferation of numerous generic and genuine walking - based measures for ms patients . They are either clinician - based, patient - based, timed over fixed distance, measure the maximum distance a person can walk over a specific time interval, or use motion sensors such as accelerometers . There is a considerable body of literature on the reliability and validity of walking - based measures but, regardless of their clinical importance, the studies of their responsiveness are either deficient or mostly related to rating scales [912]. Responsiveness is ability of a measure to detect clinically important change or change over time, and is evaluated by various responsiveness statistics . Typically, responsiveness is determined by comparing before and after scores of interventions expected to produce a change in health . As the interpretation of p values is somewhat binary and sample size - dependent, it has become common to report responsiveness as an effect size, or standardized change score, by converting change scores into standard deviation units . Effect size and p values are limited indicators of responsiveness because they are inseparably linked to the magnitude of change induced by the intervention . This can be partly overcome by comparing measures head - to - head in the same sample, which keeps sample and treatment effect constant and enables researchers to compare the relative responsiveness of competing measures . The aims of this study were: to examine the effects of ivmp on the recovery of walking ability in patients experiencing ms relapse; to compare, head - to - head, the responsiveness of some walking - based measures applied for ms trails; and to consider their potential implications for clinical trials . The patients involved in this study were recruited from the department of neurology, university hospital center split, from july 2008 to december 2010 . The research was approved by local ethics committee and all participants gave their informed consent . A total of 54 consecutive patients who were selected for ivmp (1000 mg / day over 3 days) due to ms relapse and who met the inclusion criteria were asked to participate in the study . Three patients refused to take part, 1 patient did not appear for control testing, and 1 patient was found to be noncompliant with wearing the accelerometer; therefore 49 patients were included in the statistical analyses . During the study, 6 patients experienced more then 1 relapse meeting the inclusion criteria . Due to minimizing the potential impact of practice effects on the results, a relapse was defined as either new nervous system deficits or worsening of previous ones lasting at least 24 hours . All patients complained of walking difficulties caused by ms relapse and had clinical involvement, individually or combined, of the corticospinal tract (paresis, hyperreflexia, spasticity, extensor plantar response), posterior columns or medial lemnisci tract (proprioception) and cerebellum (cerebellar ataxia) affecting 1 or both lower limbs . The new nervous system deficits or worsening of previous ones were assessed strictly by clinical examination . Mri studies for documenting location and size of the ms lesions responsible for the relapses were not included . The inclusion criteria were: 1) age 18 years or over; 2) definite diagnosis of relapsing - remitting ms (rrms); 3) edss 6.5 at the time of the inclusion; 4) relapse with onset of symptoms within 2 weeks prior to ivmp; 5) no spontaneous improvement prior to ivmp; 6) relapse involvement of gait with deterioration of at least 1 step in the relevant functional system (fs) (pyramidal, sensory or cerebellar) or an increase in edss of 1 point or more; and 7) ability to perform walking tests . Exclusion criteria were: 1) treatment with corticosteroids in the previous 3 months; 2) cognitive impairment; 3) vision impairment; 4) orthopedic disease; and 5) cardiac disease . Pre - attack edss and fs were well known because the patients were routinely seen in our outpatient facility every 3 to 6 months by a trained neurologist with experience in multicentre clinical trials (m.m . ). The following walking - based measures were administered before and after ivmp: the expanded disability status scale (edss); the multiple sclerosis walking scale-12 (msws-12); the 25-foot walk test (25fwt); the six spot step test (ssst) and the stepwatch activity monitor (sam, orthocare innovations, washington dc, usa). The 2-minute timed walk (2-mintw) was administered as a shorter (reduced) version of the 6-minute timed walk (6-mintw). The 2-mintw was chosen as a more appropriate walk test for patients suffering from ms relapse than the 6-mintw . The 25fwt and the ssst were repeated twice, and the average number of seconds was used in the analysis . Since test - retest reliability of the 2-mintw for the first 10 participants in both tests (before and after ivmp) was high (cronbach =0.981 and 0.992, respectively) all patients had worn the sam 1 week prior to ivmp was applied and wore it again the fourth week upon the corticosteroid therapy was completed . The sam was fitted according to the proprietary instructions and was individually programmed for cadence and sensitivity . Evaluation of the output data confirmed compliance with wearing the accelerometer for all patients except for 1 . The analysis utilized the average daily step count, the percentage of time spent inactive, the percentage of time spent in low step activity (115 steps / min), the percentage of time spent in medium step activity (1630 steps / min), the percentage of time spent in high step activity (> 31 steps / min), the average peak activity index (average steps / min of the highest 30 minutes of the day regardless of when they occurred) and the average steps in 1, 5, 30 and 60 minutes (average steps / min of the highest continuous period of 1-min, 5-min, 30-min and 60-min periods). No significant difference was found in sam output data between the first and the seventh day of monitoring before and after ivmp . Other tests were administered between 1 and 3 p.m. before ivmp and 31 day later at the same time of day . All walk tests, (25fwt, ssst and 2-mintw) participants performed in the same type of footwear and clothes . To reduce patients bias, the walking - based tests were conducted without fixed order, with the exception of continuous long - term walk monitoring . To reduce the clinicians bias, the indication for ivmp was established independently of the study and different parts of research were also performed independently by different authors (clinical examination m.m . ; wilcoxon signed rank test was used to assess the significance of changes of walking - based measures after ivmp . Responsiveness was determined from time 1 and 2 data by calculating both effect size (es: mean change score divided by sd of admission scores) and standardized response means (srm: mean change score divided by sd of change scores), since they can produce different values . They were interpreted using cohen s criteria (values of 0.20.49 were defined as small, those of 0.50.79 as moderate and that of 0.8 and greater as large). The relative responsiveness of competing walking - based measures (edss, msws-12, 2-mintw, ssst, 25fwt and sam parameters) was determined by computing their relative efficiency (re). Re estimates the extent to which 1 measure is more or less efficient at detecting change relative to another measure . We computed re as pair - wise squared z values from wilcoxon signed ranks test . The walking - based measure with the largest z value was chosen as the denominator for the pair - wise calculation . This measure has a measurement precision of 100% and the other values are estimated as a percentage of the most responsive measure . The effect of different walking - based measure responsiveness on sample size estimates was evaluated by computing the number of patients required for each measure to detect the same effect of ivmp . The computed values are relative to 100 patients using the most responsive walking - based measure [100{(z value of walking measure with largest z value / z value of other walking measure)}] (11). The patients involved in this study were recruited from the department of neurology, university hospital center split, from july 2008 to december 2010 . The research was approved by local ethics committee and all participants gave their informed consent . A total of 54 consecutive patients who were selected for ivmp (1000 mg / day over 3 days) due to ms relapse and who met the inclusion criteria were asked to participate in the study . Three patients refused to take part, 1 patient did not appear for control testing, and 1 patient was found to be noncompliant with wearing the accelerometer; therefore 49 patients were included in the statistical analyses . During the study, 6 patients experienced more then 1 relapse meeting the inclusion criteria . Due to minimizing the potential impact of practice effects on the results, a relapse was defined as either new nervous system deficits or worsening of previous ones lasting at least 24 hours . All patients complained of walking difficulties caused by ms relapse and had clinical involvement, individually or combined, of the corticospinal tract (paresis, hyperreflexia, spasticity, extensor plantar response), posterior columns or medial lemnisci tract (proprioception) and cerebellum (cerebellar ataxia) affecting 1 or both lower limbs . The new nervous system deficits or worsening of previous ones were assessed strictly by clinical examination . Mri studies for documenting location and size of the ms lesions responsible for the relapses were not included . The inclusion criteria were: 1) age 18 years or over; 2) definite diagnosis of relapsing - remitting ms (rrms); 3) edss 6.5 at the time of the inclusion; 4) relapse with onset of symptoms within 2 weeks prior to ivmp; 5) no spontaneous improvement prior to ivmp; 6) relapse involvement of gait with deterioration of at least 1 step in the relevant functional system (fs) (pyramidal, sensory or cerebellar) or an increase in edss of 1 point or more; and 7) ability to perform walking tests . Exclusion criteria were: 1) treatment with corticosteroids in the previous 3 months; 2) cognitive impairment; 3) vision impairment; 4) orthopedic disease; and 5) cardiac disease . Pre - attack edss and fs were well known because the patients were routinely seen in our outpatient facility every 3 to 6 months by a trained neurologist with experience in multicentre clinical trials (m.m . ). The following walking - based measures were administered before and after ivmp: the expanded disability status scale (edss); the multiple sclerosis walking scale-12 (msws-12); the 25-foot walk test (25fwt); the six spot step test (ssst) and the stepwatch activity monitor (sam, orthocare innovations, washington dc, usa). The 2-minute timed walk (2-mintw) was administered as a shorter (reduced) version of the 6-minute timed walk (6-mintw). The 2-mintw was chosen as a more appropriate walk test for patients suffering from ms relapse than the 6-mintw . The 25fwt and the ssst were repeated twice, and the average number of seconds was used in the analysis . Since test - retest reliability of the 2-mintw for the first 10 participants in both tests (before and after ivmp) was high (cronbach =0.981 and 0.992, respectively) all patients had worn the sam 1 week prior to ivmp was applied and wore it again the fourth week upon the corticosteroid therapy was completed . The sam was fitted according to the proprietary instructions and was individually programmed for cadence and sensitivity . Evaluation of the output data confirmed compliance with wearing the accelerometer for all patients except for 1 . The analysis utilized the average daily step count, the percentage of time spent inactive, the percentage of time spent in low step activity (115 steps / min), the percentage of time spent in medium step activity (1630 steps / min), the percentage of time spent in high step activity (> 31 steps / min), the average peak activity index (average steps / min of the highest 30 minutes of the day regardless of when they occurred) and the average steps in 1, 5, 30 and 60 minutes (average steps / min of the highest continuous period of 1-min, 5-min, 30-min and 60-min periods). No significant difference was found in sam output data between the first and the seventh day of monitoring before and after ivmp . Other tests were administered between 1 and 3 p.m. before ivmp and 31 day later at the same time of day . All walk tests, (25fwt, ssst and 2-mintw) participants performed in the same type of footwear and clothes . To reduce patients bias, the walking - based tests were conducted without fixed order, with the exception of continuous long - term walk monitoring . To reduce the clinicians bias, the indication for ivmp was established independently of the study and different parts of research were also performed independently by different authors (clinical examination m.m . ;; sam instructions and analyses of outputs i.l . ; statistics v. . ). Was used to assess the significance of changes of walking - based measures after ivmp . Responsiveness was determined from time 1 and 2 data by calculating both effect size (es: mean change score divided by sd of admission scores) and standardized response means (srm: mean change score divided by sd of change scores), since they can produce different values . They were interpreted using cohen s criteria (values of 0.20.49 were defined as small, those of 0.50.79 as moderate and that of 0.8 and greater as large). The relative responsiveness of competing walking - based measures (edss, msws-12, 2-mintw, ssst, 25fwt and sam parameters) was determined by computing their relative efficiency (re). Re estimates the extent to which 1 measure is more or less efficient at detecting change relative to another measure . We computed re as pair - wise squared z values from wilcoxon signed ranks test . The walking - based measure with the largest z value this measure has a measurement precision of 100% and the other values are estimated as a percentage of the most responsive measure . The effect of different walking - based measure responsiveness on sample size estimates was evaluated by computing the number of patients required for each measure to detect the same effect of ivmp . The computed values are relative to 100 patients using the most responsive walking - based measure [100{(z value of walking measure with largest z value / z value of other walking measure)}] (11). The group consisted of 39 women and 10 men, with a median age of 35 years (range 1856 years), median disease duration of 8 years (range 1.327), median edss of 3.0 (range 1.56.0) before treatment, and with a median body mass index of 22.7 (range 16.732.3). Most patients are well - educated (92% had a high school or college degree) and not actively employed (61% were unemployed or retired). Table 2 presents statistically significant differences of all walking - based measures applied, indicating significant improvement of walking ability 1 month after relapses following steroid pulses . Both the patient - rated (msws-12) and clinician - rated (edss) scales showed large degrees of responsiveness as determined by srm (1.05 and 1.29, respectively) and large or moderate responsiveness as determined by es (1.02 and 0.69, respectively). Walk tests (2-mintw, ssst and 25-ftw) showed large and moderate responsiveness as determined by srm (0.89, 0.69 or 0.55, respectively) and moderate or small responsiveness as determined by es (0.54, 0.31 and 0.27, respectively). The real - world ambulation measured by sam had small responsiveness of all parameters except for average steps in 1 minute and average peak activity index, which had moderate responsiveness as determined by srm (0.70 and 0.53, respectively). The edss had the largest z value and was chosen as the denominator for the pair - wise calculation . The 2-mintw, msws-12, ssst and 25fwt had measurement precision of 95.1%, 82.4%, 75% and 68.3%, respectively . Sam parameters had re between 60% for average steps in 1 minute and 15.5% for percentage of time spent in low step activity . The average steps in 1 minute had the largest z value; this was chosen as the denominator for pair - wise calculation and had measurement precision of 100% . Average steps in 5 minutes, average peak activity index and average daily step count had measurement precision of 77.5%, 70% and 68.4%, respectively . The worst re had percentage of time spent in medium step activity and percentage of time spent in low step activity, with re of 36.8% and 25.8%, respectively . Different walking - based measures as sample size estimates required for each measure to detect the same effect of ivmp are shown in table 6 . The number of patients required to detect the improvement detected by the edss ranged from 105 for 2-mintw to 453 and 647 patients for percentage of time spent in medium and low step activity and percentage of time spent in low step activity . . Walking difficulties are very common for ms patients; therefore the selection of appropriate walking - based outcome measures is essential to evaluate response to treatment, as well as disease progression sensitivity . The aim of this study was to examine the effects of ivmp on the recovery of walking ability in patients experiencing ms relapses and to compare the responsiveness of walking - based measures that might be used in ms clinical trails . As the importance of responsiveness lies in the balance between statistical power and sample size, the next step taken was to examine the potential implications for clinical trials of using walking - based measures with different levels of responsiveness . In this study all applied methods of walking assessment indicated significant improvement of walking ability in patients with walking difficulties caused by ms relapse 1 month after ivmp . Two previous randomized, double - blind and placebo - controlled studies convincingly demonstrated, by edss scoring, that ivmp accelerates clinical recovery from relapse of rrms 1 month after treatment . As the edss is strongly biased toward walking, our finding is not surprising . However, the responsiveness of different walking - based measures varied markedly in terms of effect sizes, relative efficiency and implication for sample size estimation . The highest responsiveness was obtained by msws-12, edss and 2-mintw . Regarding the re, the potential impact of different responsiveness on sample size estimation showed that the number of patients required to detect the same improvement of walking ability obtained by ivmp ranged from 1 (edds) to 6.5 (percentage of time spent in low step activity). The msws-12 is the newest multi - item rating scale of walking ability in ms patients that combines patients perspectives with psychometric methods . Msws-12, as the other patient - rated scale, has limitations in self - report biases and recall . Our study scales was unblinded, and it is possible that the high responsiveness results reflect the fact that patients expected a change in walking ability to occur with therapy . Furthermore, a relatively short period between the 2 tests using walking - based measures was set to minimize the impact of seasonality on continuous monitoring of long - term walking . Therefore, we suppose that the practice effects, particularly because of recall, familiarity and motivation, had an impact on our results . However, in this study and in the original publication of hobart et al ., the responsiveness of msws-12 is very similar . The edss is the best known and the most widely used clinician - rated scale . The edss has been criticized on several counts, including, among others, having a limited responsiveness as pointed in hobart et al . . A possible explanation for this discrepancy is likely due to the differences between the 2 cohorts, as well as therapy and inclusion criteria . Our study exclusively included rrms patients who were selected for steroid therapy (in hobart s study more than half of patients had primary or secondary progressive ms and they were treated with physical therapy), and were less disabled having single restriction of ms relapse with walking difficulties . Regardless of their advantages (quickness and inexpensiveness), there are also some disadvantages (single activity execution within a limited time frame, non - familiar environment, dependence on exact instructions and an unquantifiable impact of the observer). The 25fwt reflects only the ability to walk a given distance and walking speed . In our study, the responsiveness of 25fwt is small and is very similar to the responsiveness of 25fwt in hobart et al . . The responsiveness of the ssst has never been studied, and we suppose it is higher than the responsiveness of the 25fwt . The ssst also reflects the ability to walk a given distance and walking speed, but it depends more on coordination, balance and ease of abduction in the hip than does the 25ftw . The range of measurement of ssst is much wider and its floor effect is less pronounced than that of the 25ftw . We confirmed the higher responsiveness of the ssst according to the 25fwt in this sample of patients . However, the responsiveness of ssst determined by srm is moderate and that determined by ef is small . The results reveal that out of all applied walking tests, the 2-mintw has the best responsiveness (large responsiveness determined by srm and moderate responsiveness determined by ef). In recent years, accelerometer - based technology has enabled reliable and valid data recording of frequency and intensity of walking over continuous time intervals . Continuous walking monitoring provides a direct and objective measure of mobility in a community setting . In this study, exceptions are the 2 parameters reflecting burst walking activity (average steps in 1 minute and average peak activity index), which have moderate responsiveness determined by srm . The obtained result indicating that sam parameters show relatively small responsiveness was actually expected, because numerous personal and environmental factors affect everyday walking . This study has some limitations concerning the generalizability and direct applicability of our results to clinical trials in ms . Firstly, the data were not collected within the context of a randomized controlled trail . Secondly, we compared several walking - based measures in a small sample from 1 clinical site . Thirdly, we examined walking - based measures only in a sample of ms patients who were selected for steroid therapy and with the single restriction of having ms relapse with walking difficulties . Fourthly, the relatively short period between the 2 tests involves the potential practice effects of all walking - based tests, not just the msws-12 . Another limitation is that we studied only positive responsiveness of walking - based measures . Nevertheless, showing that the variable responsiveness of walking - based measures has substantial implications for clinical trials, we hope this study will contribute to creation of a body of knowledge for evidence - based selection of outcome measures . Further evaluations of responsiveness of walking - based measures for a more modest treatment [5,3537] of walking difficulties in ms patients are suggested . The negative responsiveness of walking - based measures for evaluation of walking ability worsening that may occur over time might be the topic of another research study . Finally, the impact of practice effects on responsiveness of walking - based measures in ms patients should be quantified.
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In recent years, magnesium alloys have attracted much attention as potential biodegradable bone implant materials due to their biodegradability in the bioenvironment as well as their favourable mechanical properties, especially the elastic modulus being close to that of the bone which will effectively decrease the stress shielding effect [1, 2]. Most of these studies are focused on the ways to improve corrosion resistance in physiological media, for example, through alloying or coating . Further in vitro and in vivo tests will provide essential data for further material and process optimization before the pre - clinical test stage is reached . Another issue of these potential biomedical materials, being as important as corrosion resistance but not always addressed, concerns their bioactivity, i.e. The ability of the implant to form bonding with the surrounding bone tissue after implantation . While a number of in vivo studies have shown good bone attachment to magnesium implants after 9 to 18 week implantation and good biocompatibility of magnesium in long - term service [5, 6], some other research on the early bone response to magnesium bone implants has led to inconsistent results . For example, only 50% of magnesium implants were fixed at 5 weeks post - implantation . In some cases, gaps could still be observed between the implant and bone tissue after 14-week implantation, even though the material showed a moderate degradation rate . It is clear that efforts are needed to improve the surface biocompatibility and bioactivity of mg - based materials and speed up the early tissue response to the implant . Introducing bioactive particles that have good ability to induce the deposition of ca p compounds from simulated body fluid into a metal matrix to form a metal matrix composite may be an effective way to elevate the surface biocompatibility and bioactivity of the matrix material, as demonstrated in a recent study . Ning and zhou reported that a ti / hydroxyapatite (ha) biocomposite (with a ti / ha ratio of 1:1 by volume) indeed had the ability to induce apatite nucleation and growth on its surface in simulated body fluid . Their results showed that the composite with 20% ha by weight was a cytocompatible biomaterial and the distribution and size of ha particles were of major importance for mechanical and corrosive properties . It is however known that ha has a minimal degradability and its bioactivity still needs to be improved . A previous study demonstrated a moderate degradation rate and good cytocompatibility of zk30 (3 wt% zn, 0.6 wt% zr and balance mg) that contained no potentially toxic elements such as aluminium in az91 . The present study was aimed at exploring the feasibility of using commonly acknowledged bioactive and biodegradable glass (bg, 45s5) as the reinforcing phase in the composites with the zk30 magnesium alloy as the matrix and confirming the surface biocompatibility of the composites . The structures of the composites, including the distribution of bioactive particles in the matrix as well as their mechanical properties were investigated . The surface biocompatibility of the composites was evaluated by characterizing the corrosion layer on samples soaked in a cell culture medium and comparing it with that of the matrix sample . 45s5 bioglass particles with a composition of 45% sio2, 24.5% na2o, 24.5% cao and 6% p2o5 by weight were introduced . Upon stirring, the mixture was cast into a cylindrical form by using a high pressure casting machine . The volume fractions of bg particles in the composites were 3.4, 6.9 and 14.3%, corresponding to 5, 10 and 20% by weight, respectively . Samples for microstructure observation were cut from the ingots, ground by sic papers up to 2000 grit, and then polished down to 1 m . A leica optical microscope and a scanning electron microscope (sem, jsm-6500f, jeol) combined with an energy dispersive x - ray (edx) spectrometer (inca energy, oxford instruments) were used . Furthermore, a jeol jxa 8900r electron probe microanalyzer (epma) was used to determine the distribution of the silicon element in the composites . The acceleration voltage was set at 15 kev and the probe current at 100 na using a focused electron beam . The size of the analysis area was 100 100 m with a step size of 1.0 m . For compressive testing, specimens with a length of 15 mm and a diameter of 10 mm were machined in accordance with din en 50106 . The immersion test was conducted by soaking samples in the cell culture medium (minimum essential medium with earle s balanced salts, safc bioscience inc . The minimum essential medium with earle s balanced salts (e - mem) was selected, because it represents the ion concentrations of blood plasma and contains some kinds of amino acid that is also present in human plasma . The concentrations of ions in e - mem in comparison with those in the human blood are listed in table 1 . The as - fabricated composites as well as the zk30 alloy were cut into disks with a diameter of 10 mm and a thickness of 2 mm . The surfaces of the samples were ground by sic papers up to 2000 grit, polished down to 1 m and dried, before they soaked in e - mem . A disk was immersed in the e - mem solution with a volume of 22 ml at 37.0c, thus the ratio of the volume of the solution to the apparent surface area of the disk being 0.1 cm .table 1ion concentrations in human blood and e - mem na (mmol / l)k (mmol / l)ca (mmol / l)mg (mmol / l)c (mmol / l)hco3 (mmol / l)hpo4 (mmol / l)so4 (mmol / l)amino acids (mg / l)dex / glu (g / l)blood plasma1425.02.51.5103271.00.5ndnde - mem1515.371.800.81112526.20.8970.8110.08601nd not determined ion concentrations in human blood and e - mem nd not determined after soaking in the cell culture medium for 24 h, the samples were taken out and dried . 45s5 bioglass particles with a composition of 45% sio2, 24.5% na2o, 24.5% cao and 6% p2o5 by weight were introduced . Upon stirring, the mixture was cast into a cylindrical form by using a high pressure casting machine . The volume fractions of bg particles in the composites were 3.4, 6.9 and 14.3%, corresponding to 5, 10 and 20% by weight, respectively . Samples for microstructure observation were cut from the ingots, ground by sic papers up to 2000 grit, and then polished down to 1 m . A leica optical microscope and a scanning electron microscope (sem, jsm-6500f, jeol) combined with an energy dispersive x - ray (edx) spectrometer (inca energy, oxford instruments) were used . Furthermore, a jeol jxa 8900r electron probe microanalyzer (epma) was used to determine the distribution of the silicon element in the composites . The acceleration voltage was set at 15 kev and the probe current at 100 na using a focused electron beam . The size of the analysis area was 100 100 m with a step size of 1.0 m . For compressive testing, specimens with a length of 15 mm and a diameter of 10 mm were machined in accordance with din en 50106 . The immersion test was conducted by soaking samples in the cell culture medium (minimum essential medium with earle s balanced salts, safc bioscience inc ., usa). The minimum essential medium with earle s balanced salts (e - mem) was selected, because it represents the ion concentrations of blood plasma and contains some kinds of amino acid that is also present in human plasma . The concentrations of ions in e - mem in comparison with those in the human blood are listed in table 1 . The as - fabricated composites as well as the zk30 alloy were cut into disks with a diameter of 10 mm and a thickness of 2 mm . The surfaces of the samples were ground by sic papers up to 2000 grit, polished down to 1 m and dried, before they soaked in e - mem . A disk was immersed in the e - mem solution with a volume of 22 ml at 37.0c, thus the ratio of the volume of the solution to the apparent surface area of the disk being 0.1 cm .table 1ion concentrations in human blood and e - mem na (mmol / l)k (mmol / l)ca (mmol / l)mg (mmol / l)c (mmol / l)hco3 (mmol / l)hpo4 (mmol / l)so4 (mmol / l)amino acids (mg / l)dex / glu (g / l)blood plasma1425.02.51.5103271.00.5ndnde - mem1515.371.800.81112526.20.8970.8110.08601nd not determined ion concentrations in human blood and e - mem nd not determined after soaking in the cell culture medium for 24 h, the samples were taken out and dried . Figure 1 shows the microstructures of the zk30-bg composites with different volume fractions of bg particles . As can be seen, black particles are homogeneously dispersed in the matrix and the number density of the black particles indeed increases with increasing volume fraction of bg particles as desired (fig . Sem revealed that the black particles had angular shapes, as those of the initial bg powder particles, suggesting little reaction took place during material preparation . The composites with 3.4 and 6.9% bg by volume were slightly porous and most of the pores had sizes below 10 m (fig . However, in the composite with 14.3% bg particles by volume, the pore sizes were considerably larger (fig . 1f). In a composite ingot made by the semi - solid casting method, in general, porosity may arise from (i) gas entrapment during mixing, (ii) hydrogen evolution, and (iii) shrinkage during solidification . For the present composites, the porosity might result from gas bubbles entering the slurry either independently or as gas envelopes of the reinforcing particles . Indeed, it was observed that the porosity in stir - cast composites increased almost linearly with particle content . Such an increase in porosity will have an adverse effect on the mechanical strength of the composite . The compressive strength of the composite indeed decreased from 330 to 240 mpa as the volume fraction of bg particles increased from 0 to 14.3% . It is however important to note that the strengths of the composites are still considerably higher than the typical strength of human cortical bone (88.3163.8 mpa) and therefore the sacrifice in strength due to the bg particles and associated porosity is not a matter of serious concern . The effect of the porosity may be more on the degradation rate than on the compressive strength and, therefore, it is necessary to take measures to eliminate the residual pores in the structure . Vacuum hot pressing, hot isostatic pressing and especially hot extrusion involving strong shearing have been proven to be effective methods capable of consolidating initially porous materials . 1surface morphologies of the zk30-bg composites with 3.4% (a, b), 6.9% (c, d) and 14.3% (e, f) bg particles by volume under optical microscope (a, c, e) and sem (b, d, f). The black arrows point at the reinforcing particles, while the white ones point at the pores in the compositesfig . 2compressive strengths of the zk30-bg composites with different volume fractions of bg particles surface morphologies of the zk30-bg composites with 3.4% (a, b), 6.9% (c, d) and 14.3% (e, f) bg particles by volume under optical microscope (a, c, e) and sem (b, d, f). The black arrows point at the reinforcing particles, while the white ones point at the pores in the composites compressive strengths of the zk30-bg composites with different volume fractions of bg particles since the biomedical property of a bioactive ceramic material is closely related to its chemical composition, it is of utmost importance to keep the compositional characteristics of bg particles in the composite unchanged . In general, it is rather difficult to ascertain the retention of the chemical composition of bg particles due to their amorphous structure . In the present study, edx analysis was performed to confirm the compositional retention of bg particles in the composites . As can be seen from fig . 3a, only mg, zn and a small amount of element o were detected by edx, and the mass ratio of mg and zn was close to that of the designed zk30 composition, which means that the matrix composition was well preserved after the casting procedure . As to the bg particles in the composites, edx analysis revealed the presence of ca, si, na, o and a small amount of mg (fig . The presence of mg could be attributed to the magnesium surrounding the bg particles or to mild reactions at the interfaces between the matrix and bg particles . For a clearer comparison, the weight percents of ca, si, na and o of the analyzed particles as determined by edx are listed in table 2 and compared with those of the theoretical elemental composition of bg particles . It can be seen that the discrepancies between the as - measured and theoretical composition data were not marked, suggesting the retention of bg particles in the composites . It is of particular importance to note the high si concentration in the bg particles, which is necessary for the excellent bioactivity of a bioactive glass . Epma analysis indeed revealed the silicon element with enhanced intensities scattered all over the matrix, which corresponded to the locations of bg particles (fig . 4). It was thus clear that the basic chemical composition of bg particles remained largely intact during semi - solid high pressure casting, which would be essential for the preservation of their bioactivity . In other words, semi - solid high pressure casting appeared to be a viable method to fabricate zk30-bg composites . 3edx analysis of the matrix (a) and particles (b) in the zk30-bg compositestable 2edx determined composition of bg particles in the composites in comparison with the designed composition of bg (wt%) casinaomgas - designed17.521.018.240.70as - measured22.329.714.230.63.2fig . The analysis area on a zk30-bg mmc sample is indicated by the square in the backscattered electron image (bei). The intensity of the si k in the framed area is represented by the gray scale (or a jet - like colour scheme in the online version). Here black represents a low signal and light gray - white higher intensity signals (pinkish - white the highest intensity in the online version) edx analysis of the matrix (a) and particles (b) in the zk30-bg composites edx determined composition of bg particles in the composites in comparison with the designed composition of bg (wt%) distribution of si in the composite as determined by epma . The analysis area on a zk30-bg mmc sample is indicated by the square in the backscattered electron image (bei). The intensity of the si k in the framed area is represented by the gray scale (or a jet - like colour scheme in the online version). Here black represents a low signal and light gray - white higher intensity signals (pinkish - white the highest intensity in the online version) the surface morphologies of the composites after immersion in e - mem for 24 h are shown in fig . 5 . During immersion, pitting corrosion indeed occurred on the surface of the zk30 alloy sample without bg reinforcement (fig . In contrast, a deposition layer was formed on the surface of the composite samples, and the area of this layer increased with increasing volume fraction of bg particles in the composite (fig . Edx analysis of the surface layer showed increases in the ca atomic percent and ca / p molar ratio, as the volume fraction of bg particles increased from 0 to 14.3% (fig . . The ca / p ratios of the composites in the range of 1.21.35, being lower than the theoretical value (1.67), show that the newly formed apatite layer is a ca - deficient apatite layer, consistent with the properties of bone - like apatite formed on an ha - bioglass composite and on bioglass . The results indicated that the bg particles present in the composite were indeed able to induce faster and more homogeneous deposition of a ca- and p - rich layer on the magnesium alloy matrix . The operating mechanism of spontaneous apatite formation on metallic magnesium may be as follows: an ionic exchange between mg from the substrate and h from the soaking medium takes place, leading to an increase in ph; higher ph values increase the supersaturation degree of the cell culture medium with respect to apatite . The result is the nucleation and growth of a ca-, p- and mg - rich ceramic layer on the magnesium substrate . With the addition of bg particles, apart from the supersaturation caused by the elevated ph environment, the apatite layer formation is accelerated due to the release of ca from the partial dissolution of bg particles . The silicon dissolved from the bg particles surface may act as a nucleating agent, thereby accelerating the formation of the apatite layer which can significantly improve the surface biocompatibility of the material [8, 9]. In combination with the consideration on the compressive strengths of the composites (fig . 2), the zk30-bg composite with 6.9% bg by volume showed the best balanced properties . Further research on the biocompatibility and bioactivity of the composites and other aspects relevant to the biomedical application will be carried out to optimize both material design and processing further . 5sem micrographs of the zk30 alloy (a) and the composites with 3.4% (b), 6.9% (c) and 14.3% (d) bg particles by volume after immersion in e - mem for 24 hfig . 6ca atomic percents and ca / p molar ratios of the surface layer formed on the composite samples with different volume fractions of bg particles after immersion in e - mem for 24 h sem micrographs of the zk30 alloy (a) and the composites with 3.4% (b), 6.9% (c) and 14.3% (d) bg particles by volume after immersion in e - mem for 24 h ca atomic percents and ca / p molar ratios of the surface layer formed on the composite samples with different volume fractions of bg particles after immersion in e - mem for 24 h for the first time, mg - based metal matrix composites with bioactive glass as reinforcing particles were fabricated by the semi - solid high pressure casting method . Accelerated formation of an apatite layer on the composite surface occurred, indicating an improved surface biocompatibility of the material . These results demonstrated the feasibility of improving the surface biocompatibility of mg - based implant materials by adding bioactive particles . Further research on material - cell interactions would be of great value to show the potential of the composites as biodegradable, bioactive orthopaedic materials.
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Materials rna oligonucleotides described in this work were obtained from dharmacon and dna oligonucleotides were obtained from invitrogen . We cloned ns3 + and ns3 - 4a+-expressing cdna from the hepatitis c viral genotype 1a (version h77, kindly donated by dr . The dna oligonucleotides used for pcr of ns3 and ns3 - 4a were ns3 1a-1 and ns3 1a-2 or ns4a(1a) bamhi . An internal xhoi site in the ns3(1a)+ gene was removed through a silent base pair change using a quikchange kit (stratagene). The dna oligonucleotides used for the quikchange reaction were ns3 1a-3 and ns3 1a-4 . The ns3 + and ns3/4a+ genes from hepatitis c viral genotype 1b (version n, kindly provided by dr . Stan lemon (24)) were amplified and cloned into pet15b according to the methods of beran et al . The upstream dna oligonucleotide used for pcr amplification was ns3 xhoi and the downstream dna oligonucleotide was ns3 bamhi or ns4a. Ns3 + and ns34a+ of both the 1a and 1b genotypes were cloned into pet - sumo (invitrogen) using extaq pcr (takara) followed by ligation with linear pet - sumo according to the manufacturer's protocol . The dna oligonucleotides used were the same as those described above for pet15b cloning, except that the 5 pcr oligonucleotide in the genotype 1a case was ns3(1a) sumo start and in the genotype 1b case was ns3(1b) 5 sumo start . To produce pet - sumo - ns4a(1a)+, the ns4a(1a)+ gene was pcr amplified using the dna oligonucleotides ns4a 1a sumo start and ns4a 1a sumo end and then cloned into pet - sumo . To produce the isolated ns3 protease domain, a stop codon was inserted in the ns3(1a)+ gene after amino acid 188 in the pet - sumo - ns3(1a)+ plasmid (13). Mutagenesis was conducted via quikchange (stratagene), using dna oligonucleotides ns3prot(1a)-1 and ns3prot(1a)-2 . To create the ns3/4a polyprotein with an ala / ala cleavage site instead of a thr / ser cleavage site, the wild - type ns34a(1b)+ construct was mutated by quikchange (stratagene), using oligonucleotides aa ns34a-1 and aa ns34a-2 . All constructs were verified initially through pcr screening as necessary and subsequently sequenced for accuracy (keck facility, yale university). Because there are a multitude of reports describing the purification of various autocleaved and reconstituted forms of the ns3 - 4a complex (17, 22, 23, 26, 27) and because all of these factors greatly influence ns3 enzymatic function (13), we believe it is necessary to describe our constructs, purification strategies, and reconstitutions in detail . All proteins were purified according to the methods described in beran et al ., (13, 25). Briefly, 4 liters of escherichia coli culture in lb (supplemented with 35 g / ml kanamycin) were grown at 37 c with shaking . Upon reaching the exponential growth phase, the temperature was shifted to 15 c, the cultures were supplemented with 1 mm isopropyl 1-thio--d - galactopyranoside (final concentration), and subsequently incubated overnight at 15 c with shaking . After lysing the cells using an emusiflex c5 cell disruptor (avestin), the cellular extract was centrifuged at 10,000 rpm (14,500 g) at 4 c for 10 min before being passed through a nickel column . The his6 and his6-sumo fusion proteins were purified through a nickel column (qiagen), treated with 10 units of sumo protease (invitrogen) overnight at 4 c, and subsequently passed through a gel filtration column (hiload superdex 200 16/60) equilibrated with a buffer containing 25 mm hepes (ph 8.0), 0.3 m nacl, 10% glycerol, 1 mm dithiothreitol, and 0.2% triton x-100 . Ninety - six 1.2-ml gel filtration fractions were collected at a rate of 0.4 ml / min . The ns3 - 4a complex eluted between fractions 45 and 55, presumably in a detergent micelle (17). Fractions containing ns3 - 4a were identified using the ret - s1 protease assay (the ret - s1 protease assay details are described later in this section) as well as through sds - page analysis . Moreover, anti - ns3 and anti - ns4a (monoclonal antibodies in both cases) western blotting were used to confirm the identity of the purified protein complex, which was then used for experimentation (see fig . 2, b and c, and the western blotting procedure described later in this section). Free ns3 protein eluted from the gel filtration column between fractions 60 and 70 . Fractions containing ns3 were identified using rna unwinding assays (25) and sds - page analysis . In addition, anti - ns3 western blotting was used to confirm the identity of the purified protein, which was then used for experimentation (see fig . 2b and the western blotting procedure described later in this section). Because there is a 13-kda size difference between his - sumo tagged and untagged protein, the untagged proteins were initially analyzed side by side using sds - page with uncleaved his - sumo ns34a or uncleaved his - sumo - ns3 to verify that the purified proteins were, in fact, untagged species (data not shown). After purification, preparations were divided into 10-l aliquots and stored at -80 c . The ns3 and ns3 - 4a proteins were examined for purity and their sizes compared using sds - page (fig . Protein samples (40 pmol) were subjected to electrophoresis on a nupage 412% bistris gel (invitrogen) in mes - sds buffer for 2 h at 200 v. the gel was subsequently stained with coomassie blue . For reconstitution of the protease complex using purified ns3 and purified ns4a proteins (ns3 + ns4a), we expressed his - sumo - ns3 as well as his - sumo - ns4a in e. coli using the pet - sumo vector system (invitrogen). After partial purification using a nickel column, his - sumo - ns3 and a 4-fold excess of his - sumo - ns4a were mixed and incubated together at 4 c overnight in the presence of 10 units of sumo protease (invitrogen). Reconstituted, untagged ns3 + 4a was subsequently isolated from untagged ns3 and untagged ns4a (based upon size differences) by passing the protein mixture through a gel filtration column (hiload superdex 200 16/60) using the same methods as described above for autocleaved ns3 - 4a . The same procedure was followed to reconstitute and purify untagged ns3 protease domain with untagged ns4a (ns3 protease domain + ns4a). In this case, ns3 protease domain alone eluted between gel filtration fractions 75 and 80 and ns3 protease domain + ns4a eluted between fractions 70 and 75 . The ret - s1 serine protease assay as well as sds - page analysis was used to identify the fractions containing reconstituted ns3 protease domain + ns4a . In all cases, sds - page was performed using a bio - rad miniprotean ii apparatus according to the manufacturer's protocols . The western blot transfer was performed using a bio - rad transfer apparatus according to the manufacturer's instructions . Anti - ns3 and anti - ns4a monoclonal antibodies were purchased from virogen (watertown, ma) and diluted according to the manufacturer's instructions . In these experiments (as shown in fig . 2, b and c), 25 pmol of each protein was subjected to electrophoresis on a 12% sds - polyacrylamide gel for 1 h at 200 v. subsequently, the proteins were transferred to a nitrocellulose membrane by applying 100 v for 1 h at 4 c . Finally, the blots were incubated with either anti - ns3 or anti - ns4a monoclonal antibody and subsequently developed using a pierce supersignal western blot analysis kit . Protease assays protease assays were performed at 37 c using the resonance energy transfer - s1 substrate (ret - s1) (anaspec) designed by taliani et al . Ret - s1 is an ns4a / ns4b junction mimic that fluoresces upon cleavage . All assays, unless otherwise indicated, were performed in 60-l reaction volumes containing 40 nm ns3 - 4a and 5 m ret - s1 . The data shown in fig . 6 and table 1 were collected using 120 nm protein with the indicated amounts of ret - s1 substrate . In table 1, the kcat values were calculated based upon the observation that 75% of the protein was active in each case (fig . 5). The buffer conditions were the same as those normally used for helicase assays (13): 25 mm \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}{\mathrm{mops - nh}}_{4}^{+}\end{equation*}\end{document} (ph 6.5), 3 mm mgcl2, 1% glycerol, 2 mm dithiothreitol, 30 mm nacl, 0.2% (v / v) data were collected using a cary eclipse spectrophotometer (varian) with a temperature - controlled cuvette holder . The initial background value (the value at time 0, just before substrate addition) was subtracted from all subsequent time points . Table 1the ns3 helicase domain enhances ns3 - 4a protease activity the proteolysis data were determined by monitoring ret - s1 cleavage on a fluorescence spectrophotometer (see experimental procedures). The data shown were determined using proteins of the 1a genotype and is the average of three experiments . The error values represent standard deviation.proteinkmvmaxkcatkcat/km mpmol ret - s1 cleaved / spmol ret - s1 cleaved / s / pmol enzymepmol ret - s1 cleaved / s / pmol enzyme/m substrate ns3/4a 10.0 3.0 17 2 3.15 0.30 0.315 ns3 + ns4a 3.0 1.0 0.50 0.05 0.09 0.01 0.030 ns3 protease domain + ns4a 4.0 1.2 0.11 0.01 0.020 0.002 0.005 the ns3 helicase domain enhances ns3 - 4a protease activity the proteolysis data were determined by monitoring ret - s1 cleavage on a fluorescence spectrophotometer (see experimental procedures). The data shown were determined using proteins of the 1a genotype and is the average of three experiments . The error values represent standard deviation . The fraction of active ns3 - 4a protein was determined for both tagged and untagged preparations by incubating a small amount of enzyme (40 nm) with a large amount of ret - s1 substrate (5 m). A line was fit to the slope of the proteolysis data during the steady - state phase of reaction . This line was extrapolated to the intersection with the y axis (sigma plot, systat software), which corresponds to the concentration of active enzyme present in the reaction (28). In all cases throughout this work, importantly, similar values for the fraction of active enzyme (75%) were observed when the ns3 - 4a concentration was raised to 120 nm or when the ret - s1 concentration was raised well above the km (10 3 m) (table 1) to 20 m (data not shown). Similarly, when reconstituted ns3 protease domain + ns4a and ns3 + ns4a were assayed with ret - s1 concentrations well above the km values (810 m ret - s1, whereas km = 4.0 1.2 and 3.01.0 m, respectively) (table 1), a similar enzymatic active fraction was observed for both complexes (75%) (data not shown). When protease activities of ns3 - 4a(1a) and ns3 - 4a(1b) were compared using the ret - s1 analyses, results were similar for both genotypes the ns3 - 4a complex can be purified in a highly active form to study ns3 - 4a protease function, we needed to purify ns3 in complex with its ns4a co - factor (i.e. Ns3 - 4a). In previous studies, ns3 - 4a has been overexpressed in insect cells (27) or in e. coli (12, 17). In all of these cases, additional amino acids were fused to the ns3 n terminus for the purpose of protein purification (12, 17) and lysine residues were added to the ns4a c terminus to increase solubility (17). To our knowledge, a recombinant ns3 - 4a complex that lacks tags or additional modifying amino acids has never been produced . We therefore designed an approach for generating native, full - length ns3 - 4a complex (e.g. Lacking modifications to the terminal sequences of ns3 or ns4a) using the pet - sumo expression system in e. coli (fig . The protein of interest is tagged at the n terminus with the sumo polypeptide, which enhances solubility of the protein and is then readily removed upon incubation with the sumo protease, thereby resulting in native protein that lacks additional amino acids (29). We expressed ns3 - 4a as a his - sumo - ns3/4a polyprotein in e. coli, designing the construct so that ns3 would be expected to spontaneously cleave ns4a from its c terminus (fig . 1b) (17, 27), thereby forming an active protease complex (4). After purification on a nickel column, the his - sumo fusion protein was cleaved by sumo protease from ns3, generating a native n terminus (fig . The protein mixture was subsequently passed through a gel filtration column to separate native protein complex from uncleaved fusion protein (see experimental procedures). The resultant cleaved, purified ns3 - 4a complex was 95% pure (fig . 2a). To determine whether the purified ns3 - 4a protein had autocatalytically cleaved properly, forming two separate proteins (ns3 and ns4a), we subjected the complex to denaturing electrophoresis side by side with ns3 and an uncleavable ns3/4a polyprotein control . The uncleavable ns3/4a polyprotein was produced by mutating the conserved junction amino acid sequence between ns3 and ns4a from thr / ser to ala / ala . Sds - page and western blot analysis show that the ns3 component of the wild - type ns3 - 4a preparation has the same electrophoretic mobility as ns3 (in fig . 2a, compare lanes 2 and 3 of the coomassie - stained gel and in fig . 2b, compare lanes 1 and 2 of the anti - ns3 western blot). However, the uncleaved ns3/4a polyprotein migrates more slowly than ns3, as expected due to its larger size (in fig . 2a, compare lane 4 to lanes 2 and 3 and in fig . Coomassie staining does not reveal any ns3/4a polyprotein in our ns3 - 4a preparation (fig . 2a, lane 3). However, western blots are more sensitive than coomassie staining, and consistent with this, the anti - ns3 western blots reveal a small fraction of uncleaved ns3/4a polyprotein in the wild - type ns34a preparation (fig . 2b, note the faint upper band in lane 2 that migrates similarly to the ns3/4a polyprotein in lane 3). Taken together, these results demonstrate that the preparation of wild - type ns3 - 4a complex is composed primarily of fully cleaved ns3 and ns4a molecules . The catalytic function and conformational homogeneity of this preparation figure 1.composition and purification of ns3 - 4a . The ns3 - 4a complex organization and a, pro refers to the serine protease domain and the roman numerals indicate the respective ns3 helicase subdomains . The regions where atp, rna, and the ns4a co - factor bind are indicated as well . The protein construct expressed in e. coli is depicted in panel b. the numbers below the map refer to the hcv polyprotein numbering of the amino acids of ns3 - 4a . In panel c, his - sumo - ns3 and his - sumo - ns4a fusion construct designs are presented . These proteins were purified separately and their his - sumo tags were removed using the same methods as for ns3 - 4a . Subsequently, the native ns3 or native ns3 protease domain was reconstituted with native ns4a (see experimental procedures). The ns3 - 4a complex organization and construct design are illustrated schematically (a and b). In panel a, pro refers to the serine protease domain and the roman numerals indicate the respective ns3 helicase subdomains . The regions where atp, rna, and the ns4a co - factor bind are indicated as well . The protein construct expressed in e. coli is depicted in panel b. the numbers below the map refer to the hcv polyprotein numbering of the amino acids of ns3 - 4a . In panel c, his - sumo - ns3 and his - sumo - ns4a fusion construct designs are presented . These proteins were purified separately and their his - sumo tags were removed using the same methods as for ns3 - 4a . Subsequently, the native ns3 or native ns3 protease domain was reconstituted with native ns4a (see experimental procedures). The presence of ns4a in the ns3 - 4a preparation was assessed using denaturing electrophoresis and western blot analysis with antibodies against ns4a . The anti - ns4a western blots indicate that the wild - type ns3 - 4a preparation consists of both free ns4a (which is difficult to detect because it is so small and diffuses rapidly from the gel) and a fraction of uncleaved ns3/4a polyprotein (in fig . The free ns4a migrates at the correct position, 6 kda, near the bottom of the gel (fig . 2c, lane 2). Taken together, the electrophoretic and immunoaffinity methods provide physical evidence that the ns3 - 4a preparation consists of cleaved ns3 and ns4a components . Nonetheless, it was still essential to demonstrate that the complex was catalytically active and to assess the fraction of active molecules in the preparation . A, purified proteins were subjected to denaturing electrophoresis on a 412% gradient gel . Purified ns3 (lane 2), ns3 - 4a (lane 3), and ns3/4a polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities . The band shown in lane 3 represents the ns3 component of a native, fully cleaved ns3 - 4a preparation . The band shown in lane 4 represents an ns3/4a polyprotein preparation produced by mutating the thr / ser cleavage site between ns3 and ns4a to a non - cleavable sequence (aa). In panels b and c, anti - ns3 and anti - ns4a western blot analysis confirm the identity of our purified proteins (see experimental procedures). B, lane 1 contains purified ns3, lane 2 contains purified, full - length ns3 - 4a, and lane 3 contains purified ns3/4a polyprotein . Truncated forms of ns3 are visible below the full - length protein in each lane in the anti - ns3 blot . These truncated forms of ns3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors . These truncated forms could represent either n- or c - terminal truncations of ns3 as the monoclonal anti - ns3 antibody binds to the central region of the helicase domain . Lane 1 contains purified ns3, lane 2 contains purified, full - length ns3 - 4a, and lane 3 contains purified ns3/4a polyprotein . Truncated forms of the ns3/4a polyprotein are visible below the full - length polyprotein in the anti - ns4a blot . These truncated forms of ns3/4a polyprotein likely represent n - terminal degraded ns3/4a as the monoclonal anti - ns4a antibody binds the final 11 c - terminal residues of ns4a . A, purified proteins were subjected to denaturing electrophoresis on a 412% gradient gel . Purified ns3 (lane 2), ns3 - 4a (lane 3), and ns3/4a polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities . The band shown in lane 3 represents the ns3 component of a native, fully cleaved ns3 - 4a preparation . The band shown in lane 4 represents an ns3/4a polyprotein preparation produced by mutating the thr / ser cleavage site between ns3 and ns4a to a non - cleavable sequence (aa). In panels b and c, anti - ns3 and anti - ns4a western blot analysis confirm the identity of our purified proteins (see experimental procedures). B, lane 1 contains purified ns3, lane 2 contains purified, full - length ns3 - 4a, and lane 3 contains purified ns3/4a polyprotein . Truncated forms of ns3 are visible below the full - length protein in each lane in the anti - ns3 blot . These truncated forms of ns3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors . These truncated forms could represent either n- or c - terminal truncations of ns3 as the monoclonal anti - ns3 antibody binds to the central region of the helicase domain . Lane 1 contains purified ns3, lane 2 contains purified, full - length ns3 - 4a, and lane 3 contains purified ns3/4a polyprotein . Truncated forms of the ns3/4a polyprotein are visible below the full - length polyprotein in the anti - ns4a blot . These truncated forms of ns3/4a polyprotein likely represent n - terminal degraded ns3/4a as the monoclonal anti - ns4a antibody binds the final 11 c - terminal residues of ns4a . To quantify the fraction of fully processed, active ns3 - 4a in the preparation, we analyzed the proteolysis of a depsipeptide substrate known as ret - s1, which was originally derived from the ns4a both free ns3 and the uncleavable mutant ns3/4a polyprotein are proteolytically inactive, as they fail to react with ret - s1 (see below). However, the wild - type ns3 - 4a preparation displays efficient protease activity against ret - s1, with a rate constant of 0.020 0.001 m product / s). This value is significantly faster than values obtained by others for ns3 - 4a with a non - native n terminus incubated with a 5ab peptide substrate (27). Importantly, burst kinetics experiments reveal that the ns3 - 4a preparation contains 75 14% active enzyme (fig . 3), indicating that the majority of the population is properly folded, assembled, and catalytically active . That this value falls short of 100% is consistent with the electrophoretic analyses, which indicated that 20% of the wild - type ns3 - 4a preparation remains uncleaved (fig . The physical and functional information provided by both the electrophoretic and proteolysis assays indicate that we have successfully overexpressed untagged, unmodified ns3 - 4a in a form that is conformationally homogeneous, appropriately cleaved, and highly reactive . This approach now makes it possible to quantitatively assess the proteolysis and helicase activities of the ns3 - 4a complex, and to compare these activities with the behavior of other ns3 constructs . Figure 3.steady-state proteolysis of ret - s1 by ns3 - 4a and his - ns3 - 4a . The y intercept of the line in each case corresponds to the active fraction of protein (see experimental procedures). For ns3 - 4a (solid circles), for his - ns3 - 4a (hatched squares), the active fraction was 25 12% . Ns3/4a polyprotein did not display any measurable serine protease activity (solid squares). Similar results were observed using ns3 - 4a(1a) and his - ns3 - 4a(1a). This data are the average of three experiments and the error values represent standard deviation . Steady - state proteolysis of ret - s1 by ns3 - 4a and his - ns3 - 4a . The y intercept of the line in each case corresponds to the active fraction of protein (see experimental procedures). For ns3 - 4a (solid circles), the active fraction was 75 14% . For his - ns3 - 4a (hatched squares), ns3/4a polyprotein did not display any measurable serine protease activity (solid squares). Similar results were observed using ns3 - 4a(1a) and his - ns3 - 4a(1a). This data are the average of three experiments and the error values represent standard deviation . Most previous work on ns3 has utilized n - terminal his - tagged variants of the protein (12, 13, 30, 31). For comparison purposes, we also constructed an ns3 - 4a complex with a his tag on the n terminus of ns3 . Interestingly, kinetic analysis indicates that the his - tagged ns3 - 4a preparation reacts 2-fold more slowly than the untagged variant (0.010 0.001 m product / s), and contains only a 25 12% active population (fig . 3 and experimental procedures). A his tag at the ns3 n terminus is therefore detrimental for folding and/or assembly of the protease domain, and it also diminishes the ultimate levels of proteolytic activity by the tagged ns3 - 4a complex . These findings are consistent with the fact that n - terminal ns3 his tags are in close proximity to the protease active site . Uncleaved ns3/4a polyprotein lacks protease and helicase activities the uncleavable ns3/4a polyprotein construct (containing 2 alanines where the threonine / serine cleavage sequence n - terminal to ns4a would have been) was synthesized originally as a tool that would provide a size standard for determining the presence of any uncleaved ns3/4a polyprotein in wild - type preparations of the complex . However, the mutant ns3/4a construct has also provided valuable additional information about the activities that we should expect for hcv polyproteins . To our knowledge, catalytic activities of uncleaved ns3/4a constructs have never been tested . Here we observed, using the mutant polyprotein, that uncleaved ns3/4a molecules are catalytically inactive (i.e. They lack both serine protease and helicase activity). They fail to display protease activity for substrates provided in trans (i.e. Ret - s1) (fig . Thus, proper assembly of the cleaved, ns3 - 4a complex is required for all the enzymatic activities of this protein . It is therefore likely that many sections of the polyprotein are not appropriately folded or functional until the individual proteins are cleaved apart . Figure 4.steady-state velocity curves for ns3 - 4a proteolysis of ret - s1 under a range of ph and salt conditions . The steady - state rates of proteolysis were 0.006 0.001 m product / s at ph 6.5, 30 mm nacl (solid circles), 0.019 0.001m product / s at ph 8, 30 mm nacl (pierced circles), 0.019 0.001 m product / s at ph 6.5, 75 mm nacl (solid diamonds), 0.020 0.001 m product / s at ph 8, 75 mm nacl (pierced diamonds), 0.010 0.001 m product / second at ph 6.5, 200 mm nacl (solid squares), and 0.010 0.001 m product / s at ph 8, 200 mm nacl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 9% for ph 6.5, 30 mm nacl, 84 16% for ph 8.0, 30 mm nacl, 84 15% for ph 6.5, 75 mm nacl, 70 12% for ph 8.0, 75 mm nacl, 95 5% for ph 6.5, 200 mm nacl, and 95 5% for ph 8.0, 200 mm nacl . The data shown were determined using ns3 - 4a of the 1b genotype and represent the steady - state data points fit to a line . This data are the average of three experiments and the error values represent standard deviation . Steady - state velocity curves for ns3 - 4a proteolysis of ret - s1 under a range of ph and salt conditions . The steady - state rates of proteolysis were 0.006 0.001 m product / s at ph 6.5, 30 mm nacl (solid circles), 0.019 0.001m product / s at ph 8, 30 mm nacl (pierced circles), 0.019 0.001 m product / s at ph 6.5, 75 mm nacl (solid diamonds), 0.020 0.001 m product / s at ph 8, 75 mm nacl (pierced diamonds), 0.010 0.001 m product / second at ph 6.5, 200 mm nacl (solid squares), and 0.010 0.001 m product / s at ph 8, 200 mm nacl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 9% for ph 6.5, 30 mm nacl, 84 16% for ph 8.0, 30 mm nacl, 84 15% for ph 6.5, 75 mm nacl, 70 12% for ph 8.0, 75 mm nacl, 95 5% for ph 6.5, 200 mm nacl, and 95 5% for ph 8.0, 200 mm nacl . The data shown were determined using ns3 - 4a of the 1b genotype and represent the steady - state data points fit to a line . This data are the average of three experiments and the error values represent standard deviation . Ns3 - 4a exhibits robust serine protease activity under a variety of salt and ph conditions it has previously been reported that ns3 - 4a only functions as a robust serine protease under conditions of high salt (150 mm nacl) and high ph (7.58.0) (17, 27). These same conditions are incompatible with those required for ns3 helicase activity (13, 17, 32), and it therefore has been suggested that the two activities (proteolysis and unwinding) might be mutually exclusive (17). We therefore sought to determine whether protease activity of the wild - type ns3 - 4a complex is restricted to the narrow range of previously reported conditions . To this end, we measured ns3 - 4a serine protease activity under a variety of ph (6.5 and 8.0) and salt conditions (30, 75, and 200 mm nacl) (fig . 4). We did not observe significant differences in the active fraction of protein (i.e. The y intercept of the velocity plots) under these varying ph and salt conditions, particularly when accounting for error (fig . The steady - state proteolysis velocities in 30 mm nacl at ph 6.5 and 8.0 were observed to be 0.006 0.001 and 0.019 0.001 m / s, respectively . In 200 mm nacl at ph 6.5 and 8.0, the steady - state proteolysis velocities were observed to be 0.010 0.001 m / s in both cases . Therefore, native, full - length ns3 - 4a is a robust serine protease under a broad range of salt and ph conditions, including those that are compatible with helicase activity . Figure 5.steady-state proteolysis of ret - s1 by reconstituted ns3 + ns4a and ns3 protease domain + ns4a . The steady - state velocity for ns3 + ns4a is 0.005 001 m / s (pierced circles) and for ns3 protease ns4a is 0.001 0.001 m / s (solid squares). The y intercept of the fitted lines show ns3 + ns4a and ns3 protease domain + ns4a to have 75 10% active fraction and 75 12% active fraction, respectively . The data shown are the average of three experiments and the error values represent standard deviation . Steady - state proteolysis of ret - s1 by reconstituted ns3 + ns4a and ns3 protease domain + ns4a . The steady - state velocity for ns3 + ns4a is 0.005 001 m / s (pierced circles) and for ns3 protease ns4a is 0.001 0.001 m / s (solid squares). The y intercept of the fitted lines show ns3 + ns4a and ns3 protease domain + ns4a to have 75 10% active fraction and 75 12% active fraction, respectively . The data shown are the average of three experiments and the error values represent standard deviation . Ns3hel enhances ns3 - 4a serine protease activity given that efficient ns3 helicase activity requires the protease domain (13), we asked whether ns3 protease activity is enhanced by the helicase domain . To answer this question, it was necessary to build new types of protein expression constructs because polyprotein precursors containing the isolated protease domain (i.e. A his - sumo - ns3 protease domain / ns4a polyprotein construct) did not undergo autocatalytic cleavage to form an ns3 protease-4a complex (data not shown). We therefore expressed the ns3 protease domain and full - length ns4a as separate his - sumo fusion proteins (fig . The partially purified his - sumo - ns3 protease domain and his - sumo - ns4a proteins were then combined in the presence of sumo protease and incubated to form an active complex of the native ns3 protease domain and native ns4a (ns3 protease + ns4a, see experimental procedures). Similarly, we reconstituted full - length ns3 + ns4a as a control for comparative purposes (fig . Similar to the preparation of co - expressed ns3 - 4a, 75 10% of the reconstituted ns3 + ns4a preparation and 75 12% of the reconstituted ns3 protease + ns4a preparations were proteolytically active (fig . Comparison of the kinetic parameters for ns3 - 4a, ns3 protease domain + ns4a, and ns3 + ns4a proteolysis of ret - s1 revealed that the km values for cleavage of substrate did not differ significantly (km = 10.0 3.0 m for ns3 - 4a, 4.0 1.2 m for ns3 protease + ns4a, and 3.0 1.0 m for ns3 + ns4a) (fig . 6 and table 1), indicating that ret - s1 binds similarly to all the constructs . Figure 6.steady-state proteolysis rates of ret - s1 by ns3 - 4a, ns3 + ns4a, and ns3 protease domain + ns4a in the presence of varying substrate concentrations . The data were fit to the michaelis - menten equation to determine km, vmax, and kcat values . Steady - state proteolysis rates of ret - s1 by ns3 - 4a, ns3 + ns4a, and ns3 protease domain + ns4a in the presence of varying substrate concentrations . The data were fit to the michaelis - menten equation to determine km, vmax, and kcat values . However, the maximal velocities of proteolysis by the three enzyme constructs were substantially different . The rates of cleavage for ns3 - 4a, ns3 + 4a, and ns3 protease + 4a were 17.0 2.0, 0.50 0.05, and 0.11 0.01 pmol of ret - s1 cleaved / s, respectively (fig . The large rate differences between the co - expressed ns3 - 4a and the reconstituted ns3 + ns4a constructs suggest that the ns4a cofactor does not associate and promote protease folding as effectively when presented in trans (i.e. 25 mm hepes (ph 8.0), 0.3 m nacl, 10% glycerol, 1 mm dithiothreitol, 0.2% triton x-100). Many conditions were explored to optimize reconstitution with ns4a in trans . Despite varying detergent concentrations (0.11%, using triton x-100 or chaps), varying glycerol concentrations from 10 to 50%, and varying nacl from 30 to 200 mm, increases in proteolytic activity were not observed (data not shown). Taken together, these data suggest that the protease domain active site is optimally formed only upon co - expression with ns4a . Nonetheless, the reconstituted complexes retain significant levels of activity and provide valuable tools for structure / function studies (5, 22). Given the inherently higher reactivity of co - expressed ns3 - 4a, it is clear that proteolytic activity of the isolated protease domain (ns3 protease + 4a) is directly comparable only with the reconstituted complex containing full - length ns3 (ns3 + 4a). When these two constructs are compared, the isolated protease domain is much less reactive than full - length ns3 (fig . Indeed, ns3 protease + ns4a is 5- and 6-fold less efficient than ns3 + ns4a when vmax and kcat / km values are compared, respectively (table 1). It is notable that the major effects are on kcat, which suggests that the helicase domain directly influences catalytic function of the protease active site . Therefore, ns3 protease activity is enhanced by the presence of the ns3hel domain, indicating that the two domains have evolved to become completely interdependent . By creating new types of ns3 constructs and monitoring their enzymatic activities, we have evaluated the structural integrity and catalytic potential of the serine protease domain . We have established that ns3 protease activity is strongly influenced by its structural context, and that positive and negative effects on catalysis are induced by the presence of adjacent domains and uncleaved polyprotein moieties, respectively . Taken together, our findings suggest that ns3 protease activity is tuned and regulated by other viral components, which have coevolved to maximize the role of the protease in the hcv viral lifecycle . The helicase and the protease are interdependent enzymes within a single protein like many viral proteins, ns3 is multifunctional and it contains different enzymatic activities within a single protein chain . It has long been known that the protease and atpase / helicase activities of ns3 are located on separate domains of ns3, and given the disparate nature of these activities, and their presumably different roles in function of the virus, it was generally assumed that the protease and helicase activities had no relationship to one another . However, structural analysis revealed that the protease domain is located in close proximity to the atpase and rna binding sites of ns3hel (33, 34). We therefore wondered whether the two enzymes in ns3 might actually be interdependent, having evolved to function optimally as a unit . Allosteric coactivation would have important ramifications for the function of the hcv replication complex and for the testing of hcv inhibitors, which tends to be studied with the isolated domains . In our first study of this problem, we showed that rna binding, atpase, and rna unwinding activities of the helicase are all stimulated by the presence of the covalently attached ns3 protease domain (13). Here we demonstrate that the ns3 - 4a serine protease activity is stimulated by the presence of ns3hel as well . Importantly, the ns3hel domain must be covalently attached to the ns3 protease domain, as adding ns3hel in trans had no effect on ns3 protease domain + ns4a protease activity (data not shown). Thus, the different enzymatic domains of ns3 are fully interdependent, which suggests that they have coevolved and that their presence on a single protein may have provided a selective advantage to the virus . Perhaps most importantly, the results suggest that these seemingly disparate enzymatic activities may somehow be coupled during some aspect of viral function . The ns3/4a polyprotein fails to catalyze proteolysis or unwinding just as the helicase domain activates serine protease activity, the presence of an uncleaved ns3/4a junction inhibits serine protease activity . The data presented herein show that polyprotein cleavage must occur before ns3 becomes proteolytically active . Even ns3 helicase activity requires cleavage of the ns3/4a junction sequence before any rna unwinding activity is observed (data not shown). Taken together, these data indicate that polyprotein processing must necessarily be a very early event, as it is required for catalytic functions of viral constituents . Proteolysis and helicase activities are not mutually exclusive previous work has suggested that ns3 - 4a protease activity occurs under conditions that are incompatible with helicase activity . This finding implies that helicase and protease activities might be mutually exclusive, or that their viral functions are completely unrelated . Importantly, it had been reported that ns3 - 4a protease activity is very sensitive to salt concentrations when the protein is studied at low concentrations (<1 nm) and less sensitive to salt concentrations at higher protein concentrations (1 nm) (22). We therefore conducted protease assays with relatively high ns3 - 4a concentrations (40120 nm) that fall within the range typically employed for rna helicase assays (12, 13, 25, 31). Our ability to observe helicase and protease activity under the same conditions may also stem from the fact that we utilized untagged ns3 - 4a, which is more reactive than his - tagged ns3 - 4a . An n - terminal his tag reduces both the rate of serine protease activity and the active fraction of protein (fig . Proteolysis by untagged ns3 - 4a is not highly salt dependent and the reaction is efficient under diverse conditions (fig . 4). Earlier work has demonstrated that ns3 functions as a robust rna helicase at nacl concentrations as high as 100 mm and it can unwind rna at even higher nacl concentrations, although less efficiently (13). The fact that ns3 can cleave protein or unwind rna in the same range of salt and ph conditions (including ionic conditions that approach physiological (150 mm)) indicates that ns3 - 4a is capable of transitioning smoothly between proteolysis and rna unwinding during various stages of hcv replication . Immediately after ns3 - 4a autocleaves from the hcv polyprotein, it may unwind hcv rna or use ns3hel as a motor to translocate along the hcv polyprotein and scan for subsequent peptide cleavage sites . Protein translocation by ns3hel has never been demonstrated, but its potential importance is underscored by the translocase activities of related proteins such as clpx (3537). Reconstituted forms of ns3 - 4a are significantly less reactive than autocleaved ns3 - 4a to perform this study, it was necessary to create reconstituted forms of the ns3 - 4a complex, in which the 4a protein was added in trans to the serine protease domain or to the full - length ns3 . This provided an opportunity to compare the reactivities of ns3 - 4a molecules that, despite identical sequences, were produced in different ways (through autocleavage of polyprotein or reconstitution from separate proteins). Here we observe that autoproteolyzed ns3 - 4a is a much more active serine protease than complexes that were reconstituted from separate ns4a molecules (ns3 + ns4a). Consistent with this, the ns3 - 4a complex that is disrupted by dilution into low salt buffers (17) cannot be restored to normal levels of the protease activity by the addition of salt (data not shown). Specifically, we observe that ns3 - 4a has a vmax for ret - s1 proteolysis that is 34 times faster than ns3 + ns4a (table 1). Intriguingly, the km values for ns3 - 4a and ns3 + ns4a proteolysis of ret - s1 do not vary significantly (table 1). Taken together, the kinetic analysis of proteolysis by ns3 - 4a, ns3 + ns4a, and ns3 protease + ns4a suggests that all of these constructs bind substrate with a similar affinity, but they have large differences in efficiency of chemical catalysis . One possible explanation is that the ns4a co - factor may not properly intercalate with the ns3 protease domain when ns3 and ns4a are simply mixed, potentially resulting in misalignment of active site residues within the ns3 protease domain . Autocleavage between ns3 and ns4a and the concurrent intercalation of ns4a into the ns3 protease domain requires a specific cleavage site sequence that slows the proteolysis reaction (thr / ser as opposed to cys / ser at other hcv polyprotein sites) (38). Presumably, this slow autocleavage between ns3 and ns4a facilitates the correct intercalation of ns4a into the ns3 protease domain as ns4a is being cleaved from the ns3 c terminus (38). Therefore, whereas our findings suggest that the helicase domain allosterically influences the protease active site, an alternative interpretation of the results is that a coexpressed helicase domain is required for proper folding of the protease domain and/or appropriate docking of the 4a cofactor . In addition, the expression strategy employed in this work may not optimally recapitulate folding of the individual proteins . But regardless of the mechanistic basis for the apparent codependence of the protease and the helicase, the data clearly show that reconstituted ns3 and 4a constructs are significantly impaired, which has major implications for the design of protein constructs used in drug screening and for structure / function efforts on hcv . Many investigators have aimed to disrupt ns3 - 4a protease activity as an approach toward antiviral therapeutics and various forms of the complex have been employed in these studies (1923, 39). Although it has been suggested that ns3hel might enhance ns3 - 4a protease activity (23), this synergy was not demonstrated until now . Our findings suggest that screens for ns3 - 4a protease inhibitors should involve full - length ns3 constructs rather than truncated protease domains . Moreover, these screens should utilize constructs in which ns3 - 4a is produced through autoproteolysis rather than reconstitution . Indeed, a recent study comparing the effects of ns3 - 4a serine protease inhibitors on full - length ns3 - 4a produced through autocleavage, ns3 + ns4a peptide, and ns3 protease domain + ns4a peptide demonstrated that biln 2061 and vx-950 are much better inhibitors of autocleaved ns3 - 4a serine protease activity than of reconstituted ns3 + ns4a peptide or ns3 protease domain + ns4a (22). The greater protease activity of autocleaved ns3 - 4a creates the potential for increased sensitivity when measuring protease activity in the presence of new drugs (i.e. More easily measured inhibition of protease activity in screening assays of compound libraries), which will lead to a greater diversity of promising lead compounds . Given the synergies observed among components of the ns3 helicase - protease, it will be interesting to monitor the influence of ns4a on rna binding, atpase, and rna unwinding activities of ns3 in future studies.
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Acute kidney injury (aki) is a common clinical problem in intensive care unit (icu) patients and independently predicts poor outcome [14]. In the icu setting, the overall incidence of aki is approximately 36% [5, 6], and an increasing trend has been reported [7, 8]. Cardiac dysfunction is also common in patients with aki in the icu, and increasing interest exists in how the interaction of these two systems affects clinical outcomes in this group of patients . B - type or brain natriuretic peptide (bnp) is a neurohormone secreted from ventricular myocardium in response to myocardial stretching and volume overload . Bnp has diagnostic and prognostic utility in patients with acute decompensated heart failure [1013], and bnp is an independent predictor for cardiovascular events and overall mortality in various patient groups including those with chronic kidney disease [1420]. However, it remains unclear whether plasma levels of bnp are useful in predicting aki in critically ill patients . Therefore, our study aimed to investigate whether bnp levels in the first 48 hours may be useful in diagnosis of established aki . We studied a cohort of 34 consecutive patients admitted to the icu of san bortolo hospital, vicenza, italy, between december 2007 and april 2008 . Patients requiring mechanical ventilation and admitted on any day from monday to wednesday were included, and we excluded patients with acute coronary syndrome or acute myocardial infarction . The primary outcome was presence of aki during admission or development of aki during icu stay . Patients were classified as having aki if any time during the first 48 hours after enrollment they had an increment of serum creatinine (scr) of 0.3 mg / dl or more or an increase of at least 50% from baseline and/or, an episode of urine output less than 0.5 ml / kg / hr for more than six hours despite fluid challenge of 500 ml or more . Aki was classified according to the rifle (risk, injury, failure, loss, and end - stage kidney disease) criteria . The sequential organ failure assessment (sofa) blood samples for plasma bnp and renal function were taken within 6 hours from admission and 24 and 48 hours later, to investigate association of bnp levels with clinical and laboratory parameters and sofa score . Plasma bnp was measured with fluorescence - based immunoassay with the triage point - of - care analyzer (biosite inc ., san diego, calif ., usa), which is a rapid quantitative measurement of bnp concentration in edta - anticoagulated whole blood or plasma . The method used single - use plastic cartridge with immobilized bnp antigen and bnp - specific monoclonal antibodies conjugated to fluorescent nanoparticles . Categorical variables are expressed as percentage and were compared with fisher's exact test . Normally or near normally distributed variables were presented as means standard deviations (sd); non - normally distributed continuous data were presented as medians and interquartile ranges (iqr). Differences between groups were analyzed using student's t and mann - whitney tests as appropriate . Statistical analysis was performed using the spss 15.0 (spss inc, chicago, ill, usa). During the study, a total of 34 patients were admitted to the icu, and, of these, 26 met the inclusion criteria and had sufficient data for analysis . Furthermore, 9 (34.6%) fulfilled criteria for aki, 5 (19.2%) had aki on admission, and 4 (15.4%) more developed aki during 48 hours . Characteristics of the patients are shown in tables 1 and 2 . Given the differences between scr, age, and bnp at baseline, we did examine for correlations between these variables . While baseline scr and bnp were not significantly correlated (r = 0.27, p = .19), there was a weak correlation between age and scr (r = 0.39, p = .048) and a stronger correlation between age and baseline bnp (r = .46, p = .02). In patients with aki on admission, we found a higher sofa score (10.0 2.4 versus 6.1 2.1; p = .002) and, as expected, higher scr levels (1.85, iqr 1.76 - 1.94, versus 0.82, iqr 0.69 - 1.00 mg / dl; p = .001) compared to no - aki patients . Moreover, aki patients tended to have higher bnp values on admission compared to no - aki patients (510, iqr 370544, versus 197, iqr 57393 pg / ml; p = .06) (table 1). Plasma bnp levels were also statistically significantly higher for aki patients at 24 and 48 hours after admission compared to no - aki patients (table 3). In addition, the increase in bnp of aki patients during 48 hours (from 510 to 1380 pg / ml) was significant (p = .012) (table 3 and figure 2). These patients had on admission higher scr (1.14 versus 0.82 mg / dl) and bnp (338 versus 197 pg / ml) levels compared to no - aki patients . We also analyzed levels of scr and bnp in all patients developing aki at any point during 48 hours (n = 9). For these patients, the difference in bnp versus no - aki patients at admission was even more pronounced (510, iqr 232832, versus 197, iqr 36353 pg / ml; p = .038). Also, for the 9 patients developing aki at any time during icu stay, scr and bnp levels at baseline and at 24 and 48 hours were significantly higher compared to no - aki patients (table 4). A large proportion of patients admitted to hospital, especially in the critical care setting, have various degrees of heart and kidney dysfunction . Primary disorders of one of these two organs often result in secondary dysfunction or injury to the other . Such pathophysiological interactions represent the basis for a clinical entity often referred to as the cardiorenal syndrome (crs). Limited data are available regarding the diagnostic and prognostic utility of bnp in patients with aki in intensive care unit . In a recent study, park et al . Demonstrated that bnp levels have the diagnostic and prognostic capability for crs type 4 in icu patients, according to the novel classification of crs [27, 28]. In our study, bnp was able to predict the presence of aki on admission or development of aki during icu stay with a roc - auc 0.830 (figure 1). No previous studies have focused on the significance of bnp in patients with aki admitted to the icu . In icu setting, emerging cardiac and renal impairment are strongly connected on neurohormonal basis via renin - angiotensin - aldosterone system bnp and nitric oxide, the sympathetic nervous system and other pathways such as coagulation and inflammation . Have shown in experimental animal models that the acute effects of aki on the heart occur as early as few hours after kidney injury, and that changes in cardiac structure are associated with increased cardiac bnp . It could also be speculated that the changes in bnp observed in our study may partly reflect the pathophysiology between kidney and heart in aki, the so - called crs type 3 or acute renocardiac syndrome . In this category, aki is believed to be the primary inciting factor, and cardiac failure is a common and in often times a fatal complication of aki . In our study, we demonstrate a dynamic interaction between aki and plasma bnp levels in a cohort of mechanically ventilated icu patients who were admitted primarily for noncardiac diagnosis . Further, the results support the need for additional study of the potential value of plasma bnp levels in discrimination between aki and no - aki in critically ill patients . To our knowledge, this is the first investigation of the association between plasma bnp levels and aki in critically ill patients . Extensive information regarding patient comorbidities was not available and could not be added to our analysis . Plasma bnp levels can be affected by other variables such as age, and in our study aki patients were significantly older than no - aki patients, and age was correlated with baseline bnp, hence some of the association between bnp and aki may have reflected the risk of aki related to age . Additionally, while patients with acute myocardial infarction and acute coronary syndromes were excluded, massive information about previous or current cardiac dysfunction was not collected and could have influenced plasma bnp levels . Furthermore, we did not perform objective assessment of cardiac function to document that increased bnp in this setting would be due to myocardial dysfunction . Finally, this study was not designed to look at the prognostic value of plasma bnp levels in critically ill patients with aki . In this pilot study, we have demonstrated for the first time an association between plasma bnp levels and aki in critically ill patients . Patients with aki have higher levels of bnp compared to no - aki patients, and in aki patients bnp levels continue to increase during the subsequent 48 hours . Additional studies are necessary to confirm our findings and to further shed light on the pathophysiologic interaction between kidney and heart during aki.
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Familial hypertrophic cardiomyopathy (fhc) is a disease of cardiac sarcomeres, with causal mutations identified in a variety of myofilament proteins including thin filament ca - regulatory proteins [15]. Many mutations in cardiac troponin i (ctni, the inhibitory subunit of the troponin complex, tn) and cardiac troponin t (ctnt, tn's tropomyosin - binding subunit) enhance contractile function at subsaturating, and in some instances at saturating [ca] [613]. Cardiac -tropomyosin (-tm) is a third component of the thin filament associated with fhc [14, 15] that could have a central role in functional enhancement by fhc mutations, at least at subsaturating levels of [ca], although wild - type tm by itself (i.e., in the absence of tn) does not enhance function [7, 16, 17]. The tm molecule is an -helical coiled - coil dimer that binds head to tail with adjacent tm's to form a continuous strand, with two tm strands associated with an f - actin . Each tm is associated with seven actin monomers and one ternary complex of tn to form a structural regulatory unit that blocks strong myosin cross - bridge formation in the absence of ca (blocked state). Ca binding to troponin c (tnc, tn's ca - binding subunit) permits tm movement on the thin filament to partially expose strong cross - bridge binding sites on actin (closed state), with further movement to the open state upon binding of myosin . Some fhc mutations in -tm have been shown to reduce tm's affinity for actin in vitro in the absence of tn [20, 21] and may additionally destabilize tn binding; they further reduce the thermal stability of -helix structure content in isolated tm [21, 23, 24] or in actin - tm . It is not known, however, to what extent these structural changes and changes in thermal stability of tm influence the function of regulated thin filaments . Several fhc mutations in tm may enhance some aspects of actomyosin function, but only at subsaturating and not saturating [ca]. V95a increased ca - sensitivity but also caused small decreases in the maximum solution atpase activity, maximum speed of filament sliding in motility assays, and maximum ca - activatable tension in thin filament - reconstituted cardiac preparations [15, 22, 25]. E180 g increased ca - sensitivity of tension in thin filament - reconstituted cardiac preparations, and both d175n and e180 g increased ca - sensitivity of the fraction of motile filaments in motility assays . Maximum filament sliding speed was unchanged by d175n or e180 g when the ternary complex of cardiac tn (ctn) was used but increased with skeletal tn (stn). Vastus lateralis fibers from patients with the d175n mutation exhibited increased ca - sensitivity of isometric force compared with control fibers, with no change in maximum force or shortening velocity . Maximum ca - activatable tension in thin filament - reconstituted cardiac preparations was decreased for d175n but not e180 g, while total maximum tension was unchanged for d175n but increased for e180 g; this was attributable to increases in ca - independent tension for both d175n and e180 g, and v95a, too . In a transgenic (tg) mouse model, the e180 g mutation leads to cardiac hypertrophy and increased mortality [28, 29]. These results from disparate assays indicate that fhc - related mutations require more thorough functional analysis at the level of single thin filaments to understand molecular mechanisms of the disease, and to allow for testing of potential therapeutic agents . We therefore examined the influence of three fhc mutants in -tm on structure and ca activation of cardiac thin filaments under unloaded conditions . Thin filaments were reconstituted from recombinant, human -gs - tm wt, mutants e180 g, d175n, or v95a, and ctn, and in vitro motility assays were used to examine function at the level of individual filaments . The three mutants increased ca - responsiveness of filament sliding speed at 30c with no change in the maximum or minimum speeds at pca 5 or pca 9, respectively, when thin filaments were saturated with regulatory proteins . Increased ca - responsiveness by fhc mutations was associated with greater structural disorder by circular dichroism spectroscopy (cd) at 25c; e180 g exhibited reduced functional stability at ~10c lower than wt when temperature was varied in motility assays at pca 5 . Cross - bridge duty ratio was lower by ~30% with regulated thin filaments at pca 5 and 30c than with unregulated f - actin; duty ratio was similarly lower for thin filaments reconstituted with ctn and mutants d175n and e180 g . In addition, enhancement of sliding speed for thin filaments reconstituted with d175n occurred at lower hmm densities than for wt or e180 g . These results support the hypothesis that structural destabilization of -tm may play a role in functional enhancement by fhc - related mutations when there are few cross - bridges and could in some cases lead to loss of regulation (diastolic dysfunction) under normally minor perturbations of cellular conditions such as slight elevation of body core temperature during exercise . X06825) cdnas were pcr amplified from a marathon - ready cdna library (clontech laboratories inc ., the - and -tm constructs were engineered with a bamhi site just before the start codon and a sali site just after the stop codon by pcr using pfu turbo dna polymerase (stratagene inc ., cedar creek, tex, usa). Inserts were ligated to maltose - binding protein (mbp) fusion expression vector pmal - c2 (new england biolabs inc ., beverly, mass, usa) in which a thrombin cleavage site had been engineered . Dh5 cells were transformed with the ligation mixtures, and the positive clones were identified by pcr with the same primers used for cloning . Three -tm mutants, -tmv95a, d175n, and e180 g, were generated by pcr using quikchange xl site - directed mutagenesis system (stratagene). Reactions were carried out per manufacturer's instructions with the template of -tm / pmal - c2 plasmid and primer pairs: ptmv95af: 5-cgcatccagctggctgaggaagagttgg-3 ptmv95ar: 5-ccaactcttcctcagccagctggatgcg-3 ptmd175nf: 5-ggtcatcattgagagcaacctggaacgtgc-3 ptmd175nr: 5-gcacgttccaggttgctctcaatgatgacc-3 ptme180gf: 5-cgacctggaacgtgcaggagagcgggctgagctctc-3 ptme180gr: 5-gagagctcagcccgctctcctgcacgttccaggtcg-3 plasmids extracted from three transformed mutants were sequenced with primers targeted at the flanks of mcs on pmal - c2 vector to confirm sequence changes in the mutants . For expression, competent e. coli bl21 (de3) cells (novagen inc ., madison, wis, usa) were transformed with cdna / pmal - c2 plasmids for -tm, -tm, or the -tm mutants . Single colonies were grown at 37c to an od600 of 0.5, followed by iptg induction . The cells were harvested, resuspended in column buffer (20 mm tris.cl, ph 7.4, 10 mm -mercaptoethanol, 200 mm nacl, and 1 mm edta), lysed with mild sonication, centrifuged at 12,000 g, passed over an amylose affinity column (new england biolabs inc . ), and eluted with column buffer containing 10 mm maltose . Louis, mo, usa) at 1 unit of thrombin per mg of protein in column buffer for 4 hrs at room temperature . The liberated recombinant tm homodimers were purified on a mono q column (pharmacia biotech, uppsala, sweden) by high performance liquid chromatography (hplc) with a gradient elution (buffer a: 20 mm triscl, ph 8.0, 25 mm nacl; buffer b: buffer a containing 500 mm nacl). The bamhi site on pmal - c2 vector is integral to the thrombin cleavage region so all recombinant tm's have two extra - amino acids (gs-) at the n - terminus; gs - represents a conservative alternative to the as - dipeptide in bacterially expressed tm that substitutes functionally for n - terminal acetylation [21, 30]. The purified recombinant proteins (figure 1) were concentrated, aliquoted, and stored at 80c in hplc elution buffer until use [22, 25, 31]. Myosin and muscle acetone powder were made from rabbit back and leg muscles as previously described [8, 3134]. Animal handling was in accordance with the current us national institutes of health / national research council guide for the care and use of laboratory animals . All procedures and protocols were approved by florida state university's institutional animal care and use committee . Adult male new zealand white rabbits were anesthetized with 10 mg / kg xylazine + 50 mg / kg of ketamine + 3 mg acepromazine in physiological saline (i m). Following verification of appropriate surgical depth of anesthesia, the animal was exsanguinated, skinned, eviscerated, and chilled on ice . Back and f - actin was prepared from muscle acetone powder; aliquots of f - actin were labeled with rhodamine - phalloidin (rhph) for fluorescence microscopy [8, 31, 32, 35]. Heavy meromyosin (hmm) was prepared by chymotryptic digestion of myosin [8, 31, 32, 35]. Native human cardiac troponin (hctn) was obtained from research diagnostics (flanders, nj, usa). Sds - page was conducted with 412% sds - page gels (invitrogen inc ., carlsbad, calif, usa). Gels were either stained with coomassie brilliant blue (sigma) to visualize proteins (figure 1) and analyzed with an edas-290 digital imaging system (kodak, rochester, ny, usa), or were used to transfer proteins to nitrocellulose (nc) membranes (bio - rad, hercules, calif, usa) at 30 ma, 4c for overnight . Nc membranes were rinsed for 2 min in tbs - t buffer (20 mm triscl, 150 mm nacl, 0.05% tween-20, ph 7.5), blocked for 1 hr at room temperature in tbs - t containing 2% bsa (sigma), then incubated with rabbit anti - tm antibody (sigma, t3651) in tbs - t (1: 3000) for 1 hr at room temperature . Bound antibody was detected using horseradish peroxidase - conjugated anti - rabbit igg, followed by enhanced chemiluminescence reaction with ecl plus kit (amersham biosciences, little chalfont, buckinghamshire, uk). Finally, the blots were exposed to kodak x - ray film (kodak, rochester, ny, usa). -tm, -tm, and three -tm mutants were dialyzed against cd buffer (20 mm sodium phosphate, 20 mm nacl, ph 7.4) and concentrated using centricon concentrators of 30,000 mw cut off (millipore corporation, bedford, mass, usa). Protein concentration was determined in cd buffer (in absence or presence of 6 m urea) from a280 using an extinction coefficient of 8700 m cm (http://us.expasy.org/). Data were collected at tm concentrations of ~39 m on an aviv model 202 spectrometer (aviv biomedical inc, lakewood, nj, usa) using a 1 mm cuvette and wavelength range of 190260 nm at 25c . Each sample was equilibrated for 10 min prior to data collection and scanned three times with 0.4 or 0.5 nm wavelength steps and averaging time of 1 s. molar ellipticity at 222 nm ([]222) was used to calculate -helix content, and the elements of the secondary structure were analyzed with cdpro using basis set 4 . The instrument was calibrated with ammonium (+) -camphor-10-sulfonate (csa) using the two - point method . In vitro motility assays with regulated thin filaments were carried out essentially as described [8, 31, 32, 35, 40]. Motion of rhph - labeled filaments was visualized by fluorescence microscopy and a sit camera and recorded on vhs videocassettes . Control assays with unregulated f - actin were conducted in actin buffer (ab: 25 mm kcl, 25 mm imidazole, 4 mm mgcl2, 1 mm egta, 1 mm dtt, and ph 7.4) with 2 mm atp and 0.3% methylcellulose (mc), or at pca 5 without ctn or tm . Solution composition for assays with regulated thin filaments was calculated as described [8, 31, 40]. Solutions contained 2 mm mgatp, 1 mm mg, 10 mm egta, sufficient ca(ch3coo)2 to achieve the desired pca (pca 94) (pca = log[ca], where [ca] is in molar), 50 mm k, 15 mm na, 20 mm mops, ph 7.00 at 30c, 0.5% mc, and ctn and tm (see below). /2 was adjusted to 0.085 m with trisoh and acetic acid . To minimize fluorophore photobleaching and photo - oxidative damage to the proteins, 3 mg / ml glucose, 100 g / ml glucose oxidase, 18 g / ml catalase, and 40 mm dtt were added to motility buffers . Regulated thin filaments were reconstituted in the flow cell [8, 31, 40]. Regulatory protein concentrations were 125 nm in the reconstitution buffer and motility buffer . At 125 nm hctn plus 125 nm tm, filaments were well regulated as demonstrated by the absence of movement at pca 9 and essentially all filaments moving rapidly at pca 5 . Motility data were collected during continuously varying temperature transients as described [41, 42]. Flow cells were modified by photolithographic fabrication of a heater (thin au film, 80100 nm thick) and a gold resistive thermometer (10 mm wide and 80 nm thick stripes) onto opposite sides of the glass coverslip . The heater and thermometer were independently biased by two dc source meters (keithley, model 2400) that also measured the voltage drop over the thermometer stripe in a four - probe configuration . Experimental control and data acquisition the thermoelectric heater covered most of the flow cell except for a small central window for observation of filament sliding, with the thermometer stripe located ~500 m from the window's edge . Flow cell heating was achieved using 0.8 - 0.9 a current which was turned off during cooling . The low starting temperature (1418c) and the cooling process were realized by chilled water flowing through a copper coil wrapped around the microscope objective . Digital movies were recorded directly from the video signal or from videotape using a dc2 interface w / b&w adapter hardware (miglia tech ., custom software for filament tracking (motion analysis) was developed in the linux environment and consists of image analysis software written in c++ with scripting and interface modules written in python . The image analysis software reads as input a series of images and outputs a list of filament paths . Initially, each frame of the digitized sequence was converted into a binary image using the canny edge detection algorithm . For all detected edges, a centroid was computed and compared to the previous frame . Paths were constructed for objects whose current centroid was within a user - defined measure of tolerance of its predicted value . If a centroid was outside of these bounds, a new path was created and a current path may be ended . Each output path thus lists every member edge's centroid, length, head, and tail for all constituent frames . Individual filament paths were retained when the centroid could be unambiguously tracked for 2 s. speed statistics were calculated for each retained path . The ratio of sd / mean speed was calculated for each path as an indicator of uniformity of motion [32, 43], and filaments were accepted as moving uniformly when this ratio was <0.3 . For each flow cell at a given condition, the fraction of uniformly moving filaments and their unweighted mean speed (sd) was computed from all retained paths; the typical number of paths analyzed per flow cell was in the hundreds . The second approach for motion analysis was used to determine the sliding speed of filaments during short time intervals (e.g., during continual variation of temperature) and to correlate sliding speed with the length of individual filaments (e.g., duty ratio experiments). This procedure utilized metamorph (universal imaging) and was carried out as described [41, 42]. Stacks of frames were created from digitized movies, and filament paths were visualized by superimposing all frames of one stack to form a projection and subtracting the image of the first frame . For each stack, 48 filaments were randomly selected, and the distance traveled (d) was determined by measuring the residual contour line for each filament path . Speed was determined as d/(t(#frames 1)), where t (t = 0.0333 s) is determined by the 30 frames - per - second video recording . Cross - bridge duty ratio (f) during unloaded filament sliding was estimated at pca 5 using the method of uyeda et al . . Motility data were obtained as described above for regulated thin filaments and unregulated f - actin as a function of the density,, of competent hmm on the flow cell surface, where was varied by applying different concentrations of hmm in the initial sequence of solutions added to each flow cell . In separate experiments, speeds of individual filaments and their contour lengths were measured as described above . The filament length dependence of speed was fitted to an equation that describes the interaction between myosin and actin in a stochastic manner, based on the idea that f is equal to the probability that a myosin head is in the strong actin - binding state of its cross - bridge cycle, and that filaments will move at their maximum speed only if there is at least one cross - bridge at any given time: (1)s=()sm{1(1f)n}, where s is the experimentally measured speed, sm is the intrinsic maximum sliding speed, and () is a number between 0 and 1 that represents the efficiency by which force generated by a single myosin head is translated into sliding speed at a given; the regression parameters are f and () sm . The number of myosin motor domains (n) available for interaction with a filament of a given length l was calculated according to either the band model: (2)n=30l, where the constant 30 has units of nm and represents twice the reach of a myosin motor domain, or the nearest neighbor model [44, 45]: (3)n = l(ln2)1/2 . Ca - dependence of filament sliding speed was assessed by fitting data to a modified form of the hill equation (4) by nonlinear least squares regression (sigmaplot ver . 8.0 richmond, calif, usa): (4)su = smax1 + 10n(pcapca50)+smin. Equation (4) has four regression parameters: smax is the maximum ca - activated speed obtained at high [ca] (low pca); smin is the speed at low [ca] (high pca); pca50 is an indicator of ca - sensitivity and is the pca needed for the value su smin to reach 50% of smax; n is an estimate of the slope around pca50 and is an indicator of cooperativity . Statistical differences between regression parameter estimates (e.g., mutant versus wt) were evaluated for (4) by determining whether the difference deviated significantly from zero (sigmaplot); parameters were considered to be significantly different when p <0.05 . All motility assays with regulated thin filaments reconstituted with hctn and -gs - tm wt, -gs - tm wt, or -gs - tm mutants were considered to be well regulated [31, 32, 40], that is, there was little or no motion at pca 9, and fast uniform sliding at pca 5 (figure 2), indicating that both wt and mutants support complete ca - regulation when thin filaments are saturated with regulatory proteins . These unloaded motility data contrast with isometric force measurements in thin filament - reconstituted cardiac preparations which exhibited active force generation in the absence of ca when v95a, d175n, or e180 g replaced wt -tm although this difference may at least partially reflect altered affinity of mutant tm for f - actin and/or tn and experimental conditions where thin filaments may not have been fully saturated with regulatory proteins; it remains to be seen which condition more closely reflects diastole in fhc patients with these mutations . Unregulated f - actin exhibited similar speeds at both high and low [ca] (figure 2) as reported previously [32, 43]. At pca 5, smax values for regulated thin filaments were 5372% faster than for unregulated f - actin figure 2; this result with hctn and -tm agrees with our previous report and compares with the enhancement of sliding speed by regulatory proteins from rabbit skeletal muscle [32, 46]. There was no significant difference between the speed at pca 5 for thin filaments reconstituted with -gs - tm wt compared with any of the three mutants or -gs - tm wt (figure 2). Tm alone (i.e., nonmutant tm in the absence of troponin) does not enhance smax [7, 16, 17], and, thus, this effect may be more intimately linked to troponin than tm [7, 47], as further implicated by fhc mutant data (figure 2). In support of this idea, mutations d175n and e180 g in human -tm were previously reported to increase smax relative to wt, but only when coupled with rabbit stn [20, 26] and not with human ctn (figure 2). The data in figure 2 are generally compatible with reports in which disparate experimental systems were used, with the exception of thin filament - reconstituted cardiac preparations at low ca, that these fhc - related mutations in -tm have generally small effects on function at very low or at saturating ca levels [15, 22, 2527, 48, 49]. Decreased maximum atpase activity and smax were found with v95a [15, 22] although these changes were small (<10%) and are not incompatible with our data (figure 2). Increased maximum isometric force was previously reported in preparations from -tm e180 g tg mice and thin filament - reconstituted cardiac preparations at saturating ca although fhc - related changes in unloaded smax in motility assays or vmax in fibers are not necessarily associated with corresponding changes in maximum isometric force [8, 11]. Thus, in conditions where thin filaments were saturated with tn and tm, the three mutations did not significantly affect the regulatory proteins' ability to inhibit actomyosin cycling at low [ca] and also had little or no effect on maximum unloaded filament sliding speed . Sliding speed increased monotonically as [ca] increased (pca decreased) for filaments reconstituted with hctn and -gs - tm wt (figure 3(a)). The data were well fit by the hill relation, (4) (r = 0.928; figure 3(a)) and regression parameter estimates for pca50 and n are given in table 1 . Ca - dependence of sliding speed for regulated thin filaments containing mutant tm was shifted leftward (increased ca - sensitivity) relative to -gs - tm wt (figures 3(b)3(d)). Regression estimates of pca50 (4) increased by 0.110.64 pca units relative to -gs - tm wt and were significant (p <0.05) for the v95a and e180 g mutations, although not for the d175n mutation (table 1). The cooperativity parameter n (4) was significantly reduced for both the d175n and e180 g mutations compared with wt (table 1); this result translated into an increase in ca - responsiveness for the d175n mutant, too, even though pca50 was not significantly affected (figure 3; table 1). Our results with regulated thin filaments containing human -tm and ctn (figure 3; table 1) are in general agreement with previous studies when direct comparisons can be made (see section 1), but there are also specific differences in reported effects of these tm mutations on contractile performance . Rat cardiac myocytes transfected with human -tm d175n exhibited no effect on the isometric force - pca relation or maximum force although tg rats expressing human d175n exhibited a reduction in ca - sensitivity of force compared with wt . Thin filament - reconstituted cardiac preparations exhibited increased ca - sensitivity of isometric force with v95a and e180 g, as observed in motility assays (figure 3; table 1), and cooperativity was reduced for all three mutants . Where differences were observed, they may depend on whether the assays test isometric or isotonic function which have different rate - limiting steps although another source of variability may derive from combining protein isoforms from different species . Differences could also stem from the use of homo - dimers of tm (both with respect to isoforms and mutations) in most in vitro experiments, while varying proportions of homo- and heterodimers are expected in many experiments with tg animal tissues or transfection of living cells . Where this has been examined however, the functional effects were linearly related to the amount of mutant present . The e180 g mutation increased pca50 of motility by 0.64 pca units (figure 3(d); table 1), which is a much greater extent than most other mutants, and similar to that observed for the same mutant's effect on ca - sensitivity of isometric force in thin filament - reconstituted cardiac preparations . In some other reports, the e180 g mutant also increased ca - sensitivity of various functional assays [26, 48, 50] but by lesser extents (~0.10.14 pca units). An exception was ventricular strips from tg rats expressing human -tm e180 g mutation (~5% of tm mrna) that exhibited no difference in ca - sensitivity of force relative to wt and also exhibited no cardiac hypertrophy . Ca - sensitivity of force increased more (~0.3 pca units) in cardiac myocytes from tg mouse hearts expressing ~65% -tm e180 g, with a linear correlation between pca50 and fraction of -tm e180 g that extrapolated to a large, ~0.4 pca unit shift at 100% -tm e180 g . These differences in ca - sensitivity due to fhc - related mutations are in the same direction as observed with three c - terminal fhc - related mutations in ctni although the ctni mutants also exhibited substantial increases in smax . -tm and ctni mutants could affect smax differently because of differences in the molecular mechanisms by which ctni and -tm mutants alter thin filament activation, or because of different sources for the regulatory proteins . Ca - sensitizing effects of the fhc mutations (figure 3; table 1) may be related to changes in tm structure and are thus downstream in the signaling pathway from ca binding to tnc . Each spectrum in figure 4 is the average of 2 - 3 independent determinations from different batches of protein and each determination is the average of 3 scans . Spectra were quantified from []222 and also by more extensive analysis (see section 2) with cdpro software (table 2). Both methods of analysis yielded similar, high estimates of -helix content for both wt - and -tm (7885%) as expected for a protein that is largely -helical coiled coil . Cdpro and []222 analyses indicated that -helix content was lower in the three mutant proteins (6173%; table 2). Lower -helix content of the mutant tm's was correlated with higher fractions of unordered structure in cdpro analysis (table 2). The v95a mutant had the lowest -helix content, and reduced -helix content of the e180 g mutant is consistent with prediction from the coils algorithm of local destabilization around the mutation . In addition to reduced -helix content at 25c (figure 4), the e180 g mutant exhibited decreased stability to thermal unfolding in the presence or absence of actin [21, 24]. To test whether these structural changes are associated with functional effects in addition to the marked increase in ca - sensitivity (figure 3(d)), we used a novel method for continuously varying temperature in motility assays [41, 42]. Speed of thin filaments reconstituted with -tm wt at pca 5 increased with increasing temperature up to at least 50c (figure 5, open circles; note that these control data are a subset of the wt - regulated thin filament data replotted from brunet et al . ). Above ~54c, thin filaments reconstituted with wt tm / tn exhibit an anomalous decline in sliding speed that is likely due to dissociation of regulatory proteins from actin and/or denaturation of tm or tn at elevated temperatures . Filament speed declines to that of unregulated f - actin because actin and hmm denature at higher temperatures than tm / tn during these brief excursions to high temperatures . When the assay was repeated with thin filaments reconstituted with -tm e180 g (figure 5, solid squares), the temperature dependence of speed deviated from wt at temperatures slightly above body temperature . At these higher temperatures, sliding speed was slower with mutant tm than with wt . Above ~44c, there is a deflection in the speed - temperature relation for thin filaments reconstituted with -tm e180 g (figure 5). This deflection is likely indicative of structural destabilization, denaturation, and/or dissociation of mutant regulatory proteins at a substantially lower temperature (~10c lower) than for wt and is also indicative of a strong functional correlate of differential scanning calorimetry (dsc) measurements in solution . The substantial increase in ca - sensitivity associated with the e180 g mutation (figure 3; table 1) is thus correlated with reduced affinity for actin in vitro in the absence of tn [2022], destabilization of tn binding to f - actin - tm, reduced -helix content (figure 4; table 2), and reduced thermal stability of structure [21, 24] and function (figure 5). Structural destabilization of -tm by fhc mutations could reduce flexural rigidity, as suggested by heller et al . When considering other fhc - related mutations in -tm; modeling suggests that changes in protein compliance within sarcomeres could markedly affect muscle function [5356]. The details of this mechanism are therefore distinct from the likely molecular basis for ca - sensitization by fhc - related mutations in the cooh - terminus of ctni [8, 57]. Flexibility of tm is clearly important for its structure and function and changes related to fhc mutations could lead to a decrease in the already low - energy barrier for movement of tm on actin; detailed structural studies are needed to clarify the influence of fhc mutations on tm structure and flexibility . In addition, coupling between adjacent regulatory units could be affected, as suggested by reduced cooperativity with the e180 g mutation (figure 3(d); table 1). Significantly, the reduced thermal stability of thin filaments containing the e180 g mutant -tm illustrated in figure 5 suggests that patients with the fhc mutation could be more susceptible to diastolic dysfunction, and possibly sudden cardiac death, due to thin filament dysregulation during heavy exercise . Mutations in -tm could modulate the dependence of thin filament activation on cross - bridges and cross - bridge number . To examine this possibility for the d175n and e180 g mutations the two mutations associated with the largest effects on ca - responsiveness (figure 3)we measured sliding speed of regulated thin filaments at pca 5 by varying the density of functional hmm () on the flow cell surface . For each condition (tm and), speed (s) and the length (l) of individual filaments were used to determine () sm and actomyosin duty ratio (f; (1)) as described (see section 2). Figure 6 shows that f was lower for wt - reconstituted thin filaments than for unregulated f - actin, and that f was similar for reconstituted thin filaments containing wt tm or either of the two tm mutants examined . Estimates of f in figure 6 were obtained using the band model (2) and were averaged for all . These results show that the structural and functional changes associated with mutants d175n and e180 g of -tm do not lead to substantial changes in actomyosin duty ratio relative to wt - regulated thin filaments, consistent with regulated thin filament sliding speed being similar at saturating hmm density (figure 2), and the distribution of cross - bridge states from sinusoidal analysis of isometric force in thin filament - reconstituted cardiac preparations . For regulated thin filaments at pca 5 sm was indistinguishable for -tm e180 g compared with -tm wt (figure 7). Thin filaments reconstituted with -tm d175n, on the other hand, exhibited motility and achieved maximum sliding speed at lower's than either e180 g or wt (figure 7). These results indicate that the d175n and e180 g mutations increase ca - responsiveness of filament sliding (figure 3; table 1) by very different mechanisms even though the two mutations reside in close proximity and have similar effects on overall -helical content as detected by cd spectroscopy (figure 4). The data in figures 3 and 7 suggest that effects of the d175n would be most evident at low levels of thin filament activation where the number of cross - bridges is small, and additional cross - bridges have the greatest impact . Functional and structural differences in three fhc - related mutations in -tm (v95a, d175n, and e180 g) were found using both conventional and modified in vitro motility assays and cd spectroscopy . Mutant tm's exhibited lower -helical content and more unordered structure than -gs - tm wt; these structural changes could result in alterations in flexural rigidity that, in turn, could be responsible for increased ca - responsiveness of regulated filament sliding that was observed with the mutants . -gs - tm e180 g exhibited the greatest enhancement of ca - responsiveness of the mutants studied due to both a large increase in pca50 and a reduction in n, and dysregulation at ~10c lower temperature than wt . -gs - tm d175n exhibited lower n and a marked reduction in the minimum density of hmm necessary for initiating filament sliding and for achieving maximum sliding speed at saturating ca . Increased ca - responsiveness of cardiac myofibrillar function appears to be a common feature of fhc - related mutations in -tm, ctni, and ctnt (figure 3) [8, 13, 15, 23, 2527, 29, 48, 50, 62]. The extent of enhancement of ca - sensitivity is generally associated with clinical prognosis in fhc patients [57, 63] although it is not generally known the extent to which function in vivo with variable ratios of wt and mutant homo - dimers, and also presumptive hetero - dimers, might differ from that found using in vitro assays with pure homo - dimers such as those described here . Functional consequences of increased ca - sensitivity in the living heart, in the absence of adaptive responses, are expected to be slowed relaxation and increased duration of systole because the intracellular ca transient does not normally achieve sufficiently high ca concentrations to saturate tnc . Observations on intact hearts from tg mice with -tm mutation e180 g are consistent with these expectations [28, 29, 49]. Thus, there are clear possibilities for direct links between thin filament mutations and hypertrophic signaling through altered mechanical and energetic load, and/or altered ca - cycling in myocytes.
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Worldwide over 400 million people are carriers of hepatitis b; in the united states an estimated 1.25 million people are chronically infected and an estimated 51,000 new cases occurred in 2005 [1, 2]. Although the incidence of hbv has declined since the 1980s in all age groups, the decline has been slower in adults, particularly in males from ages 2544 . Immunization guidelines from the centers for disease control (cdc) published in 1991 and updated in 1995 and 1999 call for universal vaccination of all persons younger than 18 years of age and adults older than 18 who are at risk for hepatitis b infection . In december 2006, the advisory committee on immunization practices from the cdc released the first comprehensive statement on hepatitis b immunization since universal vaccination was advocated in 1991 . These updated guidelines apart from reemphasizing the recommendations from previous iterations emphasized the importance of administering hepatitis b vaccination in primary care clinics as part of routine clinical care and to remove barriers for this care . Although guidelines abound, 10% of practitioners are completely unaware of the existence of 78% of the guidelines and current vaccination rates for adults and children at risk are less than optimal . A national survey of adults at risk for hbv found that only 30 - 31% had received the first dose of the hbv vaccine although 80% reported visiting a clinician during the past year . There are missed opportunities for hbv vaccination in patients at risk as many of these subjects had multiple visits in one year . Data on vaccination rates for patients with specific risk factors tell a similar story . Among noninjection drug users, predictors of hbv seroconversion include females who engage in unprotected receptive anal sex, men who have sex with men (msm), persons having a sex partner known for less than 6 months, and males and females who receive money or drugs for sex . Vaccination rates in groups at risk of sexual transmission of hbv however remain low although recommendations for vaccination have been longstanding . In young men who have sex with men (msm) the vaccination rates were only 9% with a prevalence of infection of 11% over the last two decades . In recent years, the vaccination rates have improved somewhat although they are still far below acceptable levels . In a survey of msm from 1999 to 2000, the incidence of hbv is high in immigrants to the us from endemic areas of the world . Approximately 40,000 immigrants infected with chronic hbv are granted permanent residence in the united states annually . A demographic group at particular risk is thus children of immigrants born in the united states, particularly those born before universal vaccination of neonates was instituted in 1991 . Furthermore access to perinatal and neonatal healthcare is limited and these groups may not seek healthcare at these times reducing the opportunity for screening and vaccination and leading to increased rates of perinatal and horizontal transmission of hbv . In a study of korean - americans from 1988 to 1990, males had a carrier rate of 8% for hbv while females had a rate of 4.4% . The rate of hbv carrier status in children of immigrants born in the united states was almost 3% . Furthermore, all mothers who had hbsag and hbeag had offspring who were hbsag positive, underscoring the risk of perinatal transmission in immigrant populations . In the hmong population in wisconsin who migrated from laos, the overall prevalence of chronic hbv in hmong children born in the united states between 1984 and 1989 was 14% . Eleven percent of the hbsag positive children were born to mothers who were hbsag negative . Vaccination rates in this population vary widely from 12% to 86% [13, 14]. Household contacts of hbsag carriers should be considered for screening and subsequent vaccination if they are susceptible [3, 4]. The risk in prison inmates is thought to be related to the high risk behaviors associated with this demographic including injection drug use and having multiple sex partners . In a correctional facility in georgia, the prevalence of hbv infection was 2 percent, with an incidence in the facility in one year of almost 3 percent in inmates identified as high risk . The annual incidence of hbv in this facility (3579 per 100,000) was 120 times the national incidence . Additionally, clustering of unique hbv nucleotide sequencing in groups of newly infected inmates suggested that there was transmission between individuals at the correctional institution . Investigation of two community outbreaks in tennessee found that 60% of those infected had a history of incarceration . There was a prevalence of 4% for chronic hbv and 3% for acute hbv in this cohort . In dialysis patients the incidence and prevalence of hbv is high due to multiple parenteral exposures during dialysis and the immunosuppressed state associated with end stage renal disease [4, 16, 17]. Since 1982, although there have been significant improvements in the vaccination rates since that time, hbv vaccination rates in hemodialysis patients actually decreased in 14 of the 18 end stage renal disease network states, districts, and territories between 2001 and 2002 . Dialysis centers are settings in which practitioners see high risk patients, and therefore further efforts should be undertaken to achieve 100% compliance with vaccination and to encourage periodic screening for hbv . Primary care practitioners have a critical role in identifying and screening patients at high risk for acquisition for hepatitis b and subsequent vaccination of these patients . However, our review of the medical literature suggests that little is known about primary care practitioners' awareness of guidelines for hepatitis b screening and vaccination, identifying the risk factors for transmission of hepatitis b, or experience of barriers to adherence to the guidelines [3, 2022]. Our study sought to assess the knowledge, attitudes, and practices of primary care practitioners in wisconsin regarding screening and vaccination guidelines for hbv and to identify barriers to these practices . A master list of primary care physicians in internal medicine, family medicine, pediatrics, and obstetrics was obtained from the wisconsin department of regulation and licensing and used to select a random sample of 400 physicians from all over wisconsin . Forty - seven did not have a valid address or forwarding address, yielding a final response rate of 47% . A 20-question survey (available upon request) was developed to assess clinician knowledge, attitudes, and practices in regard to hepatitis b vaccination and screening . Practitioners were asked about whether they offer hbv vaccinations, the number of patients seen with hbv, the number of patients vaccinated for hbv, screening practices, and barriers to screening . In addition, the practitioners were asked about their knowledge of the hepatitis b guidelines and attitudes toward hbv as a public health problem . Demographic data collected were practice type, patient population, years in practice, and country of residency training . The survey was tested for construct and content validity by expert review and by pretesting in a local group of pcps . The study protocol was approved by the institutional review board of the university of wisconsin school of medicine and public health . The first mailing was sent out without an incentive in november 2005 . Included in the mailings were a self addressed stamped envelope, a survey with a code to permit tracking and follow - up, and a separate reply postcard . The post asked practitioners about their interest in participating in a future focus group to discuss barriers to hepatitis b vaccination and a cme program addressing hepatitis b vaccination and screening . A second mailing was sent in february 2007 to increase the response rate and included a five - dollar cash incentive . Question responses were initially reported as frequencies and percentages . For cross tabulations of discrete data chi - square statistics were used and a 2-sided p value of <0.05 was considered statistically significant . Of the 166 respondents, 95 (57%) were in private practice, 25 (15%) were in hospital - affiliated private practice, 13 (8%) were in a university hospital clinic, 25 (15%) responded other (e.g., community health clinic), and 8 (5%) did not answer the question . Thirty - one percent had been in practice for 010 years, 33% for 1120 years, 25% for 2130 years, and 11% for greater than 30 years . Forty percent of practitioners saw adults only, 40% saw both adults and children, and 20% saw children only . More than half (53%) reported that they had not seen any patients with hbv infection in the past year, 40% had seen between 1 to 5 patients, 4% had seen 610 patients, and 3% had seen more than 10 patients with hbv . Residency training was completed in the united states for 97% of the respondents and internationally for three percent . Practitioners were asked what proportion of their patients at risk for hbv were screened for infection in the past year: 19% responded that they screened all patients, 36% screened some but less than half, 31% screened more than half, and 14% did not screen any of their patients for hbv . The proportion of practitioners who screen various groups of patients is depicted in figures 1 and 2 . Ninety percent of practitioners stated they screen pregnant women . Sixty - one percent said that they screen persons with a history of more than one sex partner in six months and 80% screen men who have sex with men . The number of practitioners reporting that they screen persons with a history of sex with prostitutes was higher at 86% and for screening sex workers it was 87% . Only 37% of pcps stated that they screen everybody under the age of 18 and 65% screen us - born children of immigrants . Other groups not recommended in screening guidelines (distractor groups in the survey) such as day care workers are screened by 42% of providers, people drinking well water by 9%, and people eating sushi by 12% . Screening of patients at risk for hbv increased with provider's experience with hepatitis b such that those who saw greater than 5 patients per year were more likely to screen at risk groups including sex workers, persons who have sex with prostitutes, sex partners of hbv carriers, prison inmates, and hemodialysis patients than were their counterparts in the study who saw less than 5 patients with hbv each year (p <0.0001). Ninety - one percent of respondents were in a practice that offered hbv vaccination while 9% of the respondents were not . In the past year, 16% of practitioners had not vaccinated any patients for hbv, 14% had vaccinated 15 patients, 10% had vaccinated 610, and 60% had vaccinated more than 10 patients . When the practitioners were asked if they routinely asked their patients at risk for hbv if they had been vaccinated for hbv, 66% stated that they did, and 33% reported that they did not . Of the groups identified by the cdc guidelines as populations who should receive vaccinations for hbv, several groups were correctly identified by a majority of respondents (figures 3 and 4): persons with more than one sex partner in six months (72%), men who have sex with men (82%), intravenous drug users (92%), prison inmates (52%), and hemodialysis patients (72%). Persons who have sex with prostitutes would be vaccinated by 82% of providers and sex workers by 85% . A smaller number identified children 18 years old and younger (73%) as an appropriate group for hbv vaccination . Children of immigrants born in the united states would be vaccinated by only 50% of respondents . Other groups that are not recommended for vaccination such as persons exposed to ill - prepared food would be vaccinated by 22% of respondents, day care workers by 54%, and pregnant women by 42% . The responses to the question asking for the most common cause of chronic viral hepatitis in the united states (hcv followed by hbv) were as follows: 4% hepatitis a (hav), 39% hbv, 56% hepatitis c (hcv), and 1% hepatitis e (hev). Another question was asked regarding the most common cause of chronic hepatitis worldwide and 16% replied that it was hav, 70% hbv (correct answer), 13% hcv, and 1% hev . Identification of serum tests for hbv screening are shown in figure 5 with the majority of practitioners selecting hbsag followed by hbcab as the screening tests of choice . Regarding barriers to screening patients for hbv status, 11% percent responded that the cost of screening for hbv was too great, 7% felt that someone else was responsible for screening, and 3% felt that screening for hbv was not relevant to a patient's health care maintenance . Other barriers identified by respondents were lack of knowledge about hbv risk factors, vaccination and screening (9%), time constraints (5%), and forgetting or not making a point to ask (3%). Three percent felt that since they were pediatricians all of their patients must have been immunized and therefore it was unnecessary to screen their patients for hbv . Practitioners were also asked how important hbv is as a public health problem in general with 48% responding that it was very important, 49% responding that it was somewhat important, and 3% felt that it was not important . In contrast, when asked whether or not they felt that hbv is an important public health problem in their own practice, only 18% believed it was very important, 44% somewhat important, and 38% not important . Clearly, without adequate identification of patients who are at risk, hbv is unlikely to be eradicated . Identification of all groups at risk for hbv is difficult as there are many modes of transmission including sexual, percutaneous, and mucosal exposures . This requires an investment of time inquiring about these exposures and a process that would offer appropriate screening tests and vaccinations to eligible patients . Such a plan would rely heavily on the participation of primary care physicians who have the best opportunity for screening a broad cohort of patients . Current screening practices emphasize the importance of risk factor identification particularly high risk sexual practices, intravenous drug use, and other risk groups such as prison inmates and hemodialysis patients . However, the cohort represented in this survey of pcps was unable to consistently identify relevant risk factors . Intravenous drug use was identified as a risk factor by a majority of the respondents . The practitioners in our survey less consistently identified sexual risk factors for transmission of hbv; they were less attuned to hbv risks associated with being a child born in the united states to immigrants, a prison inmate, and a patient on hemodialysis . Clearly these at risk populations need to be highlighted in educational screening and vaccination programs as well . Even if risk factors are known, routine screening may not occur due to a variety of barriers . Many practitioners indicated that they were uncomfortable with inquiring about these risk factors from their patients, cost, time constraints, and lack of awareness and knowledge about groups at risk for hbv infection . In the medical literature, other barriers to adherence to practice guidelines are lack of self - efficacy, lack of outcome expectancy, inertia of previous practice, external barriers, and patient - related barriers . Many of those who responded to this survey did not feel that there were any areas of improvement in their practices in regard to hepatitis b vaccination and screening . Funding is another barrier as many patients at risk for hbv are uninsured or underinsured . One would be educational programs for practitioners to increase knowledge of high risk groups for hbv to consider for screening and vaccination . Programs to implement the cdc guidelines can be established in medical institutions and primary care clinics . Additionally, correctional facilities are in great need of funding to vaccinate their inmates and dialysis centers need to vaccinate 100% of their susceptible patients . Follow - up surveys are being planned in the future to determine if over time hbv screening and vaccination rates have changed . Since guidelines are difficult to keep up with and implement, an alternative strategy at a national level would be a reconsidering of the guidelines to vaccinate all persons under age of 50 to cast a wider net . The cost - effectiveness of this approach versus the current strategy (vaccinating everyone under 18 and adults at high risk) needs to be worked out . The healthy people 2020 disease reduction goals have been established for achieving the prevention of hbv transmission in the united states . Disease reduction goals for infants and children include reducing the estimated number of chronic hbv infections in infants and young children to 400 cases and the number of new hepatitis b cases reported among persons 218 years of age to zero cases . Healthy people 2020 vaccination goals for infants and children include setting a target coverage level for infants of age of 03 days receiving the initial birth dose of hepatitis b vaccine to 85% and for children of age of 1935 months completing the three - dose hepatitis b vaccination series to 90% . Disease reduction goals for adults include reducing the rate of acute hepatitis b to 1.5 cases per 100,000 in persons of age of 19 years and older . Among adults in high - risk groups, disease reduction goals include reducing the number of cases of acute hepatitis b to 215 cases in injection - drug users and to 45 new infections among men who have sex with men . Without educational intervention to increase awareness, and changing attitudes and practice, these goals will not be met.
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Despite the presence of older surgical literature referencing its use in axillary - femoral bypass and access via surgical exposure for severe descending aortic disease, the use of the axillary artery (axa) as an access site has lost favor in percutaneous intervention in large part due to the success of these procedures from a radial or brachial alternative [13]. However, these distal access points are typically unable to accommodate anything larger than a 6 or a 7 fr sheath [46]. Many operators cite the axa's proximity to the neurovascular bundle or uncertainty of its ability to reliably accommodate larger sheaths as their primary concerns in avoiding axa access . Difficulties in effecting hemostasis given the arteries lack of compressibility on a bony structure further compound the reasons why the axa is not routinely utilized as a percutaneous access site . However, in the setting of hostile aortoiliofemoral (aif) segments, we feel that the axa should remain a viable consideration and may preclude the necessity for a surgical cut - down and exposure in many instances . Although use of the subclavian artery via an open surgical approach has been well described, a small number of isolated case reports regarding the percutaneous use of the axa as an access site for transcatheter aortic valve replacement (tavr), endovascular aneurysm repair (evar) of the aorta, and impella supported high risk pci have now been published . To the best of our knowledge, to date no studies exist describing the in vivo size of the axa in relation to the common femoral artery (cfa) in a general patient population . We hypothesized that the axa will not only be of comparable size to the cfa in most patients but also be less frequently calcified or diseased and able to accommodate sheaths with up to an 18 fr outer diameter in the vast majority of patients . We retrospectively reviewed 110 contrast enhanced ct scans of the chest, abdomen, and pelvis performed at newark beth israel medical center in which the right axa, right cfa, left axa, and left cfa were visualized (figures 1(a) and 1(b)). Images were reconstructed utilizing commercially available terarecon software (figure 2) and diameter measurements made of the cfa above the level of the bifurcation of the superficial femoral artery (sfa) and profunda artery but below the level of the inguinal ligament at which percutaneous arterial access is optimally achieved as well as at the first portion of the axa just distal to the subclavian artery and prior to the origination of the lateral thoracic artery (figures 3 and 4). The arterial diameter was calculated using terarecon; however, we manipulated the diameter to include the entire contrast filled vessel and to exclude the calcifications at the narrowest portion of the vessel . Calcification severity was graded upon whether the calcification was mild, moderate, or severe . This was a gestalt on how much calcification was along the iliac arteries versus subclavian and axillary arteries . The actual detection of calcification was done via the ct scan and essentially the volume calculated by subtracting the narrowest vessel size from a normal vessel diameter . Hounsfield units would not be helpful and lengths of the calcifications would also be extremely difficult as they are usually several of all different sizes . Of the 110 ct scans reviewed, patients were imaged in the supine position with hands above their head as per standard ct imaging protocol . Patients with inadequate visualization of any or all of the arteries due to artifact or inadequate contrast penetration were excluded from the study . The demographics of the 96 patients with complete images and data available are listed in table 1: 46.8% of patients were male, aged 61 15.2 years, mean height of 168.2 9.7 cm, weight 87.5 29.5 kg, and bsa 1.95 0.3 . 83.4% were hypertensive and 45.9% had dyslipidemia, 25.7% diabetes mellitus, 29.3% coronary artery disease, 28.4% renal insufficiency, 11% end stage renal disease, and 13.8% peripheral vascular disease and 34.9% had a history of tobacco use . The mean sizes of the right and left axas were 6.38 1.57 mm and 6.52 1.52 mm, respectively, versus 8.26 2.1 mm and 8.2 2.09 mm for the right and left cfas (figure 5). A direct comparison of the sizes of the axa and cfa in the same patient yielded a mean difference of 1.69 mm 1.74 . In all patients combined, the mean difference between the axa and cfa was 1.88 mm on the right and 1.68 mm on the left . Of all of the right and left axas studied, only 1.042.1% demonstrated calcification, versus 17.819.8% of the cfas, a significantly lower percentage noted in the axas versus the cfas (table 2). Of the 96 patients studied, 19 had an axa that was of the same caliber compared to their associated cfa representing 19.8% of all patients studied and 8 of 96 or 8.3% had one that was larger in size . Prior to the popularization of the radial approach in percutaneous intervention, access from the brachial artery initially via surgical cut - down and later percutaneously was commonplace . However, the inability to safely and reliably accommodate anything larger than a 7 fr sheath has proven to be the major drawback of these approaches . With the dramatic increase in the number of percutaneous transcatheter and endovascular procedures available that necessitate the use of large caliber sheaths including impella supported high risk pci (13 - 14 fr, 4.33 to 4.67 mm), tavr (1426 fr, 4.67 to 8.67 mm), and evar (922 fr, 3 to 7.33 mm), evaluation for the presence of calcification and/or atherosclerotic plaques in the peripheral vasculature, including that of the aortoiliofemoral segment, is routinely performed [9, 10]. In many instances, severe atherosclerotic disease or calcification in these arterial segments in fact, vascular access site related complications have remained the achilles heel of many of these procedures and in patients undergoing tavr have been reported to range from 6 to 14% and have been shown to affect patient survival [7, 10, 11]. In patients with the hostile aif segments, alternative approaches include traditional open surgical repair, surgical exposure and cut - down of the common femoral or subclavian / axillary arteries [7, 1214], or transapical (ta) and transaortic (tao) access for tavr . However in many instances these patients are at extremely high risk of standard or modified surgical intervention due to their clinical instability, significantly advanced age, or a myriad of comorbidities making the avoidance of general anesthesia, circulatory arrest, thoracotomies, mini sternotomies, and chest tubes preferable if possible . The use of the subclavian and/or axillary artery has previously been well described as an alternative vascular access site after surgical cut - down in both impella placement and tavr [1215]. Recently, several small case reports have described percutaneous use of the axa for tavr, impella, and evar [1518]. In fact, schfer et al . Demonstrated a percutaneous access and closure technique of the axa in 24 patients undergoing tavr with both the medtronic corevalve and edwards sapien 3 valves without significant access site complications and a 100% procedural success rate . The vascular complications experienced were safely handled with covered stent deployment . With regard to the potential complication of stroke, use of the left axa minimizes the interference with the rest of the cerebral arteries, and between the milan experience and the hamburg series, there was only 1 stroke attributed to the axa route . While differences in the muscular and elastic composition of the cfa versus the aa have been described, to date, to the best of our knowledge, no studies exist comparing the diameter of the cfa to that of the aa . Although it is typically smaller than the cfa, we suggest that in almost all patients the aa is an acceptable alternative access site for impella placement, tavr, and evar in the setting of a hostile aif . Furthermore, our findings suggest it is also less prone to develop significant atherosclerosis and calcifications . We acknowledge that use of the percutaneous closure devices in the axa is an off - label use . Based on the results of our analysis, we suggest that the aa should be considered as an acceptable alternative access site for impella placement, tavr, and evar in the setting of a hostile aif segment as we find it less often to be significantly diseased or calcified, although typically slightly smaller in overall caliber when compared to the cfa.
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Acute gastroenteritis is a common and costly clinical condition in children . Over the past two decades, pediatric acute gastroenteritis has been the subject of considerable worldwide attention . In the past, a number of laboratory studies were used to evaluate children with acute vomiting and/or diarrhea . Since oral rehydration therapy has become the preferred method of treating dehydration, however, it may be beneficial for individual patients, when oral replacement therapy was unsuccessful or for patients who are receiving parenteral hydration . Diet should be increased as soon as it is tolerated to compensate for lost caloric intake during acute illness . Lactose restriction is usually not necessary, although it might be helpful in cases of chronic malnutrition or in children with severe enteropathy; changes in formula are usually unnecessary . Full - strength formula is typically well tolerated and allows for a more rapid return to full energy intake . However, the use of antibiotics remains controversial . Despite the growing body of evidence supporting the safety and efficacy of oral rehydration solutions, common management errors include using oral rehydration solutions in children with little or no dehydration, administering intravenous rehydration therapy to children with only moderate dehydration and inappropriately withholding oral rehydration solutions or other feeding in children with vomiting . For this reason, the american academy of pediatrics revised its recommendations concerning the treatment of acute gastroenteritis in healthy children . Therefore, the aim of our study was to determine how closely current treatment of acute gastroenteritis in children among lebanese pediatricians compared with the aap recommendations and to observe the impact of such management on healthcare costs . Prior to the survey, the institutional review board (irb) committee of the makassed general hospital in beirut granted ethical approval . We then conducted a telephone - based, anonymous questionnaire survey of lebanese pediatricians concerning the management of acute gastroenteritis in healthy children between 1 month and 5 years in age with mild or moderate dehydration . Lebanese pediatricians who were registered in the lebanese order of physician were included in our survey . We divided the 22-item questionnaire into two sections (appendix 1). In the first section, we asked the pediatricians to provide their year and country of graduation as a pediatrician, and the region and type of practice (teaching hospital, community hospital, ambulatory and private practice, or a combination). The second section included 18 questions about the management of acute gastroenteritis in healthy children aged less than five years with mild or moderate dehydration . For each of these 18 questions, a score of zero was given for answers that did not concur with aap recommendations and a 1 for answers that were compatible with the recommendations . We calculated the score for each pediatrician and then compared scores according to the year, country of graduation of the pediatrician, the region and the type of practice (table 1). The cost of laboratory investigations requested by the pediatricians including complete blood count and differential (cbcd), serum electrolytes, stool analysis and culture was calculated . In addition, the cost of medical treatment (intravenous line or nasogastric tube insertion for hydration, oral fluids administration, special formula, antimotility agents, antiemetics, antibiotics, zinc supplements, probiotics and antiseptics) was calculated (table 2). Management of acute gastroenteritis in healthy children aged 1 month - 5 years with mild to moderate dehydration - a national survey demographic data of pediatricians and their mean scores of practice . Mean cost of treatment of acute gastroenteritis in children with acute gastroenteritis with mild to moderate dehydration . Data were collected and statistically evaluated with the analysis of variance test using the spss version 16 statistical software package . Data were collected and statistically evaluated with the analysis of variance test using the spss version 16 statistical software package . Data were collected and statistically evaluated with the analysis of variance test using the spss version 16 statistical software package . A total of 863 pediatricians were registered in the 2010 logbook of the lebanese order of physicians . From this logbook, the majority of the pediatricians surveyed graduated in the 1990s (39.5%) and 2000s (37%). Among them, 38.7% graduated from lebanese university hospitals, 24% from west europe excluding france, 22% from france and 11% from east europe . Fifty - four pediatricians (22.7%) worked in teaching hospitals, while 40 practiced in private clinics (16.8%). Of the 238 pediatricians, 152 were employed in urban areas and major cities while 82 pediatricians worked in rural areas (64.7% versus 35.3%, respectively). Table 1 summarizes the mean score in the different groups of pediatricians and figure 1 shows the percentages of irrational medical acts . (a) oral fluids besides ors including juices, soda, rice water, mineral water, (b) intravenous hydration, (c) nasogastric hydration, and (d) laboratory studies for mild and moderate dehydration include complete blood count and differential in addition to serum sodium, potassium, chloride and bicarbonate . Concerning rehydration, oral rehydration solutions (ors) were prescribed alone by 49% of pediatricians and in combination with other fluids such as juice, soda, water and/or rice water by 43% . Among all pediatricians surveyed, 89% avoided parenteral hydration for mild and moderate dehydration as compared to all of the pediatricians who worked in teaching hospitals . Sixty - nine percent of pediatricians resumed early feeding as recommended by the aap; 33% of teaching hospital practitioners did not follow this practice, while the practitioners who worked in rural areas tended to allow early feeding more often (73.8%). Eighteen percent of pediatricians still discontinued breastfeeding during acute gastroenteritis, with higher rates among community hospitals practitioners (20.8%). Seventy - eight of the 238 pediatricians (32.8%) continued human or regular - strength formula; while 52.8% prescribed lactose - free formula, 13.4% a diluted one, and 1% a hydrolyzed formula . Laboratory studies for mild dehydration, including cbcd, and serum electrolytes, were requested by 24.6% of pediatricians; whereas these studies were ordered by 49.6% in patients with moderate dehydration . Fifty - eight percent of pediatricians requested microscopic stool analysis and fifty percent ordered a stool culture . Eleven percent of lebanese pediatricians prescribed antimotility medications . On the other hand, 77% of pediatricians prescribed antiemetic agents . In regard to antibiotic prescription, more than 26% of pediatricians preferred to treat acute gastroenteritis with antimicrobial agents . The rate of antibiotic use was higher in pediatricians working in rural areas or in an ambulatory setting(45.2% versus 40% respectively). The estimated mean cost of irrational management was 63,872 lebanese pound (l.p . ), which was equal to usd 42.58, for each patient . Table 3 summarizes the mean cost of treatment in relation to the different groups of pediatricians . The results of our questionnaire revealed that two thirds of the pediatricians in lebanon usually followed the aap recommendations with a mean score of 11/18 . Pediatricians who graduated in the last two decades and those working in teaching hospitals and/or in urban areas were more likely to adhere to the aap guidelines than the other groups . A similar study done in israel in 1998 showed that 60% of pediatricians followed these guidelines . Oral rehydration therapy using a commercial pediatric oral rehydration solution was the preferred approach to mild or moderate dehydration and was accepted as the standard of care for the clinically efficacious and cost - effective management of acute gastroenteritis . The french survey of pediatricians in 2004 showed that 63% allowed rehydration using ors as compared to 16% of pediatricians in a multi - center european study conducted in 2000, 87% of israeli pediatricians in 1998, and 30% of us pediatricians in 1991[69]. In our survey, the results were similar to those obtained in 2001 that involved hungarian pediatricians . However, almost half of the pediatricians in our survey used ors in combination with other oral fluids such as soda, juices, mineral water or rice water . This practice, by far, tended to increase the severity of diarrheal illness by increasing the intraluminal osmolarity of the intestines when using soda or juices and, therefore, exacerbating the course of the disease . Conversely, mineral and rice water do not contain the sufficient amount of electrolytes required to compensate their fecal losses . Breastfeeding should be continued at all times, even during the initial rehydration phase in children with acute gastroenteritis . In our survey, 82% of pediatricians continued breastfeeding compared to 84% in the hungarian and 77% in the european surveys, respectively.the aap recommended continuing a non - restrictive diet promptly after an episode of gastroenteritis in children to compensate for lost caloric intake during acute illness . It was unfavorable that three quarters of the pediatricians in our survey prescribed an antidiarrheal diet and one third delayed the introduction of feeding until 24 hours after the oral rehydration . Noted that this practice remains frequent worldwide; 66% of french pediatricians prescribed a dietary regimen and only 10% of hungarian pediatricians suggested early reintroduction of normal feeding after oral rehydration . A meta - analysis of clinical trials indicated no advantage of lactose - free formulas over lactose - containing formulas for the majority of infants, although certain infants with malnutrition or severe dehydration recovered more quickly when given lactose - free formulas . In our survey, more than 32% of pediatricians used a lactose - containing formula after successful rehydration . Although medical practice has often favored beginning feeding with diluted (e.g. Half or quarter - strength) formula, controlled clinical trials have demonstrated that this practice is unnecessary and is associated with prolonged symptoms and delayed nutritional recovery . Supplementary laboratory studies, including serum electrolytes to assess patients with acute diarrhea usually are unnecessary . Stool cultures are indicated in cases of dysentery but are not usually indicated in acute watery diarrhea for the immune - competent patient . However, certain laboratory studies might be important when the underlying diagnosis is unclear or when diagnoses other than acute gastroenteritis are possible . Laboratory studies such as cbcd and serum electrolytes were largely requested by pediatricians in our survey . More than half of pediatricians working in rural areas tended to request stool analysis and culture; this may be due to the higher prevalence of parasitic and/or bacterial gastrointestinal infections in these areas . Since viruses are the predominant cause of acute gastroenteritis in developed countries, the routine use of antibiotics may lead to increased antimicrobial resistance . Even when a bacterial cause is suspected in an outpatient setting, antimicrobial treatment usually should not be initiated because the majority of cases are self - limited . An exception may be for immune - compromised children and those with an underlying disease . . Two percent of pediatricians in bahrain prescribed an antibiotic for patients with acute gastroenteritis as compared to 81% in a french population after performing a stool culture . In our survey, more than 26% of pediatricians considered acute gastroenteritis in our country to be parasitic or bacterial in origin and, therefore, prescribed antibiotics systematically as a part of treatment . Nonspecific antidiarrheal agents (e.g. Adsorbents such as kaolin - pectin), antimotility agents (e.g. Loperamide), antisecretory drugs, and toxin binders (e.g. Cholestyramine) are commonly used among older children and adults, however, data are limited regarding their efficacy . Side effects of these drugs are well - known, in particular among the antimotility agents, including opiate - induced ileus, drowsiness, and nausea caused by the atropine effects and binding of nutrients and other drugs . Almost all french pediatricians prescribed at least one drug in managing children with acute gastroenteritis . In our survey, antiemetics were the most commonly used medication by three - quarters of pediatricians, followed by probiotics, which were prescribed by 60% of pediatricians . Their efficacy has not been proven in the literature . To our knowledge, this is the first survey that evaluated the impact of management of patients with acute gastroenteritis on the healthcare system . The costs resulting from irrational management of acute gastroenteritis in our survey was surprisingly elevated for each patient . The results from our survey suggested that, with the exception of recommending ors for rehydration and continuation of breastfeeding during acute diarrhea, only a minority of pediatricians followed aap recommendations for optimal management of acute gastroenteritis . These findings suggested that effective healthcare policies are needed to implement the recommendations and to reduce the unnecessary medical costs on the healthcare system in our country.
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The fungus (tti-0009, nrrl 66178) formed patches of white mycelial pseudostromata on the dung surface that were accompanied by bright orange conidial masses (sporodochia) (figure s1; supporting information). Isolation of the fungus from the sporodochial conidia resulted in cultures identical to those from the ascospores, and fertile perithecia and sporodochia formed in ascospore and conidial isolates (figure s1). The fungus was identified as h. rostrata based on the combination of the white mycelial pseudostromata from which aggregated black, shiny ascomatal necks protruded . We also examined the type specimen (bpi 581328) of h. rostrata and observed that the ascospores were of similar size and shape to those in our specimen . In our specimen, the ascospores ranged from 27 to 32 m long to 1418 m wide with a lateral germ slit about half the length of the spore (figure s1). Database matching with the its rdna sequence (www.ncbi.nlm.nih.gov; www.fungalbarcoding.org) yielded a very high sequence similarity (99%) to one of the few known strains of hypocopra, h. anomala cbs 124649, thus indicating that the strains were congeneric . Although h. anomala and h. rostrata have been previously noted to be very similar taxa based on the nature of the white pseudostroma, our texas specimen appeared to represent the latter species because of its larger ascospore size . Database matching with the 28s rdna retrieved a large number of sequences of xylaria species in the range of 9699% identity . Neighbor - joining analysis of the its sequences of dung - inhabiting species and other species of xylariaceae indicated that h. rostrata is highly related to other coprophilous species and several undefined environmental isolates of the xylariaceae (figure s2). Methyl ethyl ketone extracts from liquid cultures of h. rostrata were partitioned between hexanes and mecn to remove lipids, and the mecn - soluble material was subjected to silica gel column chromatography followed by separation over sephadex lh-20, leading to the isolation of 13 . Two of the initial sephadex column fractions contained 1 (fractions 5 and 6). However, when fraction 5 was stored in cdcl3 after h nmr analysis, it showed signs of degradation . Use of acetone - d6 as the solvent for subsequent nmr analysis of fraction 6 avoided (or at least substantially slowed) this degradation process . The molecular formula of 1 was determined to be c15h26o2 (3 unsaturations) on the basis of hreitofms and nmr data . The h nmr spectrum (table 1) displayed signals characteristic of a trisubstituted olefin and an oxymethine unit, along with four methyl singlets and four exceptionally upfield multiplets between 0.1 and 0.8 ppm that suggested the presence of a cyclopropane unit . C and dept-135 nmr data also supported the presence of the olefin unit and an oxygenated methine and further showed the presence of two aliphatic methines, four aliphatic methylenes, and two nonprotonated sp carbons, of which one was oxygenated . The gross structure of 1 was determined by analysis of cosy and hmbc data (figure 1). Mutual cosy correlations among h-7, h-8, and h2 - 9 enabled assembly of the cyclopropane unit . Other cosy correlations were consistent with the presence of the h-1/h2 - 11 and h-3/h2 - 4/h2 - 5 spin - systems . Hmbc correlations from h-7 to c-10, from h2 - 8 to c-6 and c-10, and from h-9 to c-6 established the connectivity of the cyclopropane unit to quaternary carbons c-6 and c-10 . Hmbc correlations from both h3 - 14 and h3 - 15 to c-15 and c-14, respectively, and to c-9, c-10, and c-11 placed these two methyl groups on c-10 and linked c-10 to c-11 . Hmbc correlations from h3 - 13 to c-1, c-2, and c-3 confirmed its attachment to the olefin unit and linked oxygenated carbon c-1 to olefinic c-2 . Further correlations from h2 - 4 to c-3 and c-5 supported the presence of the c-3c-4/c-5 unit, while correlations from h3 - 12 to c-5, c-6, and c-7 enabled the completion of the cyclodecene ring - containing structure . The chemical shift of the h3 - 12 signal (0.71) was significantly upfield relative to that expected for a typical methyl group linked to an oxygenated sp carbon, but was rationalized by its proximity to the cyclopropane ring and is consistent with shifts observed for other structures having an analogous structural subunit . Treatment of 1 with acetic anhydride afforded a monoacetate displaying a significantly downfield shifted signal for h-1, confirming the presence of a free secondary alcohol group and ruling out the possibility of an ether linkage between c-1 and c-6 . This enabled completion of the gross structure of 1, for which we propose the name hypocoprin a. key cosy (boldfaced bonds) and hmbc correlations (arrows) for 1 . The relative configuration of 1 was assigned on the basis of noesy correlations (figure 2). A strong noesy correlation between h-1 and h-3, together with the chemical shift of c-13, indicated the e - geometry for the olefin unit . A noesy correlation between h-7 and one of the diastereotopic c-8 protons placed these on the same face of the cyclopropane ring . The other c-8 proton signal showed a noesy correlation to h-9, indicating that these two protons are oriented on the same face of the cyclopropane ring and opposite that of h-7 . A noesy correlation between h-7 and h3 - 14 indicated that h3 - 14 lies on the same face of the cyclodecene ring as h-7 . H3 - 14 also correlated with h-1, indicating that both h-1 and h-7 are on the same face . A correlation between h-9 and h3 - 12 oriented h3 - 12 on the same face as h-9 . These correlations allowed the assignment of the relative configuration at the four stereocenters of compound 1 . An energy - minimized model having this relative configuration was constructed using spartan10, and the major conformer was consistent with the observed noesy correlations, including a correlation of olefinic h-3 with h-7 . The most significant (s r) values were observed for h3 - 13 (0.14 ppm), h3 - 15 (+ 0.03 ppm), and the pseudoequatorial proton on c-11 (+ 0.09 ppm). These values led to assignment of the absolute configuration of 1 as shown (1r, 6s, 7s, 9s). Hypocoprin b (2) was present in relatively limited quantities in the scale - up extract, though it was more abundant in the initial screening extract based on nmr analysis . The sephadex lh-20 column fraction from which 2 was ultimately obtained initially contained nearly pure 1 . However, upon standing in cdcl3 for 4 days, it was evident that approximately 50% of the sample of 1 had degraded, as the h nmr spectrum showed the appearance of two new singlets at 4.17 and 4.56, while other signals were doubled . An attempt to separate the components by chromatography on sephadex lh-20 was not successful . Therefore, the mixture was simply allowed to stand in cdcl3 to foster further conversion to the product . The molecular formula of the product (2) was deduced to be c15h24o (4 unsaturations) by analysis of hreitofms and nmr data, suggesting that 2 might be a dehydration product of 1 . The h, c, and dept nmr data for 2 (table 2) indicated the presence of a 1,1-disubstituted olefin unit and lacked signals corresponding to the oxygenated quaternary sp carbon and the associated methyl group found in 1 . Analysis of cosy and hmbc data confirmed these observations, independently confirming that 2 differed from 1 by elimination of a molecule of water to afford a c6c12 olefin . Hmbc correlations from both c-12 protons to c-5, c-6, and c-7 confirmed the location of the 1,1-disubstituted olefin unit . Additional hmbc correlations verified that the remainder of the structure was intact . As was the case for the c-12 methyl group in 1, the terminal olefin h signals (4.17 and 4.56) are somewhat upfield - shifted relative to those of typical units of this type, and this observation is consistent with nmr data reported for other structures having a cyclopropane unit in a similarly adjacent position . Analysis of noesy data and energy - minimized models as described for 1 enabled assignment of the expected relative configuration at the three remaining stereogenic centers of 2, providing independent support for the configuration originally assigned to precursor 1 . The absolute configuration of 2 is presumed to be analogous to that of 1 . Clearly, simple dehydration of 1 at the tertiary alcohol functionality would afford 2 . An acid - catalyzed process would be consistent with its occurrence in cdcl3 due to the presence of possible traces of dcl . The limited amount of 2 evident in the nmr spectrum of the initial extract was somewhat surprising given the facility of this process . Interestingly, the process preferentially yielded disubstituted exocyclic olefin 2, rather than the trisubstituted endocyclic olefin, which was not observed . An analogous process (and regiochemical preference) has been observed upon attempted acetylation of a marine diterpenoid with a similar moiety, but no literature precedent was found for spontaneous occurrence of the process in cdcl3 . Hypocoprin c (3) was isolated as a colorless solid with the molecular formula c15h26o3 (3 unsaturations), as determined by analysis of hresitofms and nmr data . To prevent possible decomposition analogous to that observed for 1, nmr analyses for 3 were carried out using acetone - d6 . Key differences between the h and c nmr data of 1 and 3 (table 1) included replacement of one aliphatic methylene unit with an additional oxygenated methine, suggesting that one of the methylene units in 1 is oxidized to a secondary alcohol group in 3 . The gross structure of 3 was again elucidated by analysis of hsqc, cosy, and hmbc data . Cosy data, as well as hmbc correlations from the additional oxymethine proton in 3 to c-4, c-6, c-7, and c-12, enabled location of this proton at c-5 . Treatment of 3 with acetic anhydride afforded a diacetate with downfield - shifted oxymethine signals, confirming the presence of two secondary alcohol groups in 3 and eliminating the possibility of an ether linkage between any two of the three oxygenated carbons . The relative configuration of 3 was again established on the basis of noesy data (figure 2). Similarity of the noesy correlations observed for 1 and 3 enabled the assignment of the relative configuration at the four common stereocenters shared by 1 and 3 . An additional noesy correlation of h-7 with h-5 placed these hydrogens on the same face of the system, thus enabling the assignment of the relative configuration at the additional stereogenic center as shown in 3 . The absolute configuration of 3 was assigned as shown by analogy to that of 1 . The closest known structural analogues to 13 are a group of marine invertebrate - derived diterpenoids consisting of palmatol (from the mediterranean octocoral alcyonium palmatum) and pacifins (from the formosan soft coral naphathea pacifica). These previously reported compounds all have an additional prenyl unit forming more elaborate side - chain moieties attached to the cyclodecene ring in place of one of the geminal methyl groups present in the structures reported here . Most of them have other additional features not found in 13, but the cyclopropane unit and adjacent quaternary stereocenter (where present) share the same relative configuration found in 13 . Hypocoprin a (1) showed antibacterial activity in disk assays against the gram - positive bacteria staphylococcus aureus (atcc 29213) and bacillus subtilis (atcc 6051), affording 1314 mm inhibitory zones at 200 g / disk (a gentamicin standard gave similar results at 20 g / disk), but no effects against escherichia coli (atcc 25922) or candida albicans (atcc 14053) at this level . Hypocoprin c (3) showed only a 9 mm zone in the assay against staph . Aureus, while hypocoprin b (2) was inactive in all four assays . Hypocoprins a c (1) were also tested for cytotoxicity against a human osteosarcoma cell line (u2os), but were found to be inactive (ic50> 10 m). Helvolic acid (4), a well - known fungal metabolite, was found to be the major component in the original screening extract, but was only a minor constituent of the scaled - up extract . Crude mek extracts of tti-0009 dissolved in dmso were strongly inhibitory to s. aureus, a result consistent with the presence of helvolic acid, which is known to show broad spectrum activity against both gram - positive and gram - negative bacteria as well as phytotoxic and antifungal effects . Optical rotations were measured on an autopol iii automatic polarimeter (rudolph research analytical, hackettstown, nj, usa). H and c nmr spectra were recorded using bruker avance-400, 500, or 600 spectrometers . Chemical shift values were referenced to residual solvent signals for acetone - d6 (h/c, 2.05/29.9, 206.7), cdcl3 (h/c, 7.24/77.2), or pyridine - d5 (h/c, 7.22, 7.58, 8.74/123.9, 135.9, 150.3). Hsqc, hmbc, and noesy data were recorded using the bruker avance-600 instrument . All standard nmr data were processed using the nuts 2007 software, while 2d - nmr data were processed on the avance-600 computer . Partially decomposed horse dung was collected in jack brookes gregory park, hitchcock, galveston co., texas . The dung was rehydrated in deionized h2o and incubated in deep - dish petri plates lined with filter paper to simulate natural dung decomposition . The strain of h. rostrata (tii-0009) was isolated by germinating ascospores or conidia on cornmeal dextrose agar amended with 50 g / ml chlortetracycline and streptomycin sulfate . Conidial isolates germinated directly on the agar, while ascospore germination was stimulated by overnight incubation at 44 c . A subculture was deposited at the usda nrrl culture collection at the national center for agricultural utilization research, peoria, il, usa, as nrrl 66178 . To reconstruct the approximate phylogenetic position of strain tti-0009, genomic dna was extracted from mycelia grown on malt - yeast extract agar . The rdna region containing the its region and the partial sequence of 28s rdna containing d1 d2 d3 variable domains was amplified with primers its1 and lr7 . Purified pcr products were cloned in the pjet1.2/blunt cloning vector (thermo fisher scientific, ma, usa) and sequenced using primer lr0r and sequencing primers supplied by clonejet pcr cloning kit (thermo fisher scientific) in genewiz, inc . Partial sequences obtained in sequencing reactions were assembled with genestudio 2.1.1.5 (genestudio, inc . ). Sequence alignments and a neighbor joining estimate of phylogenetic relationships were assembled with mega v6.0 . Strain tti-0009 was initially grown in 50 ml of sabouraud maltose broth supplemented with yeast extract and dilute agar (difco neopeptone 10 g, maltose 40 g, yeast extract 10 g, agar 4 g, deionized h2o 1000 ml) in a 250 ml erlenmeyer flask for 4 d at 23 c and 220 rpm . A 1 ml aliquot of this seed culture was transferred to each of 20 250 ml erlenmeyer flasks each containing 50 ml of mmk2 medium (mannitol 40 g, yeast extract 5 g, murashuge & skoog salts [sigma - aldrich m5524] 4.3 g, deionized water 1000 ml) at 23 c at 220 rpm for 14 d. antimicrobial activity was evaluated by agar disk diffusion assays using standard protocols . Cytotoxicity was evaluated with the celltiter - blue cell (promega, wi, usa) viability assay with an osteosarcoma cell line (u2os). A total of 250 cells were plated in each well of a 384-well plate and incubated overnight . Individual wells were treated with 50 nl of a 10 mm solution of each compound in dmso (in duplicate) and incubated for 48 h at 37 c, and 10 l of celltiter - blue reagent was added . After further incubation for 22 h at 37 c, absorbance was read at 530/580 nm . The combined 1 l of whole fermentation culture was extracted by addition of methyl ethyl ketone (1 l) followed by agitation at 220 rpm for 2 h. the organic phase was collected and filtered, and the organic solvent and residual h2o were removed by vacuum evaporation to afford ca . The crude extract was partitioned between hexanes and mecn to obtain fractions weighing 328 and 671 mg, respectively . The mecn - soluble partition was subjected to silica gel column chromatography using a stepwise gradient of hexanes, etoac, and meoh to afford 14 fractions . Fraction 6 (96 mg), eluted with 60% etoac in hexanes, was then chromatographed over a sephadex lh-20 column using 4:1 ch2cl2hexanes, 3:2 ch2cl2acetone, 1:4 ch2cl2acetone, and 100% meoh to afford eight fractions, among which fraction 3 (32 mg) consisted of compound 1 . Fraction 5 (187 mg), eluted with 50% etoac in hexanes, was subjected to sephadex lh-20 chromatography employing the same solvent sequence to obtain eight fractions, among which fraction 2 contained an additional sample of 1 (33 mg). Upon standing in cdcl3 over time therefore, the recovered mixture of 1 and 2 was stored again in cdcl3 . After 6 days, conversion of 1 to 2 was more than 90% complete based on nmr integration . Compound 2 was also evident in fraction 3 from the initial silica gel column, but was not purified from that source . Fraction 8 (40 mg), which eluted with 80% etoac in hexanes, was similarly chromatographed over sephadex lh-20 to afford four fractions . Fraction 2 (10 mg), eluted with 4:1 ch2cl2hexanes, consisted of compound 3 . Known compound 4 was present as a minor constituent in this extract, but was more abundant in the initial screening extract (obtained from a single erlenmeyer flask containing 50 ml of mmk2 medium cultured, extracted, and partitioned as above). The mecn - soluble fraction (49 mg) was subjected to silica gel column chromatography using a stepwise gradient of hexanes, etoac, and meoh to afford eight fractions . Fraction 2 (13 mg) was subjected to reversed - phase (c18) hplc using a gradient of 20100% mecn in h2o to afford 4 (3.6 mg), which was identified by nmr comparison to literature data . Pale yellow solid; []d + 18 (c 2.3, meoh); h and c nmr data, see table 1; hmbc data (acetone - d6) h-1 c-3, 11, 13; h-3 c-1, 13; h2 - 4 c-2, 3, 5; h-7 c-5, 8, 9, 10, 12; h2 - 8 c-6, 9, 10; h-9 c-6, 8, 11, 14; h2 - 11 c-1, 2, 14; h3 - 12 c-5, 6, 7; h3 - 13 c-1, 2, 3; h3 - 14 c-9, 10, 11, 15; h3 - 15 c-9, 10, 11, 14; hreitofms m / z 220.1833 [m h2o] (calcd for c15h26o2 h2o, 220.1821). Colorless solid; []d + 13 (c 0.58, meoh); h and c nmr data, see table 2; hmbc data (cdcl3) h-1 c-3, 11, 13; h-3 c-1, 4, 13; h2 - 4 c-2, 3, 5, 6; h2 - 5 c-3, 4, 6, 7, 12; h-7 c-5, 9, 10, 12; h2 - 8 6, 7, 9, 10; h-9 c-6, 7, 8, 10; h2 - 11 c-1, 2, 14, 15; h2 - 12 c-5, 7; h3 - 13 c-1, 2, 3; h3 - 14 c-9, 10, 11, 15; h3 - 15 c-9, 10, 11, 14; hreitofms m / z 220.1826 [m] (calcd for c15h24o, 220.1821). Colorless solid; []d + 22 (c 0.49, meoh); h and c nmr data, see table 1; hmbc data (acetone - d6) h-1 c-3, 13; h-3 c-5, 13; h2 - 4 c-2, 3, 5, 6; h-5 c-6, 7, 12; h-7 c-5, 10, 12; h2 - 8 c-6, 9, 10; h-9 c-6, 8, 11, 14; h2 - 11 c-1, 2, 9, 14, 15; h3 - 12 c-5, 6, 7; h3 - 13 c-1, 2, 3; h3 - 14 c-9, 10, 11, 15; h3 - 15 c-9, 10, 11, 14; hresitofms m / z 277.1780 [m + na] (calcd for c15h26o3, 277.1773). A solution of 1 (0.5 mg) in pyridine - d5 (500 l) was combined with a solution of pyridine - d5 (6.6 l) and r-()--methoxy--(trifluoromethyl)phenylacetyl chloride (r - mtpa - cl, 6.5 l). The resulting mixture was stirred with a teflon - coated magnetic stir bar for 24 h at room temperature in a teflon - lined screw - cap vial, resulting in formation of the crude s - mtpa ester . Analogous treatment of an additional 0.5 mg portion of 1 using s - mtpa - cl afforded the r - mtpa ester . Well - resolved h nmr signals (400 mhz, pyridine - d5) showing significant (> 0.01 ppm) (s r) values: s - mtpa ester 1.66 (s, h3 - 13), 0.95 (s, h3 - 15), 1.84 (dd, j = 14, 3.2, h-11eq); r - mtpa ester: 1.80 (s, h3 - 13), 0.92 (s, h3 - 15), 1.75 (dd, j = 14, 3.2, h-11eq). A solution of 1 (1 mg) in pyridine (0.2 ml) was treated with acetic anhydride (0.2 ml), and the reaction mixture was stirred at room temperature for 24 h followed by evaporation to dryness to obtain the monoacetylated product (1 mg). H nmr data (acetone - d6, 400 mhz) 0.14 (dt, 9.2, 4.8 hz, ha-8), 0.40 (dt, 9.2, 4.8 hz, hb-8), 0.51 (m, h-9), 0.70 (s, h3 - 14 or 12), 0.71 (s, h3 - 12 or 14), 0.85 (m, h-7), 0.93 (s, h3 - 15), 1.50 (dd, 3.2, 14 hz, ha-11), 1.70 (t, 1.2 hz, h3 - 13), 1.80 (m, 2h, ha-5 and hb-11), 1.96 (s, acetyl ch3), 2.01 (m, ha-4), 2.30 (m, hb-4), 5.35 (dd, 3.2, 12 hz, h-1), 5.52 (br d, 12 hz, h-3). A solution of 3 (1 mg) in pyridine (0.2 ml) was treated with acetic anhydride (0.4 ml), and the reaction mixture was stirred at room temperature for 24 h followed by evaporation to dryness to obtain the diacetylated product (1 mg). H nmr data (acetone - d6, 400 mhz) 0.25 (dt, 9.6, 5.2 hz, ha-8), 0.47 (dt, 9.6, 5.2 hz, hb-8), 0.60 (dt, 9.6, 5.2 hz, h-9), 0.73 (s, h3 - 14 or h3 - 12), 0.77 (s, h3 - 12 or h3 - 14), 0.87 (m, h-7), 0.95 (s, h3 - 15), 1.54 (dd, 3.6, 14 hz, ha-11), 1.73 (t, 1.2 hz, h3 - 13), 1.94 (m, hb-11), 1.97 (s, acetyl ch3), 2.00 (s, acetyl ch3), 2.29 (m, h-4a), 2.40 (m, h-4b), 4.95 (dd, 4.8, 12 hz, h-5), 5.37 (dd, 3.2, 12 hz, h-1), 5.60 (br d, 12 hz, h-3).
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If cilia provide information that serves to retain cells in their functioning differentiated g0 state, then defects in this pathway are predicted to cause proliferative disorders: cancer, cystic diseases, and fibroses of various sorts . To date, the best case for such a role for cilia comes from studies on the polycystic kidney diseases (pkds). A growing body of evidence indicates that dysfunctional ciliary signaling is the proximal cause of cyst development . Consequently, mutant genes that trigger development of the pathology are candidates for players in the normal pathways of communication between cilia and the cell cycle regulatory machinery . Renal cysts are a devastating feature of a wide range of diseases, many of which have multi - organ pathology . Monoclonal cysts form from epithelial cells in the nephron, which proliferate and detach from their neighbors, eventually leading to end stage renal disease (wilson, 2004). The idea that defective cilia are the proximal cause for pkd originated with the discovery that the causative gene of the mouse orpk pkd model, polaris, is homologous to a gene essential for ciliary assembly in chlamydomonas, ift88 (pazour et al ., 2000). Many proteins associated with pkd and related diseases or syndromes have since been shown to localize to cilia and basal bodies, and/or shown to play roles in ciliogenesis (badano et al . A direct causal connection between cilia and proliferation has been controversial because many of the implicated proteins are not exclusively ciliary . However, the idea that dysfunctional ciliary signaling is the proximal cause of aberrant cell proliferation in the kidney is strengthened by several recent lines of evidence . The polycystins pc-1 and pc-2 (products of pkd1 and pkd2) are subunits of a mechano - sensitive calcium channel that has been implicated as a flow sensor for the epithelial cells of kidney tubules (boletta and germino, 2003). Postulated mechanisms by which pc-1/pc-2 might retain the differentiated epithelial cells in g0/g1 include activation of the cyclin kinase inhibitor p21(waf1) via jak - stat (bhunia et al ., 2002) and blockade of camp - stimulated cell proliferation via atk - mediated inhibition of b - raf (yamaguchi et al ., 2004). Calcium - induced changes in proliferative state require sustained ca changes in order to affect gene transcription; current data implicates both the calcineurin nfat pathway (puri et al ., 2004) and ap-1 (bhunia et al ., 2002; it is intriguing to note that pc-1 may mediate some of its effects by regulated cleavage and nuclear translocation (chauvet et al ., 2004). It will be interesting to learn whether the cleaved product of pc-1 travels to the nucleus with kap: in the sea urchin, the kap component of the kinesin-2 motor necessary for anterograde ift moves into the nucleus as cilia resorb before mitosis (morris et al ., 2004). Nuclear kap may serve as a signal to the cell that the cilia have indeed been resorbed and cell division may now proceed . Another cystogenic protein, inversin, which localizes to centrioles and cilia, binds both calmodulin and apc2 of the anaphase - promoting complex (morgan et al ., 2002a, b), thus providing another potential link between the sensory function of the primary cilium and the regulation of the cell cycle . Beyond the kidney, other pathways important for cell growth or differentiation have been found to involve cilia (pazour and witman, 2003); for example, in neuronal cells, somatostatin receptor 3 is targeted to primary cilia (handel et al ., 1999). Mouse knockouts of ift genes essential for ciliary assembly are embryonic lethal, with phenotypes reminiscent of mutations in the hedgehog pathway, suggesting that the hedgehog receptors localize to cilia (huangfu et al ., 2003). In nih 3t3 cells, the tyrosine kinase receptor pdgfr, activation of which is sufficient for a quiescent cell to reenter the cell cycle, is expressed specifically on the primary cilium (christensen et al ., 2004). Surprisingly, bovine insulin promotes the growth of low - density cultures of the ciliate tetrahymena (christensen, 1993). The insulin signal may be received by an insulin receptor - related tyrosine kinase which localizes to the tetrahymena cilia (christensen et al ., 2003). Antennae for growth signals may be, like cilia themselves, a primitive condition of the eukaryotes . All cells lose their cilia by one of two mechanisms: resorption or deflagellation / deciliation . Resorption is the process by which the cilium is gradually retracted into the cell, and usually occurs in advance of cell division . Deflagellation is the shedding of flagella that occurs in response to a wide range of stimuli . It involves the precise severing of the nine outer doublet microtubules at the base of the flagellum, but distal to the transition zone between the basal body and the flagellum - proper, at a site known as the site of flagellar autotomy (sofa; mahjoub et al ., 2004). Deflagellation is a common cellular response: sea urchin embryos, scallop gills, rabbit oviduct, porcine respiratory tissue, and rat cerebral ependymal cells all deciliate in response to stress; cells regenerate their cilia when the stressful stimulus is removed (quarmby, 2004). The fa2 gene was uncovered in a genetic screen for chlamydomonas mutants defective in deflagellation (finst et al ., 1998). Fa2p is essential for calcium - activated axonemal microtubule severing during deflagellation, and, as it turns out, it also plays a role during cell cycle progression (mahjoub et al ., 2002). Fa2p is a member of the nima - related kinase family, which is represented by at least eleven genes in humans (the nima - related expressed kinases; neks). Nek family members are known as cell cycle kinases (o'connell et al ., 2003), thus the discovery of a nek having a ciliary function was provocative . Fa2p localizes specifically to the sofa region of the cilium of interphase cells (fig . Although fa2p's ciliary function is directly related to axonemal severing, localization of the protein shows interesting changes during premitotic flagellar resorption and during ciliogenesis . During resorption, fa2p relocates from the sofa to the proximal end of the basal bodies; during mitosis, it is associated with the polar region of the mitotic spindle . As the cells exit mitosis, fa2p accumulates at the proximal end of the basal bodies and moves out to the sofa as soon as ciliogenesis is initiated (mahjoub et al ., 2004). When expressed in imcd-3 cells (derived from murine kidney), gfp - tagged chlamydomonas fa2p shows an intriguing pattern of localization . In a confluent culture, fa2p is observed lying on the presumptive ciliary sofa and at the base of both centrioles, one of which is serving as the basal body (mahjoub et al ., 2004). During mitosis, fa2p is associated with the duplicated centrioles and then with the polar region of the mitotic spindle in the mouse cells, as it is in chlamydomonas . Perhaps a functional homologue of fa2p facilitates the timing of the final break between daughter cells via triggering severing of the acetylated microtubules . (a) fa2p - ha localizes to the sofa region of the proximal axoneme (arrowheads) and also associates with basal bodies . Indirect immunofluorescence images visualized by anti - ha (red), anti-tubulin (blue), and anti - centrin (green). We do not yet know how fa2p mediates its effects on microtubule severing during deflagellation, or on the g2/m transition . One possibility for the delay in entry into mitosis is that it is a consequence of inefficient flagellar resorption, relating to the ciliary function of fa2p . Although deflagellation has been considered a distinct pathway from premitotic flagellar resorption, recent evidence indicates that these two mechanisms of flagellar loss share important signaling components (parker and quarmby, 2003; pan et al ., 2004). It is also possible that fa2p independently affects deflagellation and progression through the cell cycle: a presumptive kinase - dead fa2p localizes correctly to the sofa, but does not rescue the deflagellation defect of fa2 cells; this same mutant protein provides full rescue of the g2/m delay (mahjoub et al ., 2004). Cnk2p is another member of the chlamydomonas nek family, and it is the second nek protein shown to be axonemal (fig . 1 b; bradley and quarmby, 2005). Although fa2p is specifically localized to the sofa, cnk2p is found along the entire length of the axoneme . The roles of cnk2p in the cilia and in cell cycle regulation are also distinct from fa2p . When wild - type chlamydomonas cells pass the commitment point of the cell cycle, two decisions are made: whether to divide and if so, how many times . Cells then undergo a rapid succession of alternating m / s phases, dividing one, two, or three times, depending on the size of the cell when it passed the commitment point (pickett - heaps, 1975). Cells with altered cnk2p expression are defective at assessing their size at the commitment point: an increase in the amount of cnk2p results in small cells, while a decrease in cnk2p results in large cells . These same manipulations of cnk2p levels produce cells with short and long flagella, respectively . Flagellar length is normally tightly controlled, at least in part by a balance in the rates of assembly and disassembly (marshall, 2004). Our data indicate that cnk2p may play a role in flagellar disassembly; whether this role is independent of cell size assessment is unknown . In tetrahymena, at least four neks localize to cilia and affect ciliary length (j. gaertig, personal communication). The relationship between the disassembly that contributes to length control, premitotic flagellar resorption and the assessment of cell size are all important connections that remain to be established . We speculate that the nek family provides an important general connection between cilia and the regulation of cell cycle progression . In all organisms where they have been studied, nima and its brethren, the neks, we have noted that this family is expanded in lineages with ciliated cells (bradley et al ., 2004), but the only direct ciliary connection so far in vertebrates comes via studies of pkd models: the causative genes of two mouse pkd models are neks . Study of kat mutant mice established that nek1 carries the causative mutation in this model of autosomal recessive pkd (upadhya et al ., 2000). Hnek1 has been found to bind to other cystogenic proteins including kinesin-2, which is also required for ciliogenesis (surpili et al ., 2003). Nek8 carries the causative mutation in the juvenile cystic kidney mouse model, and morpholinos to zebrafish nek8 cause kidney cysts in that organism (liu et al ., a kinase domain mutation of the human orthologue of nek8 affects cell cycle progression, probably at the g2/m checkpoint . It is currently unknown whether nek1, nek8, or any other mammalian neks, localize to cilia, but the work in chlamydomonas suggests that mammalian neks will be found in cilia . It is interesting to consider that if cilia are indeed sending signals that regulate cell cycle progression, by physical necessity these signals must pass near or through the centrosome, which is a gathering place for all manner of cell cycle regulating proteins (doxsey, 2001; badano et al ., 2005). We are intrigued by the possibility that signals from the cilium are mediated by calcium . Calcium is implicated in both exit from g0 and at g1/s (means et al ., 1999), as well as ciliary disassembly (quarmby, 2004). It is clear that there is a relationship between cilia, cell size, and cell cycle progression . The extent to which cilia send signals that regulate cell cycle progression (and vice versa) is not yet known, but we predict that this area of research will provide fertile ground for the discovery of new players in the regulation of cell proliferation and differentiation . Available data indicates that most vertebrate cells are ciliated and that the genesis and disassembly of these cilia is coordinated with progression through the cell cycle . We are only beginning to understand how this coordination is achieved, but its significance may be profound.
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Thus, various pharmaceutical modifications of 5-asa have been attempted . A time - dependent 5-asa releasing preparation releases 5-asa into the entire small intestine and the large intestine in a time - dependent manner because of the methylcellulose coating . Meanwhile, a ph - dependent 5-asa releasing preparation (delayed - release mesalazine) releases 5-asa from the distal ileum downwards, where the ph is 7 or above . As compared to conventional preparations (pentasa) that release 5-asa from the small intestine downwards, delayed - release mesalazine (asacol) is considered to deliver a larger amount of 5-asa into the large intestine at similar doses via a drug delivery system that releases 5-asa into the large intestine from the distal ileum downwards.2,3 since the launch of delayed - release mesalazine (asacol) in the market in japan, the prescription of the drug for the treatment of ulcerative colitis (uc) has increased dramatically at our center . While there have been a number of reports on the therapeutic efficacy of delayed - release mesalazine (asacol), there are still few studies focusing on the therapeutic effects of the drug based on endoscopic assessment . The present study was designed to perform clinical and endoscopic assessment of the efficacy of delayed - release mesalazine (asacol) in patients with uc . A total of 186 uc patients who were started on treatment with delayed - release mesalazine (asacol) and underwent periodic follow - up on an outpatient basis at the saitama medical center between 2009 and 2012 were retrospectively identified from medical records and enrolled in this study . The inclusion criteria were as follows: 1) patients who were 16 years of age and older at the time of registration and 2) patients who were under periodic follow - up on an outpatient basis for at least 1 year . The exclusion criteria were as follows: 1) patients who discontinued the treatment with delayed - release mesalazine due to the development of adverse reactions and 2) patients who were observed on an outpatient basis for less than 1 year . Finally, the data of 104 of these patients (52 men, 52 women; mean age at onset of uc, 3314 years; mean age at entry of the study, 4315 years; mean disease duration, 11.08.8 years) were analyzed and all were followed - up through december 2013 . The mean follow - up period was 2.40.6 years (range: 1.24.0 years). Adverse reactions to delayed - release mesalazine were observed in six patients, consisting of two with rash, one with pancreatitis, one with elevated serum level of pancreatic amylase, one with nausea, and one with abdominal bloating . Clinical activity index (cai): patients were assessed at months 0, 1, and 12 . The cai described by lichtiger et al4 was used for the assessment of disease severity and efficacy . The components of the cai include the following: bowel movement frequency (score 04), nocturnal diarrhea (score 01), blood in stool (score 03), fecal incontinence (score 01), use of antidiarrheal drugs (score 01), abdominal pain (score 03), general well - being (score 05), and abdominal tenderness (score 03). The sum of the score for each variable ranges from 0 to 21.4endoscopic index (ei): in the 72 patients who underwent total colonoscopy before and after the start of delayed - release mesalazine treatment, the endoscopic findings were scored by the ei described by rachmilewitz,5 for endoscopic assessment of the treatment efficacy . The first colonoscopy was performed 3 months to 2 years after the start of delayed - release mesalazine treatment . The interval between the start of delayed - release mesalazine treatment and undergoing follow - up colonoscopy was 1.00.4 years (0.31.9 years). Clinical activity index (cai): patients were assessed at months 0, 1, and 12 . The cai described by lichtiger et al4 was used for the assessment of disease severity and efficacy . The components of the cai include the following: bowel movement frequency (score 04), nocturnal diarrhea (score 01), blood in stool (score 03), fecal incontinence (score 01), use of antidiarrheal drugs (score 01), abdominal pain (score 03), general well - being (score 05), and abdominal tenderness (score 03). The sum of the score for each variable ranges from 0 to 21.4 endoscopic index (ei): in the 72 patients who underwent total colonoscopy before and after the start of delayed - release mesalazine treatment, the endoscopic findings were scored by the ei described by rachmilewitz,5 for endoscopic assessment of the treatment efficacy . The first colonoscopy was performed 3 months to 2 years after the start of delayed - release mesalazine treatment . The interval between the start of delayed - release mesalazine treatment and undergoing follow - up colonoscopy was 1.00.4 years (0.31.9 years). The four rachmilewitz score items were included: granulation scattering reflected light (score 02), vascular pattern (score 02), vulnerability of mucosa (score 04), and mucosal damage (mucus, fibrin, exudates, erosions, and ulcers, score 04). The sum of the score for each endoscopic variable ranges from 0 to 12.5 the scores for each patient were determined by assessment of the areas showing the most severe inflammation . Uc was diagnosed based on the characteristic endoscopic and biopsy findings, after excluding other inflammatory bowel disorders . Steroid dependence was defined as recurrence developing during tapering or within a short time after discontinuation of the steroid dose, and steroid resistance was defined as no response to prednisolone administered at the dose of 11.5 mg / kg / day for 12 weeks . This study was approved by the etiological study ethical review board of saitama medical center, jichi medical university . Because we produced anonymized data and used them, informed consent from all study subjects statistical analysis was performed using the student s t - test or one - way analysis of variance (anova) with post hoc turkey kramer tests . All data analyses were performed with the statview software (version 5.0; sas institute inc ., a total of 186 uc patients who were started on treatment with delayed - release mesalazine (asacol) and underwent periodic follow - up on an outpatient basis at the saitama medical center between 2009 and 2012 were retrospectively identified from medical records and enrolled in this study . The inclusion criteria were as follows: 1) patients who were 16 years of age and older at the time of registration and 2) patients who were under periodic follow - up on an outpatient basis for at least 1 year . The exclusion criteria were as follows: 1) patients who discontinued the treatment with delayed - release mesalazine due to the development of adverse reactions and 2) patients who were observed on an outpatient basis for less than 1 year . Finally, the data of 104 of these patients (52 men, 52 women; mean age at onset of uc, 3314 years; mean age at entry of the study, 4315 years; mean disease duration, 11.08.8 years) were analyzed and all were followed - up through december 2013 . The mean follow - up period was 2.40.6 years (range: 1.24.0 years). Adverse reactions to delayed - release mesalazine were observed in six patients, consisting of two with rash, one with pancreatitis, one with elevated serum level of pancreatic amylase, one with nausea, and one with abdominal bloating . Clinical activity index (cai): patients were assessed at months 0, 1, and 12 . The cai described by lichtiger et al4 was used for the assessment of disease severity and efficacy . The components of the cai include the following: bowel movement frequency (score 04), nocturnal diarrhea (score 01), blood in stool (score 03), fecal incontinence (score 01), use of antidiarrheal drugs (score 01), abdominal pain (score 03), general well - being (score 05), and abdominal tenderness (score 03). The sum of the score for each variable ranges from 0 to 21.4endoscopic index (ei): in the 72 patients who underwent total colonoscopy before and after the start of delayed - release mesalazine treatment, the endoscopic findings were scored by the ei described by rachmilewitz,5 for endoscopic assessment of the treatment efficacy . The first colonoscopy was performed 3 months to 2 years after the start of delayed - release mesalazine treatment . The interval between the start of delayed - release mesalazine treatment and undergoing follow - up colonoscopy was 1.00.4 years (0.31.9 years). Clinical activity index (cai): patients were assessed at months 0, 1, and 12 . The cai described by lichtiger et al4 was used for the assessment of disease severity and efficacy . The components of the cai include the following: bowel movement frequency (score 04), nocturnal diarrhea (score 01), blood in stool (score 03), fecal incontinence (score 01), use of antidiarrheal drugs (score 01), abdominal pain (score 03), general well - being (score 05), and abdominal tenderness (score 03). The sum of the score for each variable ranges from 0 to 21.4 endoscopic index (ei): in the 72 patients who underwent total colonoscopy before and after the start of delayed - release mesalazine treatment, the endoscopic findings were scored by the ei described by rachmilewitz,5 for endoscopic assessment of the treatment efficacy . The first colonoscopy was performed 3 months to 2 years after the start of delayed - release mesalazine treatment . The interval between the start of delayed - release mesalazine treatment and undergoing follow - up colonoscopy was 1.00.4 years (0.31.9 years). The four rachmilewitz score items were included: granulation scattering reflected light (score 02), vascular pattern (score 02), vulnerability of mucosa (score 04), and mucosal damage (mucus, fibrin, exudates, erosions, and ulcers, score 04). The sum of the score for each endoscopic variable ranges from 0 to 12.5 the scores for each patient were determined by assessment of the areas showing the most severe inflammation . Uc was diagnosed based on the characteristic endoscopic and biopsy findings, after excluding other inflammatory bowel disorders . Steroid dependence was defined as recurrence developing during tapering or within a short time after discontinuation of the steroid dose, and steroid resistance was defined as no response to prednisolone administered at the dose of 11.5 mg / kg / day for 12 weeks . This study was approved by the etiological study ethical review board of saitama medical center, jichi medical university . Because we produced anonymized data and used them, informed consent from all study subjects statistical analysis was performed using the student s t - test or one - way analysis of variance (anova) with post hoc turkey kramer tests . All data analyses were performed with the statview software (version 5.0; sas institute inc ., table 1 shows the characteristics of patients . In the 104 patients, the distribution of the age at onset of uc showed a peak in their 20s, consistent with the tendency observed in a survey carried out in japan . During the follow - up period, uc aggravation occurred in 23.1% (24/104) of patients and 10.6% (11/104) were hospitalized due to aggravation of uc . The cai at months 0, 1, and 12 were 4.62.8 (range: 112), 3.41.8 (213), and 2.81.4 (110), respectively, decreasing significantly with time (p<0.001). The ei decreased significantly from 4.53.2 (range: 010) before the start of delayed - release mesalazine treatment to 3.12.8 (010) after the start of treatment with the drug (p<0.001) (figure 1a). Further analysis was performed after excluding patients who had undergone some additional treatment for uc after the start of delayed - release mesalazine treatment . Under this condition, their cai at months 0, 1, and 12 was 4.12.5 (range: 112), 3.01.3 (27) and 2.60.9 (16), respectively, decreasing significantly with time (p<0.001). The ei was assessed in 49 patients and showed a significant decrease from 4.43.6 (range: 010) before the start to 2.52.5 (08) after the start of treatment with the drug (p<0.001) (figure 1b). Stratified analyses were performed to assess the clinical effects of delayed - release mesalazine based on the cai . The analysis by the lesion extent revealed significant decrease of the cai after the start of delayed - release mesalazine treatment in patients with all the disease types; especially in patients with left - sided colitis and proctitis, the effects of delayed - release mesalazine were observed within 1 month . The analysis by the indications for delayed - release mesalazine treatment revealed significant decrease of the cai after the start of delayed - release mesalazine treatment in patients who received delayed - release mesalazine for maintenance therapy, aggravation of uc, and initial therapy, and the analysis by the previously used drug for treatment also revealed significant decreases in the cai after the start of delayed - release mesalazine treatment in both patients who had received time - dependent release mesalazine and those who had had received sasp (figure 2). In regard to the blood test findings, the white blood cell counts and platelet counts decreased significantly after the start of delayed - release mesalazine treatment (figure 3). According to a report by dinca et al, when a ph - dependent releasing preparation (2.4 g / day) or time - dependent releasing preparation (3 g / day) was administered to patients with inflammatory bowel disease, the concentration of mesalazine in the sigmoid colonic mucosa was higher in the patients receiving the ph - dependent releasing preparation.2 the efficacy of delayed - release mesalazine has been demonstrated for all disease types in clinical studies conducted in japan, in which the decrease of the ulcerative colitis - disease activity index score at the last assessment was more pronounced in the patients with the total colitis type of uc . The european crohn s and colitis organization does not recommend oral mesalazine alone but topical mesalazine for proctitis.6 a double - blind study on active uc comparing delayed - release mesalazine (2.4 g / day and 3.6 g / day), time - dependent release mesalazine (2.25 g / day), and placebo has also been conducted in japan . An analysis in patients with proctitis - type uc revealed that delayed - release mesalazine administration at either dose significantly decreased the ulcerative colitis - disease activity index as compared to placebo, whereas no significant difference was observed between patients treated with time - dependent release mesalazine and placebo.7 in the present study, delayed - release mesalazine was found to be effective for all the disease types of uc . Because its effects were observed within a month, especially in patients with left - sided colitis and proctitis, delayed - release mesalazine is assumed to be potent especially against distal - type uc . Moreover, the effects of delayed - release mesalazine were also confirmed in patients who were switched from not only time - dependent release mesalazine, but also sasp . Furthermore, the analysis performed after excluding patients who had undergone some additional treatment for uc after the start of delayed - release mesalazine treatment also revealed significant decreases of both the cai and ei . In this study, even after the start of treatment with delayed - release mesalazine or treatment switch from other drugs to delayed - release mesalazine, aggravation of uc and the need for the use of steroids were observed in approximately 20% of all patients . Furthermore, maintenance treatment needed to be augmented by immunomodulatory drugs and infliximab in 7% of all patients . When remission cannot be achieved by oral administration of a sufficient dose of 5-asa, concomitant transanal administration of 5-asa may be useful for improving the remission rate.8,9 administration of 5-asa preparations in the remission maintenance phase is important for preventing not only relapse, but also preventing cancer caused by uc.10,11 for long - term remission maintenance, once - daily administration is preferable.12 furthermore, the effect of 5-asa on inducing remission is dependent on the intramucosal concentrations of 5-asa, and the effects of oral 5-asa preparations are dose - dependent.3,13 thus, it seems to be preferable to start administration of 5-asa at high doses . In this study, although adverse reactions to delayed - release mesalazine were observed in 5.5% (6/110) of patients, neither serious adverse events nor allergic reactions to 5-asa were observed . Because allergic reactions to 5-asa include diarrhea and fever which are often difficult to distinguish from the symptoms of uc, it is not uncommon for allergic reactions to be diagnosed as refractory uc . After oral administration of 5-asa, the allergic reactions to the drug are temporarily relieved, and approximately 2 weeks later, often manifest as fever and increased stool frequency accompanied by increased serum levels of c - reactive protein (crp). Because the therapeutic strategy for the allergic reactions differs greatly from that for uc, sufficient attention should be paid to the occurrence of allergic reactions . First, it was a single - center retrospective cohort study; thus, we could not control all confounding factors in the analysis . Second, the time of colonoscopy before and after the start of delayed - release mesalazine treatment varied among the patients . In conclusion, delayed - release mesalazine is effective for all types of uc and can be positioned as a key drug for the treatment of uc . It is necessary to identify patients unresponsive to the drug in the early stages of treatment and to augment the maintenance therapy.
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Such mechanisms can be divided into those caused by anatomical or functional disk alterations (fig . Disk prolapse is most common, reported in almost 25% of asymptomatic individuals and increasing with age.67 when painful, the most common type of pain is radicular, caused by irritation of a spinal nerve or its root . This irritation may be caused by inflammation of the roots (most common in extruded or sequestered herniation) and/or compression of the root ganglion or its blood supply . In addition to pain in their extremities, some patients may also experience axial back pain, which may arise through various mechanisms . With extruded prolapse however, it must be noted that prolapse rarely occurs without some degree of disk degeneration, which can be the generator of axial pain itself (see below). Another pain mechanism via compromised nerves due to anatomical changes of the degenerated disk is stenosis . With more severe degeneration, significant height loss and listhesis can occur, resulting in foraminal or central stenosis (i.e., compression of nerve root or spinal cord, respectively, by displaced bony structures). Such mechanisms may not always be obvious on imaging but may become more evident with certain body positions and movements.89 schematic of pertinent changes to the intervertebral disk during degeneration and the possible biomechanical mechanisms of pain generated thereof . Most of the mechanisms of indirect pain arising from anatomical changes of a degenerating disk are clear, whereas those caused by functional alterations are less so . Kirkaldy - willis and farfan first proposed that in early or mild disk degeneration, the spinal motion segment (sms) becomes less stable before further degenerative changes stabilize the segment with disease progression.10 they hypothesized that this instability of the sms was a source for back pain . Changes in sms mechanical function with mild disk degeneration have been explored both ex vivo and in silico . Ex vivo, some studies confirmed the hypothesis,111213 whereas others, including a comprehensive study with over 200 cadaveric motion segments, demonstrated the opposite (i.e., decreasing range of motion and/or increasing stiffness).1415 using a phenomenological finite element (fe) approach, the main characteristics of disk degeneration and their severity were combined to build a variety of motion segment models.16 under simulated loading corresponding to daily activities, a tendency to increase stiffness with progressing overall degeneration was demonstrated . Similar results were also obtained using a motion segment fe model with a mechanistic - based disk model, which took into account the biochemical, collagen architectural, and disk height changes associated with early disk degeneration (rijsbergen in preparation). But again, other fe models have exhibited contrasting destabilizing behavior.17 nevertheless, even if early degenerative changes did lead to segment instability, how could it result in back pain? With a more lax or compliant interbody structure, the other passive and active spinal elements may be required to compensate . Selective local analgesic administration has indicated that this may occur in a minority of patients, but controlled studies with independently correlated indicators of pain are lacking . Finally, an alternative mechanism of pain may be disk degeneration - induced zygapophyseal joint degeneration . Morphological and functional changes to the disk may result in facet joint overloading and damage leading to progressive loss of cartilage and osteoarthritis . Although a causal relationship has not been demonstrated, disk degeneration and facet joint arthritis are associated both in the same patient and spinal level,1819 and their relationship is currently a topic of debate.20 although causal evidence for many indirect back pain mechanisms in disk degeneration is still not plentiful, associative evidence exists and the postulated mechanisms are rather straightforward to comprehend . This is less so with direct mechanisms of discogenic pain (i.e., pain believed to arise from the disk itself, as a result of disk degeneration). Innervation of the lumbar intervertebral disk arises from two nerve plexi that accompany the anterior and posterior longitudinal ligaments . The anterior plexus is composed of nerves derived from the sympathetic trunk and gray rami communicans and supplies the anterior and lateral annulus fibrosus (af). The posterior plexus supplies the posterior and lateral aspects of the af and derives its nerves from the sinuvertebral nerves, formed by both somatic and autonomic roots . This plexus also receives branches from multiple levels, which perhaps is the reason that axial back pain may be so difficult to assign to a specific level . In addition to the disk, this plexus also innervates the posterior longitudinal ligament, the dural sac, and the posterior aspect of the vertebra (also see article by hiyama et al in this issue).2122 in the healthy disk, nerve fibers are only found in the outer few lamellae penetrating up to approximately 3 mm into the af.2324252627 the inner af and the central nucleus pulposus (np) are not innervated.23242528 this is perhaps because nerves require accompanying blood vessels for nutrition and the inner af and np is virtually avascular due to: (1) collapse of vessels from interstitial hydrostatic pressures greater than systolic blood pressure,29 and (2) vascular ingrowth inhibition (as well as nerve ingrowth) by an extracellular matrix rich in proteoglycans containing glycosaminoglycans.3031 the nerve fibers that are found in the peripheral af are small in diameter (1 to 3 m) and contain substance p (a nociceptive neurotransmitter),272832 consistent with myelinated a fibers that transmit sharp pain and unmyelinated c - type fibers that transmit dull pain.3233 the fibers may end in various types of mechanoreceptors . The most commonly found in the ivd are consistent with the morphology of encapsulated ruffini endings and golgi tendon organs . Some unencapsulated pacini corpuscles and abundant free nerve endings have also been reported.2434 free terminals were most often reported within the af, but partial and fully encapsulated mechanoreceptors were confined to the af surface.23 whereas golgi tendon organs, ruffini endings, and pacinian corpuscles are believed to be active in proprioception, free nerve endings are believed to be active in nociception.24 in addition to the disk itself, the vertebral end plate, particularly the central portion overlying the np, is well innervated, similar to the outer af (also see article by lotz et al, in this issue).3536 in early disk degeneration, there are subtle changes to the matrix of the np and inner af.3738 this is believed to be a result of a shift in the balance of anabolic and catabolic activities from maintenance to that of more proteolytic activity.3940 furthermore, there is evidence to support that an inflammatory process mediates this matrix breakdown.4142 it has been demonstrated that disk cells are capable of producing proinflammatory cytokines such as interleukin (il)-1,41 il-4, il-6,43 il-8,4445 il-12, il-17, interferon-,43 and tumor necrosis factor- (tnf).464748 these themselves can be nociceptive triggers or mimic noxious effects,4950 but also downstream of these signaling molecules, other agents such as nitric oxide (no), leukotrienes, and prostaglandin e are powerful direct nociceptive stimuli.4551 additionally, by - products of disk cell metabolism such as lactic acid may also be noxious (for a more complete discussion of nociceptive signaling, see article by hiyama et al, in this issue).5253 one possible mechanism of discogenic pain generation is that such noxious stimuli would reach the nociceptive receptors in the outer af and osseous end plates . These solutes range in size from very small (e.g., no) to those on the order of several hundred (e.g., prostaglandins) and tens of thousands of daltons for cytokines . In vitro measurements have shown that solutes of limited size (~400 da) have a diffusivity of ~60 m / s.54555657 in a computational model, it was calculated that with one diurnal cycle, these solutes could be transported from the np center to the end plates by diffusion alone.58 thus, such solutes could freely diffuse from their production source within the disk to nociceptive receptors in the external af and end plates . In addition, inner and, to a lesser extent, outer af cells have been shown to produce proinflammatory cytokines il-1 and tnf,48 of which particularly the latter cytokine has been shown to be directly involved in pain signaling by sensitization.59 although few studies have directly compared cytokine and inflammatory mediator expression between symptomatic and nonsymptomatic degenerated disks, comparison of symptomatic degenerated to symptomatic herniated disks suggests that similar mediators may be responsible for pain generation in both conditions.44 however, only tnf production has been shown to be higher and more strongly correlated to degeneration in disks from symptomatic compared with nonsymptomatic patients with similar degrees of degeneration.47 moreover, the concentrations of np - derived factors reaching the end plate or outer annulus by diffusion would be quite low, in particular for generally larger cytokines, and the temporal stimulation pattern quite constant, which is not always consistent with the nature of axial back pain (but could be responsible for sensitization; see below). Thus, although plausible, the direct role of preinflammatory cytokines and mediators in degenerated disk pain generation remains mechanistically unproven . With moderate to advanced disk degeneration, there is general fibrosis of the np and disorganization of the af.3760 the af also becomes stiffer and weaker with age.61 associated with aging and degeneration, various forms of af tears develop.37 similar to engineering crack propagation, these are probably the result of coalescing of micro - clefts and -tears that developed earlier . Eventually these af tears may reach the external af, with or without actual prolapse . In addition to af tears, the end plate can also become compromised through cracks and fissures . This is true for both the osseous and cartilaginous end plates.60 in a study of human osseous end plate opening sizes, it was shown that the number of larger openings, corresponding to cracks and fissures, increases with degeneration grade,62 and this was also reflected by increased osseous end plate permeability with advanced degeneration,63 as well as increased diffusion through the end plate64 (for a more detailed review of the end plate mechanisms of pain generation, see the article by lotz et al, in this issue). Thus, with such structural defects, the transport of noxious stimuli and nociceptive modulators from within the disk to nociceptive receptors in the af periphery and end plates would be enhanced . Unlike healthy disks, degenerated disks can be innervated, even in their central np.336566 these nerve fibers are positive for substance p and have nerve - ending morphologies consistent with nociception . It is hypothesized that these cracks / fissures create an environment that is favorable to neoinnervation and angiogenesis . Biomechanically, the crack / fissure relieves the stresses within the surrounding disk matrix and the pressure within the crack is substantially reduced . The inner surface of cracks / fissures is reported to be depleted of proteoglycans,67 leaving the remaining collagen, the surface of which is conducive to cell adhesion and chemotaxis . Also because the af does have the capacity to heal small tears, reactive granulation tissue, which is inductive for angiogenesis and neoinnervation, has been reported in radial fissures.66 thus, without the factors that are believed to inhibit nerve ingrowth in healthy disks, these crack / fissures may allow innervation in degenerated disk . Although the clinical efficacy of diskography and its potential complications are a topic of intense debate, the behavior of positively painful disks and their classification are quite illustrative for understanding pain generation in degenerated disks.68 if there is immediate onset of pain when contrast media reaches the outer af or the injection pressure is still below 100 kpa over the opening pressure, the disk can be classified as if concordant pain is provoked at pressures between 100 and 350 kpa above opening pressure, the disk can be classified as mechanically sensitized disk are believed to be painful because they exceed the tissue stretch threshold for nociception . This may be due to stretch at the outer af or end plates, but also internally in neoinnervated disks . Fe models have predicted strains as high as 20 to 50%,6970 but in vitro measurements only as high as 8%,71 which is more in line with the elongation at failure of 4 to 13% reported for individual af collagen fiber bundles.7273 such lower af strains are believed to be below the threshold required to illicit pain and af collagen failure . However, with af or end plate structural failure, strains may be greater, similar to when posterior elements are removed (18% strain),71 and thus sufficient to stimulate pain . Although np compression within degenerated disks is drastically lower due to loss of proteoglycans and fibrosis,74 particularly high gradients of stress have been reported internally within the af with degeneration.75 with ingrowth of nerves into the af, these gradients may directly stimulate pain . Chemically sensitized disks are believed to be painful either because of direct stimulation of nociceptive receptors (either at af periphery or ingrown) by chemically noxious stimuli (produced by inflammatory processes during degeneration) transported by flow of contrast medium injected during diskography or because of sensitization by chronic exposure to such stimuli . In the latter, in response to nociceptive stimulation by degeneration products, the somatosensory system increases its sensitivity resulting in an amplified response to normally innocuous stimuli . Hence, lower concentrations of such noxious degeneration products or lower levels of tissue strain (e.g., by low diskography pressures) that would not normally result in pain generation have now, through this process, become painful . It should be noted that this classification of pain generation in degenerated disks is purely theoretical, and most likely, pain mechanisms are not one or the other, but rather mixed in some form . However, the combination of neoinnervation within the disk via cracks / fissures, nociceptive stimuli produced during degeneration, and the correlation of discogenic pain with degenerated disks exhibiting af tears and end plate lesions supports the presented mechanisms.7677 many of the current surgical treatments used for discogenic pain (e.g., fusion and total disk replacement) are based on the commonly accepted rationale that the degenerating disk is the source of pain, and thus the disk should be removed . Although these treatments are reported to offer some pain relief, their effectiveness is still debated, and they are not unequivocally recommended . They are also not without substantial and significant complications (e.g., adjacent segment disease)78 or are of limited longevity . Furthermore, their less than desirable efficacy may be attributable to deficiencies in accurately diagnosing the specific mechanism of back pain . For example, in chemically sensitized disks, posterolateral fusion alone may not be successful because the sensitized disk (i.e., the source of pain) remains . Various treatments have also been developed based on the mechanisms of discogenic pain, some of which have been outlined in this review . In some cohort studies, intradiscal electrothermal therapy was reported to have success rates as high as 75%.7980 the mechanism of action was believed to be a sort of thermal annealing of af damage as well as thermal disruption of nerve fibers . However, ex vivo studies did not demonstrate such morphological changes,81 and later blinded, randomized placebo - controlled trials showed little benefit.8283 intradiscal steroid injections were developed to suppress the inflammatory processes in disk degeneration and thus to inhibit the production of noxious agents that would chemically sensitize the degenerating disk . However, three studies did not show any prolonged pain relief,848586 but interestingly, there was significant short - term pain relief for those patients with documented subchondral end plate and vertebral marrow changes consistent with inflammation (i.e., type i modic changes; also see article by lotz et al, in this issue). Intradiscal radiofrequency thermocoagulation or intradiscal biacuplasty is another procedure to ablate nerves within the af . This technique was shown to indeed increase temperatures in a significant portion of the af.87 however, randomized controlled trials did not show substantial benefits for treatment of axial back pain.8889 finally, two striking but unreplicated recent reports have been about the use of methylene blue for the treatment of discogenic pain . Methylene blue is a neurolytic agent that can block nerve conduction or destroy nerve endings . Hence, it was introduced into the disk to devitalize the nerves, which had grown in along the af cracks / fissures.90 in a double - blind randomized placebo - controlled trial, incredible long - term improvement in pain was reported . In these patients, 19% reported complete loss of pain and another 72% reported only slight pain that no longer required medication.91 furthermore, the rate of complications was small, although the leakage of such a neurotoxic agent near the spinal cord remains an understandable concern . Since then, only a small series study was reported that did not corroborate these findings.92 although the jury is still out on the true efficacy of this treatment, the general approach to treat discogenic pain based on a mechanistic approach is certainly worthy of further investigation . Interestingly, there has been much excitement and research on biological methods to regenerate degenerated disks, showing promising results in vivo of several growth factors such as osteogenesis protein 1 and growth and differentiating factor 5 and small molecules such as simvastatin.93949596 however, these studies all concerned acute models of disk degeneration and may not reflect the actual conditions of the human degenerated disk that are very likely to affect regeneration . In addition, pain has never been an outcome parameter in these studies . We must keep in mind that it will simply not be enough to restore some of the matrix and biomechanical function of the disk . We will also need to stop the inflammatory processes, repair internal disruptions, and restore the lack of innervation within the disk.
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About 1% of the population worldwide suffers from epilepsy, and antiepileptic drugs (aeds) are often used as a lifelong treatment . Prevalence rates of childhood epilepsy reported from different countries have shown a wide variation, with most clustering around 4 - 6 per 1,000 children . Aeds are often associated with drug interactions, complicated pharmacokinetics, and adverse drug reactions (adrs). Sodium valproate is a broad spectrum aed effective in the treatment of absence, partial and tonic - clonic seiziures . The search of literature revealed that not many reviews on sodium valproate mentioned about this adr . In pediatric patients it is difficult to distinguish between normal for age enuresis, one due to epilepsy and other due to drug itself . If it is implicated to be due to epilepsy it can lead to change in dosage of antiepileptic drugs, which itself leads to worsening of this effect . A five year old boy, known case of seizure disorder since one year presented for routine follow up in the pediatrics opd . He was on syrup sodium valproate 5ml bd (25mg / kg / day) since two month duration . Syrup was of sanofi - synthelabo (india) ltd with manufacturing date september 2010, expiry dated august 2012 and batch no 210114 . Parents of the patient noticed increased frequency of micturition and bed wetting in night one month after starting the present dose of medication and reported this on his follow up visit . Was prescribed syp sodium valproate in the dose of 2.5ml bd, @ 15 mg / kg / day . As the boy once again presented with convulsions, dose of valproate was increased to 3ml bd . Three months later, child again presented with h / o convulsions inspite of taking regular medication . Tdm was done and drug concentration was found to be 37 gm /dl, which is below the therapeutic concentration (50 - 100 gm /dl). Hence the dose was further increased to 5ml bd . One month after starting this dose (5ml bd), child's parents complained of increased frequency of micturition along with bed wetting in night (enuresis). Child had achieved bladder control at the age of three years and nocturnal enuresis was never a problem with this child . But this time the bed wetting started at this increased dose . Enuresis or bed wetting occurred twice or thrice in the night almost daily for last two months . The child was a bit disstressed due to this devolupment as this has occurred almost after two years of attaining physiological control over micturition . On investigation, complete blood count showed hb 9.4mg/ dl, wbc 17.3 /cmm, platelets 3lakh . Therapeutic drug monitoring of valproate was not done at this increased dose . After the other causes of increased frequency of micturition were ruled out child was diagnosed of valproate induced increased frequency of micturition and enuresis . Causality assessment using the naranjo's criteria revealed that the drug valproate was the probable cause of this adr (overall score, 5). Valproic acid, an eight carbon fatty acid, was found while being used as a solvent for derivative of khellin to have an anticonvulsant action . Various side effects of sodium valproate are transient gi synptoms including anorexia, nausea and vomiting . Cns side effects include sedation, ataxia and tremors; these are dose related side effects . Chronic sodium valproate therapy causes rash, alopecia, stimulation of appetite and weight gain . Enuresis is defined as the voluntary or involuntary repeated discharge of urine into clothes or bed after developmental age when bladder control should be established . Most children with a mental age of five years obtain bladder control during the day and night . Egger and brett reported seven out of 100 children on sodium valproate developed nocturnal enuresis . Panayiotopoplos reported two cases of nocturnal enuresis attributed to sodium valproate; appeared 2 - 3 days of the start of treatment, during a seizure free period and disappeared on reduction or discontinuation of sodium valproate . Other studies on use of valproate in children. [7912] have recoreded enuresis as side effect, the frequency being 1 - 7% . But surprisingly these studies are quite old and we could not find recent citations . Two most likely explanations for sodium valproate induced enuresis are that the enuresis is secondary to a central effect on the thirst center, resulting in polydipsia, or secondly is a consequence of the increased depth of sleep commonly associated with sodium valproate . Increased thirst has been demonstrated in several studies with sodium valproate. [1013] although this explanation suits well to the cause of incresesd frequency which this patient also has but in our opinion it cannot explain enuresis in a patient who has already achieved bladder control two years ago . Considering the fact that the disease itself can cause this symptom it becomes more difficult to establish a causal relationship but the absence of enuresis during period of regular epileptic attacks rules out disease induced enuresis . Dechallenge with valproate is not planned for now as the patient is currently seizure free although de - challenge can confirm the causality relationship of this adr . But de - challenge or change in drug was not attempted as it could have resulted in worsening of epilepsy . When this option (of de - challenge or changeover) was discussed with the parents they agreed to accept this adr against the possible events following the dechallege or changeover . Although the exact cause of this adr is not known, it can be attributed to increased thirst and/or increased depth of the sleep . In this patient this adr seems to be dose related as it did not occur at the previous low doses given.
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The purpose of the root canal obturation is to provide a tight seal that prevents reinfection of the canal and subsequent leakage of fluid and antigenic agents into or from the periradicular tissues . Nowadays, there is increasing demand for prompt, simple and efficient obturation technique, which improves practice and causes less stress for patients and clinicians . With the widespread use of rotary niti instruments and matched - taper gutta - percha cones, the single - cone obturation technique has become popular . With the aim of improving the marginal sealing properties of root canal filling, new root canal filling systems have been recently developed such as realseal se and guttaflow2 systems, which may encourage the practitioners to use the single - cone obturation technique . The reaseal se system consists of a self - etching methacrylate sealer and resilon core material . It is claimed to reduce the application steps of the ordinary epiphany system, thus becoming a more user - friendly material and bonds to both the resilon core and radicular dentin through hybrid layers on both substrates leading to a monoblock unit, which may prevent leakage and improve the root strength . Guttaflow2 (coltene / whaledent, altsttten, switzerland) is a cold flowable; self - curing material and composed of gutta - percha powder, polydimethylsiloxane and nano silver particles . It has a better seal and good adaptability because of its high flowability and setting expansion . The sealing ability is a basic feature that needs to be tested for every root canal filling material or technique . A variety of laboratory - based experimental methods are used to detect and measure leakage along root fillings . These methods include dye penetration, spectrometry of radioisotopes, fluorometric and electrometric methods, bacterial penetration and fluid transport model . Xu et al . Discussed a new non - destructive model that measures the leakage of glucose molecules quantitatively by using a spectrophotometer . The aim of the present study was to evaluate corono - apical microleakage along root canal fillings using glucose leakage model by comparing three matched - taper single - cone filling systems with cold gutta - percha lateral compaction technique using glucose leakage model at different time intervals . The following combinations were used as a single - cone filling technique: gutta - percha / ah plus, gutta - percha / guttaflow2 and resilon / realseal se . The crowns were cut below the cemento - enamel junction so that the length of roots was standardized at 15 mm . The working length was determined and the canals were instrumented by profile niti rotary instruments (dentsply maillefer) to size 35/0.06 using the crown - down technique . The canals were irrigated after using each file with 5 ml of 3% sodium hypochlorite (naocl) solution using vibringe ultrasonic dental irrigation syringe (medgadget) and a 27-gauge max - i - probe needle (dentsply maillefer). After finishing the instrumentation, the prepared canals were rinsed with 5 ml of 17% ethylenediaminetetraacetic acid (edta) solution for 2 min followed by 10 ml distilled water as final irrigation to remove any traces of naocl . After drying all canals, the samples were divided according to the obturation technique and materials into four experimental groups of 20 samples each and two control groups of 5 samples each [table 1]. Samples in the negative control group did not receive root canal fillings while in the positive control group they were obturated with a single - cone gutta - percha size 35/0.06 but without sealer placement . Samples grouping and materials used for root canal filling in group 1, ah plus sealer was mixed according to the manufacturer's instructions and applied into the prepared root canal using a lentulo spiral size 25 . A master gutta - percha cone of size 35/0.02 was coated with sealer and placed into the root canal to the full working length . Lateral condensation was achieved using size 25/0.02 standardized gutta - percha cones and size c finger spreader (dentsply maillefer). Excess gutta - percha was cut at the orifice level with a flame - heated hand plugger and vertically compacted . In the other experimental groups (single - cone obturation technique), the tip of the matched taper cone (gutta - percha or realseal point) was dipped into the sealer and placed slowly in up and down motion until reaching the full working length . The coronal excess of the master cone was cut to coronal orifice using a flame - heated hand plugger . In the realseal group, the coronal surface of the obturation was light cured after 5 min for 40 s. all samples were incubated for 1 week at 37c and 95% humidity to allow complete setting of sealers . The roots in the experimental and positive control groups were coated with triple layers of nail varnish, except at the coronal end and apical 1 mm of the root end . Microleakage along the root canal was evaluated using the glucose leakage model as described by xu et al . The concentrations of leaked glucose (mg / dl) were measured after 1 day and then after 1, 2, 3, 4 and 6 weeks with a glucose kit (glucose liquid, quimica clinica applicada s.a) in a spectrophotometer (beckman du 520, coulter, germany) at a wave length of 505 nm . The results were statistically analyzed by kruskal - wallis and mann - whitney tests . To compare leakage at different times within each group, the crowns were cut below the cemento - enamel junction so that the length of roots was standardized at 15 mm . The working length was determined and the canals were instrumented by profile niti rotary instruments (dentsply maillefer) to size 35/0.06 using the crown - down technique . The canals were irrigated after using each file with 5 ml of 3% sodium hypochlorite (naocl) solution using vibringe ultrasonic dental irrigation syringe (medgadget) and a 27-gauge max - i - probe needle (dentsply maillefer). After finishing the instrumentation, the prepared canals were rinsed with 5 ml of 17% ethylenediaminetetraacetic acid (edta) solution for 2 min followed by 10 ml distilled water as final irrigation to remove any traces of naocl . After drying all canals, the samples were divided according to the obturation technique and materials into four experimental groups of 20 samples each and two control groups of 5 samples each [table 1]. Samples in the negative control group did not receive root canal fillings while in the positive control group they were obturated with a single - cone gutta - percha size 35/0.06 but without sealer placement . Samples grouping and materials used for root canal filling in group 1, ah plus sealer was mixed according to the manufacturer's instructions and applied into the prepared root canal using a lentulo spiral size 25 . A master gutta - percha cone of size 35/0.02 was coated with sealer and placed into the root canal to the full working length . Lateral condensation was achieved using size 25/0.02 standardized gutta - percha cones and size c finger spreader (dentsply maillefer). Excess gutta - percha was cut at the orifice level with a flame - heated hand plugger and vertically compacted . In the other experimental groups (single - cone obturation technique), each sealer was prepared and placed into prepared canals according to manufacturer's instructions . The tip of the matched taper cone (gutta - percha or realseal point) was dipped into the sealer and placed slowly in up and down motion until reaching the full working length . The coronal excess of the master cone was cut to coronal orifice using a flame - heated hand plugger . In the realseal group, the coronal surface of the obturation was light cured after 5 min for 40 s. all samples were incubated for 1 week at 37c and 95% humidity to allow complete setting of sealers . The roots in the experimental and positive control groups were coated with triple layers of nail varnish, except at the coronal end and apical 1 mm of the root end . Microleakage along the root canal was evaluated using the glucose leakage model as described by xu et al . The concentrations of leaked glucose (mg / dl) were measured after 1 day and then after 1, 2, 3, 4 and 6 weeks with a glucose kit (glucose liquid, quimica clinica applicada s.a) in a spectrophotometer (beckman du 520, coulter, germany) at a wave length of 505 nm . The results were statistically analyzed by kruskal - wallis and mann - whitney tests . To compare leakage at different times within each group, the negative control group showed no detectable glucose leakage throughout the experiment while the positive control group had immediate substantial glucose leakage, which increased over time . The mean values and statistical comparisons between the experimental groups at each time interval are given in table 2 . After the 1 day onward, there were significant differences between the experimental groups (kruskal wallis test, p <0.05). The results of the mann - whitney test indicated that there was no significant difference between groups 1 and 2 throughout the test period . After the 1 week, the lowest glucose leakage was observed in the 4 group . Starting from the 3 week onward, the lowest glucose leakage was observed in the groups 3 and 4 . Comparison between glucose leakage mean values (mg / dl) of experimental groups at specific time interval statistical comparisons between glucose leakage values within each group are presented in table 3 . There was a progressive and significant increase in the glucose leakage values in all experimental groups (friedman test, p <0.05). Comparison between glucose leakage mean values (mg / dl) of different time intervals for each experimental group in the present study, the leakage along root canal fillings was measured by the glucose penetration method, which is simple and could give reliable quantitative leakage measurements . Single - rooted teeth with single patent root canals were selected for the current study to minimize variations of canal anatomy . The canal diameter was standardized to iso size 35/06 at apical constriction to have more uniform preparation for most canals . Furthermore, the teeth were resected at the cemento - enamel junction to simplify and standardize the instrumentation and obturation procedures . One of the methods previously described for improving the root canal seal is the removal of the smear layer before filling . For this reason, the smear layer was removed in the current study by irrigating the root canals after instrumentation with 17% edta . Passive ultrasonic irrigation was used in the current study to enhance the efficacy of the irrigating solutions and smear layer removal . The cold later compaction was used in the current study as a standard to which the other single obturation techniques were compared . The reactivity of obturating materials with glucose could affect the results of the glucose leakage test . The results of this study indicate that all obturation systems allow variable degrees of glucose leakage . The glucose leakage values of ah plus groups either with lateral condensation or single - cone technique were significantly higher at the end of the experimental period . This might be explained by the fast setting and subsequent polymerization shrinkage of ah plus sealer, the lack of bonding between this sealer and gutta - percha, the low penetration ability of this sealer within the dentinal tubules and its hydrophobic property that prevents good adaptation of to the incompletely dried canal . The current results indicated a similar glucose leakage patterns in groups 1 - 3 and showed a progressive increase in glucose concentrations with time . In group 4 (realseal se), the glucose leakage pattern was inconsistent throughout experimental periods . After 1 day, no glucose leakage was observed and this may be explained by 5 min delaying the light curing of realseal, which might allow the sealer to flow within the dentinal tubules with a subsequent decrease in the polymerization shrinkage and formation of monoblock . After the 1 week, slight increase of glucose leakage was observed, which became significantly evident after the 2 week . A significant decrease of glucose leakage was observed after the 3 and 4 week which may be due to the expansion of realseal se sealer and resilon by water absorption . After the 6 week, a significant abrupt increase in the glucose leakage was observed again which can be attributed to the higher solubility and bond deterioration of realseal se sealer with time . The glucose leakage of realseal se group was lower than that of groups 1 and 2 at the last 3 weeks . This may be due to the higher flow rate, dual curing and hydrophilicity of realseal se sealer . However, some authors did not find a significant difference between the sealing ability of gutta - percha / ah plus and resilon / epiphany combinations . However, some leakage and push - out studies found that the epoxy resin sealers had better sealability and bond strength . The causes of these differences may be attributed to the method of obturation technique and leakage assessment . The overall lowest mean values for glucose leakage of guttaflow2 group may be attributed to setting expansion, high flow rate and lower solubility of this material . In spite of using different methodologies and obturation techniques, the better sealing ability of guttaflow over ah plus was supported by the finding of a previous study . However, brackett et al . Found that the sealing ability of guttaflow / gutta - percha using the single - cone technique and ah plus / gutta - percha using the warm vertical compaction or continuous wave technique was similar . However, some authors found that the sealing ability of ah plus was better than that of guttaflow . Within the limitation of this study the following conclusions could be drawn: all obturation techniques used in the current study did not prevent leakage showed a progressive increase in the glucose leakage values over time.root canals filled with matched taper single - cone technique utilizing realseal se or guttaflow2 system allowed the lowest corono - apical glucose penetration with no significant difference between them.matched taper single - cone obturation technique could be an alternative to the cold lateral condensation technique . All obturation techniques used in the current study did not prevent leakage showed a progressive increase in the glucose leakage values over time . Root canals filled with matched taper single - cone technique utilizing realseal se or guttaflow2 system allowed the lowest corono - apical glucose penetration with no significant difference between them . Matched taper single - cone obturation technique could be an alternative to the cold lateral condensation technique.
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Although resin composite materials have improved considerably since their introduction, their polymerization shrinkage remains a problem . This shrinkage could cause tensile stress and consequent debonding at the tooth - composite interface, which may lead to recurrent caries, postoperative sensitivity, and microleakage . Several approaches have been proposed to minimize the polymerization shrinkage, such as using an initial low - intensity curing light exposure, incremental placement technique, and applying an intermediate low elastic modulus liner . Use of low - shrinkage composites is one of the other approaches to control polymerization contraction stress . Silorane, a new class of ring - opening monomers, is derived from the combination of oxiranes and siloxanes, combining the properties of both, such as hydrophobicity, biocompatibility and low shrinkage . Previous studies have indicated better enamel and dentin marginal integrity of silorane compared to methacrylate - based composites, while others reported that silorane did not provide better marginal integrity than the methacrylate - based composites . Other resin composites (kalore gc, grandio and aelite ls posterior) used in this study was low shrinkage methacrylate - based composites . Weakening of the adhesive resin due to cyclic loading is an important issue in restorative dentistry . Some studies reported increased microleakage of the composite restorations under cyclic loading while others indicated that cyclic loading did not affect the microleakage and marginal integrity of composite restorations . Scanning electron microscopic (sem) evaluation is the gold standard for determination of microleakage in indirect and directly placed adhesive restorations . Sem - investigation on marginal adaptation of class v cavities might be performed easier because of the smaller size of the cavity and is therefore used more commonly . The aim of the current study was to evaluate the effect of cyclic loading on the microleakage of silorane based composite compared with low shrinkage methacrylate - based composites in class v cavities . In this experimental study 48 extracted intact human maxillary premolars, without caries, cracks or previous restorations were used . The teeth were immersed in 0.5% chloramine t at 4c for 1 week and then stored in physiologic normal saline solution until use . Class v cavities (occluso - gingival length of 3 mm, the mesiodistal width of 3 mm, and 1 mm dentinal depth) were prepared on the buccal and lingual surfaces of the teeth using tapered fissure diamond bur (tizkavan, tehran, iran) with water - cooled high - speed handpiece . A 0.5 mm, 45c bevel was placed on the enamel margins using a flame - shaped diamond bur (diatech dental ag) while gingival margins were prepared at 90c with the external surface . The prepared teeth were randomly divided into four groups of 12 teeth each (24 cavities). Materials used in this study and their chemical composition in all groups, 37% phosphoric acid gel (total etch, ivoclar vivadent) was applied to the enamel part of the cavity for 15 s, rinsed for 15 s and excess water was removed with a light air stream to achieve a moist surface and then restored as follows: group 1 (siloran system adhesive + filtek p90): the silorane self ecth primer (3 m espe, dental product, st paul, usa) was applied and agitated on dentinal surfaces of cavity for 15 s, gently air - dried, light - cured for 20 s using a led light - curing unit (guilin woodpecker medical instrument co., china) at 900 mw / cm intensity, as checked with a radiometer (led radiometer demetron, kerr, usa) after every 10 uses, and the silorane bond was then applied on all surfaces of cavity followed by a gentle stream of air, and cured for 20 s. then each cavity was filled with filtek p90 a3.5 shade composite (3 m espe, dental product, st paul, usa). In all groups, the cavities were filled in three increments: the first increment on the axial wall, the second increment was placed from about the midpoint of the gingival wall to the occlusal cavosurface margin and the third increment filled the remaining of preparation, and each increment was cured for 40 sgroup 2 (all bond se + aelite ls posterior): all bond se (bisco inc ., schaumburg, usa) was applied and agitated for 10 s, gently air dried for 5 s and then air dried with greater pressure completely . Another layer of bonding was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of aelite ls posterior a3.5 shade composite (bisco inc ., schaumburg, usa) and each increment was cured for 40 sgroup 3 (futurabond nr + grandio): a moderately thin layer of futurabond nr (voco cuxhaven, germany) was applied for 20 s and air dried for 10 s. another layer of bonding was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of grandio a3.5 shade composite (voco cuxhaven, germany) and each increment was cured for 40 sgroup 4 (g - bond + kalore - gc): g - bond (gc corporation, tokyo, japan) was applied and left undisturbed for 10 s. then air dried for 5 s. another layer of g - bond was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of kalore - gc a3.5 shade composite (gc corporation, tokyo, japan) and each increment was cured for 40 s. group 1 (siloran system adhesive + filtek p90): the silorane self ecth primer (3 m espe, dental product, st paul, usa) was applied and agitated on dentinal surfaces of cavity for 15 s, gently air - dried, light - cured for 20 s using a led light - curing unit (guilin woodpecker medical instrument co., china) at 900 mw / cm intensity, as checked with a radiometer (led radiometer demetron, kerr, usa) after every 10 uses, and the silorane bond was then applied on all surfaces of cavity followed by a gentle stream of air, and cured for 20 s. then each cavity was filled with filtek p90 a3.5 shade composite (3 m espe, dental product, st paul, usa). In all groups, the cavities were filled in three increments: the first increment on the axial wall, the second increment was placed from about the midpoint of the gingival wall to the occlusal cavosurface margin and the third increment filled the remaining of preparation, and each increment was cured for 40 s group 2 (all bond se + aelite ls posterior): all bond se (bisco inc ., schaumburg, usa) was applied and agitated for 10 s, gently air dried for 5 s and then air dried with greater pressure completely . Another layer of bonding was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of aelite ls posterior a3.5 shade composite (bisco inc ., schaumburg, usa) and each increment was cured for 40 s group 3 (futurabond nr + grandio): a moderately thin layer of futurabond nr (voco cuxhaven, germany) was applied for 20 s and air dried for 10 s. another layer of bonding was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of grandio a3.5 shade composite (voco cuxhaven, germany) and each increment was cured for 40 s group 4 (g - bond + kalore - gc): g - bond (gc corporation, tokyo, japan) was applied and left undisturbed for 10 s. then air dried for 5 s. another layer of g - bond was applied and the process was repeated again and light cured for 20 s. then each cavity was filled with three layers of kalore - gc a3.5 shade composite (gc corporation, tokyo, japan) and each increment was cured for 40 s. all specimens were finished using fine - grit finishing diamond burs (diatech dental ag, heerbrug, switzerland) and polished with sequential disks (optidisk, kerr, usa) (15 s in each margin). After storage in an incubator (malek - teb, iran) at 37c for 24 h, all teeth were subjected to 2000 thermal cycles of 5c/55c, with a dwell time of 30 s in each bath and a transfer time of 10 s (malek - teb, iran). Then in each group, half of the teeth (n = 6) were stored in an incubator at 37c and the others were load cycled (germany, sd mekanotronik), as follows: initially, a cylindrical tube was coated with a layer of wax, then the teeth were mounted up to 1 mm apical to the gingival margin of the restoration in autopolymerized acrylic resin (acropars, iran) at the middle and parallel to walls of the tube . N, a frequency of 2 hz and a displacement of 1 mm . Before sectioning the teeth, an impression from the surface of the restoration (precise, coltene, switzerland) was taken of 32 specimens (4 randomly selected restorations in each subgroup) and positive epoxy resin replica of each specimen (epo - thin, buehler ltd . Each resin replica was mounted on a metallic stub, sputter - coated with a thin layer of gold and examined under a field emission - sem (fe - sem) (hitachi s-4160, japan) with 1000 magnification and interfacial gaps were measured [figure 1]. The whole length of the gaps was expressed as a percentage of the length of the total restoration margins (enamel and dentin margins). Scanning electron microscopic micrograph of resin - dentin interface of unloaded groups (silorane (a), aelite (b), grandio (c), kalore - gc (d)) and loaded groups (silorane (e), aelite (f), grandio (g), kalore - gc (h)). After sem replicas preparation, the root apices of the teeth were sealed with sticky wax, and all surfaces of the teeth were covered with two coats of nail polish except for 1 mm around the margins of each restoration . All specimens were then immersed in 0.5% basic fuchsine dye for 24 h at 37c, washed thoroughly with distilled water, air dried and embedded in acrylic resin . All teeth were then sectioned into two halves longitudinally from buccal to lingual surface through the center of the restored area using a low - speed diamond disk mounted in a cutting machine (presi, mecatome, t201a, france) under constant water irrigation . Dye penetration at the occlusal and gingival margins was blindly assessed in the two halves by two independent investigators using a stereomicroscope (nikon 800, tokyo, japan) at 10 and 40 magnifications; if the dye penetration score on the two halves was different, the half that showed more microleakage was selected for assessment . The degree of microleakage was scored according to the following criteria: 0 . No dye penetration.1 . Dye penetration greater than one - half of the occlusal or gingival wall, but not reaching the axial wall.3 . Dye penetration greater than one - half of the occlusal or gingival wall, but not reaching the axial wall . Wallis test, dunn procedure, and mann - whitney u - test were used for statistical analysis of the data . The difference between the occlusal and gingival dye penetration scores of each specimen before sectioning the teeth, an impression from the surface of the restoration (precise, coltene, switzerland) was taken of 32 specimens (4 randomly selected restorations in each subgroup) and positive epoxy resin replica of each specimen (epo - thin, buehler ltd ., lake bluff, il, usa) was obtained . Each resin replica was mounted on a metallic stub, sputter - coated with a thin layer of gold and examined under a field emission - sem (fe - sem) (hitachi s-4160, japan) with 1000 magnification and interfacial gaps were measured [figure 1]. The whole length of the gaps was expressed as a percentage of the length of the total restoration margins (enamel and dentin margins). Scanning electron microscopic micrograph of resin - dentin interface of unloaded groups (silorane (a), aelite (b), grandio (c), kalore - gc (d)) and loaded groups (silorane (e), aelite (f), grandio (g), kalore - gc (h)). After sem replicas preparation, the root apices of the teeth were sealed with sticky wax, and all surfaces of the teeth were covered with two coats of nail polish except for 1 mm around the margins of each restoration . All specimens were then immersed in 0.5% basic fuchsine dye for 24 h at 37c, washed thoroughly with distilled water, air dried and embedded in acrylic resin . All teeth were then sectioned into two halves longitudinally from buccal to lingual surface through the center of the restored area using a low - speed diamond disk mounted in a cutting machine (presi, mecatome, t201a, france) under constant water irrigation . Dye penetration at the occlusal and gingival margins was blindly assessed in the two halves by two independent investigators using a stereomicroscope (nikon 800, tokyo, japan) at 10 and 40 magnifications; if the dye penetration score on the two halves was different, the half that showed more microleakage was selected for assessment . The degree of microleakage was scored according to the following criteria: 0 . No dye penetration.1 . Dye penetration greater than one - half of the occlusal or gingival wall, but not reaching the axial wall.3 . Dye penetration greater than one - half of the occlusal or gingival wall, but not reaching the axial wall . Wallis test, dunn procedure, and mann - whitney u - test were used for statistical analysis of the data . The difference between the occlusal and gingival dye penetration scores of each specimen was analyzed by the wilcoxon test and p <0.05 was considered statistically significant . Microleakage score in each margin according to the tested composite and cyclic loading no statistically significant differences were observed between the microleakage of unloaded and loaded groups on both occlusal and gingival margins in all materials (p> 0.05). In both unloaded and loaded groups, occlusal microleakage among four composites was not significantly different (p = 0.092, p = 1, respectively). However, at the gingival margin, aelite showed significantly higher microleakage than silorane restorations (p = 0.03, p = 0.005, respectively), and no significant differences were detected between the other groups (p> 0.05). When comparing the microleakage between occlusal and gingival margins in each group, there were significantly more dye penetration at the gingival margin than the occlusal margin in all the tested groups (p <0.05), except unloaded and loaded silorane groups (p = 0.19, p = 0.18, respectively). Table 3 summarizes the interfacial gap formation observed by fe - sem . Due to the limited sample size (n = 32) the main cause for the clinical failure of composite restoration is marginal leakage along the tooth - restoration interface . Kalore is a nano - hybrid resin composite with the filler content of 82% by weight . This resin composite is based on dupont technology, which contains a dx511 molecule in its matrix . The dupont molecule, dx-511, is a urethane dimethacrylate monomer with a low number of c = c double bonds that is compatible with the current bonding systems and composites . The molecular weight of dx-511 is 895 which is twice that of udma or bis - gma . The low polymerization shrinkage of kalore (1.7%) is due to the presence of a low number of c = c double bonds and high molecular weight of dx511 . Aelite ls posterior is a highly filled hybrid resin composite (74% by volume and 88.5% by weight), and its low polymerization shrinkage is due to its high filler content (1.39%). Grandio is a highly filled nanohybrid resin composite (71.4% by volume and 87% by weight). Gingival microleakage in kalore, grandio and aelite (in both unloaded and loaded groups) were significantly higher than occlusal margins; this finding was in agreement with the previous studies that indicated less microleakage at the occlusal margin than gingival margin . This was expected as dentin is a less favorable bonding substrate, due to its lower inorganic material (<50%), higher water content (21%), and its tubular structure . Moreover, in the current study enamel margins were etched with 37% phosphoric acid before applying self - etch adhesives . Different studies reported better marginal integrity of self - etch adhesives when the adhesive was applied following selective etching of enamel with 37% phosphoric acid . There were no significant differences between the occlusal and gingival microleakage in loaded and unloaded silorane groups . This result is probably due to the low - shrinkage nature of silorane and the fact that at the gingival margin, low polymerization shrinkage stress cannot overcome the adhesive strength . Silorane primer with almost ph of 2.7 provides a mild etching and slight decalcification of the tooth structure and a strong and long lasting bond . Moreover, mine et al furthermore, some studies reported the higher microleakage and lower bond strength of one - step self - etch adhesives in comparison with two - step self - etch adhesives . In the current study, the silorane system adhesive was two - step self - etch, while the other adhesives were one - step self - etch . In the current study, no statistically significant differences were observed among four groups at the occlusal margin (in both unloaded and loaded groups), which is in accordance with the results of earlier studies . There was a statistically significant difference in microleakage between aelite and silorane at the gingival margin (in both unloaded and loaded groups). That reported the higher microleakage of aelite compared with the other low shrinkage composites (heliomollor, venus diamond, filtek z250 and silorane). Calherios et al . Also reported that the microleakage of class v cavities restored with aelite ls was higher than that of similar cavities restored with the other low - shrinkage composites . According to hooke's law, polymerization shrinkage stress is determined by the volumetric shrinkage and viscoelastic properties of the resin composite . The higher microleakage of aelite ls is associated with its high elastic modulus and stiffness due to its high filler levels . Its high stiffness offsets its low polymerization shrinkage that results in high - stress values . Fe - sem evaluation also confirmed these results by showing higher percentage of interfacial gaps in the specimens restored with aelite (in both unloaded and loaded groups). However, due to the inadequate sample size (n = 32), the statistical analysis of the data was not performed . In the present study, there were no significant differences in microleakage between silorane, grandio and kalore; this finding was in agreement with the results of previous studies reporting that silorane did not provide better marginal integrity than the methacrylate - based composites . However, al - boni et al . Reported lower microleakage of class i cavities restored with silorane compared with the other composites (filtek z250 and amelogen plus). Also reported that the microleakage of class ii cavities filled with filtek silorane was significantly lower than that of similar cavities filled with methacrylate - based composites . These differences in studies may be explained by differences in resin composites and bonding type, microleakage scores, and cavity type . It may also be related to the formation of an oxygen inhibition layer due to the curing of silorane primer prior to the application of bonding agent . This layer is formed between the cured primer and the silorane bond and can be observed in micro - raman spectroscopy as the intermediate zone of approximately 1 m; which may be the weakest zone of silorane adhesives (but it is controversial). In the current study, occlusal and gingival microleakage of all the tested materials this finding was in agreement with the results of previous studies, although some studies showed increased microleakage of composite restorations under cyclic loading . Campos et al . Evaluated the microleakage of a condensable composite (surefil) after cyclic loading using 4000 cycles and 150-newton forces and reported that the effect of cyclic loading on gingival and occlusal microleakage was statistically significant . Also evaluated the microleakage of spectrum tph and admira ormocer after cyclic loading using 50,000 cycles and 50-newton forces and reported that the cyclic loading significantly increased microleakage for both the materials at the gingival margin . It seems that the properties of a resin composite, rather than marginal adhesion are considered as the most influential factor on its resistance to marginal degradation . It is hypothesized that, since low - shrinkage composites provide lower polymerization stress, they would be able to withstand fatigue at the interface better than the other composites . Another possible explanation is the use of composites with nanofiller content (grandio and kalore are nanohybrid composites). Cyclic loading leads to a decrease in bonding performance due to fatigue at the adhesive interface . Moreover, grandio and aelite contain spherical filler particles that have been associated with reduced stress concentration during loading compared with the sharp edges present within irregular - shaped fillers . Irregular - shaped fillers may act as a defect center promoting the accumulation of stress - induced damage . In this study, gingival microleakage in kalore, grandio and aelite (in both unloaded and loaded groups) were significantly higher than occlusal margins; this finding was in agreement with the previous studies that indicated less microleakage at the occlusal margin than gingival margin . This was expected as dentin is a less favorable bonding substrate, due to its lower inorganic material (<50%), higher water content (21%), and its tubular structure . Moreover, in the current study enamel margins were etched with 37% phosphoric acid before applying self - etch adhesives . Different studies reported better marginal integrity of self - etch adhesives when the adhesive was applied following selective etching of enamel with 37% phosphoric acid . There were no significant differences between the occlusal and gingival microleakage in loaded and unloaded silorane groups . This result is probably due to the low - shrinkage nature of silorane and the fact that at the gingival margin, low polymerization shrinkage stress cannot overcome the adhesive strength . Silorane primer with almost ph of 2.7 provides a mild etching and slight decalcification of the tooth structure and a strong and long lasting bond . Moreover, mine et al furthermore, some studies reported the higher microleakage and lower bond strength of one - step self - etch adhesives in comparison with two - step self - etch adhesives . In the current study, the silorane system adhesive was two - step self - etch, while the other adhesives were one - step self - etch . In the current study, no statistically significant differences were observed among four groups at the occlusal margin (in both unloaded and loaded groups), which is in accordance with the results of earlier studies . There was a statistically significant difference in microleakage between aelite and silorane at the gingival margin (in both unloaded and loaded groups). That reported the higher microleakage of aelite compared with the other low shrinkage composites (heliomollor, venus diamond, filtek z250 and silorane). Calherios et al . Also reported that the microleakage of class v cavities restored with aelite ls was higher than that of similar cavities restored with the other low - shrinkage composites . According to hooke's law, polymerization shrinkage stress the higher microleakage of aelite ls is associated with its high elastic modulus and stiffness due to its high filler levels . Its high stiffness offsets its low polymerization shrinkage that results in high - stress values . Fe - sem evaluation also confirmed these results by showing higher percentage of interfacial gaps in the specimens restored with aelite (in both unloaded and loaded groups). However, due to the inadequate sample size (n = 32), the statistical analysis of the data was not performed . In the present study, there were no significant differences in microleakage between silorane, grandio and kalore; this finding was in agreement with the results of previous studies reporting that silorane did not provide better marginal integrity than the methacrylate - based composites . However, al - boni et al . Reported lower microleakage of class i cavities restored with silorane compared with the other composites (filtek z250 and amelogen plus). Also reported that the microleakage of class ii cavities filled with filtek silorane was significantly lower than that of similar cavities filled with methacrylate - based composites . These differences in studies may be explained by differences in resin composites and bonding type, microleakage scores, and cavity type . It may also be related to the formation of an oxygen inhibition layer due to the curing of silorane primer prior to the application of bonding agent . This layer is formed between the cured primer and the silorane bond and can be observed in micro - raman spectroscopy as the intermediate zone of approximately 1 m; which may be the weakest zone of silorane adhesives (but it is controversial). In the current study, occlusal and gingival microleakage of all the tested materials was not affected by cyclic loading . This finding was in agreement with the results of previous studies, although some studies showed increased microleakage of composite restorations under cyclic loading . Campos et al . Evaluated the microleakage of a condensable composite (surefil) after cyclic loading using 4000 cycles and 150-newton forces and reported that the effect of cyclic loading on gingival and occlusal microleakage was statistically significant . Also evaluated the microleakage of spectrum tph and admira ormocer after cyclic loading using 50,000 cycles and 50-newton forces and reported that the cyclic loading significantly increased microleakage for both the materials at the gingival margin . It seems that the properties of a resin composite, rather than marginal adhesion are considered as the most influential factor on its resistance to marginal degradation . It is hypothesized that, since low - shrinkage composites provide lower polymerization stress, they would be able to withstand fatigue at the interface better than the other composites . Another possible explanation is the use of composites with nanofiller content (grandio and kalore are nanohybrid composites). Cyclic loading leads to a decrease in bonding performance due to fatigue at the adhesive interface . Moreover, grandio and aelite contain spherical filler particles that have been associated with reduced stress concentration during loading compared with the sharp edges present within irregular - shaped fillers . Irregular - shaped fillers may act as a defect center promoting the accumulation of stress - induced damage . Under the limitations of the present study, silorane did not provide better marginal seal than the low shrinkage methacrylate - based composites (except aelite). The present study was financially supported by tehran university of medical sciences, tehran, iran . The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article . The present study was financially supported by tehran university of medical sciences, tehran, iran . The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article.
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This is because primarily most countries, including india follow the spontaneous or voluntary system of adr reporting . There are patient - related reasons for ur like failure to recognize adr or inability to link the adr with a drug . The commonest doctor related reasons are the feeling of guilt, fear of litigation, ignorance, lethargy, inadequate risk perception about newly marketed drugs, diffidence, insufficient training to identify adrs, and lack of awareness about pv program . Similarly, adrs often go unnoticed due to failed ability of medical teams to recognize adr or correlate precisely with biochemical, pathological or radiological abnormality studies document the widespread problems of ur in pv but, we failed to cite any indian study based on facts and figures to define this problem . Hence, the first indian experience / self - introspection, postsustainable pv programme of india (pvpi) keeping focus on problem of ur and its possible reasons was undertaken . A retrospective observational, prospective cross - sectional questionnaire - based analysis was undertaken to evaluate the extent and reasons of ur of adrs in the adr monitoring (adrm) center, working under the pvpi, in a teaching tertiary care hospital in india . The suspected drug reactions monitoring data collection form was used, and the study was conducted with prior permission by the institutional ethics committee . The adrs were reported, defined and categorized as per the defined standard operating procedures of pvpi . The severity and seriousness of the reaction, adverse drug reactions reported from outpatient department (opd) or indoor patients of any severity, duration and type were included . Any adr due to poisoning, medication error, over dosage, over or noncompliance, natural products / alternate medicines and unidentified drugs were excluded in the analysis . Detailed subgroup analysis of trends of adr reporting in adrm center of the tertiary care teaching hospital and in comparison to country wise adrs reporting trends were undertaken . Working performance of adrm center as indicated by adr reports collected and reported by through the vigiflow software 5.0 were taken as the basis to analyze extent of ur . Average individual case safety reports (icsrs) per month was compared among the amc started in the first phase . The central drugs standard control organization and indian pharmacopeia commission (ipc) monthly work report was also the basis of the current analysis . Further, a questionnaire and face to face enquiry based prospective study was undertaken at our center to analyze the reasons of ur among various stakeholders likely to contribute to pvpi, namely doctors, which included residents, registrars and consultants (n = 100), nurses (n = 100) and pharmacists (n = 100). They were identified with a code number, and privacy and confidentiality was maintained regarding data throughout the study . A 10% of total strength of the total staff was selected from various clinical departments of the hospitals within the jurisdiction of adrm center . Verbal consent was obtained from all the participants as the study involved minimal risk and fell in category - c as per icmr guidelines . Predesigned and pretested questionnaire based on the study objectives, taking guidance from the previous study was subjected to a thorough peer review and subsequently modified as per the suggestions and used . The questionnaire constituted 20 questions and consisted three parts, that is, demographic profile, knowledge, attitude and practice and the common reasons for ur . The focus was to evaluate the knowledge and awareness about pvpi, adrs and pv, ignorance, if any e.g., only severe adrs need to be reported, diffidence (fear of appearing ridiculous for reporting merely suspected adrs), lethargy (an amalgam of procrastination, lack of interest or time and other excuses), indifference (the one case could not contribute to medical knowledge), insecurity (it is nearly impossible to determine whether or not a drug is responsible for a particular adr) and complacency (only safe drugs are allowed in the market) as the factors for ur . Descriptive statistical analysis was carried out with the help of computer software spss version 15 for windows, released 2006 . Chi - square test was applied for some of the parameters to prove their statistical significance . Descriptive statistical analysis was carried out with the help of computer software spss version 15 for windows, released 2006 . Chi - square test was applied for some of the parameters to prove their statistical significance . Descriptive statistical analysis was carried out with the help of computer software spss version 15 for windows, released 2006 . Chi - square test was applied for some of the parameters to prove their statistical significance . At the time of study 90 amc were operational in india and with 25 in south zone, 28 in north zone, 20 in west zone and 17 in the east zone . There are 20 non - government institutions, and the rest are government medical colleges and hospitals with operational pvpi . While comparing average number of icsrs reported by vigiflow per month from the earliest amc started postsustainable pvpi, jss medical college and hospital, karnataka contributed at rate of 212.46 followed by institute of pharmacology, madras medical college as 204.29 . The leaders from north india are postgraduate institute of medical education and research (pgimer) chandigarh contributing at the average rate of 170.63 followed by lady hardinge medical college and all india institutes of medical sciences (aiims) new delhi . Whereas, the total number of adrs reported through vigiflow contributed by our center were 1249, the average number of icsrs reported by vigiflow per month by our center was 48.038 [figure 2]. National adverse drug reactions monitoring centres functional rate (july 2011 to july 2013) as per monthly work report comparative performance to our centre with leading contributors of individual case safety reports to national pharmacovigilance centre, new delhi via vigiflow (july 11 to august 13) in a period of the 3 years total number of adrs reported was 3024 . The average number of reports per month reported by the center was 80.08 [figure 3]. Active surveillance versus spontaneous reporting contributed 66.13% versus 33.86% (p <0.0001) of the total adrs . Opd based reports comprised 76.05% and adr reported from indoor patients was 23.94% of total adr reports (p <0.0001). Department of medicine (33%), followed by oncology (19.27%), chest disease (13.49%) contributed maximally in the current study . Total number of adrs contributed by opd of adrm of pharmacology was 490 (16.20%). However, few departments like eye, ent, surgery did not contribute any adr . Total number of adrs contributed by private medical colleges in the adjoining region and by hospitals in periphery, sub - district and district hospitals was nil . Adr detection rates based on clinical presentation, biochemical investigation and diagnostic tools were 84.33%, 14.57% and 1.09% respectively, that is detection based on clinical presentation was significantly high as compared to laboratory and diagnostic tools (p <0.0001). Total reporting by postgraduate students, registrars, consultants and nurses were 72.65%, 6.58%, 16.56% and 4.19%, respectively (p <0.0001). Pharmacology postgraduate (66.13%) in comparison to other postgraduates (6.61%) contributed most adrs (p <0.0001). Number of thesis related to adr were five during the period; three carried by department of pharmacology and one each by dermatology and hiv medicine . Number of adverse drug reactions (adr) reports submitted by adr monitoring centre, government medical college jammu to national pharmacovigilance centre, new delhi performance of adrm centre, gmc jammu, india an analysis of the reasons of ur in the pv suggested lack of awareness and knowledge about pvpi, lethargy, indifference, insecurity, complacency, overwork, and lack of proper training in pv as commonly cited factors for ur by doctors, nurses and pharmacists of the hospital [table 2]. The results suggest that ur of adr exists in india too . In india after the initiation of sustainable pvpi, amc functional rate is recorded to be 56.45% . This indicates that nearly 43 centers yet remain nonfunctional even after being operational most of the time . While comparing average number of icsrs reported to vigiflow per month from the earliest amc started after sustainable pvpi, jss medical college and hospital, karnataka contributed maximally, followed by institute of pharmacology, madras medical college . At the time this study was conducted, pgimer leads in adr reporting in north india followed by lady hardinge medical college and aiims, new delhi . Whereas, number of adrs reported through vigiflow and the average number of icsrs reported by vigiflow per month by our center was substantial . The probable reason for this is that our institute is a tertiary care teaching institute catering to less number of patients in comparison to these apex institutes . Further, some of these institutes had an infrastructural setup for pv even before the sustainable pvpi was initiated, unlike our institute . The results of this study are in accordance to a recent study by world health organization (who), where it has been shown that high - income countries had the highest adr reporting rates . There is, therefore, a need to strengthen adr reporting, especially in low - income countries by suitably modifying the organizational structure, training and economic resources of national pv centers . Active surveillance contributed to the majority of the total pool of adrs, suggesting ur of adr . Passive surveillance system is limited by gross ur (<10% reporting rate), latency, and inconsistent reporting . Making adr reporting compulsory in the institution to address ur . However, there is a concern that such a compulsion can lead to false reporting thus compromising the quality of reports . Regulators in united kingdom, france, european union, the united states and canada are developing suitable approaches to enhance passive adr reporting systems . In the current study, the lower reporting from indoor patients can be overcome by monitoring computerized medical records or by developing a system of active screening of all medical records of inpatients for adrs . Departments in private medical colleges in the adjoining region, hospitals in periphery, sub - district and district which failed to contribute in adr reporting need to be involved in a collaborative approach, through training and awareness programs, to widen the reporting base . In this study, the number of adrs contributed by pharmacology adrm opd was substantial thereby indicating that patient participation directly in adr reporting can help intensify adr reporting in india . Similar studies like ours suggest positive complementary contribution of patients to pv and drug safety . These studies have shown that direct reporting of adrs by patients have prove useful in intensifying adr reporting . Adverse drug reactions often go unnoticed due to failure of medical teams to recognize adverse drug events or to correlate precisely with biochemical, pathological or radiological abnormality . Thus, biochemical investigations and diagnostic tools can pick up substantial number of adrs and can play an important role in pv . The present study suggests that the human resource, that is, pharmacology residents can be utilized effectively to strengthen reporting and for educating the health professionals, providing feedback and personal communications with prescribers . Postgraduate exams, synopsis and thesis submission, summer and major festivals grossly affect the performance of this human resource with respect to adr reporting, due to obvious reasons . As our study suggests, these periods and activities corresponded with low reporting . These factors must be anticipated, and timely measures need to be adopted to maintain the consistency in adr monitoring and reporting . Doctors (interns, house officers), nurses, pharmacist and residents also need to be more actively involved in reporting adrs, to widen the reporter base . Various approaches have been recommended to intensify reporting, like, forming adr reporting network within hospital, encouraging and educating patients to report and making adr reporting compulsory for nurses . Studies have reported a felt need to improve knowledge and attitude for adr reporting by healthcare professionals . The periodic e - mail, sms alerts represent an effective and inexpensive way to raise the awareness of doctors about the importance of spontaneous adr reporting . However, the impact of interventions have been reported to decrease after the intervention is stopped as also noticed in the current study, hence the efforts must be ongoing, dynamic and continuous . Integration of information obtained from primary care electronic medical records, cohort event monitoring and targeted spontaneous reporting, being implemented by the who, in its public health programs, have shown to complement spontaneous reporting . . Human behavior, knowledge beliefs, and motivation play an important role in adr reporting . Ur is strongly associated with certain attitudes, that possibly could be modified through educational interventions . The results of the study suggest that lack of knowledge and awareness about pvpi, lethargy, indifference, insecurity, complacency, workload, and lack of proper training in pv were some factors responsible for ur . Ignorance in 95%; diffidence in 72%; lethargy in 77%; indifference and insecurity in 67%; and complacency in 47% of subjects were held responsible for ur in a similar study . Factors which promote adr reporting like years of work experience, participation in educational activities related to the detection and resolution of drug - related problems, have not been evaluated in the current study . Factors that discourage reporting include uncertainty about the causal relationship between the adr and the drug, forgetfulness, diffidence in reporting known adrs and lack of time . The amount of time dedicated to teaching of pv in undergraduate and postgraduate courses in pharmacology is low . Thus, appropriate interventions to include adr reporting and pv in an appropriate format should be mandated in the curriculum by the medical council of india and respective stakeholders in nursing and pharmacy education . Publication of rare and unusual adrs and various research papers from the field of adrs can serve as positive incentives for clinicians to report pv . However, clinicians tend to conceal fatal and serious adrs in the recommended time fame, and some of these adrs thus remain unreported . Seeking adrm number or i d generated in the vigibase, of case reports submitted for publication to journals is recommended to verify the authenticity of the reports, and also ensures that these reports are included in the vigibase, as a part of the data submitted from india . Under - reporting is observed in spontaneous adr reporting . A multipronged approach in necessary to overcome ur, taking in to cognizance the factors that affect spontaneous reporting.
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Known as mad honey among the turkish people and produced in turkey in the black sea region from r. luteum and r. panticum's nectar is known to cause severe cardiovascular side effects as well as gastrointestinal symptoms . Grayanotoksin (andromedotoksin), which is produced by the genus rhododendron plants is responsible for toxicity. [13] after ingestion of honey, symptoms suddenly starts and termination of symptoms seldom pass 24 hours. [13] in this article, we presented a case of slow ventricular response atrial fibrillation complaints with nausea, vomiting, dizziness and chest pain about an hour after eating honey produced in the black sea region . 54 year - old male patient presented to our emergency room with complaints of nausea, vomiting, dizziness and chest pain beginning approximately two hours after honey ingestion . Initial examination showed that the patient was conscious, cooperative, with a heart rate of 40 beats / min and blood pressure of 70/40 mmhg, the other system examinations were normal . The patient's complaints started approximately one hour after eating two tablespoons of honey produced in the black sea region . His medical history did not have a known health problem, not on any medications . The onset of patient's symptoms and history of mad honey consumption is probably associated with mad honey poisoning . Routine laboratory tests, including serum potassium and magnesium level were normal, and cardiac enzymes were not elevated . Because of intermittent recurrent chest pain, coronary angiography was performed and showed normal coronary arteries . Mad honey poisoning is more frequently seen in worldwide countries like turkey, nepal, brazil and japan. [14] the honey which was produced from rhododendron genus of plants, is known to have high concentrations of grayanotoxin and this toxins are responsible for the symptoms of poisining . This honey is also used as an alternative treatment method for gastrointestinal disorders, hypertension, coronary heart disease and also is believed that sexual activity can improve with this honey . Typical symptoms of mad honey poisoning are usually gastrointestinal system symptoms, sometimes life - threatening bradycardia and hypotension may occur. [25] the main toxin responsible for the cardiac effects of mad honey poisoning was gt - i . In addition, the gt - ii is capable of spontaneous pulse inhibition on sinoatrial node . Grayanotoxin shows the effect of binding to sodium channels in cell membranes, increases the permeability of sodium channels in the cell membranes, as a result depolarization time will get longer . Continuous action of sodium channels reduce the depolarization, this condition leads to sinus node dysfunction . The most common effects of mad honey are gastrointestinal system symptoms, but bradyarrhythmias and hypotension can also be observed . Except for bradyarrhythmias and hypotension, sweating, dizziness, altered consciousness, syncope, diplopia, blurred vision, hypersalivation are less common side effects. [258] atrial fibrillation, myocardial infarction, and complete av block due to the mad honey intoxication have been reported in the literature . Electrocardiographic monitoring, intravenous saline infusion and supportive care consisting of treatment with atropine is sufficient to remedy the symptoms of patients suffering mad honey poisoning. [25] in our case, the signs of toxicity was observed approximately one hour after ingestion of honey, . As well as nausea, vomiting and dizziness, myocardial infarction was brought to mind on differential diagnosis because of chest pain . However, the ecg was unremarkable except for the slow ventricular response atrial fibrillation, the enzyme was normal in follow - up, echocardiographic and angiographic examination was normal, therefore, we ruled out probable myocardial infarction . Rapid saline infusion applied to the patient and normal sinus rhythm was restored after 1 mg atropine and during follow - up for 48 hours, no complications were occurred . Mad honey poisoning may be presented with unexplained bradycardia, hypotension, and non spesific electrocardiographic changes including ryhthm disorders can be observed during the poisoning . In the differential diagnosis, bradycardia and very rarely slow ventricular response atrial fibrillation can be observed due to the ingestion of mad honey as in our case.
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According to the world allergy organisation about 1.9%4.9% of children suffer from cow's milk allergy, with the prevalence in adults much lower; less than 0.5% . It is known that perceived food allergy could be 10 times higher [3, 4] than that confirmed by appropriate tests . Although a large number of foods are suspected to cause food allergies, most studies have focused on 6 common foods which include cow's milk, hen's egg, soya, peanut / tree nuts, fish / shellfish, and wheat, clearly showing different patterns based on the population studied and the diagnostic methods used . Peanut allergy is one of the most common causes of food - induced anaphylaxis, with a reported or challenge proven prevalence rate between 0.06% and 5.9% depending on the country and age group studied . Tree nut allergies also show a wide range of reported or challenge - confirmed prevalence ranging between 0.2%8.6% . High rates of reported allergies to tree nuts may, however, be due to cross - sensitisation with aeroallergens, rather than a primary food allergy, with an increased number of patients with so - called oral allergy syndrome / fruit pollen syndrome seen . There is a paucity of information on the role of nutrition versus just food avoidance in the management and natural course of food allergy . Very little is also known about the effect of a nutrition consultation in this process . Furthermore, the role of the dietitian and the diagnostic and therapeutic value of the elimination diet has not been established and extensively investigated . A systematic review on the role of the dietitian and the value of the elimination diet in the diagnostic and therapeutic phase found only two papers . These only partially address the issue and focus mainly on the nutritional status of children with food allergies [7, 8]. The lack of information on how to take an allergy - focused diet history, linking symptoms of allergic disease with foods implicated in the adverse reactions, and the impact of general nutritional intake, has been recognised by the european academy of allergy . A special task force initiated by the interest group on allied the paucity of information on the role of nutrition in allergy is reflected in and may even be explained by the fact that globally seen, only a few dietitians and nutritionists have both a specialty in food allergy and are academically trained in food allergy . The level of training in dietetics in general differs between countries and the uk is the only country which offers msc degrees in allergy (http://www.southampton.ac.uk/ and http://www.imperial.ac.uk/). As a result, the level of knowledge about food allergy not only differs strongly between individual dietitians but also strongly between countries . A survey conducted in the usa and uk shows a great need for food allergy knowledge amongst dietitians . Dietitians in the usa felt more knowledgeable about the definitions of food allergies (57% scored high level of knowledge in the usa versus 31% in the uk, p <0.001) and intolerances (59% scored high level of knowledge in the usa versus 30% in the uk, p <0.001). However, uk - based dietitians indicated more confidence in designing food challenge protocols (12% uk versus 8% usa scored high, p = 0.12) and 18% in the uk and 19% in the usa indicated to have no proficiency in developing challenge protocols . Very interestingly, dietitians from both countries indicated that their most immediate need was standardised patient handouts / diet sheets (89% usa and 70% uk). Having realised this problem some time ago, the international network for diet and nutrition in allergy (indana) (http://www.indana-allergynetwork.org/) was established . The aim of indana is to encourage, support, and disseminate best evidence - based clinical practice and to influence the development of the dietitian's and nutritionist's role in the field of food hypersensitivity (fhs) at international level . In this paper, the aims and activities of indana will be further discussed . The role of the dietitian differs between countries depending on the extent of the physician's role in the dietary management . In some countries the dietitians are trained to be involved in the diagnosis, while in other countries the dietitians are involved in the dietary management only . During 2010 and 2011, three official guidelines have been published on the diagnosis and management of food allergies by international organizations or by comprehensive working groups . These are the world allergy organisation guidelines on the diagnosis and management of cow's milk allergy (dracma guidelines), the usa guidelines on the diagnosis and management of food allergies in adults and children and the uk nice guidelines on the diagnosis of food allergies in children . However, the uk nice guidelines were the only guidelines that recognized the role of the dietitian in diagnosis and management of food allergies . The dracma guidelines mention the role of the dietitian (referred to as a nutritionist) in the management of cow's milk allergy . This paper sets out to present nutritional aspects in diagnosis and management of food allergies, comprising the aims and activities of the international network for diet and nutrition in allergy (indana); principles for taking an allergy - focused diet history in the diagnosis of food allergy; principles for the nutritional and dietary management of food allergy, including the impact of food allergy on an individuals diet and nutritional status, unnecessary avoidance diets and dietary restrictions, and psychosociological aspects of food allergy . The aims and activities of the international network for diet and nutrition in allergy (indana); principles for taking an allergy - focused diet history in the diagnosis of food allergy; principles for the nutritional and dietary management of food allergy, including the impact of food allergy on an individuals diet and nutritional status, unnecessary avoidance diets and dietary restrictions, and psychosociological aspects of food allergy . Acknowledging the need for education on food hypersensitivities for both dietitians and other professionals, the international network for diet and nutrition in allergy (indana) was established in 2009 by a group of academic dietitians and food scientists specializing in food allergies and intolerances . This nonprofit international network aims todevelop the role of the dietitian / nutritionist in the field of food allergy and to enhance the focus on diet and nutrition when dealing with food allergy;provide a platform for nutritional and dietary management advice for all those involved in dietary care of food allergy;educate health care professionals (hcps) involved in food allergy on nutrition and dietary management of food allergy; encourage international collaboration and research;encourage a membership that is representative of all countries and continents where food allergy is highly prevalent . Develop the role of the dietitian / nutritionist in the field of food allergy and to enhance the focus on diet and nutrition when dealing with food allergy; provide a platform for nutritional and dietary management advice for all those involved in dietary care of food allergy; educate health care professionals (hcps) involved in food allergy on nutrition and dietary management of food allergy; encourage international collaboration and research; encourage a membership that is representative of all countries and continents where food allergy is highly prevalent . Indana aims to bridge the gap in science between food hypersensitivity, immunology, nutrition, and food science to improve the prevention, nutritional diagnosis, and management of those living with food allergies and intolerances . The steering group consists of members with a first degree in nutrition & dietetics or nutrition, plus postgraduate training in allergy or a research degree at masters or doctorate level . The steering committee includes representation from the usa, europe, australia, and south africa . Membership is open to any hcp with a relevant first degree who is working in the field of food hypersensitivity (i.e. Dietitian, nutritionist, nurse, clinician, researcher, and industry). The importance of diet and nutrition in allergic disease and the role of the dietitian / nutritionist are now officially acknowledged by the european academy of allergy and clinical immunology (eaaci), by initiating an interest group on diet and nutrition in the allied health (ig on ah). This creates a platform within the eaaci where people, sharing an interest and expertise in diet and nutrition in allergy, can meet, teach, exchange expertise and ideas, and support the eaaci with regard to this topic . Also recently indana was officially incorporated in the american academy of allergy, asthma and immunology (aaaai). The primary goals of indana and the ig on ah are to educate hcps on food hypersensitivity, taking into account international variations in the role of the dietitian in the diagnosis and management of these conditions . The ig on ah is involved in setting up an eaaci - initiated practical training course on food allergy and a food allergy and anaphylaxis guideline . In addition, members of indana and the ig on ah are also presenting at international conferences of the eaaci, aaaai and the international congress of dietetics (icds). The first educational and research tool that is being developed is a standardized allergy - focused diet history . This tool is being developed under the funding of the eaaci by the interest group (ig) on allied health (ah). This tool could be adjusted for use in the usa pending support of national allergy organisations . This may be particularly useful for physicians who do not have access to dietitians . The next steps will be to develop educational materials for health care professionals around the globe and to initiate research projects . The aim of the allergy - focused diet history is to investigate if there is an association between food or diet and the symptoms of the patient . When patients have immediate food allergic reactions and reactions to single foods, it is often obvious to which food the patient has reacted . However, very often patients have chronic symptoms, reactions to compound foods, or sensitisation to foods of which the clinical relevance is not clear . This may be due to unknown allergens, may be caused by cross - sensitisation between pollens and foods, or due to a deficient or unbalanced diet . The outcome of the allergy - focused diet history may direct the physician to further diagnostic testing supporting the final diagnosis by the physician and may direct the dietary measures to be taken . A detailed allergy - focused diet history should consist of the following . According to the uk nice guidelines, the possibility of food allergy should be considered in children and young people who are presenting with one or more allergic signs and symptoms, in particular when there are persistent symptoms that involve different organ systems . These symptoms may be ige - mediated or non - ige - mediated or may not involve the immune system . If food allergy is suspected (by a hcp parent, carer, child, or young person), a hcp with the appropriate competencies should take an allergy - focused clinical history tailored to the presenting symptoms and age of the child, young person, or adult . This should include information on the following: any personal history of atopic disease (asthma, eczema, or allergic rhinitis); any individual and family history of atopic disease (such as asthma, eczema, or allergic rhinitis) or food allergy in parents or siblings; details of any foods that are avoided and the reasons why; an assessment of presenting symptoms and other symptoms that may be associated with food allergy, including questions about: the age of the child or young person when symptoms first started;speed of onset of symptoms following food contact;duration of symptoms;severity of reaction;frequency of occurrence;setting of reaction (for example, at school or home);reproducibility of symptoms on repeated exposure;what food and how much exposure to it causes a reaction;cultural and religious factors that affect the foods they eat;who has raised the concern and suspects the food allergy;what the suspected allergen is;the child or young person's feeding history, including, the age at which they were weaned and whether they were breastfed or formula - fed, if the child is currently being breastfed, considering the mother's diet;details of any previous treatment, including medication, for the presenting symptoms and the response to this;any response to the elimination and reintroduction of foods (nice). Any personal history of atopic disease (asthma, eczema, or allergic rhinitis); any individual and family history of atopic disease (such as asthma, eczema, or allergic rhinitis) or food allergy in parents or siblings; details of any foods that are avoided and the reasons why; an assessment of presenting symptoms and other symptoms that may be associated with food allergy, including questions about: the age of the child or young person when symptoms first started;speed of onset of symptoms following food contact;duration of symptoms;severity of reaction;frequency of occurrence;setting of reaction (for example, at school or home);reproducibility of symptoms on repeated exposure;what food and how much exposure to it causes a reaction;cultural and religious factors that affect the foods they eat;who has raised the concern and suspects the food allergy;what the suspected allergen is;the child or young person's feeding history, including, the age at which they were weaned and whether they were breastfed or formula - fed, if the child is currently being breastfed, considering the mother's diet;details of any previous treatment, including medication, for the presenting symptoms and the response to this;any response to the elimination and reintroduction of foods (nice). The age of the child or young person when symptoms first started; speed of onset of symptoms following food contact; duration of symptoms; severity of reaction; frequency of occurrence; setting of reaction (for example, at school or home); reproducibility of symptoms on repeated exposure; what food and how much exposure to it causes a reaction; cultural and religious factors that affect the foods they eat; who has raised the concern and suspects the food allergy; what the suspected allergen is; the child or young person's feeding history, including, the age at which they were weaned and whether they were breastfed or formula - fed, if the child is currently being breastfed, considering the mother's diet; details of any previous treatment, including medication, for the presenting symptoms and the response to this; any response to the elimination and reintroduction of foods (nice). Additional information on consumption of the major allergenic foods, food chemicals, and any possible cross - reactions do you regularly eat the following foods (peanuts, tree nuts, sesame seeds, celery, milk, egg, wheat, fish, shell fish, molluscs, soya, lupin, mustard, or sulphite containing foods) and do you experience any problems when eating them? Do you have hay fever or are you allergic to pollens?it is important that the person taking the allergy - focused diet history has knowledge of all aspects of food allergy . Oas or oral itch is a type of food allergy classified by a cluster of allergic reactions in the mouth in response to eating certain (usually fresh) fruits, nuts, and vegetables . However, within the spectrum of oas lies the pollen - fruit syndrome (pfs), characterised by much milder symptoms and needs much less stringent dietary restrictions . It is now widely reported that oas is becoming a widespread problem in europe, particularly in younger age groups . In the uk, the problem in the adult population is growing, but the number of children suffering from this problem is unclear . Information about oas is particularly important for successful management of fruit, vegetable, and nut allergies in order to prevent unnecessary restrictions in these people's diets . Pfs is not known to cause anaphylaxis but true ige - mediated allergies to fruit, vegetables, and nuts, which are on the other end of the oas spectrum can be potentially fatal, and it is important to distinguish between the two . Do you regularly eat the following foods (peanuts, tree nuts, sesame seeds, celery, milk, egg, wheat, fish, shell fish, molluscs, soya, lupin, mustard, or sulphite containing foods) and do you experience any problems when eating them? Do you have hay fever or are you allergic to pollens?it is important that the person taking the allergy - focused diet history has knowledge of all aspects of food allergy . Oas or oral itch is a type of food allergy classified by a cluster of allergic reactions in the mouth in response to eating certain (usually fresh) fruits, nuts, and vegetables . However, within the spectrum of oas lies the pollen - fruit syndrome (pfs), characterised by much milder symptoms and needs much less stringent dietary restrictions . It is now widely reported that oas is becoming a widespread problem in europe, particularly in younger age groups . In the uk, the problem in the adult population is growing, but the number of children suffering from this problem is unclear . Information about oas is particularly important for successful management of fruit, vegetable, and nut allergies in order to prevent unnecessary restrictions in these people's diets . Pfs is not known to cause anaphylaxis but true ige - mediated allergies to fruit, vegetables, and nuts, which are on the other end of the oas spectrum can be potentially fatal, and it is important to distinguish between the two . Do you regularly eat the following foods (peanuts, tree nuts, sesame seeds, celery, milk, egg, wheat, fish, shell fish, molluscs, soya, lupin, mustard, or sulphite containing foods) and do you experience any problems when eating them? It is important that the person taking the allergy - focused diet history has knowledge of all aspects of food allergy . Oas or oral itch is a type of food allergy classified by a cluster of allergic reactions in the mouth in response to eating certain (usually fresh) fruits, nuts, and vegetables . However, within the spectrum of oas lies the pollen - fruit syndrome (pfs), characterised by much milder symptoms and needs much less stringent dietary restrictions . It is now widely reported that oas is becoming a widespread problem in europe, particularly in younger age groups . In the uk, the problem in the adult population is growing, but the number of children suffering from this problem is unclear . Information about oas is particularly important for successful management of fruit, vegetable, and nut allergies in order to prevent unnecessary restrictions in these people's diets . Pfs is not known to cause anaphylaxis but true ige - mediated allergies to fruit, vegetables, and nuts, which are on the other end of the oas spectrum can be potentially fatal, and it is important to distinguish between the two . Factors to take into account during the decision making what is the clinical relevance of positive test results if tests have been done? Are there any extrinsic factors involved: medication, stress, exercise, alcohol, hormonal, infection, vitamin and mineral supplements and herbal medication . What is the clinical relevance of positive test results if tests have been done? Is there any cross - sensitisation between foods and aeroallergens? Are there any extrinsic factors involved: medication, stress, exercise, alcohol, hormonal, infection, vitamin and mineral supplements and herbal medication . What is the clinical relevance of positive test results if tests have been done? Is there any cross - sensitisation between foods and aeroallergens? Are there any extrinsic factors involved: medication, stress, exercise, alcohol, hormonal, infection, vitamin and mineral supplements and herbal medication . Chronic symptoms may also be caused by other nonallergenic factors or may be caused by an unbalanced or deficient diet with similar presentation to food intolerance . Ask general questions focusing on nutrients of importance, particularly if a dietitian is in post . This can be done by dietitians using 27 days food diaries, a 24 hours recall diet history, or a more general diet history [18, 19]. The allergy specialist dietitian could be the ideal hcp to deal with this part of the diet history, having knowledge on food allergy, nutrition, and foods . When to refer to a dietitian the more foods avoided the more important it is to refer to the dietitian; general growth monitoring and nutritional assessment, particularly children with growth issues either faltering growth or malnutrition; for help and advice on infant formula / milk substitutes and weaning; for advice on the general nutritional aspects of the diet and for psychological support; for information on life - style issues when living with food allergies such as, eating out, travelling, school trips / camps . The more foods avoided the more important it is to refer to the dietitian; general growth monitoring and nutritional assessment, particularly children with growth issues either faltering growth or malnutrition; for help and advice on infant formula / milk substitutes and weaning; for advice on the general nutritional aspects of the diet and for psychological support; for information on life - style issues when living with food allergies such as, eating out, travelling, school trips / camps . The more foods avoided the more important it is to refer to the dietitian; general growth monitoring and nutritional assessment, particularly children with growth issues either faltering growth or malnutrition; for help and advice on infant formula / milk substitutes and weaning; for advice on the general nutritional aspects of the diet and for psychological support; for information on life - style issues when living with food allergies such as, eating out, travelling, school trips / camps . Although this sounds very simple, dietary management encompasses more than advice on avoidance of the allergenic foods . Scientific data are scarce, but there is general agreement that the aims and principles of the dietary management of food allergy are multiple and include the following: obtaining relief of symptoms by avoidance of the allergenic foods; preventing inadvertent exposure to the allergenic foods; preventing the patient from unnecessary avoidance of foods; supporting normal growth and development for age and gender in children; providing an adequate, healthy, nutritionally dense, and balanced diet with appropriate alternatives for the excluded food allergens to minimize the impact on quality of life . Obtaining relief of symptoms by avoidance of the allergenic foods; preventing inadvertent exposure to the allergenic foods; preventing the patient from unnecessary avoidance of foods; supporting normal growth and development for age and gender in children; providing an adequate, healthy, nutritionally dense, and balanced diet with appropriate alternatives for the excluded food allergens to minimize the impact on quality of life . The dietary management of food allergies can be complex and difficult to follow in most cases and input from a dietitian is very important . For optimal dietary management it is very important to know which foods should be avoided in order to give appropriate avoidance advice . Without clear identification of the allergenic foods dietary management of food allergy becomes difficult . Additionally, a clear diagnosis enhances appropriate coping strategies and determines the level / degree of avoidance required . In general, stringent dietary advice may be needed for food - allergic patients, including the avoidance of the following: foods containing allergenic ingredients, even in small amounts; foods having a high risk for cross - contamination, such as chocolate in the case of peanut or cow's milk allergy / foods with precautionary labeling, although an individual risk assessment is recommended by some clinicians;unrefined oil derived from allergenic foods; meals or foods of uncertain composition, (for example, away from home). Foods containing allergenic ingredients, even in small amounts; foods having a high risk for cross - contamination, such as chocolate in the case of peanut or cow's milk allergy / foods with precautionary labeling, although an individual risk assessment is recommended by some clinicians; unrefined oil derived from allergenic foods; meals or foods of uncertain composition, (for example, away from home). Regulatory legislation is very helpful when allergenic foods are to be avoided as these allergenic foods must be declared on the label of prepacked foods . For the eu these are milk (including lactose), egg, soy, wheat or gluten, peanut, tree nuts, sesame, fish, crustaceans, molluscs, celery, lupin, mustard, and sulphites . However, dietary management becomes much more complex when other foods leading to severe reactions have to be avoided . The dietitian should educate the patient about this legislation, about reading labels, about high - risk foods, and high - risk settings such as eating out, to be stringent if medical care is not nearby and about early signs of severe reactions . Advice for school meals, day care, birthday parties, holidays, and other social circumstances should also be addressed . All of these points should be included and discussed in an adequate dietary management plan . Another aspect of food hypersensitivity is that many patients choose to avoid too many foods and therefore overrestrict their diets [3, 25]. Patients may avoid foods related to the allergenic food, for example, peanut allergic patients may avoid all other nuts and sesame, while this is not always indicated . Anxiety of unfamiliar foods and fear of inadvertent allergic reactions may also lead to unnecessary avoidance . Anecdotal evidence indicates that food allergic patients live with a permanent alertness as to what they are eating in numerous situations and settings . Successful avoidance of foods requires an understanding of label reading, meal preparation, and effective communication to friends and restaurant personnel providing food [20, 26]. The knowledge of the potential dangers of an accidental exposure may lead to a heightened level of anxiety and negatively impacts the quality of life, as well as affecting lifestyle issues and welfare . Previous research studies have shown that food allergic consumers experience difficulties when eating out and while shopping [30, 31]. The number of studies looking at health - related quality of life (hrqol) in those suffering from food allergies has increased over the years . These studies showed that hrqol in patients with food allergy and their families was significantly reduced [29, 3235]. They found that several areas of hrqol are affected, such as, family and social activities, emotional issues, and family economy, basically all aspects of family life . Food - hypersensitive children are to a large extent also limited in performing social activities without adult supervision . Patients should therefore be encouraged to expand their diet using foods which have been proven to be safe in a graded fashion . Specific ready - to - use introduction schedules for use at home for first exposure to foods have been developed and published and could also be used for reintroduction of foods . Several patient groups deserve specific attention in dietary management, such as, lactating women and young children who need weaning / introduction of solids advice and advice on the most appropriate choice of hypoallergenic formula, picky / faddy eaters, and vegetarians . It is recommended that lactating mothers with food allergic children should remove the offending food from their own diet for a period of time . These foods can be reintroduced once the infant / child improves in order to establish whether they (the mother) need to continue with the food avoidance [1, 12, 37]. Mothers avoiding cow's milk from their diet should be supplemented with calcium and vitamin d according to the national guidelines for each country . In most countries, vitamin d supplementation is suggested for all breastfeeding women, irrespective of avoiding cow's milk or not . Choosing the right formula for the patient based on the clinical presentation is a matter of huge debate with clear differences between countries . This choice should ultimately be based on clinical presentation, nutritional composition of the formula, residual allergenicity of the formula, and other components added to the formula . The dracma guidelines performed a comprehensive review of the literature and their different indications for formulas suggest the use of an aminoacid - based formula for anaphylaxis, heiner syndrome, and esinophilic eosophagitis, with the use of extensively hydrolysed formula for all other clinical presentations . Three additional papers, however, suggest the use of aminoacid - based formulas for growth faltering, severe atopic dermatitis, multiple food allergies, and infants not responding to maternal avoidance of cow's milk [37, 39, 40]. One should, however, always take national differences into account as some countries may use soya formula as a first - line approach for some clinical conditions in infants over six months . Weaning is a particular problem in infants suffering from food allergies, and the most difficult question is when and how to introduce other highly allergenic foods into the diets of infants with milk / egg allergies with parents understandably being cautious about introducing these foods . For both nutritional and developmental reasons, over restriction and delayed introduction of these foods are not recommended . Another clinical dilemma is whether to screen for other food allergies in children with one food allergy . There are difficulties with interpreting the results of these tests in young infants which may lead to over restriction of foods to which an infant is sensitised but not allergic . The national institute of allergy and infectious diseases (niaid (usa)) guidelines states that there is insufficient evidence to suggest whether, or which, foods should be tested prior to introduction in children at risk of food allergies (either from a high - risk family or with other existing food allergies). Testing prior to introduction could potentially prevent allergic reactions, but there is currently no practical consensus on which (if any) foods should be tested . Until further clear evidence becomes available, each clinician may have their own preference in dealing with the problem, but it makes sense to start with low - allergenic foods first, try the cooked version of a food first, increase the amount given, and expand the diet as soon as possible . Parents should be clear on how to deal with any unexpected reactions and realise that children with coexisting asthma / wheeze are more likely to have (perhaps) severe reactions . New allergens can emerge or patients can outgrow one or more of their food allergies . It should be obvious that in multiple and complex food allergies the management of food allergy is tailored to the individual patient and requires sophisticated skills and knowledge about food allergy and composition of foods from the dietitian . Further education activities should be undertaken to educate more dietitians in diet history taking and the management of food allergy . It is one of the aims of indana to enhance the food allergy skills of dietitians around the world . Dietary antigens induce a local hypersensitivity reaction impairing the intestine's barrier function, leading to continuation of inflammation . The consequences of the inflammatory responses may be severe and manifested as impaired growth, increased symptoms, and poor quality of life . The cornerstone of the management of documented food allergies is an elimination diet and when appropriately designed and accomplished it dampens the inflammation and ensures optimal growth and well - being of the child . Nutritional inadequacies in food allergic individuals may result particularly from the elimination of multiple foods or nutritionally key foods, such as, milk or cereals [7, 8]. Early onset of symptoms, manifested during the first few months of life, compared to late onset, 6 to 10 months of age, appears to result in more seriously affected disease and the delay in growth may be more pronounced . A brazilian study demonstrated the impact of food allergy on a child's nutritional status as the prevalence of poor growth was seen in as many as a quarter of children diagnosed with cow's milk allergy at the age of 24 months or less . A weight - for - age z - score below 2 was demonstrated in 15.1% and a height - for - age z - score below 2 in 23.9% of the children . Importantly, delay in diagnosis and thus delay in initiation of an appropriate dietary management may slow down growth . Conversely early diagnosis is associated with an appropriate growth for age, along with shorter duration of symptoms, fewer food allergies, and improved prognosis . There are a number of factors that could lead to poor growth in children with food allergy as summarised in table 1 . Nevertheless, severe cases of growth failure are rare, affecting only a minor proportion of children following an appropriate care plan and may relate to the severity of the disease, like a large skin surface area affected in atopic eczema . Lack of appropriate care or poor compliance may result in severe nutritional inadequacies, for example, where small children have been fed with rice beverages with an inappropriate nutritional composition for the needs of a small child . There is little evidence on the impact of food allergy on a child's body composition or bone health, but these may be potentially affected . The mechanisms for impaired growth may rise from a sustained inflammation and subsequent reduced bioavailability or loss of nutrients in the gastrointestinal tract, while metabolic requirements may be increased . Patients may consciously or unconsciously regulate symptoms of the disease by (unnecessary) elimination of foods . A limited diet may also be of psychosocial origin, particularly if food allergy is potentially life threatening as in some cases of peanut allergy . Patients may also develop food aversions and anxiety, resulting in inadequate dietary intake or replacement of allergenic foods by foods that are not nutritionally equivalent . Some parents seek help for their child's symptoms from alternative practitioners, and unfortunately they may prescribe very limited diets with subsequent impact on dietary variety and nutritional status . In this case, appropriate allergy testing and reintroduction of foods to the child's diet under dietetic supervision resulted in regain in weight and general improvement of health . Differences in food and nutrient intakes between children with and without food allergy have been reported . Dietary intake studies in children with food allergy compared to figures for healthy children or reference nutrient intakes have demonstrated lower intakes of energy and protein as well as that of zinc, iron, calcium, vitamin d, and vitamin e . Deviations in growth may reflect a lower dietary intake of energy and nutrients or relate to allergy itself through inflammation . Indeed, a recent study showed that despite a deviant growth between healthy children and those with food allergy, no difference in dietary intake was detected . This study again points to the need for nutritional evaluation of children with food allergies, preferably by allergy specialist dietitians . Poor growth does not seem to be a feature of allergy per se but rather a matter of inadequate dietary intake with reference to the requirements . Faltering growth may culminate in patients not being regularly monitored by hcps specializing in allergy, including a dietitian, as the reasons for growth failure are various . Prospective follow - up studies have demonstrated that with careful monitoring of growth and management of allergy in early childhood, the growth of children remains within population reference values and only a minor proportion evince poor growth . In addition, an early diagnosis of food allergy and subsequent initiation of dietary management enable catch up growth and normal adult height . Nevertheless, nutritional risks persist as a study reported that at the age of 7 to 15 years lower height and weight z - scores were detected in children who had avoided two or more foods, particularly milk, at the age of three years due to allergic symptoms . This is indeed demonstrated by a range of studies, including previously presented cases as well as the case study of a boy with multiple food allergies presented in the next chapter . An early diagnosis and appropriate care of food allergy are necessary to allow a good quality of life and nutritional status of the patient . An allergy - focused diet history assisted by the allergy - specialist dietitian may direct the physician to further diagnostic testing; supporting the final diagnosis dietetic expertise is important to conduct a dietary assessment to ensure appropriate intake of energy and essential nutrients and to provide patient - oriented counselling . This includes education on reading labels, safe eating at restaurants, risks of cross - contamination, and potential sources of hidden allergens but also informing about support groups and online resources . These patient - oriented tools facilitate the appropriate management and followup of patients with food allergies . Nonprofit organisations, such as the recently founded indana, focus on increasing awareness, education, and improvement of nutritional and dietary care in food allergy . Case 1 (a teenage girl with anaphylaxis to peanut)lisa was referred to the allergy specialist dietitian by the paediatrician because of episodes of abdominal pain and cramps with increasing severity and several food allergic reactions over the years . The family attributed these reactions to peanut, because she was diagnosed with peanut allergy since she was young . Lisa was referred to the allergy specialist dietitian by the paediatrician because of episodes of abdominal pain and cramps with increasing severity and several food allergic reactions over the years . The family attributed these reactions to peanut, because she was diagnosed with peanut allergy since she was young . Sensitization to inhalant allergens was negative, sensitization to foods was positive for the following foods: hazelnut 0.61 ku / l, peanut 76.2 ku / l, pistachio 7.14 ku / l, soy 2.72 ku / l . Sensitisation to coconut, almond, cashew nut, milk, egg, fish, and wheat was negative . There is a positive family history of atopy and asthma in her father's family . The clinical history revealed that she has suffered from abdominal pain since she was very young . She was carrying an epipen for her peanut allergy, however, was not confident about using it . Firstly, the diet history focused on the clinical relevance of the foods sensitized to and on excluding dietary errors on peanut ingestion . Foods with advisory labeling for peanuts and nuts were avoided . Over the years, several allergic reactions occurred after eating a food or meal, of which the family thought that peanut must have been the causative ingredient . Remarkably, most reactions, except the reaction to m&ms, were preceded by a form of exercise after eating (gym, playing outside, karate lessons) or stress (school party).when she was much younger she reacted to m&ms with vomiting.when she was 11 years old, she had an anaphylactic reaction following a home made indonesian meal without nuts or peanut, for which she used her epipen . The meal included tofu and soy sauce.last year, at a school party, she reacted with swollen lips, itchy ears, and dyspnoea after eating a chicken nugget.a few months ago she reacted with swollen lips, tachycardia, and presyncope after eating meat coated with bread crumbs.over the years there were several reactions of tachy- cardia, abdominal pain with cramps, and lip swelling having had commercially prepared meat products . On one occasion she had a hazelnut and a cookie with almonds without symptoms . When she was 11 years old, she had an anaphylactic reaction following a home made indonesian meal without nuts or peanut, for which she used her epipen . Last year, at a school party, she reacted with swollen lips, itchy ears, and dyspnoea after eating a chicken nugget . A few months ago she reacted with swollen lips, tachycardia, and presyncope after eating meat coated with bread crumbs . Over the years there were several reactions of tachy- cardia, abdominal pain with cramps, and lip swelling having had commercially prepared meat products . On one occasion she had a hazelnut and a cookie with almonds without symptoms . Secondly, the diet history focused on the nutritional composition of the food to rule out any over or under consumption of foods, because cramps and nausea may be related to fibre consumption . The dietitian also suspected the sensitisation to soy to be of clinical relevance and suspected exercise - induced anaphylaxis to soy . The paediatrician agreed with this, and the dietitian advised lisa and her family to avoid not only peanuts and nuts, but also soy protein . Soy protein was incorporated in the indonesian meal and could have been incorporated in the chicken nuggets, coated meat with bread crumbs, and commercially prepared meals . Results . No anaphylactic reactions occurred from that point forward, and the complaints of abdominal pain, cramps, and nausea disappeared . The dietitian advised to continue avoiding peanuts, nuts, and soy protein and to take particular care when exercising . The paediatrician / allergy nurse updated the information and the use of the adrenaline autoinjector . Teaching points an allergy specialist dietitian can sustain the diagnosis in taking an allergy - focused diet history . The diet history is the cornerstone of the diagnosis of food allergy and may direct the nature of the foods to be avoided.the dietary history may reveal if chronic symptoms may be caused by a deficient or unbalanced diet and if external factors may play a role, such as, exercise . An allergy specialist dietitian can sustain the diagnosis in taking an allergy - focused diet history . The diet history is the cornerstone of the diagnosis of food allergy and may direct the nature of the foods to be avoided . The dietary history may reveal if chronic symptoms may be caused by a deficient or unbalanced diet and if external factors may play a role, such as, exercise . Reason for referral to an allergy specialist dietitian . In many countries, access to the dietitian is limited, and allergy specialist dietitians are scarce . Referral is specifically important: when an allergy - focused diet history is required to examine if certain foods provoke symptoms or, in case of chronic symptoms, these complaints may be caused by a deficiency or imbalance in the diet;when nutritional adequacy of the diet is questionable and needs to be checked, for example, in case of avoidance diets in young infants and toddlers (specifically cow's milk free and wheat free diets), multiple food allergy, picky eaters, faltering growth, and when symptoms do not improve despite adequate medication;when counselling and nutritional management are required, for example, in patients having questions about the practical implication of the avoidance diet (replacements of foods, social activities, school meals, school camps, holidays), allergic reactions despite following an avoidance diet, anxiety to food, and overrestriction of foods . When an allergy - focused diet history is required to examine if certain foods provoke symptoms or, in case of chronic symptoms, these complaints may be caused by a deficiency or imbalance in the diet; when nutritional adequacy of the diet is questionable and needs to be checked, for example, in case of avoidance diets in young infants and toddlers (specifically cow's milk free and wheat free diets), multiple food allergy, picky eaters, faltering growth, and when symptoms do not improve despite adequate medication; when counselling and nutritional management are required, for example, in patients having questions about the practical implication of the avoidance diet (replacements of foods, social activities, school meals, school camps, holidays), allergic reactions despite following an avoidance diet, anxiety to food, and overrestriction of foods . Case 8 (11 months old boy with multiple food allergies) referral oscar was referred to the allergy specialist dietitian by the paediatrician because of acute urticaria and angioedema after ingestion of egg and growth faltering . Skin prick test results indicated; egg allergy (8 mm spt); peanut sensitisation (spt 4 mm); no other positive spt / ige for milk, wheat, fish, soy . Referral oscar was referred to the allergy specialist dietitian by the paediatrician because of acute urticaria and angioedema after ingestion of egg and growth faltering . Skin prick test results indicated; egg allergy (8 mm spt); peanut sensitisation (spt 4 mm); no other positive spt / ige for milk, wheat, fish, soy . Oscar was referred to the allergy specialist dietitian by the paediatrician because of acute urticaria and angioedema after ingestion of egg and growth faltering . Skin prick test results indicated; egg allergy (8 mm spt); peanut sensitisation (spt 4 mm); no other positive spt / ige for milk, wheat, fish, soy . The clinical history reveals that he has suffered from eczema from about 2 - 3 months allergy - focused diet history: enquire about breastfeeding, formula feeding and weaning onto solids, and if any reactions occurred . Oscar was breastfed until 6 months and received a top - up drink of cow's milk formula from 1 month of age . Solids were introduced at 5 months starting with baby rice, vegetables, and fruit . At six months gluten enquire about the introduction of the major allergenic foods milk given from 1 month and mother did not avoid milk during lactation with no noticeable reactions . Milk given from 1 month and mother did not avoid milk during lactation with no noticeable reactions . He has not had peanut, tree nuts, sesame seeds, shell fish, molluscs, mustard, celery (although she thinks it might have been in a stew she made), lupin (although difficult in the uk to know which foods contain these), soya (not sure but as she cooks everything at home it seems to be unlikely). General diet history . Mother offers 3 meals per day which are nutritionally balanced, but he has always been a difficult feeder . He has breakfast most days (baby rice with apple) but often refuses lunch (usually have 12 banana) and needs distraction most meal times but may eat about 90 g of dinner with pasta / rice / potato, meat, and mixed vegetables . He has 350 ml / day of formula, which he drinks in 5 - 6 bottles of 60 ml / bottle . There are some concerns about his intake of protein and energy (60% of required calculated intake), iron (75% of uk rni), calcium (51% of uk rni), and vitamin d (60% of uk rni). Dietary management initial dietary interventionthe initial dietary consultation included advice on an egg and nut - free diet . The peanut result clearly only indicated sensitisation at this stage rather than clinical allergy as he has not consumed peanut until now, but it was felt that he was too young for a peanut challenge.the formula was changed to an energy dense formula of 100 kcal/100 ml and 2.5 g protein/100 ml . A normal infant formula contains approx 6779 kcal / ml and 1.41.7 g of protein . Mother was also advised to further increase his protein, kcal, and calcium intake with cheese and to add double cream to sauces . Meal times should be kept to 30 min and a long consultation about dealing with faddy eating, including, practical tips follows . No force feeding was allowed and mother was asked to allow time for messy play . Inclusion of red meat (iron) and fatty fish (vitamin d) when possible was recommended, but these deficiencies should be corrected by taking sufficient formula, to initially aim to increase formula to 500 ml per day, which would not provide all the nutrients he needs but a step in the right direction . The initial dietary consultation included advice on an egg and nut - free diet . The peanut result clearly only indicated sensitisation at this stage rather than clinical allergy as he has not consumed peanut until now, but it was felt that he was too young for a peanut challenge . The formula was changed to an energy dense formula of 100 kcal/100 ml and 2.5 g protein/100 ml . A normal infant formula contains approx 6779 kcal / ml and 1.41.7 g of protein . Mother was also advised to further increase his protein, kcal, and calcium intake with cheese and to add double cream to sauces . Meal times should be kept to 30 min and a long consultation about dealing with faddy eating, including, practical tips follows . No force feeding was allowed and mother was asked to allow time for messy play . Inclusion of red meat (iron) and fatty fish (vitamin d) when possible was recommended, but these deficiencies should be corrected by taking sufficient formula, to initially aim to increase formula to 500 ml per day, which would not provide all the nutrients he needs but a step in the right direction . After dietary interventionafter the dietary intervention, his volume of feed was reduced and he was only managing 30 mls per bottle that is about 180 ml per day . Difficult feeding behaviour turned into extreme food refusal, and he was crying during and after feeds showing clear signs of discomfort . After the dietary intervention, his volume of feed was reduced and he was only managing 30 mls per bottle that is about 180 ml per day . Difficult feeding behaviour turned into extreme food refusal, and he was crying during and after feeds showing clear signs of discomfort . His eczema also seemed to flare after every meal . Results: followup and current interventionhe started on a cows' milk and soya - free diet and changed formula to amino - acid - based formula . The decision about soya avoidance was based on the concomitant soya allergy seen in children with gut symptoms and a non - ige - mediated (in this case) milk allergy . His eczema cleared completely, feeding standard improved, and he started to gain weight again . The patient is currently on egg- and nut - free diet with plans to review the nut - free diet and possible challenge after 12 months . Both the suspected milk and soya allergies also need to be confirmed by challenge, but there are currently no standardized procedures for performing food challenges in children with non - ige - mediated food allergies . Teaching points an allergy specialist dietitian can support the diagnostic process and may identify other nutrition - related issues.the diet history forms an important part of the diagnosis of food allergy and may direct the nature of the foods to be avoided particularly in the case of infants foods involved in delayed symptoms . The diet history may reveal any deficiencies which may relate to growth and development problems in infants . He started on a cows' milk and soya - free diet and changed formula to amino - acid - based formula . The decision about soya avoidance was based on the concomitant soya allergy seen in children with gut symptoms and a non - ige - mediated (in this case) milk allergy . His eczema cleared completely, feeding standard improved, and he started to gain weight again . The patient is currently on egg- and nut - free diet with plans to review the nut - free diet and possible challenge after 12 months . Both the suspected milk and soya allergies also need to be confirmed by challenge, but there are currently no standardized procedures for performing food challenges in children with non - ige - mediated food allergies . Teaching points an allergy specialist dietitian can support the diagnostic process and may identify other nutrition - related issues.the diet history forms an important part of the diagnosis of food allergy and may direct the nature of the foods to be avoided particularly in the case of infants foods involved in delayed symptoms . The diet history may reveal any deficiencies which may relate to growth and development problems in infants . An allergy specialist dietitian can support the diagnostic process and may identify other nutrition - related issues . The diet history forms an important part of the diagnosis of food allergy and may direct the nature of the foods to be avoided particularly in the case of infants foods involved in delayed symptoms . The diet history may reveal any deficiencies which may relate to growth and development problems in infants . Reasons for referral to dietitianthe dietitian can assist in and plays a crucial role in: taking an allergy - focused diet history;identifying any additional feeding problems either behavioural or nutritional;assessing nutritional intake and nutritional status;dietary management of any issues surrounding growth;practical advice on food avoidance and suitable replacements which can include: foods to avoid;label reading;suitable substitute foods for example, egg replacers / suitable infant formulas;recipe adaptation and suitable cook books;internet resources;support groups;assistance with design of food challenges;with young children having feeding difficulties when being weaned;in pregnant and lactating women following an avoidance diet for more than a few weeks, when losing weight involuntarily or when they consider stopping breastfeeding due to lack of dietary counselling and/or decrease in breast milk production . The dietitian can assist in and plays a crucial role in: taking an allergy - focused diet history;identifying any additional feeding problems either behavioural or nutritional;assessing nutritional intake and nutritional status;dietary management of any issues surrounding growth;practical advice on food avoidance and suitable replacements which can include: foods to avoid;label reading;suitable substitute foods for example, egg replacers / suitable infant formulas;recipe adaptation and suitable cook books;internet resources;support groups;assistance with design of food challenges;with young children having feeding difficulties when being weaned;in pregnant and lactating women following an avoidance diet for more than a few weeks, when losing weight involuntarily or when they consider stopping breastfeeding due to lack of dietary counselling and/or decrease in breast milk production . Taking an allergy - focused diet history; identifying any additional feeding problems either behavioural or nutritional; assessing nutritional intake and nutritional status; dietary management of any issues surrounding growth; practical advice on food avoidance and suitable replacements which can include: foods to avoid;label reading;suitable substitute foods for example, egg replacers / suitable infant formulas;recipe adaptation and suitable cook books;internet resources;support groups; suitable substitute foods for example, egg replacers / suitable infant formulas; recipe adaptation and suitable cook books; assistance with design of food challenges; with young children having feeding difficulties when being weaned; in pregnant and lactating women following an avoidance diet for more than a few weeks, when losing weight involuntarily or when they consider stopping breastfeeding due to lack of dietary counselling and/or decrease in breast milk production . Case 8 (a boy with severe eczema associated with multiple food allergies) referral jack presented at the allergy centre at age 3 months with severe eczema covering a large area of his body and face . A detailed history revealed that he had been born at full term, with a normal delivery . Following consultation with a paediatrician and a dietitian, jack's mother was given information on skin care for eczema, in line with nice guidelines . Although she did not want him to have skin prick tests (spt) at this stage, she was advised on avoidance of foods that commonly cause allergic reactions in breastfed infants, and she agreed to begin a diet excluding cow's milk and egg . In addition, advice on low - allergen weaning was provided, as jack's mother wanted to start introducing solid foods in the coming months . When jack returned to the clinic at age 6 months, his mother was successfully avoiding dairy and egg . She had read advice that rice milk should be avoided for babies and toddlers, and she was drinking soya milk instead, consuming more than 500 ml each day . Jack's eczema was improving but not completely cleared, and his weight had dropped below the 25th centile . Jack was otherwise well, with a normal physical examination and full blood count . Jack presented at the allergy centre at age 3 months with severe eczema covering a large area of his body and face . A detailed history revealed that he had been born at full term, with a normal delivery . Following consultation with a paediatrician and a dietitian, jack's mother was given information on skin care for eczema, in line with nice guidelines . Although she did not want him to have skin prick tests (spt) at this stage, she was advised on avoidance of foods that commonly cause allergic reactions in breastfed infants, and she agreed to begin a diet excluding cow's milk and egg . In addition, advice on low - allergen weaning was provided, as jack's mother wanted to start introducing solid foods in the coming months . When jack returned to the clinic at age 6 months, his mother was successfully avoiding dairy and egg . She had read advice that rice milk should be avoided for babies and toddlers, and she was drinking soya milk instead, consuming more than 500 ml each day . Jack's eczema was improving but not completely cleared, and his weight had dropped below the 25th centile . Jack was otherwise well, with a normal physical examination and full blood count . Jack had begun weaning at age 17 weeks, with baby rice, fruit, and vegetables . He had accidentally been given fromage frais (contains milk) by a relative and had experienced a severe immediate reaction, involving swelling of his lips and tongue, red facial flush, hives on his chest, and wheezing . Indeed, spts at age 6 months confirmed that jack was sensitized to a number of foods, including milk, egg, and soya . Given jack's ongoing symptoms and his positive spt to soya, the paediatrician advised his mother to begin excluding soya from her diet, while continuing to avoid egg and dairy . Jack's mother was feeling worn out by the constant effort of checking food labels and managing jack's eczema and irritability . Consequently, she wanted to introduce a formula feed at bedtime, and an amino - acid - based formula was prescribed . Written information was provided explaining that it could be expected to taste and smell different from other formulas, and also that the change in diet may alter stool colour and consistency . If required, to aid introduction, we advised that it could be mixed with breast milk, pointing out that mixed feeds must be used immediately to avoid possible digestion by enzymes in the breast milk . Additionally, all foods, excluding dairy, egg, soya, and peanut, could be introduced into jack's diet, one at a time . Jack's mother initially introduced amino - acid - based formula in a 30: 70 mix with breast milk . The proportion of formula was gradually increased and within a few days, jack was taking full formula feeds . At age 9 months, jack's weight had increased to just below the 50th centile and he had a varied diet of solid foods, with breast milk and night - time feeds of amino - acid - based formula . Jack's mother was also adhering well to the exclusion diet and was taking calcium and vitamin d supplements . At age two days prior to the appointment, he had a reaction after eating hummus, despite the fact that his mother was eating hummus regularly and he was still receiving breast milk . The symptoms included swollen lips and a red facial flush, though there was no wheezing this time . Spts at age 12 months confirmed that he was sensitized to sesame, among other foods . We advised that jack and his mother should exclude sesame from their diets, while continuing to avoid dairy, egg, and soya . Jack's progress will be monitored at 6 to 12 monthly intervals, as appropriate . At each follow - up visit, spts and records of accidental ingestion deciding when to challenge can be difficult and should take into account both the clinical factors discussed here and the family's readiness, as the process can cause anxiety and emotional distress . A negative oral challenge for a given food will then enable recommendation of its introduction to jack's diet . Clinical reactivity to milk and sesame has been confirmed by the reactions during oral exposure . The diagnosis of egg and soya allergy at this stage is based on sensitisation and improvement of symptoms after avoidance but will need to be confirmed by oral food challenges under medical supervision.
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Oculocutaneous albinism is a group of autosomal recessive disorders featuring hypopigmentation of the hair, skin and eyes . Ocular signs associated with the disease are nystagmus, decreased visual acuity, hypopigmentation of the retina, foveal hypoplasia, translucency of the iris, macular transparency, photophobia and abnormal decussation of nerve fibers at the chiasm . An 8-year - old caucasian girl presented to our clinic referral center for hereditary retinopathies of the second university of naples with a diagnosis of stargardt disease and a progressive reduction in visual acuity in both eyes . She underwent a complete ophthalmic examination including standard electroretinography and optical coherence tomography (oct). Best - corrected visual acuity was 20/30 in the right eye and 20/40 in the left eye . Biomicroscopy of the anterior segment revealed a transparent cornea, in situ and transparent lens and normally pigmented iris . The typical oct pattern led us to direct the molecular analysis towards the genes involved in oculocutaneous albinism . In this case, the role of oct was crucial in guiding the molecular analysis for the diagnosis of albinism . Oct is therefore instrumental in similar cases that do not present typical characteristics of a disease . The case also proves the relevance of molecular analysis to confirm clinical diagnoses in hereditary retinal diseases . Oculocutaneous albinism (oca) is a group of autosomal recessive disorders featuring hypopigmentation of the hair, skin and eyes ., four types of oca (oca14), attributed to mutations in tyr, oca2, tyrp1 and matp, respectively, have been identified . Ocular signs associated with the disease are nystagmus, decreased visual acuity, hypopigmentation of the retina, foveal hypoplasia, translucency of the iris, macular transparency, photophobia and abnormal decussation of nerve fibers at the chiasm . This article describes an atypical case of oca in which the use of optical coherence tomography (oct) was crucial for establishing the diagnosis . An 8-year - old caucasian girl presented to our clinic referral center for hereditary retinopathies of the second university of naples . The patient complained of a progressive reduction in visual acuity in both eyes, leading to the diagnosis of stargardt disease . Her past medical history was significant for exotropia and visual acuity impairment . On examination, visual acuity was 20/30 in the right eye and 20/40 in the left eye . Biomicroscopy of the anterior segment revealed a transparent cornea, in situ and clear lens and normally pigmented iris in both eyes . A slightly diffuse depigmentation in the midperiphery and a pinkish optic disk with clear margins and mild macular dystrophy at the posterior pole were observed at fundus examination . Oct revealed the absence of normal foveal depression (foveal thickness was 295 m in the right eye and 299 m in the left eye; normal range 168239 m) and of the small elevation of the inner segment / outer segment junction in the fovea . Furthermore, the high reflectivity across the fovea suggested a persisting nerve fiber layer and multiple inner retinal layers across the fovea (fig . 1). Hence, all oct findings were in contrast with the diagnosis of juvenile macular dystrophy [2, 3]. The clinical pattern of foveal hypoplasia revealed by oct was instead similar to that observed in albinism [4, 5], even if the patient featured normal pigmentation of the skin, hair and iris (fig . 2) and did not show typical ocular albinism signs such as nystagmus and iris transillumination . A molecular analysis was then performed on the genes involved in albinism and revealed the presence of r402q and 1177delg mutations in the tyr gene . Written consent was obtained from the patient for the publication of this case report and the accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal . Oct has been shown to provide detailed morphometric information on retinal structure and to be an important tool for the diagnosis and follow - up of diseases such as age - related macular degeneration, choroidal neovascularization and macular edema [6, 7, 8]. In recent years, the use of this technique has been applied to other types of retinal diseases aiding in the differential diagnosis of hereditary retinal dystrophies . Today, oct has become an important examination method for the diagnosis and follow - up of retinitis pigmentosa and its complications and can be considered a marker in cases of stargardt disease [2, 3 and albinism [4, 5]. In this case report oct can therefore be considered an important diagnostic tool in cases that do not present typical ocular and cutaneous characteristics of a disease . In fact, oct showed a thicker fovea in our patient compared to the general population, which may be due to the absence of a foveal pit as part of foveal hypoplasia associated with oca . Moreover, the oct images revealed the absence of the small elevation of the inner segment / outer segment junction in the fovea and the persistence of a nerve fiber layer and multiple inner retinal layers across the fovea; these two features may be explained by the foveal hypoplasia as previously described in the studies by craft et al . And chong et al . Relating these features to foveal hypoplasia in oca patients . Our case also proves the relevance of molecular analysis to confirm clinical diagnoses in hereditary retinal diseases . Furthermore, the phenotype described in this report suggests that patients affected by oca associated with r402q and 1177delg tyr gene mutations may present less severe clinical manifestations including good visual acuity, absence of nystagmus and an evident iris, skin and hair pigmentation . In conclusion, this case confirms the relevance of oct in the diagnosis of hereditary retinal dystrophies, providing characteristic patterns that can be considered reliable disease markers.
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Malaria is a vector borne disease, caused by the plasmodium parasite . According to who report, there were estimated 216 million episodes of malaria in 2010, of which approximately 81%, or 174 million cases, were in the african region . There were estimated 655,000 malaria deaths in 2010, of which 91% were in africa . In addition to acute disease episodes and deaths in africa, malaria also contributes significantly to anaemia in children and pregnant women, adverse birth outcomes such as spontaneous abortion, stillbirth, premature delivery, and low birth weight, and overall child mortality . Included in the who report was the fact that resistance to artemisinin, a vital component of drugs used in the treatment of p. falciparum malaria, has been reported in a growing number of countries in southeast asia . Resistance to pyrethroids, the insecticides used in itns and most commonly used in irs, has been reported in 27 countries in africa and 41 countries worldwide . The search for new antimalarial drugs requires identification of new biochemical targets for drug development and development of new chemical entities [2, 3]. Epidemiological studies have provided convincing evidence that natural dietary compounds, which humans consume as food, possess many biological activities . One plant food that has been shown to be therapeutic against a number of diseases is pigeon pea, cajanus cajan l. (fabaceae), an important grain legume crop in the tropics and subtropics . The extracts of pigeon pea are commonly used to treat diabetes, fever, dysentery, hepatitis, and measles worldwide [5, 6]. Cajanus cajan has been used traditionally as a laxative and was identified as an antimalarial remedy . In continuation of our study of the nigerian ethnomedicine for the discovery of new antimalarial drugs [7, 8], the present report is on the bioassay - guided fractionation and isolation of antiplasmodial compounds from cajanus cajan leaf extract . Cajanus cajan leaves were collected from otu, oyo state of nigeria, in the month of january and authenticated at the herbarium of botany department, university of ibadan (ui), and that of the forestry research institute of nigeria (frin), ibadan, where a voucher specimen was deposited as fhi 106560 . Leaves of c. cajan were air dried at rt (2631c) and pulverized with a hammer mill . 500 g of plant material was extracted in redistilled methanol (2.0 l) by maceration at rt (30c) for 72 h. after determination of yield of crude methanol extract, the sample was stored in the fridge (4c) till needed for analysis . 2.0 g of dry weight of crude methanol extract was fractionated by suspension in meoh: h2o in a ratio of 70: 30 to yield 0.35 g of hexane, 0.46 g of dichloromethane (dcm), 0.41 g of etoac, and 0.73 g of aqueous methanol fractions, respectively . The hexane and dcm fractions were combined based on the analysis and chromatographed on flash column using silica gel (merck). It was eluted with increasing polarity of hexane - dcm, and 50 ml portions were collected, respectively . The fractions that eluted with hexane: dcm (50: 50 to 20: 80; 130 mg) indicated the presence of predominantly 3 compounds on tlc analysis . This was subjected to ptlc using chcl3: etoac (17: 3) (merck, 20 20 cm, 12 plates) to give compounds 1, 2, and 3 with rf 0.45, 0.55, and 0.80, respectively . The compounds were subjected to structural analysis using nmr and ms . The asexual stages of plasmodium falciparum (multidrug resistant strain k1) obtained from dr . Warhurst, london school of hygiene and tropical medicine, were cultured continuously according to the modified candle jar method . The method of makler and hinrichs was used in the estimation of parasite growth inhibition . Cultures were cryopreserved to contain at least 5% ring - form parasites and were maintained at 24% hematocrit; this was used in preparing 2% hematocrit and washing with phosphate buffered solution (pbs) 3 times . Stock solutions of extracts were prepared by dissolving known quantities of dried extracts (500 g) in 1: 1 dimethyl sulphoxide (250 l) and distilled water (250 l). Serial dilutions (10 dilutions, 0.5500 g / ml) of the extracts / fractions were made in quadruplicates in 96-well microtitre plates . The drug plate was placed in the chamber with a little sterile water in a petri dish . This was placed in the laminar flow chamber (envair, uk) gassed with prefiltered mixture of 3% o2, 4% co2, and 93% n2, and then swiftly sealed and incubated at 37c for 48 hours . After incubation, acetylpyridine adenine dinucleotide (apad) regent was added to each well, followed by n - bromosuccinimide (nbs) and then incubated at 37c for 20 min . Optical density was measured in a plate reader at 550 nm and analysed with a wallac counter using an ms excel program . Ic50 values were estimated by plotting the% inhibition against the log drug concentration at 95% confidence limits using the linear and nonlinear regression analyses . The crude methanol extract (dry weight yield of 8.6 g) had an ic50 of 53.5 g / ml, the hexane fraction had ic50 of 62.5, and both dcm and aqueous meoh had ic50 of 31.3 g / ml, while ethyl acetate fraction had ic50 of 15.6 g / ml compared to chloroquine dipo4 with ic50 0.21 g / ml (0.66 m). Yields of compounds 13 were 5.0 mg (3.8%), 7.0 mg (5.3%), and 11.3 mg (8.7%) with ic50 values of 2.0 g / ml (7.40 m), 5.4 g / ml, and 5.6 g / ml, respectively (see table 1 for details). Compound 1 obtained from chromatographic analysis of the ethyl acetate fraction had an ic50 of 2.0 g / ml . The ei - ms of compound 1 had the [m+] at m / z 270, and c-13 nmr broad band indicated the presence of 16 carbon atoms and in agreement with c16h14o4 . Comparison of the spectroscopic data with those obtained from the literature identified the compound as 2,6-dihydroxy-4-methoxy chalcone (cajachalcone) (figure 1). Compound 2 with an ic50> 5 g / ml also obtained from the ethyl acetate fraction, its ei - ms had a molar mass of 294, the c-13 nmr, indicated 19c atoms and a formula of c19h17o3, suggestive of a phenanthrone furandione derivative . The data available were not sufficient to confirm the structure of compounds 2 and 3 . Cajanus cajan l., fabaceae, has been used locally as part of ethnotherapy for malaria infection in south western nigeria; its utilization as an antimalarial agent cuts across the whole of sub - saharan africa as well as other tropical countries as reported by some authors [7, 11]. From the result of this study, the crude methanol extract of this plant had an ic50 of 53.5 g / ml; subsequently, bioassay - guided fractionation and chromatographic separations led to the isolation of the compound responsible for the displayed antimalarial activity 2,6-dihydroxy-4-methoxy chalcone (cajachalcone); the compound displayed significant antimalarial activity ic50 of 2.0 g / ml (7.4 m). Chloroquine diphosphate (10 g / ml) was used as control and had ic50 value of 0.21 g / ml (0.66 m). Its structure was confirmed by comparison with h - nmr data reported for licochalcone a and 2,4-dimethoxy-4-butoxychalcone as shown in table 2 [12, 13]. Naturally occurring chalcones (1,3-diaryl-2-propen-1-one) are the key intermediates for various plant metabolites . They are biologically active compounds with known antibacterial [14, 15], antifilarial, antiviral [17, 18], antileishmanial, and cytotoxic [20, 21] activities . Licochalcone a, an oxygenated chalcone (figure 1) first isolated from roots of chinese licorice, showed antimalarial activity in both in vitro and in vivo systems . Since then, investigators have been searching for new more - potent lead molecules based on chalcone scaffolds as potential antimalarial agents [23, 24]. The simple structure and unambiguous synthesis of chalcones have attracted the attention of chemists to develop different analogs of this novel scaffold for various infectious diseases including malaria . A series of alkoxylated, hydroxylated, prenylated, oxygenated, quinolylated chalcones from natural sources and syntheses have been evaluated for antiplasmodial activity with encouraging results [25, 26]. Synthesized 4-methoxy; 2,4-dimethoxy; 2,5-dimethoxy; 3,4-dimethoxy and 3,4,5-trimethoxy benzaldehyde series of chalcone derivatives . In the 4-methoxy series, with ic50 of 1.6 g / ml, the antimalarial activity compared favourably with licochalcone a (ic50 of 1.43 g / ml) against chloroquine - sensitive 3d7 strain . In 2,4-dimethoxy series, ic50 values of between 1.1 and 7.68 the antimalarial activity of 2,4-dimethoxy chalcone ic50 2.1 g / ml (a naturally occurring 4-methoxy derivative) in our study was also compared favourably with the result of synthesized 4-methoxy series (ic50 1.6 g / ml). Meanwhile, yadav and coworkers concluded that the presence of methoxy groups at positions 2 and 4 in chalcone derivatives (figure 1) appeared to be favorable for antimalarial activity as compared to other methoxy - substituted chalcones; thus, we can infer that the isolated chalcone could be a template for the synthesis of 2,4-dimethoxy substituted derivatives, with methoxy substitution at position c-4 . It is believed that chalcone derivatives that possess antimalarial activity interact with parasite p. falciparum enzyme cysteine protease, one of the key enzymes involved in hemoglobin degradation within the acidic food vacuole of the intraerythrocytic parasite . Inhibition of this enzyme hampers digestion of hemoglobin within the food vacuole and proves fatal for the parasite . The world health organization 2011 has advised that the development of new tools is a necessary priority, particularly for vector control, diagnostic testing, treatment, and surveillance . It is our belief that 2,4-dimethoxy chalcone isolated from cajanus cajan l could be a lead for antimalarial drug development . Chalcones and derivatives are small bioactive molecules that have been synthesized and so have a high potential as leads for discovery and development of antimalarial agents.
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Prion diseases are a group of fatal neurodegenerative disorders that are sporadic, inherited, or transmissible . These include kuru and creutzfeldt - jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle . These pathologies are caused by the conformational transition of the native and predominantly -helical cellular prion protein (prp) into a significantly more -sheet - containing pathogenic isoform (prp), which unlike prp, is insoluble in mild detergents and partially resistant to digestion with proteinase k . Prp is a cell surface glycosylphosphatidylinositol - anchored protein that is mainly expressed in neurons and glial cells and to a lesser extent in several peripheral tissues [4, 5]. The normal physiological function of prp remains elusive, although it has been related to signaling, neuroprotection, neuritogenesis, synaptic transmission, oxidative stress, and copper metabolism [611]. Prp binds copper ions with low micromolar affinity via histidine and glycine - containing peptide repeats in its n - terminal region [1217]. This cu binding domain is located between residues 6091 and consists of four identical repeats of the peptide sequence pro - his - gly - gly - gly - trp - gly - gln . Although the number of octapeptide repeats varies in different species, in mammals this region is one of the most highly conserved and therefore, very likely defines a functional domain of prp . In vitro, the octarepeat region has the capacity to reduce cu(ii) to cu(i) [19, 20]. In addition, there is another cu binding site outside the octarepeat region [2124] of higher affinity, in the order of nanomolar, that involves his96 and his111 . Prp is localized presynaptically at central synapses [2527] and is found in synaptic membranes and in synaptic vesicles [9, 28]. Furthermore, prp - null mice show an impaired long - term potentiation, suggesting that prp is involved in normal synaptic function, and moreover, it has been shown that prp is involved in regulating the presynaptic cu concentration and synaptic transmission . The p2x family of nucleotide receptors forms non - selective cationic channels activated by extracellular adenosine triphosphate (atp). These receptors are widely expressed in the central nervous system (cns) [3032] and are involved in synaptic transmission and plasticity including long - term potentiation as recently shown by us . Interestingly, trace metals modulate p2x receptors, particularly, the p2x4 receptor subtype is differentially modulated by trace metals at physiological concentrations [3437]. While zn facilitates the atp - evoked currents, cu inhibits it in a concentration - dependent manner . Previously, we demonstrated that the n - terminal octarepeat fragment of the prp prevents and reverses the inhibitory action of cu on the p2x4 receptor when added to the media . Herein, in an attempt to determine whether the prp - cu interaction is relevant to synaptic activity, we extended our investigations to test whether the full - length prpco - expressed with the p2x4 receptor may modulate in situ the cu - induced inhibition of the atp current gated by the p2x4 receptor . Copper chloride, atp (as the tetrasodium salt), collagenase ia, and penicillin - streptomycin were purchased from sigma chemical co (st louis, mo). All the salts used to prepare the barth's incubation media and the recording solutions were analytically graded and were purchased from merck (darmstadt, germany). A segment of the xenopus laevis ovary lobe was surgically removed from adult anesthetized frogs; stages v - vi oocytes were manually defolliculated and then incubated with collagenase ia (1 mg / ml) for 30 min . Oocytes were manually injected with 7.512.5 ng cdna coding for the rat p2x4 receptor with or without cdna coding for the hamster prion protein (prp-3f4), both cdnas in plasmid pcdna3, at 250 ng/l . After 4872 h of incubation at 15c in barth's solution (in mm): 88 nacl, 1 kcl, 2.4 nahco3, 10 hepes, 0.82 mgso4, 0.33 ca(no3)2, ph 7.5, supplemented with 10 iu / l penicillin/10 mg streptomycin, oocytes were clamped at 70 mv using the two - electrode voltage clamp technique with an oc-725c oocyte clamper (warner instrument corp, hamden, ct). Atp and cucl2, dissolved in barth's solution, were superfused at 2 ml / min . Atp - evoked currents were recorded with a 10 s atp exposure applied regularly at 1015 min intervals . These intervals were increased up to 25 min for maximal atp concentrations in concentration - response curves protocols to decrease desensitization . Copper was applied for 30 s prior 10 m atp (coapplied with cucl2). To study the distribution of prp, oocytes were coinjected with the cdna coding for the rat p2x4 receptor with the cdna coding for mouse prp - gfp (mmprp - egfp[25 - 266]-cdna3). Oocytes, where p2x4 receptor expression was validated electrophysiologically, were directly analyzed on a zeiss lsm 5 pascal confocal microscope . After electrophysiological protocols, each oocyte injected with cdna coding for p2x4 and prp-3f4 was homogenized for 30 min in ice, using 40 l of lysis buffer per oocyte (100 mm nacl, 20 mm tris - hcl ph 7.4, 1% triton x-100) supplemented with a protease inhibitors cocktail . The extracts were centrifuged for 30 s at 14000 r.p.m . At 4c and the supernatant was removed and resolved by 12% sds - page and transferred to nitrocellulose . Nonspecific binding sites were blocked with 5% (w / v) milk in tris - buffered saline (tbs) 0.1% tween (tbst) for 1 h. after blocking, blots were incubated with monoclonal anti-3f4 antibody, diluted 1: 5000 in 3% (w / v) milk in tbst for 1 h at room temperature, followed by three 15 min washes in tbst at room temperature . The reactions were followed by incubation with anti - mouse antibody peroxidase labeled (pierce, rockford, il) and developed by enhanced chemiluminescence . The atp and cu concentration - response curves were fitted to a sigmoid function using the graphpad prism software (san diego, cal). The median effective (ec50) or median inhibitory concentrations (ic50) for atp or copper, respectively, were interpolated from these curves . Each protocol was performed in separate oocytes coming from at least two separate batches of oocytes . Mann - whitney nonparametric student's t - test was used for statistical analysis . A p value <0.05 was considered significant . To evaluate whether the expression of prp modulates the inhibition of the p2x4 receptor by cu, we first evaluated the expression of prp in oocytes co - injected with the cdna coding for the hamster prion protein (prp-3f4) and the cdna coding for the rat p2x4 receptor . Figure 1(a) shows the detection by western blot of p2x4 receptor and prp-3f4 using an antibody that recognizes the 3f4 epitope . Both proteins are strongly detected in an injected oocyte and not in the control noninjected oocyte . Then we analyzed the distribution of prp in oocytes co - injected with the cdna coding for the rat p2x4 receptor and the cdna coding for prp - gfp . Oocytes in which the expression of p2x4 receptor was verified electrophysiologically were analyzed in a confocal microscope to study the localization of prp - gfp . As observed in figure 1(b), prp - gfp is located on the surface of injected oocytes . Then, we evaluated the atp concentration - response curves in oocytes expressing the p2x4 receptor and coexpressing the p2x4 receptor and prp-3f4 . The presence of prp-3f4 caused a slight, but not significant, reduction in the potency of atp, reflected as an increase in its ec50 from 11.2 1.1 m for p2x4 alone to 45.2 9.4 m for p2x4/prp-3f4 (n = 4, p = 0.0571, figure 2), this slight displacement of atp concentration - response curve in the presence of prp-3f4 could represent a minor regulation of prp-3f4 on p2x4 receptor activity . We assess the cu - induced inhibition of 10 m atp currents in oocytes expressing p2x4 receptors . The magnitude of the inhibition by 10 m cu, preapplied during 30 s, was 51.5 5.3% of the 10 m atp - evoked currents however, the 10 m cu - induced inhibition was reduced only to 71.9 5% of the 10 m atp - evoked currents in oocytes co - expressing p2x4 receptors and the prp-3f4 (n = 12, p <0.05 compared to p2x4 alone, figures 3(a) and 3(b)), showing that prp-3f4 prevented the cu - induced inhibition of p2x4 receptors compared to the cu inhibition elicited in oocytes expressing only this receptor . Furthermore, the presence of prp-3f4 in the oocytes caused a rightward displacement of the cu concentration - response curve obtained in oocytes expressing only p2x4 receptor, an ic50 of 11.5 1.9 m was obtained for p2x4 and 34.1 7.6 m for p2x4/prp-3f4 (n = 57, p <0.01, figure 3(c)), confirming that prp-3f4 prevented the cu - induced inhibition not only at low micromolar concentrations of cu, but even at higher physiological concentrations of the metal . Several functions have been attributed to prp, including immunoregulation, signal transduction, copper binding, neurite outgrowth, induction of apoptosis or prevention of apoptosis against apoptotic stimuli, and others . In addition, prp has been related to synapse formation and maintenance and synaptic transmission [9, 10, 42], although the mechanisms by which it exerts its role is still unknown . One of the proposed targets for prp in synapse is to modulate cu homeostasis, based on a highly conserved cu - binding sequence located on its n - terminal domain, which includes four identical repeats of the peptide sequence pro - his - gly - gly - gly - trp - gly - gln [12, 15, 16]. It is known that prp binds cu with high affinity [1417], and the octarepeat region of the human prp (prp59 - 91) reduces cu(ii) to cu(i) in vitro, which depends on the tryptophan residues present in the octapeptide repeats [19, 20]. Cu modulates synaptic transmission at micromolar concentrations by a wide range of mechanisms, be one of the most relevanting modulations of neurotransmitter receptors within glutamatergic, gabaergic, and purinergic synapses, among others [43, 44]. In a previous study, we demonstrated that cu at micromolar concentrations inhibits the atp - evoked currents of p2x4 receptors . Here we show that the full - length prion protein - expressed in xenopus oocytes localizes in the cell surface and modulates the cu interaction with p2x4 receptor; oocytes which coexpressed prp-3f4 and p2x4 receptors have a diminished cu - induced inhibition of the atp - evoked currents compared with oocytes which only expressed the p2x4 receptor . This reduced inhibition by cu was observed on cu concentration - response curves, where the ic50 of cu was significantly increased in the presence of prp-3f4, indicating that prp-3f4 can exert its modulatory role even at high micromolar concentrations of cu, reached in the synaptic cleft after depolarization . These results, together with our previous findings showing that coapplication of cu with the n - terminal prp fragment (prp59 - 91) prevents the inhibitory effect of copper on p2x4 receptors and even reverts the established cu - induced inhibition of the p2x4 receptors, strongly support the idea that prp could modulate synaptic copper and therefore affect the function of p2x4 receptors and synaptic transmission . In addition to the potential synaptic role of prp driven by its ability to bind cu, a known modulator of neuronal excitability [43, 44], there is increasing evidence of direct interaction between prp and neurotransmitter receptors . Prp directly interacts with the nr2d subunit of the nmda receptor, inhibiting it and preventing nmda - induced excitoxicity in the hippocampus . On the other hand, prp also exerts a neuroprotective role against kainate - induced neurotoxicity in the hippocampus, probably by regulating differentially the expression of glur6 and glur7 kainate receptor subunits . Moreover, prp can modulate the activity of serotoninergic receptors signaling pathways in 1c11 cells . We observed a slight, although not significant, reduction on atp affinity of p2x4 receptor in the presence of prp-3f4, this might suggest an interference with atp binding or stabilization of closed states, although further experiments are required to evaluate this hypothesis . Altogether, these studies and the presented here highlight the modulatory role of prp at synaptic transmission in cns, involving direct regulation of neurotransmitter receptors and/or their signaling cascade, or indirectly, by controlling the synaptic levels of cu . The understanding of the physiological function of prp on synaptic transmission may clarify the pathogenic processes underlying prion diseases . Based on our results, it is possible to suggest that the resulting cognitive deterioration of prion diseases could involve a loss of the modulatory role of prp on brain function, as it is converted to the pathogenic isoform.
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The patient in this report was suffering from diabetic neuropathy, end - stage renal disease, restless legs syndrome (rls), and depression . He had injuries of both basal ganglia and the thalamus as a result of hypoglycemia, causing chorea and more severe leg pains and dysesthetic sensations . Rls is a sensorimotor disorder characterized by unpleasant leg sensations together with an irresistible inner urge to move . Chorea is a neurological syndrome characterized by abrupt involuntary movements resulting from a continuous flow of random muscle contractions . The first - line pharmacological treatment for rls is a dopaminergic agent, whereas anti - dopaminergic drugs are the main pharmacological treatment for chorea . In general, greater d2-receptor blocking action results in greater anti - choreic efficacy (1). Dopaminergic drugs for rls would aggravate the choreic movements, while anti - dopaminergic agents for the treatment of chorea would exacerbate the rls . In this report, the association between the complex medical conditions and symptoms of a patient with two coexisting, opposing diseases is discussed . A 44-yr - old man with insomnia and depression was referred to the psychiatric department of our institution . He complained that he rarely fell asleep because of dysesthetic sensations and pain in the lower legs and feet and that he was depressed and feeling hopeless . Although he was aware of having had a high blood sugar level for 10 yr, he never sought treatment . About 3 yr earlier, in summer 2003, he began to experience cold, numbing, and pins - and - needles sensations on his toes that eventually spread throughout his feet . About a year later, he started taking hypoglycemic agents . However, the pain in his feet became severe, and restlessness as a result of creeping sensations in his lower legs gradually started to occur when he was lying down or sitting awake . The unpleasant sensations and restlessness were temporarily relieved by massage and walking, but the pain in his feet remained little changed . These dysesthetic sensations were most severe when he lay down to sleep, increasing his sleep latency to longer than one hour . He woke repeatedly and had a difficulty falling back asleep because of further dysesthetic sensations . In july 2005, the patient received hemodialysis for end - stage renal disease . In those days his serum hemoglobin, iron, and ferritin levels were 7.4 g / dl, 29 g / dl, and 97 ng / ml, respectively, so oral iron supplementation was administered . He gradually became depressed, experienced feelings of worthlessness, lost his appetite, and eventually stopped eating . Subsequently, he showed irregular, spasmodic, involuntary movements in his limbs and on his face . His blood sugar level was very low (49 mg / dl). T2- and fluid - attenuated inversion recovery (flair)-weighted magnetic resonance imaging (mri) scans showed high signal changes over the bilateral basal ganglia and right thalamus, although abnormal enhancing lesions were not found after contrast infusion (fig . The patient was diagnosed with hypoglycemic injury of both basal ganglia and the right thalamus and received conservative medical treatment . One week later, he presented to the psychiatric department complaining of depression and insomnia . He was depressed and feeling hopeless and had severe sleep disturbance and thoughts of suicide . He complained that the most distressing problem was the difficulty in falling asleep and staying asleep because of the dysesthetic restlessness . He satisfied the diagnostic criteria for rls in the international classification of sleep disorders (2). He was prescribed citalopram 20 mg for depression, gabapentin 900 mg, and clonazepam 0.5 mg for peripheral neuropathy and rls, oral iron supplements for iron deficiency anemia and rls, and haloperidol 1.5 mg for choreic movements, and received daily supportive psychotherapy . The dysarthria and involuntary hyperkinetic movements gradually improved over his whole body, except for his left arm . The pain in his feet remained severe, and the dysesthetic restlessness continued in his lower legs . Four weeks later, follow - up t2-weighted mri scans showed resolution of previously noted basal ganglia lesions, residua of the hyperintensity change in the left putamen, and an infarction as an old lesion in the right thalamus (fig . The patient showed increased deep tendon reflexes on neurological examination as well as delayed latencies and small amplitudes for complex muscle action potential and sensory nerve action potential on a nerve conduction test, suggesting peripheral polyneuropathy . He then underwent a nocturnal polysomnography to examine the causes of his insomnia . When he lay down for polysomnographic evaluation, he experienced dysesthetic sensations and an irresistible inner urge to move his legs, as usual . The patient complained that he could not endure the dysesthetic sensations and was given his usual medications before bed: gabapentin 300 mg, clonazepam 0.5 mg, citalopram 10 mg, and haloperidol 1.5 mg . There were 55.2 periodic limb movements per hour in sleep, and about 36% of them led to arousal . The complete diagnoses of the patient included chorea, peripheral polyneuropathy, rls, periodic limb movement disorder, insomnia, and depression . During those days, his serum hemoglobin, iron, and ferritin levels were nearly normal: 11.4 g / dl, 62.0 g / dl, and 210.0 ng / ml, respectively . Although gabapentin, clonazepam, oral iron supplements, citalopram, and haloperidol were prescribed for 4 weeks, the pain, dysesthetic sensations, insomnia, and depression continued . To make matters worse, however, the choreic movements did not improve, and the dysesthetic restlessness, insomnia, and depression worsened . The haloperidol was reduced to 1.5 mg, and the gabapentine was replaced with carbamazepine 400 mg and topiramate 25 mg . However, there was no improvement after 2 weeks . Consequently, carbamazepine and topiramate were replaced with tramadol 150 mg, while maintaining haloperidol 1.5 mg, citalopram 20 mg, and clonazepam 0.5 mg . After the first administration of tramadol, the pain, dysesthetic restlessness, difficulty in initiating sleep, frequent awakenings, and difficulty in falling back to sleep after an awakening slightly improved, and then gradually improved further, followed by improvements in the depressed mood, lethargy, markedly decreased volition and appetite, and feelings of hopelessness . Peripheral neuropathy is a disorder that needs to be differentiated from rls because both conditions include some similar symptoms; however, it is also a main cause of secondary rls (3). The patient's dysesthetic sensations in the present case improved with movement but worsened in the evening . The findings in his nerve conduction test also supported the presence of peripheral polyneuropathy . As a result although its pathophysiology is not completely understood, circumstantial evidence exists for the role of the dopaminergic system in the pathophysiology of rls (4). A positron emission tomography (pet) (5) study and a single - photon emission computed tomography (spect) (6) study showed significantly decreased striatal d2 receptor activity . Also, there is circumstantial evidence for the role of iron in the pathophysiology of rls (7). Tyrosine hydroxylase is the rate - limiting enzyme in the production of dopamine and requires iron as a cofactor for hydroxylation of tyrosine (8). Therefore, iron deficiency may reduce dopamine production in the brain indirectly, while dopaminergic agents are effective in therapy for idiopathic rls (9). The patient had conditions that would cause rls, such as end - stage renal disease, iron deficiency, and peripheral neuropathy, and then to make matters worse, he was injured in the basal ganglia and thalamus by hypoglycemia . Following the hemodialysis treatment for the end - stage renal disease, his symptoms of rls were aggravated, and after the brain injury, choreic movements developed and symptoms of rls became extremely severe . In this case, iron deficiency due to esrd with hemodialysis and poor nutrition might lead to decreased production of dopamine, and it would have caused rls . In addition, the injury of the basal ganglia might have raised a further decrease of dopamine, aggravating rls . Chorea results from various dysfunctions within the complex neural network interconnecting the basal ganglia - thalamus - motor cortex (10). In the present case, the choreic movements developed after injuries to the basal ganglia and thalamus and decreased as the injuries recovered, although the movements remained owing to the thalamic infarct . Because anti - dopaminergic drugs are used to treat chorea, dopaminergic agents for rls could not be used in this case . Most studies have reported that gabapentin improved pain and dysesthetic sensations without the risk of dyskinesia, but some studies found that gabapentin induced dyskinesia or even chorea . These reports hypothesized that increased gabanergic input may precipitate more motor facilitation through the thalamus (11), augment dopaminergic activity in the striatum (12), or indirectly raise the concentration of serotonin (13). The patient experienced alleviation of his pain, dysesthetic sensations, and insomnia after being prescribed tramadol, which produces both opioid and non - opioid analgesic effects . Tramadol acts on the central nervous system and is known to have an effect on opioid receptors, especially the receptor, and serotonergic and noradrenergic transporters . The positive pharmacological response to an opioid and the exacerbation of rls by the opiate receptor blocker naloxone support the hypothesis that dysfunction of the endogenous opiate system is connected to the pathophysiology of rls (14). The present case was remarkable because of the risk associated with using dopaminergic or gabanergic drugs given the patient's complicated medical condition and the observation that tramadol was an effective treatment . The positive effect of tramadol may originate not only from its action on opioid receptors but also through increased activities of dopamine, serotonin, and noradrenalin . However, not only the effect of tramadol but also the recovery from the injury of the basal ganglia, iron supplement, and improvement of depression may contribute to the alleviation of the symptoms of rls . This case suggests that the dopaminergic system participates intricately with the opioid, serotonergic, and noradrenergic systems in the pathophysiology of rls and pain and indirectly of depression and insomnia.
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Wide hybridisation between b. rapa (, genome: aa) and b. nigra (, genome: bb) was performed to produce allodiploids (f1, genome: ab), and subsequently allotetraploids (f1, genome: aabb) were obtained by treating the allodiploids with 0.2% colchicine for 16 h, as described in our pervious study reported by ghani et al .,, . After self - crossing of the allotetraploids, all of the plants were grown in vermiculite mixed with 30% soil in a growth chamber under growth conditions of 22/18 c (day / night) and 16 h of illumination per day . The leaves from three plants of each type were collected 45 days after sowing in the vegetative stage for the analyses of small rnas . To determine small rna populations in b. rapa, b. nigra and their progenitors (both the allodiploid and the allotetraploid), small rna libraries were generated from the leaves of the four genotypes, i.e., b. rapa (aa), b. nigra (bb), the allodiploid (ab), and the allotetraploid (aabb). The total rna was isolated using the trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions and was sent to beijing genomics institute (bgi) for sequencing . After treatment of the raw data, the clean sequences were subjected to further analyses as previously described . The sequences were then matched to the genome of all of the plants to identify the repeat - associated srnas and to assess the expression of srnas . The differential expression of mirnas, the abundance of mirnas in all of the libraries was normalised . The normalisation values were compared between the two libraries and were calculated in the form of fold - changes (fold - change = log2 (treatment / control)). Moreover, the p - value was obtained using a previously described formula . For the prediction of targets, the gene function, including the biological process, cellular component localisation, and molecular function of the genes were analysed (fig . 2). Wide hybridisation between b. rapa (, genome: aa) and b. nigra (, genome: bb) was performed to produce allodiploids (f1, genome: ab), and subsequently allotetraploids (f1, genome: aabb) were obtained by treating the allodiploids with 0.2% colchicine for 16 h, as described in our pervious study reported by ghani et al .,, . After self - crossing of the allotetraploids, all of the plants were grown in vermiculite mixed with 30% soil in a growth chamber under growth conditions of 22/18 c (day / night) and 16 h of illumination per day . The leaves from three plants of each type were collected 45 days after sowing in the vegetative stage for the analyses of small rnas . To determine small rna populations in b. rapa, b. nigra and their progenitors (both the allodiploid and the allotetraploid), small rna libraries were generated from the leaves of the four genotypes, i.e., b. rapa (aa), b. nigra (bb), the allodiploid (ab), and the allotetraploid (aabb). The total rna was isolated using the trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions and was sent to beijing genomics institute (bgi) for sequencing . After treatment of the raw data, the clean sequences were subjected to further analyses as previously described . The sequences were then matched to the genome of all of the plants to identify the repeat - associated srnas and to assess the expression of srnas . The differential expression of mirnas, the abundance of mirnas in all of the libraries was normalised . The normalisation values were compared between the two libraries and were calculated in the form of fold - changes (fold - change = log2 (treatment / control)). Moreover for the prediction of targets, the gene function, including the biological process, cellular component localisation, and molecular function of the genes were analysed (fig . 2) this result showed that sirnas play key roles in maintaining the genomic stability through the regulation of small rna levels . Moreover, most mirnas were highly overexpressed in the allotetraploid, which might be induced by the heterosis, such as mir159, mir169, and mir164, mir165, and mir166, which have a major role in flower and leaf development in the allotetraploid . Taken together, the findings of this study demonstrated that sirnas and mirnas maintain the genomic and phenotypic stability in the allotetraploid.
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Childhood obesity has emerged as a significant health problem in a transitional society . Rapidly changing dietary practices and a sedentary lifestyle have led to a high prevalence of childhood overweight and obesity among school - aged children (defined by the international obesity task force cut - points) in developing countries between 2004 and 2010: 41.8% in mexico, 22.1% in brazil, 13.322.3% in south africa, 27.9% in argentina, and 2.828% in india . In thailand, data from the two national surveys demonstrated an increase of the obesity prevalence among the 6- to 12-year - old children from 5.8% in 1997 to 6.7% in 2001 by using the weight - for - height criteria of a local reference . This rising trend coincided with an increase of type 2 diabetes among thai diabetic pediatric patients from 5% between 1986 and 1995 to 17.9% between 1996 and 1999 . Increased energy content in diet, decreased levels of physical activity, and increased sedentary lifestyles as well as a number of cultural and environmental factors have been identified as the causes of obesity in children [46]. In the thai context, determinants of overweight and obesity among children in previous reports included having obese parents, being in a family with a high income, maternal overweight prior to pregnancy, high birth weight, being the only child, large amounts of food consumed by children, having a lesser amount of exercise than peers, and tv viewing time more than 2 hours per day [710]. Changes in several social and environmental factors have been suggested as causes of the obesity epidemic among children, for example, reduced physical education at school, increased homework loads, school vending machines, tv, larger food portion sizes, fast - food restaurants, video games, and many others . Time spent in seated sedentary behaviors (sb) (e.g., electronic media use) reduces time allocation for physical activity (pa) and hence increases risk of overweight and obesity . Moreover, snack and sugar - sweetened beverage consumption while watching tv further augments the positive energy balance . Children worldwide have increasing access to electronic media, such as tv, computer / video games, cell phones, and the internet in daily life . In thailand, the number of internet cafs which provide online game service increased 1.8 times over 2 years from 2008 to 2010 . The fourth national health examination survey in thailand revealed that 5% of the 6 to 9 year olds and 12% of the 10 to 14 year olds engaged in computer games more than 1 hour each day, while 57.1% and 73.1%, respectively, watched tv more than 2 hours / day during the weekdays . A report from the child and adolescent mental health center in bangkok demonstrated an increase in the game addiction rate from 5% in 2005 to 9% in 2009 . In regard to media use, an association of sedentary behavior, primarily through tv viewing, with body mass index (bmi) and obesity has been well documented [14, 15], while the relationship of seated computer game use with obesity was unclear . Most of the studies investigated total screen time including tv viewing, working on a computer, and playing video games [15, 16]. A limited number of studies that looked at computer games independently had mixed results [1619]. In order to gain an understanding of the current rising prevalence of childhood overweight, we used recent data from the fourth national health examination survey (nhes iv) to investigate the associations of sb (i.e., time spent at computer gaming and television viewing) with overweight among children aged 6 to 14 years in thailand . In addition, we examined whether the effect of screen time on overweight varied by physical activity status and gender . The national health examination surveys have been conducted every 5 years since 1991 . The fourth national health examination survey (nhes iv) 2008 - 2009 conducted by the national health examination survey office was designed to represent the noninstitutionalized thai population using a multistage stratified sampling based on 2008 thai population registers . For the first stage, 5 provinces were randomly sampled by proportion to size (pps) from each of the 4 regions, except bangkok . In the second stage, 3 to 5 districts were selected by pps from each province . In the third stage, in each province, 13 - 14 electoral units (eus) or villages were selected by pps from each of the urban and rural areas . In the final stage, for each eu / village, 8 to 10 males and 8 to 10 females were selected by systematic random sampling from population registers from each of the six broad age and gender groups (114-, 1559-, and 60-year - old males / females). In bangkok, 5 to 6 eus were randomly selected by pps from each of the 12 districts . The final sample size of the 1- to 14-year - old subjects was 9,035 individuals (response rate of 92.8%). This report presents an analysis of the data from the school - aged population: 5,999 children aged 6 to 14 years . Subjects were weighed in light clothes using a tanita scale and recorded to the nearest 0.1 kg . Standing heights were measured by trained research assistants using a locally made stadiometer and recorded to the nearest 0.1 cm . Information regarding dietary intake and physical and sedentary activities was taken by interviewing the parents of children under 10 years of age and the subjects themselves for those aged 10 years and older . The questions for media use were how many hours a day during the past month did you watch television? And how many hours a day during the past month did you play computer games? For physical activity, the question was how many days during the past week did you have physical activity that increased your breathing rate and heart rate for at least 60 minutes? Overweight was defined using the age- and gender - specific bmi cut - points of the international obesity task force . For media use, tv time was categorized into 2 hours / day or less and more than 2 hours / day according to the american academy of pediatrics recommendation and time spent in playing computer games was classified into 1 hour / day or less and more than 1 hour / day . Exercise of moderate intensity was grouped into having 60 minutes for 3 days / week or more and less than 3 days / week . Since gender difference has been noted in the studies of obesity especially in relation to physical activity behaviors [15, 23], the analysis was thus computed separately for boys and girls . Logistic regressions were computed to examine the association of media use and physical activity level with overweight . Consumption of high energy snacks (examples given were potato crisps and rice - shrimp crisps), which was highly associated with overweight in both male and female subjects, was selected as a covariate in the logistic regression analysis . In addition, family income, which was reported to be associated with overweight in thai children, was also included in the regression models . The analyses were performed using stata 11.0 . The national ethical review committee for research in human subjects, ministry of public health, approved the study . The sampled families were informed of the data collection process and verbal permission was obtained . Table 1 describes overweight prevalence, media use, physical activity, consumption of high energy snack, and family income characteristics of the subjects . Prevalence of overweight using age- and gender - specific bmi cut - points of the international obesity task force was 15.2% for girls versus 16.7% for boys . Most subjects watched tv more than 2 hours / day . On the other hand, only 5.2% engaged in computer games more than 1 hour / day; male subjects played computer games twice as much as the females (p <0.001). Male subjects also engaged in moderate - intensity physical activity significantly more than the females (p <0.001). No gender difference was noted in the frequency of having high energy snacks and family income levels . The associations of media use and physical activity with overweight are shown in table 2 . From the multiple logistic regression analysis, computer game use and physical activity behavior had a significant effect on the likelihood of overweight among the 6- to 14-year - old subjects . With the 1 hour / day or less as the referent group, use of computer games more than 1 hour / day increased the likelihood of overweight (adjusted odds ratio (aor) = 1.40; 95% confidence interval (ci): 1.021.93), whereas children who engaged in 60 minutes of moderate - intensity physical activity for more than 3 days / week were less likely to be overweight (aor = 0.75; 95% ci: 0.660.84). Watching tv more than 2 hours / day significantly increased the risk for overweight (aor = 1.67; 95% ci: 1.172.40), while engagement in moderate - intensity physical activity significantly decreased the risk among girls (aor = 0.62; 95% ci: 0.510.76). For boys, associations of computer game use and engagement in moderate - intensity pa with overweight the relationship between media use and overweight was further explored by a subgroup analysis according to the pa level (table 3). The effect of media use was significant only among the girls with a lower level of pa . Female subjects who spent 3 days / week in 60 minutes of moderate - intensity pa had a greater risk for overweight if they spent more than 2 hours / day viewing tv (aor = 1.72; 95% ci: 1.092.74 versus 2 hours / day or less) or more than 1 hour / day playing computer games (aor = 1.99; 95% ci: 1.153.47 versus 1 hour / day or less), while those having more than 3 days / week of moderate - intensity pa and engagement in either tv viewing or computer gaming did not pose significant risks . For boys, media use did not exert significant risk for overweight in either group of pa . By using the data from the recent national health examination survey, we found that computer game use for more than 1 hour a day was associated with an increased risk for overweight while engagement in moderate - intensity pa significantly decreased the likelihood of overweight among the 6- to 14-year - old female subjects . Further analysis showed that the effect of computer game use and tv viewing on the risk for overweight was significant among girls, but not boys, who spent 3 days / week in 60 minutes of moderate - intensity pa . The association between the level of pa and weight status has been shown to be nonuniform in the recent systematic review of cross - sectional studies . Like ours, most of the reports that found significant negative associations, the outcomes were different for boys and girls . Gender differences in health behaviors relating to obesity have been documented in other studies [23, 25, 26]. These results may be due to the differences in physiological responses or gender role expectation in the society for boys and girls [2729]. On the other hand, child weight status itself may influence the intensity and frequency of pa and this may give rise to the mixed findings of their associations . Moreover, the self - report of pa which was used in most surveys may affect the accuracy and lead to inconsistent results . The positive association between sedentary behaviors, namely, tv viewing or playing video / computer games, and child weight status found in this study is consistent with the majority of previous studies in the recent systematic review . Research on the association between screen time and overweight / obesity in children mostly used total screen time (i.e., combining tv viewing, video / computer game use, and non - leisure computer use). Nowadays, the media such as computer games and the internet substantially occupies children's pastime . An investigation of the independent effect of this electronic media use on overweight / obesity in children will have public health importance . Like studies of physical activity, research assessing the effect of video / computer game use on the weight status of children showed mixed results . A meta - analysis by marshall et al . And three other recent studies [3032] among children and adolescents found no association, while a positive relationship between electronic game use and weight status were found to be curvilinear with a difference in ages in a study of us children using 24-hour time - use diaries to record the amount of video game use . Children under the age of 8 with higher weight status played moderate amounts of electronic games, while children with lower weight status played either very little or a lot of electronic games . This study also found that girls, but not boys, with higher weight status played more video games . Video game use was also found to be associated with an increased cardiometabolic risk score, blood pressure, and lipids [17, 18]. As children usually engage in multiple sb, an effect of each activity on child weight status should be disentangled to provide evidence for development of effective interventions to promote healthy behaviors for obesity control . We found physical activity to be a moderator of the relation between sedentary activities (i.e., tv viewing time and computer game time) and overweight . Girls who had 60 minutes of moderate - intensity pa 3 days / week were more likely to be at risk for overweight if they watched tv> 2 hours / day or played computer games> 1 hour / day than those who had such level of pa> 3 days / week . This moderating effect was not observed in boys . In a study of 9,278 taiwanese adolescents, yen et al . Demonstrated that the relationship between increased bmi and a high level of tv viewing was found only in adolescents who exercised less than 1 hour / day, but not in those who exercised 1 hour / day or more . These findings indicated that adequate exercise may be protective for children who spend a lot of time on sedentary activities from increased adiposity, particularly among girls . These moderators, namely, age, gender, and pa, should be taken into account in developing interventions for children and adolescents . However, in the present study, it is not clear whether the findings of differences in the associations by gender are due to a variation in energy expenditures pattern during tv watching or in the type of computer game use between boys and girls . Given the publicized untoward effect of tv viewing and computer game use, the bias may be likely towards underreporting . Nevertheless, this study has its strength as a national representative study with a relatively large sample size . The findings could reflect the behavioral pattern of thai children and a meaningful relationship of these behaviors to child overweight problem . The lives of children nowadays have been considerably affected by societal changes and media technologies . We found that computer game use for more than 1 hour a day and tv viewing for more than 2 hours a day increased the risk for overweight among girls who had a low pa level . These findings are helpful for developing effective interventions to promote healthy behaviors for the control of obesity . As computers are being used in schools and at home for educating children at a younger age, the risk of this electronic media should be communicated to the families and the public as well . Parents should be informed to discipline the use of a computer in order to control sedentary time . Tracking societal changes on lifestyles should be carried out regularly to identify potential areas for targeted interventions.
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Colonic lipomas are benign mesenchymal tumors that might not require treatment if asymptomatic, whereas large colonic lipomas that cause abdominal pain, hemorrhage, constipation and intussusception require surgical resection . Endoscopic submucosal dissection (esd) is considered to be effective for treating large lipomas; however, esd is also associated with the risk of perforation and bleeding . Endoscopic unroofing is a simple, safe and effective procedure that comprises the excision of the upper third of a large lipoma to allow remaining adipose tissue to gradually extrude from the resected stump [1, 4, 5, 6]. Nonetheless, the outcomes of this procedure are not always positive, and we describe the failure of unroofing to treat two large colonic lipomas . The colonoscopy of an 82-year - old woman with abdominal pain revealed a 40-mm pedunculated lipoma in the ascending colon and a 20-mm sessile lipoma in the transverse colon (fig 1a). The pedunculated mass in the ascending colon was resected en bloc by esd using an electric knife (dual knife, olympus, tokyo, japan), and the sessile mass in the transverse colon was treated by unroofing . The upper third of the tumor was resected using an electrocautery snare (olympus, tokyo, japan) (fig 1b). Follow - up colonoscopy 5 days later revealed an open surface on the unroofed stump and residual lipoma (fig 1c) that persisted for up to 1 month . By that time, the regenerated surface had become covered with a mucosa, and the lipoma looked the same as it had been before unroofing (fig 1d). The residual lipoma was not resected because the patient no longer had abdominal pain and requested only follow - up observation . A 74-year - old man who presented with abdominal pain and melena was diagnosed with a lipoma in the transverse colon and referred to our department . The colonoscopy revealed a 50-mm - wide lipoma accompanied by mucosal hypertrophy and erosion in the transverse colon (fig 2a). The mucosa of the upper third of the tumor was circumferentially incised using an electric knife (dual knife, olympus) and resected using an electrocautery snare (olympus) (fig 2b, c). Seven days later, the unroofed surface had become coated with a white substance (fig 3a). Since residual adipose tissue was not likely to drain, the lesion was resected piecemeal using an electrocautery snare (fig 3b, c). One month thereafter, the patient was free of abdominal pain and melena, and the colonoscopy showed that the resected region had become a scar without residual lipoma (fig 3d). The colonoscopy of an 82-year - old woman with abdominal pain revealed a 40-mm pedunculated lipoma in the ascending colon and a 20-mm sessile lipoma in the transverse colon (fig 1a). The pedunculated mass in the ascending colon was resected en bloc by esd using an electric knife (dual knife, olympus, tokyo, japan), and the sessile mass in the transverse colon was treated by unroofing . The upper third of the tumor was resected using an electrocautery snare (olympus, tokyo, japan) (fig 1b). Follow - up colonoscopy 5 days later revealed an open surface on the unroofed stump and residual lipoma (fig 1c) that persisted for up to 1 month . By that time, the regenerated surface had become covered with a mucosa, and the lipoma looked the same as it had been before unroofing (fig 1d). The residual lipoma was not resected because the patient no longer had abdominal pain and requested only follow - up observation . A 74-year - old man who presented with abdominal pain and melena was diagnosed with a lipoma in the transverse colon and referred to our department . The colonoscopy revealed a 50-mm - wide lipoma accompanied by mucosal hypertrophy and erosion in the transverse colon (fig 2a). The mucosa of the upper third of the tumor was circumferentially incised using an electric knife (dual knife, olympus) and resected using an electrocautery snare (olympus) (fig 2b, c). Seven days later, the unroofed surface had become coated with a white substance (fig 3a). Since residual adipose tissue was not likely to drain, the lesion was resected piecemeal using an electrocautery snare (fig 3b, c). One month thereafter, the patient was free of abdominal pain and melena, and the colonoscopy showed that the resected region had become a scar without residual lipoma (fig 3d). Endoscopic unroofing is simple and safe, and a useful method of treatment that also enables pathological examinations . The procedure is applicable to a diagnosis not only of lipomas, but also of submucosal masses such as gastrointestinal stromal tumors, leiomyomas, and neuroendocrine carcinomas [7, 8]. We treated colonic lipomas in 2 patients using unroofing, but the outcomes were insufficient in both . We therefore investigated why the remaining lipoma failed to drain . In the 1st patient, the unroofed area of the transverse colon lipoma was small, and only the surface was resected, which might have prevented the drainage of adipose tissue despite it being exposed . Adipose tissue partially drained immediately after unroofing in the 2nd patient, but a hard, thickened mucosa was encapsulated by a submucosal layer that might have prevented the spontaneous drainage of residual fat . Adipose tissue can take several days to weeks to drain from lipomas, but this did not occur within a period of about 1 week in either patient . The mucosa regenerated on the unroofed surface after 1 month in the 1st patient, whereas endoscopic findings showed that adipose tissue had not drained in the 2nd patient within 1 month, and additional endoscopic mucosal resection (emr) was required . More investigation is needed about lipomas treated by endoscopic unroofing, particularly those from which adipose tissue does not drain . Large colonic lipomas can spontaneously detach, but treating those 2 cm using endoscopic procedures is associated with increased risk of complications such as perforation and hemorrhage . Therefore, in addition to standard emr, attempts have been made to reduce complications by applying a detachable nylon endoloop or a long clip to the base of large colonic lipomas [12, 13]. In addition, recent improvements in esd have allowed the safe resection of large lesions, and large colonic lipomas are now included among the indications for this procedure . However, the risk of complications remains high for endoscopic procedures, and surgical resection is recommended for lipomas with a base that is 4 cm or patients with an unclear diagnosis, intussusception - induced obstruction, expansion into the muscle layer and serosa, and incomplete endoscopic resection . However, patients with abdominal pain and hemorrhage should be treated in consideration of complete resection, but not by unroofing, which could leave a residual tumor . Drainage should be confirmed after unroofing and any residual lipoma should be treated by additional resection.
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Ascites is a major complication associated with portal hypertension in decompensated cirrhotic patients (1,2) and is commonly treated with aldosterone antagonists and loop diuretics (3). However, patients with cirrhosis have decreased serum sodium and hypoalbuminemia (4), which reduce the effects of diuretics (4). This requires an dosage adjustment, resulting in limitations in treatment (5,6) due to concerns of exacerbating hyponatremia and nephropathy by high - dose diuretics (7,8). Tolvaptan is an oral vasopressin v2 receptor antagonist that was approved for the indications of fluid retention associated with cirrhosis in september 2013 . Tolvaptan has a mechanism of action that differs completely from other diuretics (9) and eliminates excess water without increased electrolyte excretion . Therefore, tolvaptan may be an innovative therapy for ascites that is resistant to treatment with existing diuretics in patients with hepatic edema . Tolvaptan is a diuretic drug that functions independently of liver functions, particularly the level of serum albumin . Clinical trial data in japan showed that tolvaptan is effective for ascites complicated with decompensated cirrhosis with decreased albumin synthesis (10 - 12). The efficacy of tolvaptan has been found to be about 60% (13), but in clinical practice some patients with cirrhosis experienced earlier relief and improved quality of life (qol), whereas other patients have a delayed response or are nonresponsive to tolvaptan and require other treatment . Therefore, in the current study we compared patients with cirrhosis who did and did not show an early response to tolvaptan, with the goal of identifying predictors of the effect of tolvaptan that would allow another therapeutic intervention to be used as soon as possible in non - responders . The subjects included 26 patients with cirrhosis who were treated with tolvaptan in our hospital from september 2013 to april 2015 . Tolvaptan was administered to liver cirrhosis patients who had ascites despite treatment with combination therapy of a loop diuretic and an anti - aldosterone agent . Tolvaptan was initially administered at a dose of 3.75 mg and increased to 7.5 mg if the effect was insufficient on administration day 3 . Height, body weight, urine volume, blood test and urinalysis data were collected and are presented as the mean standard deviation (sd). Statistical analyses were conducted using the jmp9 software program (sas institute, cary, usa). A chi - square test or fisher's exact test was used to evaluate differences between two groups . Changes from baseline in the sample data from the same group were evaluated by the t - test . Cut - off values were determined by receiver operating characteristic (roc) analyses to build categorical valuables from consecutive data . The study was performed according to the declaration of helsinki and the clinical research guidelines in japan and approved by the institutional review board at yamaguchi university hospital (no . Data are shown as median (range), number (%) or mean sd tolvaptan was initially administered to all patients at a dose of 3.75 mg and increased to 7.5 mg if the effect was insufficient on administration day 3 . Body weight decreased significantly from 55.511.8 kg before treatment to 52.110.5 kg after 1 week of treatment . Urine osmolality significantly decreased from 488170.4 to 355.5138.6 mosm / kg after 1 week of treatment; however, serum albumin, creatinine and sodium concentrations were not significantly changed after 1 week (table 2). Body weight decreased by approximately 2.4 and 3.5 kg after 1 and 2 weeks of treatment, respectively, with respective reductions of 6.2% and 8.0% from baseline (fig . Data are shown as mean sd changes in body weight after the administration of tolvaptan . Body weight significantly decreased from 55.5 11.8 kg before treatment to 52.1 10.5 and 5.1 11.7 kg after 1 and 2 weeks of treatment (p<0.001). The respective body weight decreases of approximately 2.4 3.3 and 3.5 4.7 kg after 1 and 2 weeks represented reductions of 6.2% and 8.0%, respectively, from baseline . Data were compared between patients with weight loss 2 kg (early responders, group r) and <2 kg (early non - responders, group n) after 1 week of treatment with tolvaptan (fig . 2). Four patients in group n subsequently had a decrease in body weight of 2 kg after reduction of loop diuretics (2 patients), fluid replacement (1 patient) and ascites removal (1 patient), leading to remission and hospital discharge . There were no significant differences in age, height, body weight, sex, child - pugh score, hepatic function including albumin, model for end - stage liver disease (meld) score, meld - na score, rate of complication with hepatocellular carcinoma (hcc), or dose of diuretics between groups r and n. however, blood urea nitrogen (bun) and serum creatinine were significantly lower and the renal function was better maintained in group r, suggesting that these conditions were requirements for an early effect of tolvaptan (table 3). Data are shown as mean sd after initiating treatment with tolvaptan, urine osmolality 4 h after oral administration was significantly lower (23696 vs. 364122 mosm / kg, p<0.05) and the% decrease in osmolality from baseline was significantly higher (48.023.3% vs. 15.417.3%, p<0.01) in group r. the patients in group r also had higher serum sodium and lower brain natriuretic peptide (bnp) levels, however, the levels were not significantly difference from that of group n (table 3). Treatment outcomes for hcc were compared between the two groups using the response evaluation criteria in solid tumors (recist). There were no significant differences in the rate of complication with hcc, clinical stage, or cancer treatment between groups r and n (table 3). 3), and the complete response (cr) + partial response (pr) rate did not differ significantly between groups r and n (40.0% vs. 14.3%, p=0.28) (fig . 4). The prognosis in group r was better due to a significant increase in the kaplan - meier survival curve (p<0.05) (fig . Kaplan - meier curves for patients with hcc (solid line) and without hcc (dotted line). Significant differences were analyzed by the log - rank test (cochran - mantel - haenszel). Association between therapeutic effects of tolvaptan on ascites and the response evaluation criteria in hepatocellular carcinoma . The results were evaluated using the response evaluation criteria in solid tumors solid tumors (recist). The y - axis indicates the complete response (cr)+partial response (pr) rates (%) for hcc . Kaplan - meier curves for early responders (solid line) and early non - responders (dotted line) to tolvaptan . Significant differences were analyzed by the log - rank test (cochran - mantel - haenszel). The treatment of 26 patients in our hospital with tolvaptan at a dose of 3.75 mg for 1 week resulted in an overall response rate of 61.5% . Within 1 week, 16 of these patients had achieved a weight loss 2 kg . Therefore, the results of this study were similar to or better than those in a previous clinical trial that showed an efficacy of 55% at a dose of 7.5 mg (13). There was no significant difference in the hepatic reserve between responders and non - responders in the current study; however, the renal function in early responders was better than that in early non - responders . Diuretics cause renal impairment (14 - 16) and the effect of tolvaptan is poor in patients with heart failure, an increased bun / cre ratio and decreased estimated glomerular filtration rate (egfr) (15 - 18). Because the renal function is involved in the prognosis of hepatic cirrhosis, it is important to maintain the renal function and control diuretics (19 - 22). However, the non - responder group may have included many patients with potential renal insufficiency . Therefore, further prospective studies are needed to evaluate whether the efficacy of tolvaptan improves by early administration . In the current study, non - responders were likely to have higher bnp levels, however, none had clinical heart failure . Non - responders were found to have significantly poorer renal functions and there was a correlation between the renal function and bnp level (18,19). Therefore, the bnp level in non - responders may be increased by renal impairment . In responders, urine osmolality 4 h after tolvaptan administration was significantly lower and the rate of reduction of urine osmolality was high . The cut - off values determined by the roc analysis to achieve a weight loss 2 kg were bun 29 mg / dl, serum creatinine 1.35 mg / dl, and decreased urine osmolality 4 h after administration 34.7% . A clinical study showed that tolvaptan was effective in patients with urine osmolality> 352 osm / l before administration and decreased urine osmolality of> 26% at 4 to 6 h after administration (19,20). For patients with hepatic cirrhosis, the change in urine osmolality after administration is an indicator of efficacy in early administration . Determination of urine osmolality 4 h after the administration of tolvaptan can predict the subsequent efficacy and appropriate dosage of tolvaptan . If tolvaptan is effective, the qol is improved due to reduced ascites and edema; however, such a prognosis has not been shown in patients with hepatic cirrhosis . Some cardiovascular studies have shown an improved prognosis in responders, whereas others have indicated that the prognosis is not improved (21 - 24). In this study, there was no significant difference in the treatment outcome of hcc between responders and non - responders and no significant difference in the prognosis between patients with and without hcc . However, the prognosis of responders was significantly improved in comparison with that of non - responders . This result suggests that tolvaptan can improve the prognosis in patients with hepatic cirrhosis; however, the responder group included many subjects with good renal functions . Consequently, it is possible that their prognosis was already good before the administration of tolvaptan . Because of the small numbers of patients in the multivariate analysis, there were no significant differences; however, the prognosis tended to improve in the effective group (data not shown). A potential limitation associated with this study therefore, prospective randomized comparative trial (rct) studies are needed in more subjects without complication of hcc and in those with relatively well - maintained hepatic functions . Tolvaptan is effective for diuretics - resistant ascites regardless of the hepatic function and albumin level and may improve the prognosis . An accumulation of cases for prospective rct studies including multivariate analyses is needed to evaluate the long - term safety and efficacy of tolvaptan, the effect on prognosis, and the appropriate administration period.
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The nutmeg plant, myristica fragrans houtt, is a member of the small primitive family called myristicaceae, taxonomically placed between the annonaceae and lauraceae . At present, myristicaceae is considered as a member of magnotiales or its taxonomical equivalents . Nutmeg has long been known for its psychoactive properties (producing anxiety / fear, hallucination), from as early as 16th century writings to current internet based site . Nutmeg is widely accepted as a flavouring agent, and was used in higher doses (500mg / kg) as aphrodisiac and psychoactive agent in male rat . Nutmeg and its oleoresin are used in the preparation of meat products, soaps, sauces, baked foods, confectioneries, puddings, seasoning of meat and vegetables, to flavor milk dishes and punches . Powdered nutmeg is rarely administered alone, but enters into the composition of numerous medicines such as aromatic adjuncts . Medicinally, nutmeg is known to be a stimulant and has carminative properties . In pregnancy and lactation, traditionally nutmeg has been used as an abortificient; though this has largely been discounted, but it remains a persistent cause of nutmeg intoxication in women . The active ingredient in nutmeg is called myristicine and is a naturally occurring insecticide and acaricide with possible neurotoxic effects on dopaminergic neurons and a monoamine oxide . Cytotoxic and apotoxic effects of myristicine have been reported such that cell viability was reduced by exposure to myristicine in a dose dependent manner . Olaleye et al . Reported that the phytochemical constituent of nutmeg includes alkaloids, saponins, anthraquinones, cardiac glycosides, flavonoids and phlobatanins, while tannins were absent in the aqueous extract . The phytate content was reported to be 564.11 mg/100 g while the antioxidant indices of 100 mg/100 g, 44% and 0.6 were obtained for the ascorbic acid value, free radical scavenging activity and reducing power, respectively . The kidney is a paired organ located in the posterior abdominal wall, whose functions include removal of waste products from the blood and regulation of the amount of fluid and electrolytes balance in the body . As in humans, the majority of drugs administered are eliminated by a combination of hepatic metabolism and renal excretion . The kidney also plays a major role in drug metabolism, but its major importance to drugs is still its excretory functions . It would therefore be worthwhile to examine the effects of nutmeg on the kidneys of adult wistar rat thereby either corroborating previous work done by other researchers, or disproving the toxic effects of nutmeg in this organ, with a view to advising the consumers on the inherent dangers of excessive consumption of the spice / aphrodisiac . This study was given approval for the methodology and other ethical issues concerning the work by the university of benin research ethics committee . Twenty - four adult wistar rats of both sexes with average weight of 220 g were randomly assigned into three groups: a, b and c of (n=8) in each group . Group a and b served as treatment groups (n=16) while group c (n=8) served as the control . The rats were obtained and maintained in the animal holding of the department of anatomy, school of basic medical sciences, university of benin, edo state, nigeria . The animals were fed with growers mash obtained from edo feeds and flour mill limited, ewu, edo state, nigeria and given water liberally . The nutmeg seeds were obtained from new benin market, benin city, edo state, nigeria . They were dried and graded into powder at the department of pharmacognosy, faculty of pharmacy, university of benin, benin city, nigeria . The rats in the treatment groups (a & b) were given 0.1 g (500mg / kg body weight) and 0.2 g (1000mg / kg body weight) of nutmeg thoroughly mixed with the feeds respectively on a daily basis for forty - two days (6 weeks). The control group (c) received equal amount of feeds without nutmeg added for the same period . Blood samples were collected and analyzed for blood urea nitrogen (bun) and serum creatinine (scr) by using the commercial kits on the forty- third day of the experiment . After bleeding, the rats were sacrificed by cervical dislocation and the abdominal cavity was opened up using a pair of forceps to expose the kidneys which were quickly dissected out and fixed in 10% formal saline for routine histological techniques . The tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax . Sequential differences among means were calculated at the level of p <0.05, using turkey contrast analysis as needed . Twenty - four adult wistar rats of both sexes with average weight of 220 g were randomly assigned into three groups: a, b and c of (n=8) in each group . Group a and b served as treatment groups (n=16) while group c (n=8) served as the control . The rats were obtained and maintained in the animal holding of the department of anatomy, school of basic medical sciences, university of benin, edo state, nigeria . The animals were fed with growers mash obtained from edo feeds and flour mill limited, ewu, edo state, nigeria and given water liberally . The nutmeg seeds were obtained from new benin market, benin city, edo state, nigeria . They were dried and graded into powder at the department of pharmacognosy, faculty of pharmacy, university of benin, benin city, nigeria . The rats in the treatment groups (a & b) were given 0.1 g (500mg / kg body weight) and 0.2 g (1000mg / kg body weight) of nutmeg thoroughly mixed with the feeds respectively on a daily basis for forty - two days (6 weeks). The control group (c) received equal amount of feeds without nutmeg added for the same period . Blood samples were collected and analyzed for blood urea nitrogen (bun) and serum creatinine (scr) by using the commercial kits on the forty- third day of the experiment . After bleeding, the rats were sacrificed by cervical dislocation and the abdominal cavity was opened up using a pair of forceps to expose the kidneys which were quickly dissected out and fixed in 10% formal saline for routine histological techniques . The tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax . Sequential differences among means were calculated at the level of p <0.05, using turkey contrast analysis as needed . Twenty - four adult wistar rats of both sexes with average weight of 220 g were randomly assigned into three groups: a, b and c of (n=8) in each group . Group a and b served as treatment groups (n=16) while group c (n=8) served as the control . The rats were obtained and maintained in the animal holding of the department of anatomy, school of basic medical sciences, university of benin, edo state, nigeria . The animals were fed with growers mash obtained from edo feeds and flour mill limited, ewu, edo state, nigeria and given water liberally . The nutmeg seeds were obtained from new benin market, benin city, edo state, nigeria . They were dried and graded into powder at the department of pharmacognosy, faculty of pharmacy, university of benin, benin city, nigeria . The rats in the treatment groups (a & b) were given 0.1 g (500mg / kg body weight) and 0.2 g (1000mg / kg body weight) of nutmeg thoroughly mixed with the feeds respectively on a daily basis for forty - two days (6 weeks). The control group (c) received equal amount of feeds without nutmeg added for the same period . Blood samples were collected and analyzed for blood urea nitrogen (bun) and serum creatinine (scr) by using the commercial kits on the forty- third day of the experiment . After bleeding, the rats were sacrificed by cervical dislocation and the abdominal cavity was opened up using a pair of forceps to expose the kidneys which were quickly dissected out and fixed in 10% formal saline for routine histological techniques . The tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax . Sequential differences among means were calculated at the level of p <0.05, using turkey contrast analysis as needed . The result of this experiment revealed that nutmeg consumption caused significant (p<0.05) increase in functional nephrotoxicity indicators such as bun and scr in nutmeg - treated rats compared with control (table 1). Effects of long time consumption of nutmeg (0.1 g and 0.2 g) on bun and scr concentration the control sections of the kidneys showed normal histological features . The section indicated a detailed cortical parenchyma and the renal corpuscles appeared as dense rounded structures with the glomerulus surrounded by a narrow bowman's spaces (fig . 1) control section of kidney; this shows cortical parenchyma to consist of dense rounded structures, the glomeruli (g), surrounded by narrow bowman's capsular spaces (bcs). The kidneys of the animals in group a treated with 0.1g / day of nutmeg revealed some level of cyto - architectural distortion of the cortical structures as compared to the control (fig . 2) photomicrograph of treatment section of the kidney of rats that received 0.1 g of nutmeg revealing some level of cyto - architectural distortion (cad). The kidney sections of animals in group b treated with 0.2g / day of nutmeg revealed marked distortion of cyto - architecture of the renal cortical structures, and degenerative and atrophic changes . The renal corpuscles were less identified and the bowman's spaces were sparsely distributed as compared to the control group (fig . Treatment section of the kidney of rats that received 0.2 g of nutmeg, revealing some level of cyto - architectural distortion (cad), degenerative and atrophic changes (adc), and vacuolations (v) appearing in the stroma . The results (h & e) reactions revealed that chronic consumption of nutmeg caused varying degree of cyto - architectural distortion and reduction in the number of renal corpuscle in the treated groups compared to the control group . There were several diffuse degeneration and necrosis of the tubular epithelial cells in the kidneys of the treated animals . The degenerative and atrophic changes where observed more in the kidneys of rats that received the higher dose (0.2 g) of nutmeg . It may be inferred from the present results that higher doses of nutmeg consumption may have resulted in degenerative and atrophic changes observed in the renal corpuscle . The possible deduction from these results is that secondary metabolites, which are largely responsible for therapeutic or pharmacological activities of medicinal plants, may also account for their toxicity when the dosage is abused . The actual mechanism by which nutmeg induced cellular degeneration observed in this experiment needs further investigation . The necrosis observed is probably due to the high concentration of nutmeg on the kidney . Pathological or accidental cell death is regarded as necrotic and could result from extrinsic insults to the cell as osmotic thermal, toxic and traumatic effect . Physiological cell death is regarded as apoptotic and organized programmed cell death (pcd) that is mediated by active and intrinsic mechanisms . The process of cellular necrosis involves disruption of membranes, as well as structural and functional integrity . Cellular necrosis is not induced by stimuli intrinsic to the cells as in programmed cell death (pcd), but by an abrupt environmental perturbation and departure from the normal physiological conditions . In cellular necrosis, the rate of progression depends on the severity of the environmental insults: the greater the severity of the insult, the more rapid the progression of cellular injury . It may be inferred from the present study that prolonged administration and higher doses of nutmeg resulted in increased toxic effect on the kidney . Our results are in agreement with previous studies that indicated nutmeg to be toxic to the kidneys when abused . The results obtained in this study revealed that nutmeg consumption could affect the histology of the kidney of adult wistar rat; causing disruptions and distortions of the cyto - architecture of the kidneys . This resulted in the cellular necrosis, and sparsely distribution of the bowman's spaces . It is recommended that caution should therefore be advocated in the intake of this product and further studies be carried out to examine these findings.
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A 74 year old male with a past medical history significant for hypertension, hypercholesterolemia, hypothyroidism, and barrett's esophagus presented with gradually progressive bilateral lower extremity weakness and urinary retention . The patient gradually developed difficulty with ambulation to the point that he needed assistance getting up and with transfers . Upon electrolytes were within normal limits: sodium 145 meq / l, potassium 3.5 meq / l, chloride 107 meq / l, bun 28 mg / dl, cr 0.9 mg / dl, glucose 102 mg / dl, calcium 9.6 mg / dl . Cbc showed wbc 4.6 thou / cumm, hemoglobin 13.4 gm / dl, hematocrit 39%, mcv 96 fl, platelet count 120 thou / cumm, polys 64% . Folate level was> 20 ng / ml, vitamin b12 level was 667 pg / ml, htlv antibody was negative, rpr was nonreactive . Unenhanced magnetic resonance imaging (mri) of the cervical spine showed no evidence of cord signal abnormality or evidence of fracture . Unenhanced mri of the thoracic spine showed abnormal hyperintense t2 signal and hypointense t1 signal extending from the t6 vertebral body level down to the conus medullaris with smooth mild cord expansion at the conus medullaris (figure 1). A flame shaped hyperintense t2 signal abnormality sparing the chord peripherally with a low peripheral t2 signal throughout the intradural central canal . The findings were compatible with type i dural arteriovenous fistula . Combined, pre- and post - contrast mri of the thoracic spine revealed enhancement of all the serpiginous intradural blood vessels along the posterior aspects of the thoracic spinal cord (figure 2). This strengthened the leading diagnosis of type i spinal dural arteriovenous fistula (sdavf). There were multiple flow voids noted on the t2 sequence within the intradural space and surrounding the cauda equina nerve roots . Follow - up post - contrast study of the lumbar spine showed evidence of enhancing vessels in an intradural location, surrounding the conus medullaris (figures 3 and 4). Figure 1sagittal t2-weighted image of the thoracic spine demonstrate hyper intense t2 signal abnormality within the central area of the thoracic spinal cord extending from t6 to the conus medullaris . Sagittal t2-weighted image of the thoracic spine demonstrate hyper intense t2 signal abnormality within the central area of the thoracic spinal cord extending from t6 to the conus medullaris . Figure 2post - contrast image of the thoracic spine demonstrates multiple intradural flow voids (arrows). Post - contrast image of the thoracic spine demonstrates multiple intradural flow voids (arrows). Figure 3sagittal t1 fat saturated sequence post - contrast shows prominent tortuous intradural veins from level t1112 down to the cauda equina nerve roots (arrow) sagittal t1 fat saturated sequence post - contrast shows prominent tortuous intradural veins from level t1112 down to the cauda equina nerve roots (arrow) figure 4axial t1 fat saturated post contrast image shows enhancement of prominent intradural veins (arrow). Axial t1 fat saturated post contrast image shows enhancement of prominent intradural veins (arrow). Figure 5axial t2 sequence shows hyper intense t2 signal in the central aspect of the thoracic spinal cord with several prominent intradural flow voids (arrow). Axial t2 sequence shows hyper intense t2 signal in the central aspect of the thoracic spinal cord with several prominent intradural flow voids (arrow). Figure 6axial t2 sequence shows hyper intense t2 signal in the central aspect of the lumbar spinal cord with several prominent intradural flow voids (arrow). Axial t2 sequence shows hyper intense t2 signal in the central aspect of the lumbar spinal cord with several prominent intradural flow voids (arrow). Figure 7sagittal t2-weighted sequence shows hyper intense abnormality throughout the conus medullaris with sparing of the periphery . Multiple intradural flow voids are seen from the level of the conus medullaris down to the cauda equina nerve roots (arrows). Sagittal t2-weighted sequence shows hyper intense abnormality throughout the conus medullaris with sparing of the periphery . Multiple intradural flow voids are seen from the level of the conus medullaris down to the cauda equina nerve roots (arrows). Figure 8stir sequence of the lumbar spine shows abnormality throughout the conus medularis with sparing of the periphery (arrows). Stir sequence of the lumbar spine shows abnormality throughout the conus medularis with sparing of the periphery (arrows). Figure 9stir image of the thoracic spine demonstrate hyper intense t2 signal abnormality within the central area of the thoracic spinal cord extending from t6 to the conus medullaris (arrow). Stir image of the thoracic spine demonstrate hyper intense t2 signal abnormality within the central area of the thoracic spinal cord extending from t6 to the conus medullaris (arrow). Sdavf is a rare spinal vascular malformation which can lead to significant morbidity when left untreated . A number of classifications that reflects the complex anatomy of these vascular malformations have been proposed . The point at which a meningeal artery pierces the dura represents a potential site for the development of dural arteriovenous fistula . Based on a review of the records of 81 patients with spinal arteriovenous lesions, rosenbaum et al . Classified spinal arteriovenous lesions into four types, the most common of which is the sdavf, type i. depending on the number of arterial feeders, type i spinal arteriovenous fistulas are divided into type ia and type ib . A type ia fistula has one feeder and a type ib fistula has multiple feeders . The nidus of a sdavf is located in the dura covering the proximal nerve root and adjacent spinal dura . Additionally, intradural arteriovenous malformations (avms) are classified into glomus (type ii), juvenile (type iii), and direct (type iv). In juvenile avms,, there is no intervening glomus of vessels . In a retrospective analysis of 156 sdavf cases, these patients often present with progressive myelopathy, claudication, sensory loss, bowel and bladder dysfunction . In sdavfs, a dural branch of a segmental spinal artery fills the pial coronal venous plexus by draining into a medullary vein . The median time from symptom onset to diagnosis falls in the range of 1244 months . A sdavf appears as a hyperintense lesion on t2-weighted images, and often a corresponding hypointense signal can be found on t1-weighted images . Additional mri findings include prominent intradural veins, spinal cord enhancement, enlargement, scalloping and irregular cord surface . Although the above mri findings are nonspecific, they should prompt further investigation with arteriography . Myelography is recommended by some authors for patients whose clinical findings are consistent with sdavf but have negative or equivocal mri findings . Postgadolinium mr angiography can aid in the diagnosis of sdavfs by supplementing the information provided by the mri . It can depict abnormal intradural vessels, can be helpful in determining the level of the fistula and can be used for treatment follow - up . It allows for more detailed characterization of the anatomy of the fistula and its feeding vessels . There are no exact recommendations and at this time optimal treatment is planned on an individual basis . Endovascular embolization can sometimes lead to incomplete obliteration of the fistula and has been reported to have higher failure rates when compared to surgery . Sdavf is an important treatable condition that should be considered in the differential diagnosis of patients with progressive myelopathy . Due to the rarity of the condition and its non - specific clinical presentation, a high index of suspicion, the condition can be often misdiagnosed as a spinal cord tumor leading to surgical exploration . Although no concrete guidelines exist for the treatment of spinal sdavfs, treatment options include surgery and embolization.
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The fact that exercise and physical activity have positive effects on health is well known . Most of the research on exercise - induced changes carried out during the past years has mainly focussed on its impact on cardiovascular and musculoskeletal diseases . Only recently it has been noted, that exercise also leads to neural alterations that increase brain function and mental health . Neurobiological functioning in the human brain seems to depend upon an active or non - active lifestyle . Neuronal alterations can be induced lifelong but already in the fetal state movements of the unborn child and the mother can induce growth, development and networking of nerve cells . Therefore physical activity seems to be an important stimulus for neural adaptations of the brain in all age groups . The main effects of exercise on brain function are found in an altered blood flow (which might explain the lower risk of cerebrovascular diseases in an active population), reduced risk of neurodegenerative and age - related cognitive deficits [3, 6, 7] as well as improved learning and memory functions . Many studies show benefits due to exercise such as reduced age - related neuronal loss and an increase in cell proliferation and neurogenesis, the process by which new neurons are generated . In addition, recent studies have shown that exercise produces antidepressant responses in rodent models and moodelevating actions in humans [11, 12]. The antidepressants effects of exercise are of special interest, since major depressive disorder is a life threatening disease accompanied by a high risk of suicide and is a major cause of morbidity worldwide [13 - 15]. Therefore, the aim of this paper is to review the relationship between physical activity and depressive disorders and the potential neurobiological alterations induced by exercise that might lead to the relief of mental disorders like depression . To do so, we searched electronic databases for literature and reviewed articles concerning the latter phenomenon from 1963 until 2009 . Since mdd is a major health problem and the effectiveness of current medical antidepressants is only about 65%, the antidepressant actions of exercise are of immense interest . According to the global burden of disease study mild to moderate major depressive disorder (mdd) ranks now second behind ischemic heart disease for years of life lost due to early death or disability . Mdd is the most prevalent of all psychiatric disorders, affecting up to 25% of women and 12% of men during their lifetimes . According to greden et al . The pan - european study depres showed in 1997 that 13359 out of 78463 adults who participated in screening interviews across six countries in europe suffered from depression . The resulting economic burden is about $83.1 billion per year only in the usa . The main symptoms of mdd are depressed mood, anhedonia (lost of interest or pleasure), increased tiredness, irritability, difficulties in concentrating, abnormalities in appetite and sleep and suicidal intentions . Depressive symptoms are correlated with the presence of chronic disease, inability to work, increased mortality risk, increased use of medical services, decreased well being and lowered functioning . Ten percent of those diagnosed with mdd commit suicide [28, 29], depressed patients tend to develop coronary artery disease and type 2 diabetes . Today only 50% of all patients show complete remission, although up to 80% demonstrate partial responses . Furthermore, the medications require long - term treatment for weeks to months before a therapeutic response is achieved . Therefore, there is an enormous demand for more effective methods to treat depressive disorders . Although the prevalence of depression and its impact is high, knowledge about the pathophysiology of mdd is still not completely understood . That is primarily due to difficulties in observing pathological changes within the human brain and that most depressions occur idiopathically . The risk factors of depression are diverse like stressful life events, endocrine abnormalities (hypothyroidism and hypercortisolism), cancers and side effects of drugs [22, 32, 33]. The diagnosis of mdd bases on symptomatic criteria set forth in the diagnostic and statistical manual . It becomes clear from the criteria s that the diagnosis of depression is not based on objective diagnostic tests, but rather on a set of symptoms . It is a syndrome that consists of numerous diseases of different causes and pathophysiologies that makes the diagnosis of mdd subjective and is based on the documentation of certain symptoms over a time of at least two weeks . The diagnostic criterias overlap with other conditions such as anxiety disorders, which have substantial co - morbidity with depression [35, 36]. Epidemiological studies show that 40%50% of the risk to suffer from depression is genetic [37, 38]. Despite some promising leads, there are still no confirmed genetic findings for mood disorders . Nongenetic factors are as diverse as stress and emotional trauma, viral infections, and even stochastic processes during brain development have been implicated in the etiology of depression [38, 40]. Depressive syndromes occur in the context of innumerable medical conditions like endocrine disturbances (hyper- or hypocortisolemia, hyper- or hypothyroidism), collagen vascular diseases, parkinson s disease, traumatic head injuries, certain cancers, asthma, diabetes and stroke . Several brain regions and circuits that regulate emotion, reward and executive functions are implicated in this disease . Dysfunctional changes within the interconnected limbic region have been implicated in depression and also in antidepressant action . A large body of post - mortem and neuroimaging studies of depressed patients have reported reductions in grey - matter volume, glial density in the prefrontal cortex and the hippocampus . These regions are thought to mediate the cognitive aspects of depression, such as feelings of worthlessness and guilt [33, 42, 43]. Patients with depression have shown to suffer from statistically significant smaller left hippocampal volume than non - depressive comparison subjects . In this study magnetic resonance imaging (mri) was used to measure the volume of the hippocampi in 16 patients with major depression (10 men, 6 women) and 16 case - matched non - depressed controls . Patients with a history of post - traumatic stress disorder or current medication use other than antidepressant were excluded from this investigation . The findings of this study showed that the right hemisphere suffered from a reduction of hippocampal volume by 12% but without statistical significance . The left hemisphere showed a significant reduction in volume of the hippocampus by 19% in the depressed patients compared to the matched controls . These results suggest that depression causes loss of brain volume observed in the hippocampi, especially in the left hemisphere . Data from epidemiological studies suggests an association between physical inactivity and higher levels of depressive symptoms [45, 46]. It has been shown that reduced physical activity leads to increased symptoms of depression in older adults and that depressive symptoms decrease when physical activity is resumed . Blumenthal et al . (1999) could show that the influence of a 16-week exercise training program as a therapeutic treatment of depressive patients is as effective as antidepressive medications . 156 men and women with diagnosed mdd (50 years) were randomly assigned into three groups of interest: (1) aerobic exercise, (2) antidepressants (sertraline hydrochloride) and (3) combined exercise and medication group . The subjects attended three supervised exercise sessions per week for 16 consecutive weeks at an intensity of 70% to 85% of heart rate reserve that was calculated from the maximum heart rate . The maximum heart rate was achieved during a treadmill test every participant had to fulfil in advance . Each aerobic exercise session began with 10-minutes warm - up exercise, followed by 30 minutes of continuos walking or jogging at the described intensity . The heart rate was monitored and recorded 3 times per session by a trained exercise physiologist via radial pulses . The study could show that 16 weeks of treatment exercise was equally effective in reducing depression among patients with mdd as antidepressants . Several meta - analyses [49 - 54] studied the impact of exercise on depression and all concluded that exercise had positive effects . Two studies concluded that more intense exercise led to larger improvements in mood [55, 56]. There is evidence that physical activity induces physiological changes in endorphine and monoamine levels, and also reduces the levels of the stress hormone cortisol . Recent studies suggested that exercise stimulates the growth of new nerve cells and induces the release of proteins and peptides, which are known to improve health and survival of nerve cells, such as brain - derived neurotrophic factor (bdnf), vascular endothelial growth factor (vegf), insulin - like growth factor (igf-1) and the gene vgf (nerve growth factor inducible) [58 - 62]. Even though the effectiveness of exercise in decreasing symptoms of depression has been well established, mead et al . Concluded in 2009, after reviewing articles concerning the influence of exercise on depressive symptoms, that the effect of exercise was not significant . Lepore infered that exercise may only be a diversion from negative thoughts and social contacts might influence the positive outcome . Especially the determination regarding the optimum type, frequency and duration of exercise is questioned by mead et al ., 2008 . He points out that future research has to consider the design of exercise to determine more specifically what kind of exercise is of benefit and what not, e.g. Whether exercise should be performed supervised or unsupervised, indoors or outdoors, or in a group or alone . The monoamine hypothesis of depression, which postulates that depression is caused by decreased monoamine function, especially serotonin (5-hydroxytryptamine 5-ht) and norepinephrine (ne) in the brain, originated from early clinical observations [41, 65]. Today s antidepressant and anxiolytic drugs such as tricyclic antidepressants (tcas), monoamine oxidase inhibitors (maois), serotonin - norepinephrine reuptake inhibitors (snris) and selective serotonin reuptake inhibitors (ssris) are still designed to increase monoamine transmission acutely . They primarily affect the serotonergic and/or the norepinephrine system, whether by inhibiting the reuptake of serotonin and/or norepinephrine into the presynapse or by inhibiting the activity of monoamine oxidase, thus preventing the breakdown of monoamine neurotransmitters and thereby increasing the availability of serotonin and/or norepinephrine in the synaptic cleft [67, 68]. Although these monoamine - based agents are potent antidepressants, the cause of depression is far from being due to a simple deficiency of central monoamines . The problem is that the maois and ssris produce immediate increases in monoamine transmission, whereas their mood - enhancing properties require weeks of treatment . Because of this delay in time it is thought that the acute increases in the amount of synaptic monoamines induced by antidepressants produce secondary neuroplastic changes that occur over a longer timescale and involve transcriptional and translational changes that mediate molecular and cellular plasticity [22, 65]. Nevertheless monoamine - based antidepressants remain the first line of therapy for depression, but their long therapeutic delays in time and low remission rates (about 30%) have encouraged the search for more effective agents [41, 69]. One of the mechanisms through which exercise produces the antidepressant effects might be similar to that of the antidepressant drug treatment since exercise also affects the central serotonergic system . The synthesis of brain 5-ht depends on two main variables, the neuronal concentration of its precursor, tryptophan (trp), and the activity of its rate - limiting enzyme, tryptophan hydroxylase (tph; converts tryptophan into 5-hydroxytryptophan). Acute physical exercise increases blood free tryptophan and decreases albumin bound tryptophan both in animals [71 - 73] and humans [74 - 76] by increasing the rate of lipolysis . It was shown in humans that an increase in levels of the serotonin metabolite, 5-hydroxyindoleacetic acid follows physical exercise . Since trp is competing with other amino acids like valine, leucine and isoleucine to enter the brain, it has also been demonstrated that exercise decreases the levels of these amino acids leading to higher availability of the serotonin precursor trp in the brain [78 - 80]. Therefore the higher concentrations of trp in blood plasma and also in the cerebrospinal fluid following exercise enhance the serotonin neurotransmission in the brain . Other experiments with animals have demonstrated an immediate increase in the activity of brain cells that produce norepinephrine after acute exercise [81 - 83]. Since serotonin - norepinephrine reuptake inhibitors is a common choice of treatment that acts antidepressive by inhibiting the reuptake norepinephrine into the presynapse and thereby increasing the availability of norepinephrine in the synaptic cleft [67, 68], it is noteworthy that the same effects can be achieved by exercise . Increased levels of norepinephrine and its metabolites as well as the activation of tyrosine hydroxylase, an enzyme that is involved in the production of norepinephrine is also observed after acute [81 - 83] and chronic exercise in animals [84 - 86]. Therefore it can be presumed that excercise produces the same mood - elevating effects as antidepressants by altering the availability of norepinephrine . Although not as consistent yet nevertheless notable is the effect of exercise on the levels of dopamine as an antidepressant factor . It has been demonstrated that dopamine activity is increased following exercise [77, 87]. Dopamine seems to play an important role in patients with parkinson s disease but has also been described to correlate to motivational problems and anhedonia seen in patients affected by mdd . A common feature of addictive drugs is that they alter the levels of dopamine in the nucleus accumbens . It has been observed in rodents that running increases levels of dopamine in the nucleus accumbens and that those animals can be trained to lever press for access to running wheels to get their reward . Similar behaviour can be observed in humans that train excessively which can result in fatigue and mood disturbances as been reported in overstrained humans . Therefore dopamine seems to be of certain relevance why exercise can be addictive and reinforcing, and also why it has its antidepressant effect on humans . As in the case of antidepressants exercise induces higher concentrations of serotonin and/or norepinephrine but this cannot explain the observed mood - elevating delay in time . Therefore neuroplastic changes that involve transcriptional and translational changes would appear to play a critical role in the treatment of mdd (see chapter: 1.6 neurotrophic factors and neurogenesis). Depression is often described as a stress - related disorder, and there is evidence that episodes of depression occur in the context of some form of stress . Even though, stress per se is not sufficient to cause depression but early clinical studies identifying reproducible but small increases in serum glucocorticoid concentrations in depression [92, 93] led to a significant interest in the role of a dysfunctional hypothalamic physical or psychological stress increases serum glucocorticoid concentrations, and some depression - like symptoms can be produced in rodents by chronic administration of glucocorticoids . High levels of glucocorticoids can reduce hippocampal subgranular zone (sgz) proliferation rates and produce atrophic changes in hippocampal subregions . Patients with cushing s syndrome, who have extremely high concentrations of circulating cortisol, also show depressive features and atrophic changes in the hippocampus [22, 97]. Several metabolic abnormalities that are often associated with depression, such as insulin resistance and abdominal obesity, can be at least partly explained by an increase in glucocorticoids [32, 98]. Hypercortisolaemia in depression is manifested at several levels, including impaired glucocorticoid - receptor - mediated negative feedback, adrenal hyper - responsiveness to circulating adrenocorticotropic hormone (acth) and hypersecretion of corticotrophin - releasing factor (crf), the hypothalamic activator of acth release from the pituitary . Chronic antidepressant administration has shown to increase the concentration of corticosteroid receptors, which can restore hpa negative feedback and normalize cortisol levels and hpa function . Therefore it appears that there is an interrelationship between stress, high glucocorticoid levels and depression . But not only antidepressants, also exercise can induce changes on the functioning of the hpa axis . Although acute high intensity physical activity leads to increased levels of stress hormones corticotropin and cortisol, long - term exercise (meaning that the body adapts to training stimuli) attenuates the human stress response [101 - 103]. Exercise can be a stressful stimulus itself depending on the intensity and duration of the activity so that stressful stimulations like exercise need to be followed by adaptations of the organism . If the organism becomes adapted to exercise, then the subsequent response of catecholamine release to stressful intensities of exercise is less than that observed in nontrained subjects . After a training program undertaken at moderate intensities for 4 weeks, the organism already reacts with lower concentrations of acth and cortisol to exercise [104, 105]. Furthermore, the effects of exercise in trained subjects indicate that after ending the exercise, the concentrations of cortisol reach their basic levels faster than in untrained subjects . Whether these effects of lower reactivity to stressful exercise events can be related to stressful events in daily life remains unclear . A meta analysis of crew and landers including 34 studies, 92 effect strengths (es), n=1.449 demonstrated a correlation between the level of fitness and reactivity to stressful events (es=.48) this study demonstrated that trained subjects do not react as strongly to stress as untrained subjects exposed to stress . The problem with the latter study was the measured outcome of stress e.g. Cardiovascular parameters . In nearly all stress - exercise - related situations, untrained individuals react with higher heart frequencies but data regarding physiological parameters such as noradrenaline, adrenaline or acth levels are generally missing . In many reviews and meta analyses [108 - 110] that have investigated the correlation between the level of fitness (by maximal and submaximal exercise tests) and stressors it was shown that trained subjects exhibit a higher reactivity to stress (es=.08, p<.001) and recover faster from stress too (37 studies, 118 es, n=1.092). Most effects were demonstrated in heart frequency, blood pressure, blood flow and vascular resistance . Animal studies have indicated that animals that exercised voluntarily show improved stress - coping abilities in physically demanding and psychological challenges . The latter improved stress - coping abilities appeared as adaptive responses of the hpa axis [110 - 112], improvements in sleep quality and increased stress resistance of sleep / eeg profiles, and also reduced anxiety - related behaviour in voluntary exercised mice and rats compared to sedentary control animals . Decreases in volume observed in the hippocampi and other regions of the forebrain in depressed patients have supported a hypothesis for depression involving decrements in neurotrophic factors [115, 116]. Neurotrophic factors are known to regulate neural growth and differentiation during development and are also regulators of plasticity and survival of adult neurons and glia . Support for the bdnf hypothesis of depression has come from a large preclinical literature showing that stress can reduce bdnf - mediated signalling in the hippocampus, whereas chronic treatment with antidepressants increases bdnf - mediated signalling . Similar changes have been observed in the post - mortem hippocampus, as well as in serum bdnf- concentrations of humans with depression . The second support for the theory that neurotrophic factors are of importance in treating depression is based upon the time delay of the mood - elevating effects of antidepressants, which is only seen after prolonged administration (several weeks to months). The cellular effect of antidepressants is the induction of hippocampal neurogenesis - the process by which neural progenitors of the sgz divide mitotically to form new neurons that differentiate and integrate into the dentate gyrus [65, 118]. Blockade of hippocampal neurogenesis inhibits the therapeutic - like effects of most antidepressant treatments in rodent models . Moreover, antidepressant treatment, possibly through the actions of transcription factor camp response element binding protein (creb) or other transcriptional regulators [15, 65], increases the amounts of several growth factors in the hippocampus that influence neurogenesis . These include bdnf as well as vegf and the recently discovered neuropeptide vgf, which themselves have antidepressant and pro - neurogenic properties in rodents [119 - 121]. Furthermore, both central and systemic administration of igf-1 increases hippocampal cell proliferation and neurogenesis in the adult rat [122, 123]. Central administration of igf-1 has shown to produce antidepressant - like effects in the rat forced swim test . This data supports the neurotrophic hypothesis of depression, which means that neuronal adaptations induced by antidepressant drugs are necessary to produce mood - elevation effects . This supports the theory that neurotrophic factors play a key role in the relief of depressive symptoms . Like antidepressants, exercise can also increase the synthesis of new neurons in the adult brain and therefore induce mood - elevating effects . Van praag et al . (1999) observed an increase in hippocampal neurogenesis in rats with regular access to a running wheel . Recent studies demonstrated that adult neurogenesis can be influenced by stress, ageing, environmental enrichment [127, 128] and physical activity [9, 129]. Kempermann et al . In 1997 showed the positive effects of environmental enrichment on neurogenesis in mice . These mice were also tested in a spatial memory task, the morris water maze, in which the enriched animals learned faster than control animals suggesting the possibility that the new neurons cause enhanced cognition . Experiments comparing animals undergoing exercise (wheel running) and animals raised in an enriched environment without exercise showed more bromodeoxyuridine (brdu; a synthetic nucleoside, used in the detection of proliferating cells)-positive cells in the runners group than in the group that was exposed to enriched environment without exercise . Further investigations demonstrated that already 10 days of wheel running increases cell genesis in rodents [131 - 133]. The increase of hippocampal neurogenesis by running became strongly manifested [134 - 139] that is also associated with improved hippocampal synaptic plasticity . The mechanisms by which exercise induces neurogenesis is based on the increase of following molecules: bdnf, vegf, igf-1, the neuropeptide vgf, 5-ht and -endorphins [119, 134, 141]. As already mentioned several days of voluntary wheel running enhance the levels of bdnf mrna in the hippocampus as has been shown in several studies [141 - 147]. The changes in the mrna were found in neurons of the dentate gyrus (dg), the hilus and the ca3 region of the hippocampus . In addition to the hippocampus, exercise also augmented levels of bdnf mrna in the lumbar spinal cord, the cerebellum and the cortex . Other growth factors like nerve growth factor (ngf) and it is well known that -endorphins are increased after exercise [150, 151]. It has been shown that the infusion of opiates induces an increase in cell proliferation and also that antagonists of the opiate receptor decrease cell proliferation in the dentate gyrus [152, 153]. Infusion of recombinant protein in mammals to elevate the levels of vegf, a protein secreted from blood that acts on endothelial cells to stimulate the formation of bloodvessels, has been shown to increase cell proliferation in the adult hippocampus and ventricular zone . It was demonstrated that the levels of vegf are also elevated following exercise [61, 155]. Pointed out in 2003 that vegf is necessary for the effects of running on adult hippocampal neurogenesis whereas peripheral blockade of vegf neutralizes running - induced neurogenesis . Another growth factor that is up - regulated in the brain and in the periphery after exercise is the insulin - like growth factor igf-1 . Igf-1, structurally related to pro - insulin, plays an important role in depressive disorders by contributing to neural development through neurogenesis and synaptogenesis, facilitating oligodendrocyte survival and stimulating myelination [157 - 159]. Igf-1 promotes cell proliferation and inhibits cell death during healthy but also during stressed or diseased states . Peripheral administration of igf-1 has been shown to induce up - regulation of bdnf mrna levels in the brain . Therefore it is suggested that igf-1 initiates growth factor cascades in the brain that can alter mechanisms of plasticity . Furthermore, carro et al . Could show in three experiments that exercise has neuroprotective effects by its increased passage of circulating igf-1 into the brain since after blocking the passage exercise no longer worked neuroprotective in simulated brain insults in rodents . Further evidence comes from fernandez et al . Who could show that systemic administration of igf-1 to brain - damaged sedentary mice or rats is sufficient to elicit functional recovery after simulated brain insult in rodents . Based on these findings circulating igf - i has a physiological neuroprotective tonic effect on the brain that is depressed in sedentary subjects . Hunsberger et al . Used a microarray technique to show that exercise upregulates a primary signaling cascade for neurotrophic factors and a peptide precursor, vgf . Furthermore, it was demonstrated that vgf induces synaptic plasticity genes that are also altered after exercise (nrn1 and syn1) [162, 163]. It is remarkable that exercise regulates so many genes especially in the hippocampus and underscores that exercise can be a potent tool to influence brain metabolic functions . Recent research has shown that pro - inflammatory cytokines not only induce " sick symptoms ", but also impinge on physically ill patients by leading to depressive disorders . In approximate 33% of patients who are treated by recombinant human cytokines interleukin-2 (il-2) and interferon- (ifn-) major depressive disorder is observed . It has been shown in animal models of inflammation that existing states of decreased reactivity to reward (anhedonia) and reduced social exploration can be reversed by antidepressant treatment . Sickness is basically an adaptive response to infection . As in the case of depressive disorders, but unlike depression, sickness is completely reversible once the disease - causing agent has been eliminated . Van den biggelaar et al . Studied 267 people at the age of 85 without any psychiatric history . In this study certain mediators like pro - inflammatory cytokines are produced in an infection that contain interleukin-1 and (il-1, il-1), tumor necrosis factor- (tnf-) and interleukin-6 (il-6). These in the periphery produced cytokines also act on the brain causing behavioral symptoms postulated as " sickness behavior " [166, 167]. It has been repeatedly observed in patients suffering from major depression that the levels of pro - inflammatory cytokines, acute - phase proteins, chemokines and adhesion molecules are increased [168 - 175]. The most frequently observed alterations are increased levels of il-6 in the plasma as in the serum and/or elevations of c - reactive protein [166, 168 - 171]. Further alterations were observed in elevated concentrations of il- - and tnf- in peripheral blood and in the cns of patients suffering from mdd [172, 175, 176]. Major depressive disorders caused by immunotherapy in cancer or hepatitis c patients who were receiving immunotherapy supported the theory of cytokine - induced depression first postulated by smith and later by maes . Behavioral data in animal studies have indicated a relationship between cytokines and depression . Systemic administration of lipopolysaccharide (lps) induced the expression of il-1 and other pro - inflammatory cytokine mrnas and proteins in the brain in many studies [179 - 182] in addition showing that depressive - like behaviour remained after sickness behaviour had already retreated . Frenois et al . Observed a decrease in the preference for a sucrose solution, a phenomenon that was still apparent when food intake and drinking had already normalized . If the animals received antidepressants before lps - treatment the reduced intake of a sweetened solution was neutralized . Another link in favour of relationship between cytokines and depression stems from the fact that immunotherapy reduces the plasma levels of tryptophan which determines the rate of serotonin synthesis in the brain . A key role in the context of inflammation and depressive disorders seems to play il-1- that inhibits the expression of bdnf in the hippocampus of rats after undergoing social isolation . Stress - induced neuronal cell loss in animals is also associated with increased levels of tnf- and nf- b (nuclear factor' kappa - light - chain - enhancer' of activated b - cells). Over - expression of tnf- is observed in decelerated brain growth and neural damage, which is associated with reduced igf-1 activity, in this case especially in the cerebellum . This finding is of great interest since igf-1 can therefore act as an anti - inflammatory cytokine in the brain and can also be induced by exercise . Exercise has been shown to influence the immune system and seems to play an important role in the relationship between the immune function and depressive disorders . During exercise, the cascade in cytokine response differs from the " classical " response to infections represented by the onset of circulating il-6 during exercise . Epidemiological data suggests a relationship between physical inactivity and low - grade inflammation in healthy subjects [190 - 192]. Could (2003) show that exercise in the form of 3 hours ergometer cycling can suppress endotoxin - induced tnf- production . Exercise works as an anti - inflammatory agent by leading to higher levels of il-6 which is followed by raising il-1ra and il-10 levels and also by suppression of tnf- production as demonstrated in animals and in vitro studies . Exercise gives rise to high levels of epinephrine that has also been shown after infusion to inhibit tnf- production in response to endotoxin in vivo . Except for strenuous exercise which is mainly pro - inflammatory, the exact dose of exercise that has anti - inflammatory effects has not been clearly established . However, the data suggests that moderate aerobic exercise seems to induce the most promising effects considering the anti - inflammatory and antidepressive outcomes . To summarize the relationship between depressive disorder, cytokines and exercise, epidemiological data shows the correlation between physical inactivity and low - grade inflammation [190 - 192]. Since immunotherapy reduces plasma levels of tryptophan, it is noteworthy that levels of tryptophan can be directly influenced by exercise . As already mentioned acute physical exercise increases blood free tryptophan and in animals [71 - 73] and humans [74 - 76]. And also igf-1, which counteracts the behavioral depressing effects of cytokines, can be influenced by physical activity [60, 156]. Since mdd is a major health problem and the effectiveness of current medical antidepressants is only about 65%, the antidepressant actions of exercise are of immense interest . According to the global burden of disease study mild to moderate major depressive disorder (mdd) ranks now second behind ischemic heart disease for years of life lost due to early death or disability . Mdd is the most prevalent of all psychiatric disorders, affecting up to 25% of women and 12% of men during their lifetimes . According to greden et al . The pan - european study depres showed in 1997 that 13359 out of 78463 adults who participated in screening interviews across six countries in europe suffered from depression . The resulting economic burden is about $83.1 billion per year only in the usa . The main symptoms of mdd are depressed mood, anhedonia (lost of interest or pleasure), increased tiredness, irritability, difficulties in concentrating, abnormalities in appetite and sleep and suicidal intentions . Depressive symptoms are correlated with the presence of chronic disease, inability to work, increased mortality risk, increased use of medical services, decreased well being and lowered functioning . Ten percent of those diagnosed with mdd commit suicide [28, 29], depressed patients tend to develop coronary artery disease and type 2 diabetes . Only 50% of all patients show complete remission, although up to 80% demonstrate partial responses . Furthermore, the medications require long - term treatment for weeks to months before a therapeutic response is achieved . Therefore, there is an enormous demand for more effective methods to treat depressive disorders . Although the prevalence of depression and its impact is high, knowledge about the pathophysiology of mdd is still not completely understood . That is primarily due to difficulties in observing pathological changes within the human brain and that most depressions occur idiopathically . The risk factors of depression are diverse like stressful life events, endocrine abnormalities (hypothyroidism and hypercortisolism), cancers and side effects of drugs [22, 32, 33]. The diagnosis of mdd bases on symptomatic criteria set forth in the diagnostic and statistical manual . It becomes clear from the criteria s that the diagnosis of depression is not based on objective diagnostic tests, but rather on a set of symptoms . It is a syndrome that consists of numerous diseases of different causes and pathophysiologies that makes the diagnosis of mdd subjective and is based on the documentation of certain symptoms over a time of at least two weeks . The diagnostic criterias overlap with other conditions such as anxiety disorders, which have substantial co - morbidity with depression [35, 36]. Epidemiological studies show that 40%50% of the risk to suffer from depression is genetic [37, 38]. This makes depression a highly hereditary disorder . Despite some promising leads, there are still no confirmed genetic findings for mood disorders . Nongenetic factors are as diverse as stress and emotional trauma, viral infections, and even stochastic processes during brain development have been implicated in the etiology of depression [38, 40]. Depressive syndromes occur in the context of innumerable medical conditions like endocrine disturbances (hyper- or hypocortisolemia, hyper- or hypothyroidism), collagen vascular diseases, parkinson s disease, traumatic head injuries, certain cancers, asthma, diabetes and stroke . Several brain regions and circuits that regulate emotion, reward and executive functions are implicated in this disease . Dysfunctional changes within the interconnected limbic region have been implicated in depression and also in antidepressant action . A large body of post - mortem and neuroimaging studies of depressed patients have reported reductions in grey - matter volume, glial density in the prefrontal cortex and the hippocampus . These regions are thought to mediate the cognitive aspects of depression, such as feelings of worthlessness and guilt [33, 42, 43]. Patients with depression have shown to suffer from statistically significant smaller left hippocampal volume than non - depressive comparison subjects . In this study magnetic resonance imaging (mri) was used to measure the volume of the hippocampi in 16 patients with major depression (10 men, 6 women) and 16 case - matched non - depressed controls . Patients with a history of post - traumatic stress disorder or current medication use other than antidepressant were excluded from this investigation . The findings of this study showed that the right hemisphere suffered from a reduction of hippocampal volume by 12% but without statistical significance . The left hemisphere showed a significant reduction in volume of the hippocampus by 19% in the depressed patients compared to the matched controls . These results suggest that depression causes loss of brain volume observed in the hippocampi, especially in the left hemisphere . Data from epidemiological studies suggests an association between physical inactivity and higher levels of depressive symptoms [45, 46]. It has been shown that reduced physical activity leads to increased symptoms of depression in older adults and that depressive symptoms decrease when physical activity is resumed . Blumenthal et al . (1999) could show that the influence of a 16-week exercise training program as a therapeutic treatment of depressive patients is as effective as antidepressive medications . 156 men and women with diagnosed mdd (50 years) were randomly assigned into three groups of interest: (1) aerobic exercise, (2) antidepressants (sertraline hydrochloride) and (3) combined exercise and medication group . The subjects attended three supervised exercise sessions per week for 16 consecutive weeks at an intensity of 70% to 85% of heart rate reserve that was calculated from the maximum heart rate . The maximum heart rate was achieved during a treadmill test every participant had to fulfil in advance . Each aerobic exercise session began with 10-minutes warm - up exercise, followed by 30 minutes of continuos walking or jogging at the described intensity . The heart rate was monitored and recorded 3 times per session by a trained exercise physiologist via radial pulses . The study could show that 16 weeks of treatment exercise was equally effective in reducing depression among patients with mdd as antidepressants . Several meta - analyses [49 - 54] studied the impact of exercise on depression and all concluded that exercise had positive effects . Two studies concluded that more intense exercise led to larger improvements in mood [55, 56]. There is evidence that physical activity induces physiological changes in endorphine and monoamine levels, and also reduces the levels of the stress hormone cortisol . Recent studies suggested that exercise stimulates the growth of new nerve cells and induces the release of proteins and peptides, which are known to improve health and survival of nerve cells, such as brain - derived neurotrophic factor (bdnf), vascular endothelial growth factor (vegf), insulin - like growth factor (igf-1) and the gene vgf (nerve growth factor inducible) [58 - 62]. Even though the effectiveness of exercise in decreasing symptoms of depression has been well established, mead et al . Concluded in 2009, after reviewing articles concerning the influence of exercise on depressive symptoms, that the effect of exercise was not significant . Lepore infered that exercise may only be a diversion from negative thoughts and social contacts might influence the positive outcome . Especially the determination regarding the optimum type, frequency and duration of exercise is questioned by mead et al ., 2008 . He points out that future research has to consider the design of exercise to determine more specifically what kind of exercise is of benefit and what not, e.g. Whether exercise should be performed supervised or unsupervised, indoors or outdoors, or in a group or alone . The monoamine hypothesis of depression, which postulates that depression is caused by decreased monoamine function, especially serotonin (5-hydroxytryptamine 5-ht) and norepinephrine (ne) in the brain, originated from early clinical observations [41, 65]. Today s antidepressant and anxiolytic drugs such as tricyclic antidepressants (tcas), monoamine oxidase inhibitors (maois), serotonin - norepinephrine reuptake inhibitors (snris) and selective serotonin reuptake inhibitors (ssris) are still designed to increase monoamine transmission acutely . They primarily affect the serotonergic and/or the norepinephrine system, whether by inhibiting the reuptake of serotonin and/or norepinephrine into the presynapse or by inhibiting the activity of monoamine oxidase, thus preventing the breakdown of monoamine neurotransmitters and thereby increasing the availability of serotonin and/or norepinephrine in the synaptic cleft [67, 68]. Although these monoamine - based agents are potent antidepressants, the cause of depression is far from being due to a simple deficiency of central monoamines . The problem is that the maois and ssris produce immediate increases in monoamine transmission, whereas their mood - enhancing properties require weeks of treatment . Because of this delay in time it is thought that the acute increases in the amount of synaptic monoamines induced by antidepressants produce secondary neuroplastic changes that occur over a longer timescale and involve transcriptional and translational changes that mediate molecular and cellular plasticity [22, 65]. Nevertheless monoamine - based antidepressants remain the first line of therapy for depression, but their long therapeutic delays in time and low remission rates (about 30%) have encouraged the search for more effective agents [41, 69]. One of the mechanisms through which exercise produces the antidepressant effects might be similar to that of the antidepressant drug treatment since exercise also affects the central serotonergic system . The synthesis of brain 5-ht depends on two main variables, the neuronal concentration of its precursor, tryptophan (trp), and the activity of its rate - limiting enzyme, tryptophan hydroxylase (tph; converts tryptophan into 5-hydroxytryptophan). Acute physical exercise increases blood free tryptophan and decreases albumin bound tryptophan both in animals [71 - 73] and humans [74 - 76] by increasing the rate of lipolysis . It was shown in humans that an increase in levels of the serotonin metabolite, 5-hydroxyindoleacetic acid follows physical exercise . Since trp is competing with other amino acids like valine, leucine and isoleucine to enter the brain, it has also been demonstrated that exercise decreases the levels of these amino acids leading to higher availability of the serotonin precursor trp in the brain [78 - 80]. Therefore the higher concentrations of trp in blood plasma and also in the cerebrospinal fluid following exercise enhance the serotonin neurotransmission in the brain . Other experiments with animals have demonstrated an immediate increase in the activity of brain cells that produce norepinephrine after acute exercise [81 - 83]. Since serotonin - norepinephrine reuptake inhibitors is a common choice of treatment that acts antidepressive by inhibiting the reuptake norepinephrine into the presynapse and thereby increasing the availability of norepinephrine in the synaptic cleft [67, 68], it is noteworthy that the same effects can be achieved by exercise . Increased levels of norepinephrine and its metabolites as well as the activation of tyrosine hydroxylase, an enzyme that is involved in the production of norepinephrine is also observed after acute [81 - 83] and chronic exercise in animals [84 - 86]. Therefore it can be presumed that excercise produces the same mood - elevating effects as antidepressants by altering the availability of norepinephrine . Although not as consistent yet nevertheless notable is the effect of exercise on the levels of dopamine as an antidepressant factor . It has been demonstrated that dopamine activity is increased following exercise [77, 87]. Dopamine seems to play an important role in patients with parkinson s disease but has also been described to correlate to motivational problems and anhedonia seen in patients affected by mdd . A common feature of addictive drugs is that they alter the levels of dopamine in the nucleus accumbens . It has been observed in rodents that running increases levels of dopamine in the nucleus accumbens and that those animals can be trained to lever press for access to running wheels to get their reward . Similar behaviour can be observed in humans that train excessively which can result in fatigue and mood disturbances as been reported in overstrained humans . Therefore dopamine seems to be of certain relevance why exercise can be addictive and reinforcing, and also why it has its antidepressant effect on humans . As in the case of antidepressants exercise induces higher concentrations of serotonin and/or norepinephrine but this cannot explain the observed mood - elevating delay in time . Therefore neuroplastic changes that involve transcriptional and translational changes would appear to play a critical role in the treatment of mdd (see chapter: 1.6 neurotrophic factors and neurogenesis). Depression is often described as a stress - related disorder, and there is evidence that episodes of depression occur in the context of some form of stress . Even though, stress per se is not sufficient to cause depression but early clinical studies identifying reproducible but small increases in serum glucocorticoid concentrations in depression [92, 93] led to a significant interest in the role of a dysfunctional hypothalamic pituitary adrenal axis (hpa) in the pathophysiology of depression . Physical or psychological stress increases serum glucocorticoid concentrations, and some depression - like symptoms can be produced in rodents by chronic administration of glucocorticoids . High levels of glucocorticoids can reduce hippocampal subgranular zone (sgz) proliferation rates and produce atrophic changes in hippocampal subregions . Patients with cushing s syndrome, who have extremely high concentrations of circulating cortisol, also show depressive features and atrophic changes in the hippocampus [22, 97]. Several metabolic abnormalities that are often associated with depression, such as insulin resistance and abdominal obesity, can be at least partly explained by an increase in glucocorticoids [32, 98]. Hypercortisolaemia in depression is manifested at several levels, including impaired glucocorticoid - receptor - mediated negative feedback, adrenal hyper - responsiveness to circulating adrenocorticotropic hormone (acth) and hypersecretion of corticotrophin - releasing factor (crf), the hypothalamic activator of acth release from the pituitary . Chronic antidepressant administration has shown to increase the concentration of corticosteroid receptors, which can restore hpa negative feedback and normalize cortisol levels and hpa function . Therefore it appears that there is an interrelationship between stress, high glucocorticoid levels and depression . But not only antidepressants, also exercise can induce changes on the functioning of the hpa axis . Although acute high intensity physical activity leads to increased levels of stress hormones corticotropin and cortisol, long - term exercise (meaning that the body adapts to training stimuli) attenuates the human stress response [101 - 103]. Exercise can be a stressful stimulus itself depending on the intensity and duration of the activity so that stressful stimulations like exercise need to be followed by adaptations of the organism . If the organism becomes adapted to exercise, then the subsequent response of catecholamine release to stressful intensities of exercise is less than that observed in nontrained subjects . After a training program undertaken at moderate intensities for 4 weeks, the organism already reacts with lower concentrations of acth and cortisol to exercise [104, 105]. Furthermore, the effects of exercise in trained subjects indicate that after ending the exercise, the concentrations of cortisol reach their basic levels faster than in untrained subjects . Whether these effects of lower reactivity to stressful exercise events can be related to stressful events in daily life remains unclear . A meta analysis of crew and landers including 34 studies, 92 effect strengths (es), n=1.449 demonstrated a correlation between the level of fitness and reactivity to stressful events (es=.48). This study demonstrated that trained subjects do not react as strongly to stress as untrained subjects exposed to stress . The problem with the latter study was the measured outcome of stress e.g. Cardiovascular parameters . In nearly all stress - exercise - related situations, untrained individuals react with higher heart frequencies but data regarding physiological parameters such as noradrenaline, adrenaline or acth levels are generally missing . In many reviews and meta analyses [108 - 110] that have investigated the correlation between the level of fitness (by maximal and submaximal exercise tests) and stressors it was shown that trained subjects exhibit a higher reactivity to stress (es=.08, p<.001) and recover faster from stress too (37 studies, 118 es, n=1.092). Most effects were demonstrated in heart frequency, blood pressure, blood flow and vascular resistance . Animal studies have indicated that animals that exercised voluntarily show improved stress - coping abilities in physically demanding and psychological challenges . The latter improved stress - coping abilities appeared as adaptive responses of the hpa axis [110 - 112], improvements in sleep quality and increased stress resistance of sleep / eeg profiles, and also reduced anxiety - related behaviour in voluntary exercised mice and rats compared to sedentary control animals . Decreases in volume observed in the hippocampi and other regions of the forebrain in depressed patients have supported a hypothesis for depression involving decrements in neurotrophic factors [115, 116]. Neurotrophic factors are known to regulate neural growth and differentiation during development and are also regulators of plasticity and survival of adult neurons and glia . Support for the bdnf hypothesis of depression has come from a large preclinical literature showing that stress can reduce bdnf - mediated signalling in the hippocampus, whereas chronic treatment with antidepressants increases bdnf - mediated signalling . Similar changes have been observed in the post - mortem hippocampus, as well as in serum bdnf- concentrations of humans with depression . The second support for the theory that neurotrophic factors are of importance in treating depression is based upon the time delay of the mood - elevating effects of antidepressants, which is only seen after prolonged administration (several weeks to months). The cellular effect of antidepressants is the induction of hippocampal neurogenesis - the process by which neural progenitors of the sgz divide mitotically to form new neurons that differentiate and integrate into the dentate gyrus [65, 118]. Blockade of hippocampal neurogenesis inhibits the therapeutic - like effects of most antidepressant treatments in rodent models . Moreover, antidepressant treatment, possibly through the actions of transcription factor camp response element binding protein (creb) or other transcriptional regulators [15, 65], increases the amounts of several growth factors in the hippocampus that influence neurogenesis . These include bdnf as well as vegf and the recently discovered neuropeptide vgf, which themselves have antidepressant and pro - neurogenic properties in rodents [119 - 121]. Furthermore, both central and systemic administration of igf-1 increases hippocampal cell proliferation and neurogenesis in the adult rat [122, 123]. Central administration of igf-1 has shown to produce antidepressant - like effects in the rat forced swim test . This data supports the neurotrophic hypothesis of depression, which means that neuronal adaptations induced by antidepressant drugs are necessary to produce mood - elevation effects . This supports the theory that neurotrophic factors play a key role in the relief of depressive symptoms . Like antidepressants, exercise can also increase the synthesis of new neurons in the adult brain and therefore induce mood - elevating effects . Van praag et al . (1999) observed an increase in hippocampal neurogenesis in rats with regular access to a running wheel . Recent studies demonstrated that adult neurogenesis can be influenced by stress, ageing, environmental enrichment [127, 128] and physical activity [9, 129]. Kempermann et al . In 1997 showed the positive effects of environmental enrichment on neurogenesis in mice . These mice were also tested in a spatial memory task, the morris water maze, in which the enriched animals learned faster than control animals suggesting the possibility that the new neurons cause enhanced cognition . Experiments comparing animals undergoing exercise (wheel running) and animals raised in an enriched environment without exercise showed more bromodeoxyuridine (brdu; a synthetic nucleoside, used in the detection of proliferating cells)-positive cells in the runners group than in the group that was exposed to enriched environment without exercise . Further investigations demonstrated that already 10 days of wheel running increases cell genesis in rodents [131 - 133]. The increase of hippocampal neurogenesis by running became strongly manifested [134 - 139] that is also associated with improved hippocampal synaptic plasticity . The mechanisms by which exercise induces neurogenesis is based on the increase of following molecules: bdnf, vegf, igf-1, the neuropeptide vgf, 5-ht and -endorphins [119, 134, 141]. As already mentioned several days of voluntary wheel running enhance the levels of bdnf mrna in the hippocampus as has been shown in several studies [141 - 147]. The changes in the mrna were found in neurons of the dentate gyrus (dg), the hilus and the ca3 region of the hippocampus . In addition to the hippocampus, exercise also augmented levels of bdnf mrna in the lumbar spinal cord, the cerebellum and the cortex . Other growth factors like nerve growth factor (ngf) and it is well known that -endorphins are increased after exercise [150, 151]. It has been shown that the infusion of opiates induces an increase in cell proliferation and also that antagonists of the opiate receptor decrease cell proliferation in the dentate gyrus [152, 153]. Infusion of recombinant protein in mammals to elevate the levels of vegf, a protein secreted from blood that acts on endothelial cells to stimulate the formation of bloodvessels, has been shown to increase cell proliferation in the adult hippocampus and ventricular zone . It was demonstrated that the levels of vegf are also elevated following exercise [61, 155]. Pointed out in 2003 that vegf is necessary for the effects of running on adult hippocampal neurogenesis whereas peripheral blockade of vegf neutralizes running - induced neurogenesis . Another growth factor that is up - regulated in the brain and in the periphery after exercise is the insulin - like growth factor igf-1 . Igf-1, structurally related to pro - insulin, plays an important role in depressive disorders by contributing to neural development through neurogenesis and synaptogenesis, facilitating oligodendrocyte survival and stimulating myelination [157 - 159]. Igf-1 promotes cell proliferation and inhibits cell death during healthy but also during stressed or diseased states . Peripheral administration of igf-1 has been shown to induce up - regulation of bdnf mrna levels in the brain . Therefore it is suggested that igf-1 initiates growth factor cascades in the brain that can alter mechanisms of plasticity . Furthermore, carro et al . Could show in three experiments that exercise has neuroprotective effects by its increased passage of circulating igf-1 into the brain since after blocking the passage exercise no longer worked neuroprotective in simulated brain insults in rodents . Further evidence comes from fernandez et al . Who could show that systemic administration of igf-1 to brain - damaged sedentary mice or rats is sufficient to elicit functional recovery after simulated brain insult in rodents . Based on these findings circulating igf - i has a physiological neuroprotective tonic effect on the brain that is depressed in sedentary subjects . Hunsberger et al . Used a microarray technique to show that exercise upregulates a primary signaling cascade for neurotrophic factors and a peptide precursor, vgf . Furthermore, it was demonstrated that vgf induces synaptic plasticity genes that are also altered after exercise (nrn1 and syn1) [162, 163]. It is remarkable that exercise regulates so many genes especially in the hippocampus and underscores that exercise can be a potent tool to influence brain metabolic functions . Recent research has shown that pro - inflammatory cytokines not only induce " sick symptoms ", but also impinge on physically ill patients by leading to depressive disorders . In approximate 33% of patients who are treated by recombinant human cytokines interleukin-2 (il-2) and interferon- (ifn-) major depressive disorder is observed . It has been shown in animal models of inflammation that existing states of decreased reactivity to reward (anhedonia) and reduced social exploration can be reversed by antidepressant treatment . Sickness is basically an adaptive response to infection . As in the case of depressive disorders, but unlike depression, sickness is completely reversible once the disease - causing agent has been eliminated . Van den biggelaar et al . Studied 267 people at the age of 85 without any psychiatric history . In this study certain mediators like pro - inflammatory cytokines are produced in an infection that contain interleukin-1 and (il-1, il-1), tumor necrosis factor- (tnf-) and interleukin-6 (il-6). These in the periphery produced cytokines also act on the brain causing behavioral symptoms postulated as " sickness behavior " [166, 167]. It has been repeatedly observed in patients suffering from major depression that the levels of pro - inflammatory cytokines, acute - phase proteins, chemokines and adhesion molecules are increased [168 - 175]. The most frequently observed alterations are increased levels of il-6 in the plasma as in the serum and/or elevations of c - reactive protein [166, 168 - 171]. Further alterations were observed in elevated concentrations of il- - and tnf- in peripheral blood and in the cns of patients suffering from mdd [172, 175, 176]. Major depressive disorders caused by immunotherapy in cancer or hepatitis c patients who were receiving immunotherapy supported the theory of cytokine - induced depression first postulated by smith and later by maes . Behavioral data in animal studies have indicated a relationship between cytokines and depression . Systemic administration of lipopolysaccharide (lps) induced the expression of il-1 and other pro - inflammatory cytokine mrnas and proteins in the brain in many studies [179 - 182] in addition showing that depressive - like behaviour remained after sickness behaviour had already retreated . Frenois et al . Observed a decrease in the preference for a sucrose solution, a phenomenon that was still apparent when food intake and drinking had already normalized . If the animals received antidepressants before lps - treatment the reduced intake of a sweetened solution was neutralized . Another link in favour of relationship between cytokines and depression stems from the fact that immunotherapy reduces the plasma levels of tryptophan which determines the rate of serotonin synthesis in the brain . A key role in the context of inflammation and depressive disorders seems to play il-1- that inhibits the expression of bdnf in the hippocampus of rats after undergoing social isolation . Stress - induced neuronal cell loss in animals is also associated with increased levels of tnf- and nf- b (nuclear factor' kappa - light - chain - enhancer' of activated b - cells). Over - expression of tnf- is observed in decelerated brain growth and neural damage, which is associated with reduced igf-1 activity, in this case especially in the cerebellum . This finding is of great interest since igf-1 can therefore act as an anti - inflammatory cytokine in the brain and can also be induced by exercise . Exercise has been shown to influence the immune system and seems to play an important role in the relationship between the immune function and depressive disorders . During exercise, the cascade in cytokine response differs from the " classical " response to infections represented by the onset of circulating il-6 during exercise . Epidemiological data suggests a relationship between physical inactivity and low - grade inflammation in healthy subjects [190 - 192]. Could (2003) show that exercise in the form of 3 hours ergometer cycling can suppress endotoxin - induced tnf- production . Exercise works as an anti - inflammatory agent by leading to higher levels of il-6 which is followed by raising il-1ra and il-10 levels and also by suppression of tnf- production as demonstrated in animals and in vitro studies . Exercise gives rise to high levels of epinephrine that has also been shown after infusion to inhibit tnf- production in response to endotoxin in vivo . Except for strenuous exercise which is mainly pro - inflammatory, the exact dose of exercise that has anti - inflammatory effects has not been clearly established . However, the data suggests that moderate aerobic exercise seems to induce the most promising effects considering the anti - inflammatory and antidepressive outcomes . To summarize the relationship between depressive disorder, cytokines and exercise, epidemiological data shows the correlation between physical inactivity and low - grade inflammation [190 - 192]. Since immunotherapy reduces plasma levels of tryptophan, it is noteworthy that levels of tryptophan can be directly influenced by exercise . As already mentioned acute physical exercise increases blood free tryptophan and in animals [71 - 73] and humans [74 - 76]. And also igf-1, which counteracts the behavioral depressing effects of cytokines, can be influenced by physical activity [60, 156]. Exercise induces physiological changes that make it a potentially powerful agent for use as a therapeutic method of intervention in many health disorders such as diabetes, stroke, certain cancers, coronary heart disease and or obesity . It seems that neurobiological health and functioning depends on the physical activity level of each person s life . The observed behavioural and biological influence of exercise training on depressive disorders suggests that it induces the same neurobiological alterations as antidepressant drug treatment by elevating the levels of serotonine [79, 80, 197], increasing central norepinephrine neurotransmission [81 - 83], altering the hypothalamic adrenocortical system [110 - 112] and raising -endorphin concentrations [150, 151]. Furthermore, exercise stimulates the growth of new nerve cells and the induction of the release of proteins and peptides that improve the health and survival of nerve cells like bdnf, vegf, igf-1 and vgf [57, 59, 60, 119, 141]. Increased inflammatory biomarkers seem to appear before the onset of depression, but the cytokine - response to exercise and its effect on depressive disorders needs to be further investigated . There is no accurate published information concerning dosage, duration, frequency, intensity or type of exercise to be used as an antidepressive treatment . . Therefore it would be interesting to answer following questions in the near future: do the behavioural results correlate with the molecular changes in neurotrophic factors or monoamine, cytokine or cortisol alterations? Can there be observed changes in the neuronal morphology, e.g. Dendritic atrophy and spine reduction after the induction of depression and their possible modification after an exercise therapy? What specific role are cytokines playing in depression and are they related to and contribute to positive outcomes when exercise is used as an intervention in depressive disorders? How should the exercise be designed for it to be useful as an intervention in brain - related disorders like depression? Since mdd is a major health problem and the effectiveness of current antidepressants is limited, the antidepressant actions of exercise are of great interest and could represent more than just an alternative to current treatments . In all, these findings support the theory that brain health is activity dependent and that exercise training should be further promoted as a preventive and rehabilitative strategy to avoid or treat brain - related disorders.
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Physical inactivity is one of the major factors behind the increase in the death rate related to cardiovascular diseases since the introduction of modern work styles1 . In addition, due to sedentary lifestyles, physical inactivity has become a major risk factor of death regardless of sex2 . Also, aerobic exercise helps cardiopulmonary functions to function smoothly and prevents aging, providing enough energy for everyday activities3 . Specifically, compared with some vigorous exercises, walking has been recommended as an optimal and practical type of exercise regardless of age, time, and location with less risk of hurt during exercise and high exercise effects5, 6 . In particular, one large - scaled study of 73,500 postmenopausal women showed that brisk walking for about 30 minutes prevented cardiovascular related diseases6.the pedometer has recently been used to monitor walking . It has given people a direct motivation to continue as they can measure and see directly how many steps they have taken7 . The pedometer has been strongly recommended as a useful tool for everyone as its cost is low and it has high reliability and validity8 . Also, it can be of great use in regular exercise9 as it serves as a monitor and provides instant feedback10 . Sedentary workers are expected to have higher risk of diseases related to inactivity as they might neglect walking due to their working style . Step count a day (step / day), which is measured and recorded by a pedometer, provides a basis for providing feedback and establishing walking goals in a walking program . Using step count a day, thus, it is a minimal way of implementing a strategy to increase step count11 . Precise understanding and analysis of individuals' condition and walking patterns in everyday activity . In addition, in the analysis of walking pattern in which a pedometer is used, it is necessary to discriminate between different walking patterns, such as during the week and over the weekend, as the walking patterns are different, except for small groups of people and age ranges12 . A number of studies in which a pedometer was used have focused on every day activities within a day in all ranges of age groups, but there is a lack of research about sedentary workers . Therefore, the purpose of this study was to analyze the step counts of inactive sedentary workers in their twenties on different days of the week according to gender and degree of obesity by using a pedometer focusing on different days of the week and the weekend . Criteria of classification: low weight (bmi <18.5 kg / m), standard weight (18.524.9 kg / m), overweight (25.029.9 kg / m) the participants of this study were enrolled from office workers in their twenties in u city . Thirty - six participants 18 males and 18 females, voluntarily participated in this study . They were instructed to keep a journal of step count for 2 weeks and were given a pretest explanation of the method and direction of the experiment . All of the subjects participated in this study without dropouts till the end of the study . Bmi (body mass index) is used to determine obesity and is the body mass divided by the square of the subject's height13 (table 1). A pedometer and a journal of step count were provided to each participant in the pretest session with instructions on how to keep the journal and how to use the pedometer . The subjects were instructed to record their step count during average work hours, or between 9 am to 6 pm, on different days of the week . Participants' journal- keeping status and pedometer use were checked every day by phone and text message . The participants' journals were collected after 2 weeks and their average step counts were calculated . The pedometer used to measure sedentary workers' step count objectively was a mp-500 (yamasa co, tokyo, japan) pedometer, and the step counts over 2 weeks were recorded daily . The pedometer was worn at the joint of the right thighbone and pelvis, or the anterior superior iliac spine (asis) point where belt line of the waist and the center - line of the legs meet vertically14 . Individual participants' step counts at the workplace between 9 am to 6 am were recorded in daily journals which were collected for further analysis . The average and standard deviation of the step count collected during the period of the experiment were calculated . Step counts on different days of the week were analyzed according to sex and obesity degree using the independent t - test and anova . *: p<0.05, * *: p<0.01, msd: mean standard deviation *: p<0.05, msd: mean standard deviation table 2 showed the results of sedentary workers' step count during weekdays according to gender . Females had higher step counts than males on every day of the week except fridays and sundays . Both males and females had higher step counts over the weekends and males had the highest step count on sundays, while female did on saturdays . In addition, there was a statistically significant difference between the step counts of males and females on thursdays using the independent t - test (p<0.01). Table 3 provides the results of analysis of sedentary workers' step counts during days of the week for the different bmi groupings (table 3). The step count of the low weight group was higher on every day of the week than that of the overweight group . Specifically, the step count of the standard weight group was lower than that of the overweight group on tuesdays and thursdays and the highest step counts of the standard weight group was reported on sundays . There were statistically significant differences between the step counts of the different bmi groupings on both mondays and sundays using the anova (p<0.05). Pedometers have been used in a variety of physical programs, especially for low - active adolescent girls15, sedentary workers16, overweight adults17 . They is because they can present concrete goals for physical activity during the day, and individuals are able to easily check their physical activities on their own . Also, it has been reported that a long - term pedometer - determined ambulatory activity helped to decrease risk factors18 . Therefore, this study was conducted to see if there were any differences in the number of steps taken by sedentary workers in their twenties based on sex and their degrees of obesity . Against the expectation that males' step counts would be higher than females' step counts on each day of the week according to sex showed that females' step counts were higher than males', except on fridays and sundays . This might be attributable to females' working habit of moving more frequently than males during work hours . Comparing step counts between weekdays and weekends, we found that both males and females showed higher step counts during weekends than weekdays.the target step count in walking programs for adults is generally 10,000 steps a day10, 19 . This study counted daily steps of weekdays only during work hours, which led to lower step counts than the average adult's step count . Thus, the 10,000 step count may have been achieved if activities during rest hours at the workplace when participants didn't wear a pedometer were included . As predicted, step counts on each day of the week differed among different degrees of obesity . It was highest for the low weight group, and lower for the standard weight, and overweight groups in rank - order . In particular, the difference in step counts between the low weight and overweight groups was approximately double during the weekend, compared with that of during weekdays . In addition, the step counts during the weekend of the low weight and standard weight groups were 2,000 to 4,000 step counts greater than those of weekdays . This indicates that the average physical activity during the weekend was higher in the low weight and standard weight groups than in the overweight group . The sedentary workers' lower than average step count on each day of the week indicates low physical activity, which may lead to a variety of health problems such as obesity . Thus, future study should cover interventions to help sedentary workers' to increase their physical activity . Also, more effort should be made seeking practical ways to improve physical activity at the workplace to keep workers in good health.
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Cell nuclei in two - dimensional (2d) pathology images can yield quantitative information about the presence or absence of disease processes and also help evaluate disease progress . Detecting and segmenting nuclei correctly with minimum human effort is important for cell nuclei analysis . Jung and kim showed that improved segmentation accuracy led to better classification performance using the unique classifier for thyroid follicular lesions . However, automatic nuclei segmentation in pathology images still remains a difficult problem due to the high variability in images caused by differences in slide preparation, image acquisition, and nuclei heterogeneity . Cell nuclei detection plays a critical role in the overall segmentation procedure, which requires a point per nucleus and close to nucleus center, referred to as seed . Many approaches have been described in the literature to locate cell nuclei in 2d microscopy images . The combination of finding peaks in the euclidean distance map and watershed, though often resulting in overseeding, can be applied to locate seeds . The circular - shaped nuclei can be effectively located using hough transformation methods at the cost of expensive computation . H - maxima / minima transform is a powerful approach to detect nuclei by finding the local maximums in images . The multiscale laplacian - of - gaussian (log) filtering constrained by the distance map - based adaptive scale selection can be used to detect cell nuclei . This method improves seed accuracy but is sensitive to minor peaks in the distance map and thus leads to overseeding . Qi et al . Proposed a method based on single - path voting followed by mean - shift clustering to find seeds for touching and overlapping nuclei . Speaking of segmentation methods more broadly, a wide range of methods has been described in the literature . Thresholding, morphological operation, watershed, region growing, active contour model, clustering, and graph cut are the cornerstones of the segmentation methods proposed in the literature . The simplest thresholding method followed by morphological operation works well for objects with little intensity variations and high foreground / background contrast . Watershed is a nonparametric method widely applied in nuclei segmentation, with the disadvantage of over - segmentation . Active contour models can be used to obtain a smooth contour by minimizing an energy function and can be utilized in pathology images where nuclei appear unclear . The well - known chan and vese model was used to obtain the outer contours of nuclei, followed by a watershed - like algorithm to separate the clustered nuclei . Al - kofahi et al . Utilized a graph - cut - based binarization to extract the foreground and then a second graph - cut - based algorithm to refine the initial contours obtained by constrained multiscale log filter, which was shown to perform well in pathology images with dense nuclei . Proposed a method called ovuscule which was a modified snake model taking the shape of an ellipse . Its evolution is driven by the combination of the integral of data over the inner ellipse and outer elliptical shell . More nuclei segmentation methods including active contour model, watershed - based method, and markov random field are described in irshad paper . Here, we show a comparison between the method we propose and a few such modern methods . In this paper, we describe an unsupervised nuclei segmentation method, which we call multiscale edge selection in polar space (mesps). Specifically, a filter bank consisting of rings with various sizes is first constructed to locate nuclei by finding the local maximums in the response map . In the segmentation step, the nuclei contours are iteratively refined by selecting the correct edges in polar space at different smoothing levels . We believe the accurate nature of the segmentation procedure, simplicity of use, and computational efficiency are key advantages of our method as will be demonstrated . An edge pyramid is then constructed, where edge maps are generated using a set of smooth parameters . Edge selection is performed at each level and the nuclei contour evolves across the edge pyramid to delineate the spatial content of cell nuclei tissue blocks and cytology slides were obtained from the archives of a local hospital (approved as an exempt protocol by the institutional review board). Cases for analysis included liver resection specimens and cytology slides prepared from fine - needle aspiration biopsies of thyroid nodules . All tissues were fixed in 10% neutral - buffered formalin and processed on a conventional tissue processor using a series of graded alcohols and xylenes before paraffin embedding . Tissue sections were cut at 5 thickness from the paraffin - embedded block and placed on conventional 25 mm 75 mm 1.0 mm superfrost plus microscope slides using fisherbrand superslip coverslips (50 mm 24 mm 0.17 mm; fisher scientific, thermo fisher scientific, inc ., all tissue sections for imaging were stained using conventional hematoxylin and eosin (h and e) protocol used in the histology laboratory . For the thyroid cytology preparations, aspirate smears were fixed in 95% ethanol and then stained with the papanicolaou (pap) staining technique . Briefly, the pap stain uses hematoxylin, og-6, and eosin azure (combination of eosin y, light - green sf, and green fcf dyes) to stain cytological preparations . Nuclei stained with this technique have blue - green color and excellent chromatin detail that can be visualized by light microscopy . Whole slide digital images of the liver slides were acquired using an omnyx vl4 digital whole slide scanner (omnyx, llc, waterfront pi, pittsburg, pa, usa) equipped with a 60 dry objective . Images obtained had a resolution of 0.1375 /pixel and were saved in the proprietary format and then converted to lossless jpeg format . All thyroid cytology slide images were acquired using an olympus bx51 microscope equipped with a 100 uis2 uplanfl oil immersion objective (numerical aperture 1.30; olympus america, central valley, pa, usa) and 2 megapixel spot insight camera (diagnostic instruments, sterling heights, mi, usa). Image specifications were 24-bit rgb channels and 0.074 /pixel, 118 89 m field of view . The basic idea of nuclei detection is to find local evidence for the presence or absence of a nucleus in the image . To that end, we construct a filter bank composed of rings of different sizes modeled by the function: r x + y (r +), where r is the radius and is the thickness . Given a certain dataset, prior information such as, the size of the smallest and largest nuclei can be reasonably estimated; thus, the size range of the filters can be defined according to image resolution . We note that the shape of filters can be changed and modeled by different functions to adapt the nuclei appearance in different datasets . In our experiment, the sampled locations = [xi, yi] can be obtained from a set of centered coordinates [x1,, x2r + 1], r xi r + the filter image patch with size r denoted as fr () is convolved with a gaussian function, which is meant to be an approximation of point spread function . Given an image i (), the likelihood of the pixel at being the center of an underlying nucleus is defined as follows: where denotes the normalized cross correlation (ncc) between the filter fr () and the image i. and with being the neighborhood of pixel of the same size as the filter fr (). The maximization procedure above is performed pixel by pixel searching for the filter fr within the filter bank which best matches the appearance of the potential nucleus at location . Pixels having ring - shaped surrounding pixels with similar radius as that of a filter will have strong responses and are likely to be nuclei centers . On the contrary, irrelevant tissue structures or noisy background thus, the method is able to yield size estimation for each nucleus by searching for the best - matched filters . Here, we note that most nuclei take the shape of an ellipse and thus, theoretically, elliptical filters would generate stronger responses compared with ring - shaped filters . However, since more parameters (e.g., length of major and minor axis, rotation angle) would be required to control an ellipse, leading to a larger searching space when performing ncc operation, we use the ring - shaped filters instead . As we can see from the simulation experiment [figure 2], ring - shaped filters are able to generate the strong responses when applied to detect both circular and elliptical nuclei in noisy background . (a) constructed filter bank with filters of different sizes (magnified for viewing purpose). (e) response map for (d) to locate the cell nuclei, the standard k - means clustering method is applied to classify the responses into three classes based on the response intensity: (1) background; (2) weak responses from nonnuclei structures; (3) strong responses from potential nuclei . Using connected component analysis, nuclei seeds can be obtained by computing the mass center of each isolated pixel cluster classified as strong responses . (d) detected nuclei seeds (green dots) in practice, the false positive nuclei seeds can be filtered out by postprocessing operations, such as thresholding the area of isolated pixels clusters . With detected nuclei seeds, it is required that the subsequent segmentation algorithm delineate the nuclei contours efficiently and accurately with minimal manual intervention . Our goal is to segment nuclei correctly in the complex background (existence of a large variety of nuclei morphology, chromatin texture, staining procedure as well as tissue heterogeneity). As well known, edge detection produces outlines at locations where large gradient exists, for example, nuclei borders and noisy background structures [figure 4a]. To obtain the initial segmentation, a blurred version of the nuclei image is required, which describes the nuclei outlines and excludes noisy details hindering the delineation of nuclei contours . The multiscale strategy enables the nucleus contour to refine iteratively from the initial segmentation by changing the blur parameter smoothly . The proposed method is designed to discriminate the nuclei borders pixels from remaining garbage pixels . (a) original image with a subwindow showing the edge map for one nucleus (detected by canny edge detector, i = 3) with the seed in the center (red dot). (b) edge pixels are transformed into polar space with the nucleus seed being the origin . Red points are the locations with locally maximal number of pixels; green points show the edge pixels along the optimal path searched by dijkstra's algorithm . (c) constructed undirected graph with nodes being the red points in (b) and edge weights being the cost defined by the combination of distance and intensity metrics . (d) final contour (red), and optimal path (green) are shown in the image patch specifically, the input image is first convolved with the 2d gaussian function with zero mean at multiple scales = [0,,n1], with 0 and n1 being the minimum and the maximum of, respectively . Next, an edge pyramid is constructed consisting of a set of edge maps generated by the edge detector (e.g., canny detector), where the top level and the bottom level correspond to 0 and n1, respectively . We aim to select the correct edges in polar space starting from the bottom level and then take it as guidance for edge selection in the next higher level . The algorithm refines the contour iteratively and produces the final contour when edge selection is performed at the top level of the edge pyramid . The first step is to select correct edges from the edge map obtained at the largest scale n1, where artifacts are the least prevalent . The size of the image patch for each nucleus can be determined adaptively according to the size estimation from nuclei detection step . In the nucleus edge map, edges can be classified into three categories: correct edges (forming the nucleus contour), edges inside the nucleus, and edges outside the nucleus . One prominent feature to discriminate these three kinds of edges is that, intuitively, correct edge pixels on the nucleus counter often have smoother distance changes away from the seed in comparison to the drastic distance fluctuations of pixels on noisy edges inside or outside the nucleus . In addition, considering the intensity, edge pixels along the nucleus border have relatively consistent intensity compared with that of incorrect edge pixels . With these simple observations in mind, the solution of delineating nucleus contour becomes finding the path with the minimal transportation cost (nonzero) starting and ending at any chosen point on the nucleus border based on both distance and intensity metrics . The polar coordinate system provides a natural space to search for the optimal path connecting the start point and the end point for each nucleus . Given the edge map ei detected at the i level, edge pixel = [x, y] in cartesian coordinate can be transformed into polar space by, where = [x, y] is the coordinate of nucleus seed in the image patch . Figure 4b shows that the edge map in the subwindow of figure 4a is transformed in polar space . In the polar coordinate system, the transportation cost between any two neighbor edge pixels pm = [rm,] and pn = [rn, +] is defined as follows: where vm and vn denote pixel intensities for pm and pn; is infinitesimal; a, b are the weights for the distance term and intensity term, respectively; rmax and vmax are the maximal distance difference and the maximal intensity difference, respectively, between pm and pn in the edge map, enabling both two metrics to have the same scale . The optimal path * can be found by minimizing the function defined as follows: where l denotes the length for the path . In the discrete setting, the function above can be rewritten as follows: where n is the number of edge pixels along the path . Dijkstra's algorithm is an algorithm widely applied to find the shortest path between the source node and the terminal node in a graph, such as road networks and telephone network . We represent the edge pixels in polar space in the form of undirected graph g = [v, e] [figure 4c], denote the edge pixels and the graph edges with weights c(, pm, pn) in e are only feasible for any two adjacent angle nodes pm, pn . To make the algorithm robust against noisy edge pixels, the edge pixels are further represented / discretized by finding the locations with locally a maximal number of edge pixels for each angle in the polar space . A sliding window with width w and height h, moving along the distance direction for each, the number of edge pixels in the sliding window centered at [rj, i] is denoted as n (rj, i) and locations with locally maximal number of pixels at angle i are denoted as (i). These detected locations are the approximate representation of the original edge pixels in polar space [red dots in figure 4b]. The benefit of discretizing edge pixels is that it helps reduce the number of possible paths between the source node and the terminal node . Thus, the computational cost is reduced dramatically when searching for the optimal path using dijkstra's algorithm . In practice, it is not easy to select the source node and terminal node along the nucleus border . In this paper, we propose that the source node and the terminal node can be chosen as edge pixels with the same distances away from the seed at angle 0 and 2. however, there might be multiple source - terminal pairs in the graph, and thus multiple optimal paths are found by dijkstra's algorithm . This is so especially when a small is applied, where many noisy edge pixels exist at the angle 0 and 2. when noisy edge pixels are selected as the source or terminal node, the optimal path would be searched by dijkstra's algorithm at the cost of passing through edges with large weights in the constructed graph . Thus, the real nucleus contour can be found by choosing the path with the minimal cost connecting source - terminal pairs . One potential issue is that some detected edges may not be complete due to the weak gradient at the nucleus border and thus some pixels along the optimal path are not necessarily on the real nucleus contour . Here, we apply the ransac algorithm and the spline curve fitting method to estimate the nucleus contour . Given the edge pixels on the optimal path, a subset of pixels is selected to generate a fitted curve model describing the rough shape of the optimal path . The points fit the estimated model well are called inliers and points with large errors are called outliers . The step repeats for a fixed number of times, and the model with the maximal number of inliers is kept . The curve fitting step takes as input the pixels along the optimal path and outputs the final smooth contour c connecting the isolated and incomplete detected edges when performed at the k level of the edge pyramid . The smooth contour generated from the k level is taken as the initial nucleus border and helps guide the edge selection for the k + 1 level . Specifically, at the k + 1 level, edge pixels within the distance range [c d, c + d] are chosen as edge candidates and edge pixels outside the range are discarded in the sense that a more refined contour at the k + 1 level should be close to the contour obtained from the k level with a distance tolerance d. when the blur parameter is changed slightly, the edge locations change smoothly and would not shift too much . Therefore, for the k + 1 level, the edge pixel locations at angle should be within the range [c()d, c() + d]. With the set of edge candidates, the edge selection is performed as described above to generate a more accurate nucleus contour c. as the contour is refined iteratively from the bottom level of the edge pyramid up to the top level, it gradually attaches to the real border of nucleus . Given the small blurring 0 at the top level, our algorithm can delineate the nucleus spatial extent precisely . Tissue blocks and cytology slides were obtained from the archives of a local hospital (approved as an exempt protocol by the institutional review board). Cases for analysis included liver resection specimens and cytology slides prepared from fine - needle aspiration biopsies of thyroid nodules . All tissues were fixed in 10% neutral - buffered formalin and processed on a conventional tissue processor using a series of graded alcohols and xylenes before paraffin embedding . Tissue sections were cut at 5 thickness from the paraffin - embedded block and placed on conventional 25 mm 75 mm 1.0 mm superfrost plus microscope slides using fisherbrand superslip coverslips (50 mm 24 mm 0.17 mm; fisher scientific, thermo fisher scientific, inc ., all tissue sections for imaging were stained using conventional hematoxylin and eosin (h and e) protocol used in the histology laboratory . For the thyroid cytology preparations, aspirate smears were fixed in 95% ethanol and then stained with the papanicolaou (pap) staining technique . Briefly, the pap stain uses hematoxylin, og-6, and eosin azure (combination of eosin y, light - green sf, and green fcf dyes) to stain cytological preparations . Nuclei stained with this technique have blue - green color and excellent chromatin detail that can be visualized by light microscopy . Whole slide digital images of the liver slides were acquired using an omnyx vl4 digital whole slide scanner (omnyx, llc, waterfront pi, pittsburg, pa, usa) equipped with a 60 dry objective . Images obtained had a resolution of 0.1375 /pixel and were saved in the proprietary format and then converted to lossless jpeg format . All thyroid cytology slide images were acquired using an olympus bx51 microscope equipped with a 100 uis2 uplanfl oil immersion objective (numerical aperture 1.30; olympus america, central valley, pa, usa) and 2 megapixel spot insight camera (diagnostic instruments, sterling heights, mi, usa). Image specifications were 24-bit rgb channels and 0.074 /pixel, 118 89 m field of view . The basic idea of nuclei detection is to find local evidence for the presence or absence of a nucleus in the image . To that end, we construct a filter bank composed of rings of different sizes modeled by the function: r x + y (r +), where r is the radius and is the thickness . Given a certain dataset, prior information such as, the size of the smallest and largest nuclei can be reasonably estimated; thus, the size range of the filters can be defined according to image resolution . We note that the shape of filters can be changed and modeled by different functions to adapt the nuclei appearance in different datasets . In our experiment, the sampled locations = [xi, yi] can be obtained from a set of centered coordinates [x1,, x2r + 1], r xi r + the filter image patch with size r denoted as fr () is convolved with a gaussian function, which is meant to be an approximation of point spread function . Given an image i (), the likelihood of the pixel at being the center of an underlying nucleus is defined as follows: where denotes the normalized cross correlation (ncc) between the filter fr () and the image i. and with being the neighborhood of pixel of the same size as the filter fr (). The maximization procedure above is performed pixel by pixel searching for the filter fr within the filter bank which best matches the appearance of the potential nucleus at location . Pixels having ring - shaped surrounding pixels with similar radius as that of a filter will have strong responses and are likely to be nuclei centers . On the contrary, irrelevant tissue structures or noisy background thus, the method is able to yield size estimation for each nucleus by searching for the best - matched filters . Here, we note that most nuclei take the shape of an ellipse and thus, theoretically, elliptical filters would generate stronger responses compared with ring - shaped filters . However, since more parameters (e.g., length of major and minor axis, rotation angle) would be required to control an ellipse, leading to a larger searching space when performing ncc operation, we use the ring - shaped filters instead . As we can see from the simulation experiment [figure 2], ring - shaped filters are able to generate the strong responses when applied to detect both circular and elliptical nuclei in noisy background . (a) constructed filter bank with filters of different sizes (magnified for viewing purpose). (b) (e) response map for (d) to locate the cell nuclei, the standard k - means clustering method is applied to classify the responses into three classes based on the response intensity: (1) background; (2) weak responses from nonnuclei structures; (3) strong responses from potential nuclei . Using connected component analysis, nuclei seeds can be obtained by computing the mass center of each isolated pixel cluster classified as strong responses . (d) detected nuclei seeds (green dots) in practice, the false positive nuclei seeds can be filtered out by postprocessing operations, such as thresholding the area of isolated pixels clusters . With detected nuclei seeds, it is required that the subsequent segmentation algorithm delineate the nuclei contours efficiently and accurately with minimal manual intervention . Our goal is to segment nuclei correctly in the complex background (existence of a large variety of nuclei morphology, chromatin texture, staining procedure as well as tissue heterogeneity). As well known, edge detection produces outlines at locations where large gradient exists, for example, nuclei borders and noisy background structures [figure 4a]. To obtain the initial segmentation, a blurred version of the nuclei image is required, which describes the nuclei outlines and excludes noisy details hindering the delineation of nuclei contours . The multiscale strategy enables the nucleus contour to refine iteratively from the initial segmentation by changing the blur parameter smoothly . The proposed method is designed to discriminate the nuclei borders pixels from remaining garbage pixels . (a) original image with a subwindow showing the edge map for one nucleus (detected by canny edge detector, i = 3) with the seed in the center (red dot). (b) edge pixels are transformed into polar space with the nucleus seed being the origin . Red points are the locations with locally maximal number of pixels; green points show the edge pixels along the optimal path searched by dijkstra's algorithm . (c) constructed undirected graph with nodes being the red points in (b) and edge weights being the cost defined by the combination of distance and intensity metrics . (d) final contour (red), and optimal path (green) are shown in the image patch specifically, the input image is first convolved with the 2d gaussian function with zero mean at multiple scales = [0,,n1], with 0 and n1 being the minimum and the maximum of, respectively . Next, an edge pyramid is constructed consisting of a set of edge maps generated by the edge detector (e.g., canny detector), where the top level and the bottom level correspond to 0 and n1, respectively . We aim to select the correct edges in polar space starting from the bottom level and then take it as guidance for edge selection in the next higher level . The algorithm refines the contour iteratively and produces the final contour when edge selection is performed at the top level of the edge pyramid . The first step is to select correct edges from the edge map obtained at the largest scale n1, where artifacts are the least prevalent . The size of the image patch for each nucleus can be determined adaptively according to the size estimation from nuclei detection step . In the nucleus edge map, edges can be classified into three categories: correct edges (forming the nucleus contour), edges inside the nucleus, and edges outside the nucleus . One prominent feature to discriminate these three kinds of edges is that, intuitively, correct edge pixels on the nucleus counter often have smoother distance changes away from the seed in comparison to the drastic distance fluctuations of pixels on noisy edges inside or outside the nucleus . In addition, considering the intensity, edge pixels along the nucleus border have relatively consistent intensity compared with that of incorrect edge pixels . With these simple observations in mind, the solution of delineating nucleus contour becomes finding the path with the minimal transportation cost (nonzero) starting and ending at any chosen point on the nucleus border based on both distance and intensity metrics . The polar coordinate system provides a natural space to search for the optimal path connecting the start point and the end point for each nucleus . Given the edge map ei detected at the i level, edge pixel = [x, y] in cartesian coordinate can be transformed into polar space by, where = [x, y] is the coordinate of nucleus seed in the image patch . Figure 4b shows that the edge map in the subwindow of figure 4a is transformed in polar space . In the polar coordinate system, the transportation cost between any two neighbor edge pixels pm = [rm,] and pn = [rn, +] is defined as follows: where vm and vn denote pixel intensities for pm and pn; is infinitesimal; a, b are the weights for the distance term and intensity term, respectively; rmax and vmax are the maximal distance difference and the maximal intensity difference, respectively, between pm and pn in the edge map, enabling both two metrics to have the same scale . The optimal path * can be found by minimizing the function defined as follows: where l denotes the length for the path . In the discrete setting, the function above can be rewritten as follows: where n is the number of edge pixels along the path . Dijkstra's algorithm is an algorithm widely applied to find the shortest path between the source node and the terminal node in a graph, such as road networks and telephone network . We represent the edge pixels in polar space in the form of undirected graph g = [v, e] [figure 4c], denote the edge pixels and the graph edges with weights c(, pm, pn) in e are only feasible for any two adjacent angle nodes pm, pn . To make the algorithm robust against noisy edge pixels, the edge pixels are further represented / discretized by finding the locations with locally a maximal number of edge pixels for each angle in the polar space . A sliding window with width w and height h, moving along the distance direction for each, the number of edge pixels in the sliding window centered at [rj, i] is denoted as n (rj, i) and locations with locally maximal number of pixels at angle i are denoted as (i). These detected locations are the approximate representation of the original edge pixels in polar space [red dots in figure 4b]. The benefit of discretizing edge pixels is that it helps reduce the number of possible paths between the source node and the terminal node . Thus, the computational cost is reduced dramatically when searching for the optimal path using dijkstra's algorithm . In practice, it is not easy to select the source node and terminal node along the nucleus border . In this paper, we propose that the source node and the terminal node can be chosen as edge pixels with the same distances away from the seed at angle 0 and 2. however, there might be multiple source - terminal pairs in the graph, and thus multiple optimal paths are found by dijkstra's algorithm . This is so especially when a small is applied, where many noisy edge pixels exist at the angle 0 and 2. when noisy edge pixels are selected as the source or terminal node, the optimal path would be searched by dijkstra's algorithm at the cost of passing through edges with large weights in the constructed graph . Thus, the real nucleus contour can be found by choosing the path with the minimal cost connecting source - terminal pairs . One potential issue is that some detected edges may not be complete due to the weak gradient at the nucleus border and thus some pixels along the optimal path are not necessarily on the real nucleus contour . Here, we apply the ransac algorithm and the spline curve fitting method to estimate the nucleus contour . Given the edge pixels on the optimal path, a subset of pixels is selected to generate a fitted curve model describing the rough shape of the optimal path . The points fit the estimated model well are called inliers and points with large errors are called outliers . The step repeats for a fixed number of times, and the model with the maximal number of inliers is kept . The curve fitting step takes as input the pixels along the optimal path and outputs the final smooth contour c connecting the isolated and incomplete detected edges when performed at the k level of the edge pyramid . The smooth contour generated from the k level is taken as the initial nucleus border and helps guide the edge selection for the k + 1 level . Specifically, at the k + 1 level, edge pixels within the distance range [c d, c + d] are chosen as edge candidates and edge pixels outside the range are discarded in the sense that a more refined contour at the k + 1 level should be close to the contour obtained from the k level with a distance tolerance d. when the blur parameter is changed slightly, the edge locations change smoothly and would not shift too much . Therefore, for the k + 1 level, the edge pixel locations at angle should be within the range [c()d, c() + d]. With the set of edge candidates, the edge selection is performed as described above to generate a more accurate nucleus contour c. as the contour is refined iteratively from the bottom level of the edge pyramid up to the top level, it gradually attaches to the real border of nucleus . Given the small blurring 0 at the top level, our algorithm can delineate the nucleus spatial extent precisely . The first step is to select correct edges from the edge map obtained at the largest scale n1, where artifacts are the least prevalent . The size of the image patch for each nucleus can be determined adaptively according to the size estimation from nuclei detection step . In the nucleus edge map, edges can be classified into three categories: correct edges (forming the nucleus contour), edges inside the nucleus, and edges outside the nucleus . One prominent feature to discriminate these three kinds of edges is that, intuitively, correct edge pixels on the nucleus counter often have smoother distance changes away from the seed in comparison to the drastic distance fluctuations of pixels on noisy edges inside or outside the nucleus . In addition, considering the intensity, edge pixels along the nucleus border have relatively consistent intensity compared with that of incorrect edge pixels . With these simple observations in mind, the solution of delineating nucleus contour becomes finding the path with the minimal transportation cost (nonzero) starting and ending at any chosen point on the nucleus border based on both distance and intensity metrics . The polar coordinate system provides a natural space to search for the optimal path connecting the start point and the end point for each nucleus . Given the edge map ei detected at the i level, edge pixel = [x, y] in cartesian coordinate can be transformed into polar space by, where = [x, y] is the coordinate of nucleus seed in the image patch . Figure 4b shows that the edge map in the subwindow of figure 4a is transformed in polar space . In the polar coordinate system, the transportation cost between any two neighbor edge pixels pm = [rm,] and pn = [rn, +] is defined as follows: where vm and vn denote pixel intensities for pm and pn; is infinitesimal; a, b are the weights for the distance term and intensity term, respectively; rmax and vmax are the maximal distance difference and the maximal intensity difference, respectively, between pm and pn in the edge map, enabling both two metrics to have the same scale . The optimal path * can be found by minimizing the function defined as follows: where l denotes the length for the path . In the discrete setting, the function above can be rewritten as follows: where n is the number of edge pixels along the path . Dijkstra's algorithm is an algorithm widely applied to find the shortest path between the source node and the terminal node in a graph, such as road networks and telephone network . We represent the edge pixels in polar space in the form of undirected graph g = [v, e] [figure 4c], denote the edge pixels and the graph edges with weights c(, pm, pn) in e are only feasible for any two adjacent angle nodes pm, pn . To make the algorithm robust against noisy edge pixels, the edge pixels are further represented / discretized by finding the locations with locally a maximal number of edge pixels for each angle in the polar space . A sliding window with width w and height h, moving along the distance direction for each, the number of edge pixels in the sliding window centered at [rj, i] is denoted as n (rj, i) and locations with locally maximal number of pixels at angle i are denoted as (i). These detected locations are the approximate representation of the original edge pixels in polar space [red dots in figure 4b]. The benefit of discretizing edge pixels is that it helps reduce the number of possible paths between the source node and the terminal node . Thus, the computational cost is reduced dramatically when searching for the optimal path using dijkstra's algorithm . In practice, it is not easy to select the source node and terminal node along the nucleus border . In this paper, we propose that the source node and the terminal node can be chosen as edge pixels with the same distances away from the seed at angle 0 and 2. however, there might be multiple source - terminal pairs in the graph, and thus multiple optimal paths are found by dijkstra's algorithm . This is so especially when a small is applied, where many noisy edge pixels exist at the angle 0 and 2. when noisy edge pixels are selected as the source or terminal node, the optimal path would be searched by dijkstra's algorithm at the cost of passing through edges with large weights in the constructed graph . Thus, the real nucleus contour can be found by choosing the path with the minimal cost connecting source - terminal pairs . One potential issue is that some detected edges may not be complete due to the weak gradient at the nucleus border and thus some pixels along the optimal path are not necessarily on the real nucleus contour . Here, we apply the ransac algorithm and the spline curve fitting method to estimate the nucleus contour . Given the edge pixels on the optimal path, a subset of pixels is selected to generate a fitted curve model describing the rough shape of the optimal path . The points fit the estimated model well are called inliers and points with large errors are called outliers . The step repeats for a fixed number of times, and the model with the maximal number of inliers is kept . The curve fitting step takes as input the pixels along the optimal path and outputs the final smooth contour c connecting the isolated and incomplete detected edges when performed at the k level of the edge pyramid . The smooth contour generated from the k level is taken as the initial nucleus border and helps guide the edge selection for the k + 1 level . Specifically, at the k + 1 level, edge pixels within the distance range [c d, c + d] are chosen as edge candidates and edge pixels outside the range are discarded in the sense that a more refined contour at the k + 1 level should be close to the contour obtained from the k level with a distance tolerance d. when the blur parameter is changed slightly, the edge locations change smoothly and would not shift too much . Therefore, for the k + 1 level, the edge pixel locations at angle should be within the range [c()d, c() + d]. With the set of edge candidates, the edge selection is performed as described above to generate a more accurate nucleus contour c. as the contour is refined iteratively from the bottom level of the edge pyramid up to the top level, it gradually attaches to the real border of nucleus . Given the small blurring 0 at the top level, our algorithm can delineate the nucleus spatial extent precisely . Before our algorithm is applied to pathology images, the nuclei channel should be extracted from rgb color space by color deconvolution (e.g., extracting hematoxylin channel from h and e - stained images). After that, all image data are normalized to fit the intensity range [0, 1]. We demonstrate the proposed method on two real datasets including thyroid dataset (35 representative images, pap - stained, 903 cell nuclei) and liver datasets (25 images, h and e - stained, 2145 cell nuclei). In our experiment, the sliding window width w and height h were set as 15 degrees and 2 pixels, respectively, and were fixed for both two datasets . The only parameter to be changed differently for the two datasets is the maximal smooth level max which was set to be 3 and 5 for the thyroid dataset and the liver dataset, respectively . The minimal smooth level min is usually set to be 1 to capture the precise nucleus border . For comparison, we choose the following state - of - the - art algorithms used for nuclei segmentation including the ovuscule, level set and template matching . Template matching has the ability of detecting nuclei in the image while level set and the ovuscule need detected nuclei seeds for segmentation . In our experiment, level set and the ovuscule adopted the seeds detected by mesps for comparing the segmentation performance . For qualitative comparison, sample segmentation results by approaches listed above are shown in figure 5, where the rows from the top to the bottom correspond to the results from level set, the ovuscule, template matching, and our method, respectively, and the columns from left to right correspond to sample images from liver dataset and thyroid dataset, respectively . First column: liver dataset; second column: thyroid dataset . From the top row to the last row are the results by level set, the ovuscule, template matching, and multiscale edge selection in polar space, respectively . Note that segmentation flaws are pointed out by black arrows in addition, we evaluated the nuclei detection efficiency of template matching and mesps using the miss rate (mr) defined as follows: where sa are the seeds detected by the algorithms and sm are the seeds selected manually; sasm and sasm are the number of seeds in the union set and the intersection set of sa and sm, respectively . The segmentation accuracy was measured by the area error rate (aer) focusing on the number of incorrectly segmented pixels and the spatially - aware evaluation metric normalized sum of distances (nsd) with the ground truth labeled manually . Quantitative analysis of nuclei detection and segmentation efficiency of different approaches is shown in table 1 . Quantitative evaluation of different approaches on nuclei detection and segmentation efficiency from the quantitative evaluation of different approaches, we note that the proposed method showed similar or superior performance compared with existing segmentation methods validated on two datasets . For the thyroid dataset, level set segmented nuclei with the highest accuracy with aer and nsd being 8.31% and 0.29, respectively . Our method generated similar results as that of level set, showing that mesps has the nuclei segmentation ability comparable to the state - of - art method . Moreover, for the liver dataset in the complex setting (nonuniform illumination, noisy background, and nuclei heterogeneity), mesps could still be able to find the nuclei borders accurately and perform the best compared with the listed approaches . Considering the comprehensive performance over the two validation datasets, mesps archived the best segmentation accuracy with 10.34% aer and 0.33 nsd on average . For the overall nuclei detection efficiency, both template matching and mesps could detect most nuclei labeled manually with the similar mrs . However, we should be aware of the supervised fashion of template matching method that needs users to select a set of nuclei for training and then finds the templates that best match the testing nuclei from the constructed statistical model . This paper proposed an unsupervised method to detect and segment cell nuclei automatically from the 2d pathology images based on ncc and mesps . The validation experiment showed that the method could segment the cell nuclei accurately when applied to real pathology images with different stainings (e.g., h and e, pap staining) and image qualities (e.g., blurring, noise, texture heterogeneity). The multiscale strategy ensures that the ill effects caused by noise, nonuniform intensity, etc . The small smooth level at the top of the edge pyramid helps the generated contour gradually cling to the real nucleus border at the pixel level . With the small step size of, the contour changes smoothly as it iterates from the bottom level up to the top level of the edge pyramid . Second, it is designed in an unsupervised way that does not need users to train a model used for segmentation . Once the parameters are set, the algorithm could detect and segment the cell nuclei in the dataset automatically . The performance of mesps depends on the image gradient; thus, it is not sensitive to staining techniques or imaging modalities, which makes it useful and applicable to various datasets in clinic settings . Finally, the proposed algorithm is light weight, consisting of several basic but effective algorithms including ncc, edge detection, and dijkstra's algorithm . The proposed framework is mathematically simpler than the mentioned state - of - the - art methods . In addition, we proposed some ways to reduce the computational cost by reducing the number of both nodes and edges when dijkstra's algorithm is applied . First, the edge pixels in polar space are represented by the points which have locally maximal number of pixels within a sliding window for each angle . This operation reduces the number of nodes greatly in the constructed undirected graph with the additional benefit of denoising . Moreover, edges between two nodes in the graph would be deleted if the distance difference is over a certain threshold, in the sense that pixels at adjacent angles on the nucleus contour should be near each other and the distance away from the nucleus center changes little . Each node only connects a few nodes at adjacent angles, which prominently cut down the number of possible paths between the source and the terminal nodes . We should note that there are parameters introduced in our proposed method, including filter size, min, max, smooth step size, cost weights (a, b), and the threshold for edge deletion used to reduce computational cost . Here, the parameters to be set to adapt to various datasets are filter size and max . As described in the previous section, the filter size depends on the image resolution as well as the cell nuclei type in study, whose size could be usually found in the literature . It is desired to keep the correct nuclei edge pixels in the edge map, at the same time, be able to filter out the noisy edges . In practice, the optimal max can be set experimentally by randomly selecting a few sample images in the dataset and observing the edge maps so that the edge detector could describe the outlines of most nuclei . Our algorithm is not sensitive to other parameters and they were fixed when validated on two datasets . In our experiment, the values of min, smooth step size, a, b, and the threshold were set to be 1, 0.5, 0.4, 0.6, and 20 pixels, respectively . Besides the advantages mentioned above, the method also has some limitations that are noteworthy of discussion . First, the method is designed for segmenting convex - shaped cell nuclei . In the polar space, parts of the contour for nonconvex shape nucleus are mapped to multiple locations at the same angle, which violates our assumption that there is only one optimal location along the nucleus contour per angle . Even though most cell nuclei have the shape of sphere or ellipsoid, highly concave cell nuclei can be sometimes observed under microscopy due to the sectioning of nuclei at odd angles or tissue distortion in slides preparation procedure, or both . Second, the method could not handle overlapping cell nuclei even if the nuclei seeds are detected correctly . Because of the blurring of border in the overlapping area, the edge detector usually does not generate the edge pixels delineating the two nuclei . The method would treat the two nuclei as one and produce the outer border of them . However, we should note that (1) the ultimate goal of nuclei segmentation is for exploring the correlation between nuclei morphology and cellular / disease progress, (2) overlapping cell nuclei provides limited information for analysis due to the difficulty of recovering inherent information within the overlapping area . Therefore, with plenty of isolated cell nuclei available in the dataset, nuclei overlapping problem is negligible in nuclei based analysis . The proposed method locates cell nuclei by measuring the matching degree between local image patches and the predefined nucleus - shaped filters . Afterward, the method transforms the object segmentation problem into a classic shortest path problem . The accurate delineation of cell nuclei is based on the detected edge pixels on the border which can be correctly selected by the well - known dijkstra's algorithm . The multiscale strategy enables the contour generated at each level evolves smoothly to the actual nuclei border . In the future, the method could be further automated by enabling the algorithm to select the optimal maximal smooth parameter based on image gradient statistics . Nih awards ca188938, gm090033, and pa state health department award 4100059192 supported the study . Nih awards ca188938, gm090033, and pa state health department award 4100059192 supported the study.
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We calculated the risk that a given pet in western europe is contagious for rabies on a given day by the equation we describe factors associated with rabid pets (https://zenodo.org/record/49670 #) and define pet transport as any noncommercial movement of a live cat, dog, or ferret and its owner or an authorized person across an administrative border . During 20012013, a total of 21 animal rabies cases attributed to pets from rabies - enzootic countries were reported in western europe (https://zenodo.org/record/49670 #), which represented 1.6 pets / year and 23 days / year of potential contagiousness . All owners except 1 (a spanish man living in a van) were official residents of western europe . Circumstances that led to pet examination and rabies diagnosis were clinical suspicion (14 pets), bitten humans (3 pets), border quarantine (2 pets), and retrospective data (2 pets with indigenous secondary cases during the alert in france in 2008). Average contagious period was 16 days / pet: 14 days in western europe (8 days without signs of rabies and 6 days with signs of rabies) and 2 days before arriving in western europe . For 1 dog, signs of rabies appeared before the animal entered western europe . For each rabid animal, an average of 34 (range 0187) persons and other animals received peps . The maximum value of this range corresponds to an alert in france in 2004 . After this alert, human and pet vaccinations led to vaccine shortages that required importing of vaccines not authorized for use in france (5). We identified animal origin and mode of entry into western europe (table 1). Three cases were imported from eastern europe to germany, 1 from the gambia to france, and 1 from sri lanka to the united kingdom . Customs officials could not identify any of 11 cases in animals transported mainly by road (e.g., after a ferry trip from morocco to spain, portugal, or france). Seven pets were transported through other countries in western europe before arriving in the country of diagnosis (https://zenodo.org/record/49670 #). Six puppies and 1 kitten were transported by air, of which only 2 were identified by customs officials (in the united kingdom and germany). * na, not applicable . We considered only countries in western europe with a population> 1 million persons . Pa, calculated risk that a given pet is rabid on a given day in a country in western europe relative to pet transport . Pac, calculated risk that a given cat is rabid on a given day in a country in western europe relative to pet transport . Pad, calculated risk that a given dog is rabid on a given day in a country in western europe relative to pet transport . #pad was lower if the dog had no signs of rabies (9.25 x 101.03 x 10 for dogs with no signs of rabies and 6.44 x 106.44 x 10 for dogs with signs of rabies assuming that 90%99.99% had no signs of rabies on a given day). Of 19 transported rabid pets, 8 (42%) had no rabies vaccination, pet passport, or health certificate . Only 6 were vaccinated (0/2 infected in france, 3/3 imported but raised in western europe, 3/7 imported by air, and 0/8 imported by road). Most vaccinated pets did not comply with recommended age for vaccination (> 12 weeks of age) or time between vaccination, serologic analysis, and transport . No reports mentioned valid rabies serologic analysis included in european pet movement policy (figure) for unlisted third countries (e.g., morocco, the gambia, sri lanka, or azerbaijan) (6). Using data for 20012013, we calculated that, for contact on a given day with a pet in western europe, the probability of the pet being contagious for rabies attributed to pet transport was 7.52 10 (table 2). 576/2013, http://eur-lex.europa.eu/legal-content/en/txt/?uri=celex:32013r0576) on movement of cats, dogs, and ferrets, 20032013 . Before 2003, national rules applied (e.g., animal checked at destinations, rabies vaccination, animal identification, quarantine, health certification). A health certificate provided by an official veterinarian is mandatory for pets transported from outside the eu . We observed a significant correlation between number of contagious days for dogs in a country and number of tourists traveling from this country to morocco (= 0.73, p = 0.017). We found no correlation with other variables tested (total dog population, dog population density, number of dogs per inhabitant). Risk for indigenous rabies has decreased in western europe . During 20012013, because of appropriate control of imported rabid pets, only 4 indigenous cases of human rabies were reported (3 in recipients of organs from a donor infected in india and 1 from a rabid bat in scotland) (https://zenodo.org/record/49670 #). Since 2011, no indigenous rabies cases have been reported in terrestrial mammals in western europe . Because of increased travel (7), rabies imported by trips to rabies - enzootic countries has increased, and travel became the main source of rabies in humans (1.46 patients / year) (8) and pets (1.6 rabid pets / year) in 20012013 . However, because of improved surveillance, although the number of imported rabies cases increased, the number of secondary cases decreased (https://zenodo.org/record/49670 #). Illegal importation of rabid animals is not limited to western europe (9) or dogs and cats (10). This finding highlights the need for a global approach for regulation of animal movement worldwide and strengthening real - time reporting for animal and human rabies . Risk for dog rabies being reintroduced into the european union from morocco was estimated as 0.21 cases / year (11). However, we estimate that 1.1 pets / year are entering western europe after being infected in morocco . Morocco has become the main source of pet rabies in western europe, often through ceuta and melilla (spanish enclaves in northern morocco). Because no prophylaxis or specific vaccinations are needed for travel to northern africa, few travelers seek pretravel advice and most have little knowledge of pet rabies (12,13). Lack of awareness also increases importation of human rabies . Despite an efficient policy for preventing entry of rabid pets, the united kingdom reported the highest number of patients with imported rabies during the study period (https://zenodo.org/record/49670 #). Patients returning to this country did not believe that a correct pep was needed after exposure abroad . None of the transported rabid pets fully satisfied european pet movement policy, which raised questions about how to improve the current regulation application . Increasing international travel, expansion of the schengen area (26 countries in europe that have a common visa policy) into rabies - enzootic countries in eastern europe, and development of internet animal trade (source of illegal importation) (14) are new challenges for ensuring compliance . Because bat rabies is more difficult to control than dog rabies, and some developing countries still have difficulties controlling rabies, eradication of rabies is not a realistic objective . Awareness should be increased, and current regulations for pet transport should be applied to reduce rabies importation and ensure that risk in western europe remains low . To avoid unnecessary and costly pep and optimize resource allocation, it should be clearly stated which who recommendations, public health england recommendations, or other practices most relevant after pet exposure should be applied . Low risks (<10-6) are usually considered acceptable or essentially 0 (3,15). The risk of a fatal car crash while traveling to pep consultations was higher than the risk of rabies after exposure to a pet in france in 20012011 (3). The most pertinent policy in areas at low risk for rabies is probably that of the united kingdom (i.e., no pep outside alert areas that do not have asymptomatic animals or exposure to bats) (https://zenodo.org/record/49670 #).
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Attention deficit hyperactivity disorder (ad / hd) is a psychiatric condition affecting an estimated 57% of children . Ad / hd's core symptoms of elevated impulsivity, increased motor activity, impaired concentration, and short - term memory deficits are often chronic with their symptomatic expression changing with development . While stimulant pharmacotherapy is the evidence - based treatment of choice for ad / hd, complementary and alternative (cam) therapies are becoming increasingly common treatments for the condition . In addition to chronicity, ad / hd has several other characteristics that make it a focus for cam . While ad / hd is generally believed to stem from neurophysiological deficits, a precise etiology has yet to be established . The treatment of choice for the condition is a schedule ii stimulant medication with addiction potential . Stimulant medication, while efficacious, may be associated with side effects including facial tics, hypertension, and anorexia . Finally, an estimated 2030% of children with ad / hd do not respond to stimulant medication . Surveys suggest that up to 60% of us patients use complementary and alternative medicine with half of this group using cam in the past year . Children with chronic conditions are far more likely than the general pediatric population to use cam . In an australian survey, 64% of parents of children with ad / hd reported using some form of cam . As a result of these factors, physicians diagnosing and treating adhd are likely to be asked about the types, availability, and effectiveness of alternative therapies . Cam therapies vary widely and include homeopathy, dietary restriction and supplements, herbal products, biofeedback, and attention training . While randomized placebo - controlled trials (rcts) are the standard for judging therapeutic efficacy, few of the cam therapies this paper's purpose is to familiarize mental health professionals and primary care clinicians with common complementary and alternative ad / hd therapies as well as the current evidence of their effectiveness . Hopefully, this current paper will help health care providers be better able to respond to patient and parent queries about the varieties, possible mechanisms of action, and benefits of cam therapies for childhood ad / hd . Abnormally low levels of dopaminergic activity in the prefrontal cortex have been implicated in childhood adhd . In animal studies, ginkgo biloba increases dopaminergic activity . In an open label, four - week trial, children receiving a combination of ginseng and gingko biloba were rated by parents as improved [10, 11]. Besides the limited treatment period and the absence of teacher ratings, the fact that some participants were taking concomitant stimulant medications limits drawing conclusions . Pycnogenol (pyc), the trade name for french maritime pine bark extract, is believed to act as a vasodilator improving cerebral blood flow to brain regions involved in ad / hd . Additionally, pyc may regulate and modulate possibly elevated catecholamine levels among ad / hd children . In a one - month rct, however, a small clinical study of pyc with ad / hd adults did not indicate that the herbal was superior to placebo [11, 13]. While probably being best known as a potential alternative treatment for major depressive disorder, hypercurium perforatum was the subject of one of the most rigorous cam trials on ad / hd conducted to date . In an eight - week rct with pediatric patients, neither parent ratings of symptoms nor clinician ratings of global behavioral improvement significantly differed for st . While findings are inconsistent, some investigators report abnormal levels of minerals among children with ad / hd including magnesium, copper, calcium, zinc, and iron deficiencies [15, 16]. A possible mechanism is that many minerals are cofactors for neurotransmitter synthesis, uptake, and breakdown . Zinc, magnesium, iron, and lead have been the most commonly studied . While screening for lead is a common part of the initial evaluation of children with ad / hd symptoms, there is little evidence that mineral supplementation decreases lead absorption [11, 15, 17]. For example, iron and zinc supplements did not significantly reduce overactivity or inattention among lead - exposed children . As a cofactor, iron is a modulator for synthesis of both norepinephrine and dopamine [11, 19]. However, in the absence of anemia, iron supplementation in children with ad / hd has not been found to be associated with consistent improvement in parent or teacher behavior ratings . Magnesium, as a cofactor for enzyme production, is involved in neurotransmitter synthesis and some studies have found magnesium deficiencies associated with ad / hd . An rct found that, compared with placebo, children with adhd symptoms and magnesium deficiency receiving magnesium supplementation demonstrated significant improvement after six months of treatment . Of the mineral supplements studied, the strongest evidence for clinical efficacy is for zinc . Low levels of zinc have been associated with deficits in several cognitive functions including information processing [11, 21]. Two studies of children with adhd receiving zinc supplementation have found improvement based on rating scales [21, 22]. There are suggestions that when added to conventional drug therapies such as methylphenidate, zinc may augment medication effects . Compared with other nutrients, the role of efas in ad / hd and their therapeutic application has been subjected to several rcts . Some investigations have found ad / hd to be associated with lower phospholipid levels and red cell membranes deficient in omega-3s such as docosahexaenoic acid (dha). While the exact mechanism for efas is not yet firmly established, it has been postulated that omega-3 fatty acids may act upon central nervous system cell membranes and phospholipid composition . Increased omega-3 levels in cellular membranes, in turn, impact dopaminergic and serotonergic activity . One study found improvement in parent - rated behavior and attentional tasks following 15 weeks of fish oil supplements while another report indicated improved attention and global behavioral improvement for children receiving combined omega-3 and omega-6 supplements compared with olive oil . A third rct found significant sustained improvement for 30 weeks on cognitive tasks as well as on parent ratings with efas . However, teacher rated symptoms did not change significantly nor was there any additional benefit from adding a multivitamin supplement as augmentation . A 30-week placebo - controlled trial of phosphatidylserine contained omega-3, followed by an open - label period, found a similar pattern of parent - reported benefits for restless - hyperactive symptoms in the absence of teacher - reported effects . However, a subgroup of treated ad / hd children demonstrating particularly pronounced hyperactivity / impulsivity as well as oppositional behavior demonstrated a significant reduction in both parent- and teacher - rated restlessness and emotional lability . Despite these positive findings, two other rcts found that efas had minimal effect on ad / hd symptoms . Among children on stimulant therapy, four months of dha supplementation an additional study did not find any benefit from two months of efa supplementation on either teacher or parent behavioral ratings or standardized cognitive tasks . In several rcts reporting benefit from efas, the investigators employed a number of behavior ratings and cognitive tasks with few advantages for efas on most outcome measures . Another area of concern is that while biochemical measures indicated increases in omega-3 levels, a systematic association between efa levels and behavioral outcomes has not been established . In one study, omega-3 fatty acid supplements however, the frequent use of olive oil, a source of oleic acid which when converted to oleamide, has some psychoactive effects, as a placebo in this research may underestimate omeaga-3's benefits . A meta - analysis found a modest effect (.31) for omega-3 ffa supplementation on ad / hd symptoms . Finally, a cochrane paper, while reporting tentative benefits for combined omega-3 and omega-6 supplements, concluded that there was little evidence that efa's reduced ad / hd symptoms . Currently, while demonstrating some promise as a complement to stimulant medication, the evidence base for efas is inconclusive . First, children with ad / hd may have a specific allergy to refined sugar . A second view is that sugar ingestion influences cognition and behavior through a form of functional hypoglycemia . The latter hypothesis is supported by evidence of abnormal glucose metabolism among several groups of hyperactive children . A meta - analysis, conducted 15 years ago, concluded that there was no evidence that sugar ingestion caused hyperactivity or impacted cognitive performance . However, a more recent paper was slightly more equivocal with 25% of studies finding some evidence of increased hyperactivity and inattention following sugar ingestion . Mothers believing that sugar triggered symptoms were more critical and directive and more likely to rate their child as disruptive after being told that the child had ingested sugar rather than a placebo . In the 1970s, feingold concluded that 50% of ad / hd children demonstrated particular sensitivity to dietary food colorings, preservatives, and natural salicylates and recommended diets free of these additives as treatment . While early papers and a meta - analysis concluded that the feingold diet had little impact on ad / hd symptoms, recent research suggests that subgroups of children may be particularly sensitive to these substances . Additionally, nonclinical pediatric samples have demonstrated increases in hyperactivity after ingesting food dye and a preservative . Among preschool children with ad / hd, a preservative- and artificial - flavoring - free diet was associated with decreased parent - rated hyperactivity . Presently, it appears that subgroups of children may be particularly sensitive to artificial additives but this association is not specific to those with ad / hd [3638]. Several studies have suggested that food restriction followed by a systematic gradual reintroduction of offending foods may have some benefit in reducing hyperactive behavior [3941]. While multiple few foods diets exist, a commonly employed restrictive diet was developed by egger et al . And includes a limited number of foods from each group including meats (lamb, turkey), carbohydrates (rice, potatoes), vegetables (carrots, cabbage), and fruits (apples, bananas). After the benefits of this usually well - tolerated diet are apparent, new foods are gradually reintroduced and in the case of ad / hd, behavior is carefully observed . While there are some suggestions that children with food allergies may show some behavioral improvement with the oligoantigenic approach, the clinical effects of this dietary intervention appear small particularly when compared with methylphenidate . Additionally, methodological issues such as small sample size, absence of blinding, and overreliance on parent ratings make the results difficult to interpret . It is likely that among children with both ad / hd and food allergies, dietary intervention may be a useful complementary therapy . Any substance that may cause a specific illness is administered in reduced and/or diluted doses as a means of treating the condition . The treatment is often individualized based upon specific patients' symptoms and personality characteristics . The active treatments were either verum or mixtures of homeopathic substances including selenium and sodium phosphate . There was no evidence that homeopathic treatment was effective in reducing symptoms as reported on parent - completed behavioral rating scales or cognitive tasks . Frei and colleagues, who conducted one of the trials, raised questions about the adequacy of evaluating outcomes based only on several months of treatment . They noted that homeopathic treatments required an average of 6 1/2 months to reduce symptoms by 50% . Frei and colleagues also found that children treated with methylphenidate responded more slowly to homeopathic treatment even after stimulant medication had been discontinued . Quantitative eeg studies suggest that children with ad / hd frequently exhibit a pattern of cortical hypoarousal in brain regions associated with alertness, attention, and self - control . While less common, a smaller group of patients demonstrate hyperarousal with an increase in fast eeg frequencies over the same regions . These abnormalities may reflect structural differences in neuroreceptor density as well as altered neurotransmitter activity for dopamine and norepinephrine with possible serotonin and acetylcholine involvement . While eeg monitoring occurs, children perform tasks while information about their neuroelectrical signals is translated into graphic displays, lights, or tones . In addition, patients may move cartoon - like video characters through cognitive - behavioral activity to maintain specific eeg patterns . The overall goal is for the patient to demonstrate a cortical activation pattern comparable to typical age peers for a 45-minute period . Sessions are typically scheduled on a weekly or biweekly basis for 45 minutes to an hour . The average number of training sessions is 43 with a range from 34 to 50 [4547]. Results of nonrandomized trials and a recent meta - analysis indicated that children receiving neurofeedback demonstrated improved performance on attentional tasks such as the test of variable attention (tova) as well as reduced parent - rated inattention and impulsivity [4749]. A comparison of neurofeedback with another alternative therapy, attention skills training, found both treatments to be effective in reducing parent - rated ad / hd symptoms . However, neurofeedback was associated with greater reductions in both parent- and teacher - rated symptoms . At least two studies have found neurofeedback to be equally effective as stimulant medication [51, 52]. Monastra found that those patients receiving neurofeedback appeared to maintain their cognitive - behavioral gains better when medication was withdrawn for a one - week washout . Additionally, among children taking stimulant medication, addition of neurofeedback was associated with dosage reduction . While appearing promising, neurofeedback's evidence basis has been questioned . To date, most neurofeedback studies are based on self (i.e., parent-)-selected samples and the majority rely on unblinded parent ratings and laboratory task performance as outcome measures . The absence of consistent teacher - rated improvement, together with the nonrepresentativeness of cognitive tasks to real world academic demands and the few posttreatment follow - up studies, raises concern about neurofeedback's generalizability . Nonspecific factors occurring during neurofeedback therapy such as the multiple months of regular therapist contact with children, repeated assessment of attentional skills, parent education, and behavior training make it difficult to conclude that eeg feedback is specifically responsible for the reported 6075% improvement rates [52, 53]. In the recent meta - analysis, neurofeedback's comparative effectiveness was substantially less in the few randomized studies involving a semiactive control intervention the time and cost (30 to 40 sessions at $75.00 to $150.00) may be prohibitive to many parents particularly when not covered by insurance . Neuropsychologically, ad / hd is characterized by impaired executive functioning which includes planning, reasoning, and response inhibition . There are several commercially available computerized training programs designed to improve working memory and concentration among patients with ad / hd . Cogmed, a commercial program, includes visual - spatial and verbal tasks in which the patient briefly views a pattern or verbal material such as phonemes or letters on a screen and then applies it to a pattern on the subsequent screen . Once the child and their parents are familiar with the procedure, training sessions can be conducted on a home computer . Several clinical studies indicate significant pre - post training improvement on computerized tasks generalizing to novel cognitive tests [5557]. In a study with a six - month followup after training, however, six - month posttreatment ratings by psychologists did not indicate clinically significant improvement in cognitive skills . In terms of effect size, the authors concluded that the benefits of training were comparable to methylphenidate . While memory training has been less well - studied than neurofeedback, the time commitment and cost are comparable . Commercial programs such as cogmed require that a specially trained coach be present physically or by phone during training . While promising, cognitive training is likely to be more effective for symptoms of inattention with significantly less benefit for hyperactivity . Research on yoga indicates that it impacts neurophysiological functions including oxygen consumption, lateralization patterns, and cognition all of which often demonstrate atypical patterns in children with ad / hd . While yoga is not a unitary set of techniques, a foundational procedure for ad / hd is respiratory training with a focus on rhythmic inhalation and exhalation that reduces sympathetic nervous system activity while providing an attentional focus . In concert with breathing, finally, yoga may also include visual fixation on an object such as a candle flame or mental visualization of a word or shape . Two studies applying yoga to children with ad / hd have suggested that it may have some mild benefits that may additive with medication effects [58, 59]. There does appear to be a dose - response relationship with more sessions of yoga associated with greater improvement on teacher ratings of hyperactivity / impulsivity . Parents participating in yoga along with their children reported reductions in stress and improvement in managing their child . After treatment, parents rated their children as demonstrating improved self - esteem, and reduced behavioral problems . Several studies have examined the impact of therapeutic massage on adolescents with ad / hd [60, 61]. While the rationale for this therapy has not been well articulated, there is evidence that massage increases eeg patterns associated with attention as well as vagal ton [60, 62]. Increased vagal cardiac control a randomized controlled trial indicated that adolescents receiving weekly or biweekly massage therapy demonstrated improved mood as well as teacher rated classroom behavior [60, 61]. In addition, students with ad / hd were found to have significantly improved task focus moving from being on task 47% of the time to 75% at the end of ten consecutive school days of 15-minute massage sessions . The length of the intervention a total of 10 days to four weeks is unlikely to have enduring benefits after regular massage has ended . Additionally, in the few studies conducted, medication status was unclear . Mindfulness meditation involves training people to be observers of their ongoing thoughts and feelings without attempting to change these internal experiences . Several studies of mindfulness meditation in adults have suggested that it may have beneficial effects on cognitive activities such as shifting set and possibly, in improving working memory . There are relatively few studies on meditation techniques applied to childhood ad / hd and the reports are based on small sample sizes . Results to date have been mixed with one study indicating improvement in parent ratings of impulsivity and improved performance on an attentional measure and another indicating improved classroom behavior in the absence of parent reported improvement [64, 65]. Green space is the term used to describe a natural green setting including trees, and grass . Green space exposure as a form of treatment is based on attention restoration theory (art). As with rest or sleep, activities that draw upon involuntary attention permit voluntary attention to recover . Those environments, such as the classroom requiring more effortful forms of attention, are fatiguing . In contrast, outdoor environments with green space are gently absorbing and draw upon involuntary attention while restoring voluntary attention . In a survey, parents of children with adhd reported greater symptom improvement after children participated in activities in natural " settings versus indoor or artificially built outdoor settings such as cement playgrounds . While direct pre - post green space exposure studies are limited, a recent study found that children with adhd performed better on a verbal task sensitive to concentration after taking a walk in a park versus a residential or downtown setting . Effect sizes were comparable to those associated with methylphenidate treatment . While interesting, there are little data to indicate the duration of green space exposure required or the duration of improved cognitive functioning after exposure . To date, complementary and alternative therapies for childhood attention deficit hyperactivity disorder have not been consistently supported . Relatively few studies include randomized controlled placebo trials . Conducting randomized controlled trials is particularly challenging in complementary and alternative medicine since an important therapeutic factor appears to be participants' belief in cam efficacy as well as their relationship with the provider . Additionally, interventions such as neurofeedback and memory training, requiring multiple months of individualized sessions, may be beneficial to patients and their parents because of the nonspecific factor of individualized attention from a mental health provider . Because of self - selection and expectancy effects as well as the current unknown efficacy of cam versus the established effectiveness of stimulant medication, it has been difficult to conduct true randomized cam trials for ad / hd . In addition, many of the trials papered had particularly large attrition rates . It is noted, however, that most of the published trials of this treatment, while including some of the limitations noted immediately above, are also authored by those with a strong allegiance to this therapy . Of the nutritional supplements papered, omega-3 oils and possibly zinc the possible negative effects of nearly all of the treatments paper are negligible . Given that very few of these treatments have been tested in head to head trials with established therapies such as methylphenidate, it is difficult to make definitive conclusions regarding possible clinical efficacy . Nonetheless, the consistent finding that up to 30% of children with ad / hd do not respond to stimulant medication and the physical side effects of pharmacotherapy suggests that there is a definite role for efficacious alternative and complementary therapies . However, investigations to date suggest that as the rigor of research design increases, the likelihood of finding a positive effect for alternative therapies for ad / hd decreases . Therapies such a neurofeedback, omega-3s, or zinc supplements, which may be beneficial, may demonstrate optimal benefit when complementing established treatments including stimulant medication.
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Ewing's sarcoma (ews) accounts for 10% of malignant bone tumors and commonly arises from the long bones, pelvis, and ribs . Primary ews arising from cranial bones is rare and accounts for only 1%4% of all ews . We report the case of a 15-year - old girl with ews of the skull and discuss the treatment of this rare entity . A 15-year - old girl presented with swelling on the vertex of the head since 1 year, rapidly enlarging since few days . Computed tomogram (ct) showed an expansile lytic lesion with elevation of periosteum in the mid frontoparietal calvarium projecting into the scalp with heterogenous soft - tissue component [figure 1]. The lesion was extending intracranially . A magnetic resonance (mr) angiogram showed lytic destructive mass lesion of frontal calvarium breaching the dural lining and partially encasing the superior sagittal sinus [figure 2]. Histopathological examination showed a highly cellular and vascular tumor composed of uniform round cells with scanty cytoplasm and round vesicular nucleus with periodic acid on immunohistochemistry, the cells showed strong membrane positivity for mic 2 and were negative for leukocyte common antigen (lca) and synaptophysin [figure 4]. The picture was suggestive of ews . Computed tomography scan showing the expansile lytic lesion in frontoparietal calvarium projecting into the scalp with soft - tissue component magnetic resonance angiogram showing the destructive mass lesion in frontoparietal calvarium section showing cellular small round cell tumor with vesicular nuclei and vacuolated cytoplasm . A mitotic figure is seen toward the center of the field (h and e, 400) tumor cells showing diffuse and strong membrane staining for mic 2 (200) her hematology and serum chemistries were normal . A cerebrospinal fluid study, ct of the chest and bone marrow examination were normal . She received combination chemotherapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide, and vp16 for 1 year . Ews is a highly malignant bone tumor seen in children and young adults, and primary involvement of the skull is rare . Ews of the skull affects the frontal, parietal, and temporal bones and sometimes the skull base . Ews typically grows extradurally and often reaches a large size before it is clinically detectable . In a retrospective study, among 332 cases of ews, about 90% of the cases occur in the first two decades of life, peak incidence is between 5 and 13 years of age, and males are affected more frequently . The most common symptoms are headache and scalp swelling, the duration of which ranges from 2 weeks to 2 years . Plain x - ray of the skull shows a lytic lesion with mottling and erosion, and the classical onion peel appearance is rare . Ct and mr imaging are useful to understand the size, extent, and involvement of dura and brain parenchyma . Ct scan is useful to delineate bone involvement, and on mri, the mass is hypo- to iso - intense on t1-weighted and iso- or hyper - intense on t2-weighted with heterogeneous enhancement on contrast . On bone scintigraphy histologically, these tumors are characterized by sheets of small round blue cells with a high nuclear - cytoplasmic ratio . Immunohistochemistry helps differentiate ews from other small round cell tumors such as rhabdomyosarcoma, neuroblastoma, and lymphoma . Ews is positive for cd99 and vimentin and negative for desmin, lca, and synaptophysin . Ews is characterized by a balanced translocation between chromosome 11 and 22 which fuses ews gene on chromosome 22q12 with fli1 gene on 11q24 . Rarely, translocation t(21;22) or t(7;22) is observed . Fli1 fusion transcript type 1 being associated with improved outcome compared with that in patients with other fusion transcript types . The incidence of metastasis is less, radiologically the typical onion peel appearance may not be seen, and the prognosis is better than the disease in other sites . Like extracranial ews, multimodal therapy is the treatment of choice for cranial ews also . Surgical resection is crucial in the management of ews of the skull and is the preferred approach if the lesion is resectable . A radical excision should be attempted to minimize the tumor mass . In calvarial ews, as majority of patients present with rapidly enlarging lesion and raised intracranial pressure, upfront surgical resection and decompression become imperative unlike ews of extremities where presurgical chemotherapy is used to shrink the tumor . In calvarial ews, it is usually not possible to get a margin free of microscopic disease and hence local radiotherapy is recommended at a dose of 4050 gy . Current chemotherapy regimen consisting of a combination of vincristine, cyclophosphamide, doxorubicin alternating with ifosfamide, and etoposide used for the treatment of ews has given a 5-year survival rate of about 70% . With total excision, radiation, and standard chemotherapy, the 5-year survival ranges from 39% to 65%.
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Reagents and chemicals pfu dna polymerase was from stratagene (la jolla, ca). Qiagen (valencia, ca) kits were used for plasmid dna purification and the extraction of dna from agarose gels . C - palmitoyl coenzyme a (c - pcoa), c - palmitic acid, c - butyryl coenzyme a, c - crotonoyl coenzyme a, and c - docosanoyl coenzyme a were purchased from arc radiolabeled chemicals (st . Louis, mo; 5055 mci / mmol). Unlabeled palmitoyl coenzyme a (pcoa) was purchased from avanti polar lipids, inc . Trehalose 2-palmitate (t2p) and trehalose 2-sulfate were synthesized as described previously (1214). The synthesis and structural characterization of trehalose 3-palmitate (t3p) are reported in the supplemental material . Glass - backed silica gel 60 hptlc plates were purchased from emd chemicals (gibbstown, nj). Electrospray ionization fourier transform ion cyclotron mass spectrometry (esi - ft - icr ms)mass spectra were obtained on an apex ii ft - icr mass spectrometer equipped with a 7-tesla actively shielded superconducting magnet (bruker daltonics, billerica, ma). Ions were introduced into the ion source via direct injection at a rate of 1 l / min . Ions were generated with an apollo pneumatically assisted electrospray ionization source (analytica, branford, ct) operating in the negative ion mode and were accumulated in an rf - only external hexapole for 0.52 s before being transferred to the ion cyclotron resonance cell for mass analysis . Mass spectra consist of 512,000 data points and are an average of between 28 and 128 scans . The spectra were acquired using xmass version 6.0.0 or 7.0.8 (bruker daltonics). Additional mass spectra were obtained on an ltq ion trap mass spectrometer equipped with an esi source (thermofinnigan) operating in either the negative or positive ion mode . Ions were introduced into the ion source via direct injection at a rate of 5 l / min . For linear ion trap tandem mass spectrometry (ms) experiments, the precursor ions were isolated with an isolation width of 13 da; the ions were activated with a 1320% normalized collision energy for 100 ms, and the qz value was maintained at 0.250 . Spectra are an average of 100 scans, acquired using xcalibur, version 1.4 (thermofinnigan). Preparation of protein expression vector the papa3 gene (rv1182, encoding residues 2472) was amplified from m. tuberculosis h37rv genomic dna with the primers 5-tactgtagtcgaattcttgcgggttggaccgttgac-3 (ecori) and 5-gattacaggtctgcagtcaggcaacattctgctgct-3 (psti). Rv1182 was ligated into a modified pmal - c2x vector (new england biolabs) encoding an n - terminal his7 tag and tev cleavage site . The tev cleavage site was introduced by site - directed mutagenesis using the quikchange pcr mutagenesis kit (stratagene), and the his7 tag was annealed to the 5 end of the maltose - binding protein (mbp)-coding region . Dna sequencing was performed to confirm the successful construction of the protein - encoding plasmid . Transformants were used to inoculate 1-liter cultures of lb medium containing 100 mg / liter ampicillin and 2 g / liter glucose . The cultures were incubated at 37 c for 2.5 h with shaking until an a600 of 0.60.8 was attained . Protein expression was induced by the addition of isopropyl -d-1-thiogalactopyranoside to a final concentration of 100 m . After 1820 h at 18 c, cells from each liter of culture were harvested and suspended in 30 ml of lysis buffer (20 mm tris, ph 7.4, 200 mm nacl, 1 mm edta, 1 mm dtt, 1 mm tcep) supplemented with 5 g / ml lysozyme and 5 g / ml dnase . Cells were lysed using a high pressure homogenizer (avestin emulsiflex - c5). The lysate was cleared by centrifugation and applied to amylose resin (new england biolabs) equilibrated with lysis buffer . After washing the resin with additional lysis buffer, mbp - papa3 was eluted in lysis buffer containing 10 mm maltose . The eluted protein was diluted 1:15 in low salt buffer (50 mm tris, ph 7.4, 1 mm dtt, 1 mm tcep, 10% glycerol) and loaded onto a monoq hr 5/5 column (ge healthcare) equilibrated with the same buffer . The protein was purified using a gradient of 1.5100% high salt buffer (50 mm tris, ph 7.4, 1 m nacl, 1 mm dtt, 1 mm tcep, 10% glycerol) over 40 min at a constant temperature of 4 c . Fractions (0.5 ml each) were analyzed by sds - page, and those containing pure protein were pooled and analyzed by the bradford protein assay to determine protein concentration . Mbp was cleaved from papa3 using actev protease (invitrogen) and removed using nickel - nitrilotriacetic acid resin (qiagen), which bound the his - tagged mbp and protease while leaving the desired protein in solution . Papa3 was subsequently concentrated and stored at 80 c . To confirm the identity of the protein, the purified sample was desalted on a microbore reversed - phase column (bruker agilent) and characterized using a bruker hewlett - packard esi - ion trap mass spectrometer . Tryptic digestion and mass fingerprinting using an ltq mass spectrometer provided further confirmation of the identity of the protein . Protein concentration was determined by uv absorption at 280 nm using a calculated extinction coefficient of 57,340 m cm . Biochemical characterization of papa3a previously described tlc - based assay (5) was used to characterize the acyltransferase activity of papa3 . Briefly, papa3 (2 m) was incubated with 20 m c - pcoa and 1 mm of the desired sugar substrate in reaction buffer (100 mm ammonium bicarbonate, ph 7.2) for 2 h at room temperature . Alternative c - labeled acyl donors were screened at a concentration of 20 m . The reactions were quenched by the addition of an equal volume of ethanol and subsequently analyzed by tlc (35:65 methanol: chloroform) and phosphorimaging . Stocks of each sugar substrate were prepared in water with the exception of t2p and t3p, which were dissolved in dimethyl sulfoxide (dmso). The amount of dmso in any given reaction did not exceed 5% of the reaction volume . Pat and sl-1 share related structures and biosynthetic gene clusters . A, structure of pat . Papa3 (1 m) was incubated with 20 m unlabeled pcoa and 1 mm trehalose or t2p in reaction buffer for 6 h at room temperature . Samples were lyophilized and stored at 20 c prior to analysis by esi - ft - icr and linear ion trap ms . Construction of m. tuberculosis mutants m . Tuberculosis cells (erdman strain) were cultured in 7h9 medium supplemented with 10% oadc, 0.5% glycerol, and 0.05% tween 80 or on 7h10 solid agar medium supplemented with 10% oadc and 0.5% glycerol . Hygromycin (50 g / ml) or kanamycin (25 g / ml) was used when necessary . The papa3 mutant strain was created by homologous recombination as described previously (15). Briefly, specialized transduction phage phmws120 was incubated with concentrated wild - type erdman m. tuberculosis cells for 4 h at 39 c . The resulting deletion replaced 1136 bp of papa3 (amino acids 45423) with a hygromycin resistance cassette . The papa3::papa3 complementation strain was created by cloning the papa3 gene from m. tuberculosis (erdman strain) into the mycobacterial expression vector pmv261 (16) under the control of the groel promoter, resulting in the complementation plasmid pmws149 . This plasmid was electroporated into the papa3 strain, and transformants were selected on kanamycin - containing plates . The stf0 mutant was created by homologous recombination using transduction phage phmws102 as described above . The resulting deletion replaced 600 bp of stf0 (amino acids 22222) with a hygromycin resistance cassette . Cultures (50 ml) were transitioned to tween 80-free 7h9 media and grown at 37 c for 1 day . Surface lipids were extracted with hexane (1 ml) as described previously (17). The remaining cell pellets were extracted with 4 ml of chloroform: methanol (1:1). Papa3 reaction product samples were resuspended in 1 ml of 100% methanol (meoh) for esi - ft - icr ms analysis . For linear ion trap ms analysis of papa3 reaction product samples, 500 l of the resuspended volume were concentrated to dryness and resuspended in 3 ml of chloroform (chcl3):isopropyl alcohol (ipa) (2:1). M. tuberculosis cell surface extracts were concentrated to dryness under nitrogen and resuspended in 3 ml of chcl3:ipa (2:1). Lipids in each sample were separated using a modification of the method of kaluzny et al . (18). Samples were passed over a solid - phase extraction column (sep - pak vac, nh2 resin, waters) that had been pre - charged with 3 ml of 0.1 m ammonium acetate in meoh . The column was eluted using 3 ml of each of the following solvents: 1) chcl3:ipa (2:1); 2) diethyl ether: acetic acid (98:2); 3) 100% meoh; and 4) 0.1 m ammonium acetate in meoh . Column fractions from the papa3 reaction product samples were concentrated to dryness and resuspended in 1 ml of chcl3:meoh (2:1). Lithium acetate was added to the papa3 reaction products and the t2p and t3p standards to a final concentration of 1 mm . Column fractions from m. tuberculosis cell surface extracts were concentrated to dryness under nitrogen and resuspended in either 200 l chcl3:meoh (2:1) (fractions from solvents 1 and 2) or 400 l of chcl3:meoh (2:1) (fractions from solvents 3 and 4) for mass spectrometry analysis . Genomic analysis of the pat biosynthetic locus the pap gene family encodes polyketide synthase - associated acyltransferases that are involved in the synthesis of some of the complex lipids produced by m. tuberculosis (5, 11). In the m. tuberculosis genome, papa3 is clustered with the polyketide synthase - encoding gene pks3/4 . In some strains, including the sequenced h37rv strain, there is an intervening stop codon in pks3/4 that results in two separate open reading frames (termed pks3 and pks4) (7). Strains containing this mutation do not synthesize pat (19), indicating that an intact pks3/4 gene is essential for the biosynthesis of this glycolipid . Within the same gene cluster mmpl10 belongs to the same protein family as mmpl8, which is required for sl-1 biosynthesis (17, 19, 20). The genomic organization of pks3/4, papa3, and mmpl10 parallels that of pks2, papa1, and mmpl8 in the sl-1 biosynthetic gene cluster (fig . We previously demonstrated that papa1 is the acyltransferase responsible for coupling the polyketide product of pks2 to a trehalose - based acceptor (5). By analogy, we hypothesized that papa3 is essential for pat biosynthesis, catalyzing trehalose acylation . Papa3 is an acyltransferase that esterifies trehalose and t2p the papa3 gene from the h37rv m. tuberculosis strain was expressed in escherichia coli bl21(de3) as an n - terminal mbp fusion protein . Sds - page analysis revealed an apparent molecular mass of 95 kda for the purified protein . Following tev cleavage and subsequent removal of the protease and mbp by ni - affinity chromatography (supplemental fig . S1), the identity of the protein was confirmed by mass spectrometry . The measured mass (51,809 3 da) was consistent with the predicted molecular mass of the protein (51,777.5 da) oxidized at a single methionine residue . In addition, tryptic digestion and mass fingerprinting of the purified protein generated 61% sequence coverage, providing further confirmation of the identity of the protein (data not shown). Incubation of papa3 with c - pcoa and trehalose resulted in the formation of two unique products, as determined by silica gel tlc and phosphorimaging (fig . Only the less polar product was formed by the reaction of papa3 with c - pcoa and synthetic t2p (12, 13). Papa3 showed no activity against several other saccharides, including t3p, trehalose 2-sulfate,,-trehalose, glucose, and maltose, suggesting that the enzyme is selective for trehalose and t2p (table 1). C - pcoa to c - palmitic acid in the absence of another substrate (fig . Further kinetic analysis of the papa3 reaction containing trehalose or t2p was precluded by the complexity of the product mixture . Table 1substrate specificity of papa3nd means not detectable.substrateproduct formationanucleophileb trehalose yes t2p yes t3p nd trehalose 2-sulfate nd,-trehalose nd glucose nd maltose nd acyl - coac palmitoyl - coa yes docosanoyl - coa yes butyryl - coa nd crotonoyl - coa nd aproduct formation was assessed by tlc and phosphorimaging.breactions were performed with 2 m enzyme, 20 m c - palmitoyl - coa, and 1 or 10 mm of each substrate in 100 mm ammonium bicarbonate, ph 7.2, at room temperature for 2 h.creactions were performed with 2 m enzyme, 20 m c - acyl - coa, and 1 mm of trehalose or t2p in 100 mm ammonium bicarbonate, ph 7.2, at room temperature for 2 h. substrate specificity of papa3 nd means not detectable . Reactions were performed with 2 m enzyme, 20 m c - palmitoyl - coa, and 1 or 10 mm of each substrate in 100 mm ammonium bicarbonate, ph 7.2, at room temperature for 2 h. reactions were performed with 2 m enzyme, 20 m c - acyl - coa, and 1 mm of trehalose or t2p in 100 mm ammonium bicarbonate, ph 7.2, at room temperature for 2 h. interestingly, no activity was detected with c - butyryl coenzyme a or c - crotonoyl coenzyme a, which contains a trans-2-ene functionality like the mycolipenoyl groups of pat (table 1). However, product formation was observed upon incubation of papa3 with trehalose and c - docosanoyl coenzyme a, which consists of a 22-carbon saturated fatty acid conjugated to coenzyme a (data not shown). Taken together, these findings indicate that lipid chain length influences the substrate specificity of papa3 . A, papa3 was incubated with c - pcoa and either trehalose (tre) or t2p . Two new products (1 and 2) were observed in the reaction with trehalose, but only product 2 was observed in the reaction with t2p . B, esi - ft - icr ms analysis of product 1 from the papa3 reaction with trehalose . A product ion with m / z 615.32, corresponding to the m / z of a chloride adduct of synthetic t2p, was observed in the papa3 reaction . In contrast, the control reaction lacking papa3 showed no product at m / z 615 . C, esi - ft - icr ms analysis of product 2 from the papa3 reaction with tre and t2p . An ion with m / z 853.54 was observed in both reactions but was not present in control reactions lacking papa3 . A, papa3 was incubated with c - pcoa and either trehalose (tre) or t2p . Two new products (1 and 2) were observed in the reaction with trehalose, but only product 2 was observed in the reaction with t2p . B, esi - ft - icr ms analysis of product 1 from the papa3 reaction with trehalose . A product ion with m / z 615.32, corresponding to the m / z of a chloride adduct of synthetic t2p, was observed in the papa3 reaction . In contrast, the control reaction lacking papa3 showed no product at m / z 615 . C, esi - ft - icr ms analysis of product 2 from the papa3 reaction with tre and t2p . An ion with m / z 853.54 was observed in both reactions but was not present in control reactions lacking papa3 . Figure 3.linear ion trap ms of the monoacyl product ion from the reaction of papa3 with trehalose (a) is consistent with that of synthetic t2p (b) and not that of synthetic t3p (c). Ms analysis was performed in the positive ion mode using lithium - cation coordination . Shown are the ms spectra of the dissociation ions obtained from the cleavage of the glycosidic bond between the individual pyranose rings of trehalose . Linear ion trap ms of the monoacyl product ion from the reaction of papa3 with trehalose (a) is consistent with that of synthetic t2p (b) and not that of synthetic t3p (c). Ms analysis was performed in the positive ion mode using lithium - cation coordination . Shown are the ms spectra of the dissociation ions obtained from the cleavage of the glycosidic bond between the individual pyranose rings of trehalose . T2p and trehalose dipalmitate are products of papa3products from the reaction of papa3 with pcoa and either trehalose or t2p were characterized by esi - ft - icr and linear ion trap ms operating in the negative ion mode . The reaction of papa3 with trehalose yielded two unique products measured via ft - icr ms at m / z 615.3153 and m / z 853.5447, corresponding to the exact masses of the chloride adducts of trehalose palmitate and trehalose dipalmitate, respectively (fig . The accurate mass spectra were calibrated internally, and both reaction products were measured to sub - ppm accuracy . The product ion at m / z 853.54 was also observed in the reaction of papa3 with t2p (fig . Linear ion trap tandem mass spectrometry (ms) of the ion at m / z 615.32 yielded dissociation ions consistent with those derived from synthetic t2p (supplemental fig . Ms of the ion at m / z 853.54 was consistent with 2,3-dipalmitoylation of a single pyranose ring of trehalose (supplemental fig . S3), which was confirmed in the positive ion mode using lithium - ion coordination (supplemental fig . S4). Together, these data demonstrate that papa3 sequentially acylates trehalose to form trehalose dipalmitate . T3p is not produced by papa3to rule out the possibility that the initial acylation product of papa3 is t3p or a mixture of 2- and 3-palmitoylated species, we synthesized t3p for additional biochemical and structural studies . To determine whether t3p is a viable intermediate in the biosynthesis of trehalose dipalmitate, we incubated t3p with papa3 and c - pcoa . Interestingly, the presence of t3p in the reaction mixture appears to diminish the hydrolysis of c - pcoa by papa3, suggesting it may instead inhibit the enzyme . For further confirmation that the monoacyl product of papa3 is not t3p, we analyzed t3p by ms in the positive ion mode using lithiumcation coordination (21, 22) and compared its fragmentation to that of t2p and the papa3 monoacyl reaction product ion (fig . T2p, t3p, and the monoacyl papa3 reaction product were observed at m / z 587, corresponding to the lithium adducts of these molecules . Two dissociation ions at m / z 425 and 407 were observed in the ms spectra of these ions, corresponding to the cleavage of the glycosidic bond between the pyranose rings of trehalose (data not shown). The ms spectrum of the monoacyl papa3 reaction product dissociation ion at m / z 407 was identical to that derived from synthetic t2p (fig . 3, a and b) and dramatically distinct from the ms spectrum derived from synthetic t3p (fig . Combined with the finding that t3p is not a substrate for papa3, these data support the assignment of t2p as the initial acylation product of papa3 . Papa3 is required for pat biosynthesis in vivo to determine whether papa3 is required for pat biosynthesis in vivo, a papa3 deletion mutant, papa3, was generated in the erdman strain of m. tuberculosis . The h37rv strain sequenced by cole et al . (23) contains a stop codon in pks3/4, which truncates the encoded polyketide synthase and abolishes pat biosynthesis . However, sequencing confirmed that the erdman strain used in these studies encodes a single open reading frame for pks3/4, yielding a functional polyketide synthase (data not shown). Extraction of the crude lipids from wild - type erdman cells with organic solvents followed by esi - ft - icr ms analysis confirmed the presence of the pat lipid envelope in this strain of m. tuberculosis (fig . In contrast, pat was not observed in extracts from the papa3 mutant strain . Complementation of the papa3 mutant strain with a plasmid encoding papa3 restored pat production, demonstrating that papa3 is essential for the biosynthesis of this glycolipid in m. tuberculosis . Given the similarities between the pat and sl-1 genetic loci, we sought to determine whether the two biosynthetic pathways shared common intermediates . The first committed step in sl-1 biosynthesis is the sulfation of trehalose at the 2-position of one of the glucose moieties by the sulfotransferase stf0 (24). Thus, we analyzed the m. tuberculosis stf0 mutant strain, which lacks sl-1 as well as its upstream biosynthetic precursors, for the presence of pat . Ft - icr ms analysis of crude lipid extracts clearly showed the presence of pat in the stf0 mutant strain (fig . These data indicate that the pat biosynthetic pathway is independent of the sl-1 pathway and that these pathways share no intermediates other than the cellular pool of trehalose . The data presented here establish that papa3 is an acyltransferase required for pat biosynthesis in m. tuberculosis . Recombinant papa3 selectively acylates trehalose and t2p in a manner consistent with the structure of pat . Also, deletion of papa3 from m. tuberculosis prevents pat synthesis in vivo . Furthermore, despite the genetic and structural similarities between pat and sl-1, the biosynthetic pathways of these metabolites are independent . Esi - ft - icr ms analysis of lipid extracts from wild type, papa3::papa3, and stf0 m. tuberculosis strains revealed the presence of characteristic pat lipoforms that are absent from papa3 m. tuberculosis extracts . Esi - ft - icr ms analysis of lipid extracts from wild type, papa3::papa3, and stf0 m. tuberculosis strains revealed the presence of characteristic pat lipoforms that are absent from papa3 m. tuberculosis extracts . Papa3 first acylates the 2-position of one of the glucose residues of trehalose with a palmitoyl group to form t2p . A mycolipenoyl group, synthesized by pks3/4, is then transferred to the 3-position of t2p by papa3 to generate 2,3-diacyltrehalose . 2,3-diacyltrehalose may either be transported to the cell surface or serve as a biosynthetic intermediate that is further elaborated with mycolipenic acids to give pat . Papa3 first acylates the 2-position of one of the glucose residues of trehalose with a palmitoyl group to form t2p . A mycolipenoyl group, synthesized by pks3/4, is then transferred to the 3-position of t2p by papa3 to generate 2,3-diacyltrehalose . 2,3-diacyltrehalose may either be transported to the cell surface or serve as a biosynthetic intermediate that is further elaborated with mycolipenic acids to give pat . In vitro, papa3 catalyzes the sequential transfer of two palmitoyl groups onto a single glucose residue of trehalose, suggesting that papa3 installs both the palmitoyl group at the 2-position of pat and the 3-mycolipenoyl group (fig . It is possible that papa3 associates with other proteins in the pat biosynthetic pathway, forming a coordinate synthetic complex similar to that described for phthiocerol dimycocerosate biosynthesis (25). Such supramolecular assemblies may influence substrate availability and orientation in the papa3 active site, thereby conferring this unique activity . Whether papa3 is truly papa3 may only install the palmitoyl group of pat in vivo, in which case the formation of trehalose dipalmitate by the purified enzyme is an in vitro artifact . Such activity has been observed with the lauroyltransferase of e. coli lipid a biosynthesis, which transfers two lauroyl groups in vitro but only one in vivo (26). Alternatively, the mycolipenoyl groups found at the 3-, 6-, 2-, and 4-positions of pat may all be physiological products of papa3 . This hypothesis is supported by the genetic association between papa3 and the mycolipenate synthase pks3/4 and the promiscuity of papa3 toward the acylation state of trehalose . Unfortunately, our analysis of the acylation events catalyzed by papa3 is limited by the lack of a commercially available mycolipenoyl substrate and our limited capability to detect either t2p or trehalose mycolipenates in vivo . The chemical similarities between pat and sl-1 suggest that the biosynthesis of these molecules may be comparable . However, the sl-1 gene locus encodes two pap proteins, papa2 and papa1, which sequentially install a palmitoyl group and a methyl - branched hydroxyphthioceranoyl group, respectively . By comparison previously, we showed that papa2 does not recognize trehalose and cannot account for t2p synthesis (5). We initially hypothesized that the sl-1 precursor trehalose-2-sulfate-2-palmitate (termed sl659) may be desulfated by a sulfatase to form t2p, thus negating the need for a committed trehalose palmitoyl - transferase . However, the stf0 mutant strain, which lacks sl659, maintained the ability to synthesize pat . We therefore conclude that sl-1 and pat biosynthesis are independent . On the basis of our biochemical and genetic data, initially, papa3 modifies the 2-position of one of the glucose residues of trehalose with a palmitoyl group from an unknown acyl donor, most likely pcoa or an acyl pantotheine - based cofactor . A mycolipenoyl group is then transferred to the 3-position of t2p by papa3, which may associate directly with pks3/4 or an unknown acyl carrier protein to initiate this second acylation step . The resulting 2,3-diacyltrehalose may be transported to the cell surface without further modification through an unknown pathway . Alternatively, it may serve as a biosynthetic intermediate that is elaborated either intracellularly or extracellularly with the three remaining mycolipenoyl groups of pat by means of papa3 or an unidentified acyltransferase . By analogy to other m. tuberculosis lipid biosynthetic pathways, transport of pat or its precursor to the cell surface the synthesis of physiological substrates for in vitro assays and the comparative lipid analysis of a panel of pat biosynthetic mutants, including mmpl10 and pks3/4, may resolve some of these issues . Whereas genes from the pat biosynthetic gene cluster are up - regulated under various conditions of environmental stress, including phagosomal acidification and nutrient starvation (27, 28), the role of pat in m. tuberculosis pathogenesis remains a mystery . Notably, a recent study of an m. tuberculosis strain deficient in pat biosynthesis suggests pat does not contribute to virulence in mice (29). However, the phenotype of sl-1-deficient m. tuberculosis strains is also indistinguishable from wild type in the murine model of infection (5), suggesting the function of these glycolipids may be host - specific . Thus, a more appropriate model of tuberculosis may be key to elucidating the role of these lipids in m. tuberculosis pathogenesis.
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The prozone effect is most often associated with secondary syphilis, human immunodeficiency virus (hiv) co - infection, and pregnancy . The incidence of the prozone phenomenon is attaining clinical significance due to increasing population at risk for sexually transmitted diseases, especially those who are hiv positive . A 36-year - old female presented with a chief complaint of rash all over the body, of 15 days duration, associated with severe itching . On detailed history elicitation, the patient applied some native medications and developed generalized itching and presented with a picture of contact irritant dermatitis in the form of papules, vesicles, and scaling, all over the body . The patient was treated for the same with a combination of topical steroids and antihistamines . The patient's rash was persistent in the form of macules, papules, and scaling . A thorough examination revealed the presence of few lesions on the genitalia mainly on the labia majora [figure 1a], which were flat topped papules and the patient also had a mucous patch over the hard palate [figure 2a]. Thus, secondary syphilis was strongly suspected and the patient was subjected for venereal disease research laboratory (vdrl) testing . As the suspicion of secondary syphilis was high, we conducted a detailed examination of the patient's husband . He denied a history of any sexual exposure and any genital lesion, but gave a history of intermittent urethral discharge . Therefore, we requested for repeat vdrl test, with higher dilution and it was reactive with 1: 512 dilution . The patient and husband were subjected to hiv testing, and both were found to be seropositive . All skin, oral [figure 2b] lesions regressed and condyloma lesions [figure 1b] disappeared after one month of follow - up . The patients were referred to the voluntary counseling and testing center (vctc) for further evaluation and initiation of antiretroviral therapy . Genital lesions before treatment oral lesions before treatment oral lesions after treatment disappearance of lesions after treatment prozone phenomenon is an immunological event, relying on an antigen - antibody interaction such as in rapid plasma reagin (rpr) or vdrl . An agglutination or precipitation reaction will be positive (i.e., visible through lattice formation) depending on several factors that determine the size and solubility of the immune complex formed in vitro . The optimal ratio of the antigen antibody yields an insoluble precipitate that is visible, thus rendering the test positive . The zone of equivalence defines this optimal ratio . In the zone of the antibody excess (prozone) or antigen excess (post zone), false negative results will occur . The prozone phenomenon in the setting of syphilis may become prevalent because of the current acquired immunodeficiency syndrome (aids) epidemic . As syphilis and hiv mutually increase the chance of contracting other diseases, anomalous b - cell behavior can lead to hyper - responsiveness to antigenic stimulation, leading to excess antibody production . This is performed by diluting the patient's serum to bring the antibody concentration into the zone of equivalence . Therefore, it is important to notify the laboratory in this regard, when the clinical findings strongly suggest syphilis and when the nontreponemal serological test results are negative . The purpose of this article is to focus attention on the prozone phenomenon in the setting of syphilis, which may become more prevalent due to increasing hiv incidence . Thus, an astute dermatologist who maintains a high index of suspicion and continued familiarity with protean manifestations of secondary syphilis should look for the prozone phenomenon in suspected cases.
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Local environment is contaminated with parasite eggs shed by infected cats and dogs [2, 3]. Humans acquire an infection with toxocara by accidental consumption of infecting parasite eggs or larvae [4, 5]. Infection with toxocara is one of the most widespread zoonotic parasitic infections and causes a disease known as toxocariasis . The burden of toxocariasis in north america is significant; however, toxocariasis is recognized as a neglected zoonotic disease [6, 7]. The seroprevalence of toxocara infection varies substantially among population groups, that is, 2%5% in adults in urban areas, 14.2%37% in rural areas, and 63.2%92.8% in some tropical countries . Toxocara does not mature in the human intestines but instead migrates through tissues and organs of the body . Dissemination of toxocara may occur to muscles, eyes, liver, lungs, and central nervous system . Toxocara may invade the brains of humans; however, neurotoxocariasis or cerebral toxocariasis remains a poorly understood phenomenon . Toxocara infection may lead to eosinophilic meningitis, meningoencephalitis, myelitis, cerebral vasculitis, optic neuritis, epilepsy, and multiple cerebral infarction . In addition, toxocariasis has been associated with dementia [11, 15, 16] and mental confusion . A high seroprevalence of toxocara infection has been found in psychiatric patients [1820]. In a study in italy, researchers found a 13% seroprevalence of toxocara infection in psychiatric patients . In a recent study in mexico, 4.7% of 128 psychiatric inpatients were seropositive for toxocara infection, whereas, in a study in china, a 16.4% seroprevalence of toxocara infection in psychiatric patients was found . Only few infectious agents have been studied in relation with suicide attempts including influenza b and toxoplasma gondii infections [22, 23]. Several studies have shown that toxocara affects the brain of humans and rodents [2426]. However, it is unknown whether toxocara infection is associated with suicide attempts . Therefore, we performed a case - control seroprevalence study to determine whether toxocara infection is associated with suicide attempts in psychiatric outpatients in durango city, mexico . This case - control study was performed using stored serum samples from a recent toxoplasma gondii study in psychiatric patients in durango city, mexico . Subjects (n = 282) enrolled in the study were psychiatric outpatients who attended two public hospitals in durango city: the hospital of mental health miguel vallebueno and the general hospital of the secretary of health . Inclusion criteria for suicide attempters were the following: (1) psychiatric outpatients with history of one or more suicide attempts; (2) those aged 18 years and older; and (3) those who accepted to participate in the study . In total they were 1861 years old (mean 34.01 10.25 years) and included 119 females and 37 males . Inclusion criteria for psychiatric controls were the following: (1) psychiatric outpatients without history of suicide attempts; (2) those aged 18 years and older; and (3) those who accepted to participate in the study . The control group included 126 (75 females, 51 males) patients aged 1869 years (mean 38.00 11.59 years). Sociodemographic, clinical, and behavioral characteristics of the psychiatric patients were obtained with a questionnaire through a face - to - face interview . Sociodemographic items were age, gender, birthplace, educational level, occupation, and socioeconomic status . Clinical items included diagnosis of current psychiatric disease and concomitant diseases, suicidal ideation, history and number of suicide attempts, time from last suicide attempt, and method of suicide attempts . It is unclear how sensitive the face - to - face interview to detect suicide attempts used is . In addition, other clinical data including lymphadenopathy, frequent headache, impairments in memory, reflexes, hearing, and vision, and history of blood transfusion, transplant, surgery, alcohol consumption, drug abuse, or sexual history from all participants were obtained . Behavioral items were the following: contact with animals and cat excrement, traveling, type of meat consumed, consumption of raw or undercooked meat, unpasteurized milk, dried or cured meat, unwashed raw vegetables and fruits or untreated water, frequency of eating in restaurants or fast food outlets, contact with soil, and type of flooring at home . Sera of patients were kept frozen at 20c until analyzed . All serum samples were analyzed for anti - toxocara igg antibodies with a commercially available enzyme immunoassay (eia) toxocara kit (diagnostic automation, inc ., an absorbance reading 0.3 optical density units was used as a cut - off for seropositivity . We performed the statistical analysis with the software epi info version 7 and spss 15.0 (spss inc ., for calculation of the sample size, we used a 95% confidence level, a power of 80%, a reference seroprevalence of 4.7% as the expected frequency of exposure in controls, and an odds ratio of 3.5 . These values were taken as the minimum number of participants for each group . To assess the association between toxocara infection and suicide attempts and other characteristics of the patients the two - tailed fisher exact test was used to compare the frequencies among the groups . Variables with p values <0.10 obtained in the bivariate analysis were further analyzed with stratification by gender . The study was performed using only residual serum samples and questionnaires from a previous survey in psychiatric outpatients . The ethical committees of the general hospital and the hospital of mental health in durango city approved the previous study . The purpose and procedures of the survey were explained to all participants, and a written informed consent was obtained from all of them . The additional analysis of serum samples and questionnaires was approved by the ethical committee of the instituto de seguridad y servicios sociales de los trabajadores del estado in durango city, mexico . One of the 156 (0.6%) suicide attempters and 1 (0.8%) of the 126 controls were positive for anti - toxocara igg antibodies (or = 0.80; 95% ci: 0.0413.02; p = 1.00). The suicide attempter seropositive for toxocara had a low anti - toxocara igg antibody level (optical density units = 0.608). Similarly, the seropositive control has a low anti - toxocara igg antibody level (optical density units = 0.839). None of the sociodemographic characteristics including age, gender, birthplace, educational level, occupation, and socioeconomic status showed an association with toxocara seropositivity (table 1). Likewise, none of the clinical characteristics studied including psychiatric disease and concomitant diseases, number of suicide attempts, time from last suicide attempt, method of suicide attempts, lymphadenopathy, frequent headache, impairments in memory, reflexes, hearing, and vision, and history of blood transfusion, transplant, surgery, alcohol consumption, drug abuse, or sexual history showed an association with toxocara seropositivity . In contrast, bivariate analysis of the behavioral characteristics of the psychiatric patients (cases and controls together) showed four variables with p value <0.10: consumption of meat from boar (p = 0.07), pigeon (p = 0.09), and squirrel (p = 0.07) and consumption of raw dried goat meat (p = 0.06). Other behavioral characteristics of patients including contact with animals and cat excrement, traveling, consumption of unwashed raw vegetables and fruits, unpasteurized milk or untreated water, frequency of eating in restaurants or fast food outlets, and contact with soil showed p values> 0.10 in the bivariate analysis . Stratification by gender showed that toxocara seroprevalence was significantly higher in male patients with consumption of raw dried meat (1/1: 100%) than male patients without this consumption (0/88: 0%) (p = 0.01). Toxocara seroprevalence was comparable in male patients with consumption of meat from boar, pigeon, and squirrel than male patients without these consumption acts (p 0.05). Toxocara seroprevalence was similar in female patients with consumption of meat from boar, pigeon, and squirrel and raw dried goat meat than female patients without these consumption acts . Toxocara infection is one of the five more common nematodal infections of the nervous system . Toxocara infection was associated with depression in a 65-year - old woman confirmed with psychometric tests . Brain involvement during toxocara infection may lead to disease and possibly to changes in behavior . Therefore, the present study aimed to determine whether toxocara exposure was associated with suicide attempts in psychiatric patients . We found a low prevalence of toxocara exposure among psychiatric outpatients, and toxocara seropositivity was not associated with suicide attempts . In a previous study in psychiatric patients, a 4.7% seroprevalence of toxocara exposure was found . The lower prevalence found in the present study than that previously reported in psychiatric patients can be explained by differences in the characteristics of the patients; that is, we studied outpatients, whereas in the previous study only inpatients were examined . Results of the present study suggest that toxocara exposure did not represent a risk for suicide attempts in the psychiatric patients studied . However, this is the first study of its kind and results should be confirmed . Other population groups including inpatients and people living in high seroprevalence places (rural areas, tropical countries) with larger sample sizes should be studied . However, the very low seroprevalence of toxocara infection found among psychiatric patients did not allow us to obtain further statistically significant associations . Remarkably, consumption of goat meat was previously associated with toxocara exposure in psychiatric inpatients in durango city . The fact that consumption of goat meat was associated with toxocara exposure in two independent studies points towards the importance of this factor for the transmission of toxocara infection to humans . In the present study, we examined new cases and a larger sample size (n = 282) of psychiatric patients than those (n = 128) included in the previous study . In the present study, an association of toxocara exposure with the consumption of raw dried meat from goat was found . To the best of our knowledge, this is the first report of an association of consumption of raw dried goat meat with toxocara exposure . In a recent study, a clinical case of a 51-year - old man with lower motor neuron disease a 10.1% seroprevalence of anti - toxocara antibodies was found in goats in thessaly, greece . Results suggest that toxocara exposure is not associated with suicide attempts in psychiatric outpatients in durango city, mexico . However, further studies with larger samples sizes to confirm our results should be conducted . The association between toxocara seropositivity and consumption of raw dried goat meat deserves further investigation . Too few patients were seropositive to assess further associations between toxocara exposure and sociodemographic, clinical, and behavioral characteristics of patients.
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The molecular targets of selection favoring brain expansion during human evolution have been sought by identifying dramatic, lineage - specific shifts in evolutionary rate . The increase in duf1220 domains during human evolution provides one of the most dramatic increases in copy number (popesco et al . A single copy of this protein domain is found in pde4dip in most mammalian genomes . In primates, this ancestral domain has been duplicated many times over, reaching its peak abundance in humans where several hundred duf1220 domains exist across 2030 genes in the nuclear blastoma breakpoint family (nbpf) (vandepoele et al . 2005; dumas et al . 2012). The majority of these map to 1q21.1, a chromosomal region with complex, and unstable genomic architecture (obleness et al . Interspecific duf1220 counts show a pattern of phylogenetic decay with increasing distance from humans (popesco et al . 2006; dumas and sikela 2009; dumas et al . 2012). In humans, 2012), and severe neurodevelopmental disorders, including autism spectrum disorder (asd) and microcephaly (dumas et al . 2012; davis et al . The severity of asd impairments is also correlated with 1q21.1 duf1220 copy number suggesting a dosage effect (davis et al . 2014). Taken together, these observations led to the suggestion that the expansion of duf1220 copy number played a primary role in human brain evolution (dumas and sikela 2009; keeney, dumas, et al . Although functional data are limited, they provide some indication of how duf1220 domain copy number count influences brain development . Duf1220 domains are highly expressed during periods of cortical neurogenesis, suggesting a potential role in prolonging the proliferation of neural progenitors by regulating centriole and microtubule dynamics to control key cell fate switches critical for neurogenesis (keeney, davis, et al . Pde4dip, which contains the ancestral duf1220 domain, does indeed associate with the spindle poles (popesco et al . 2006) and is homologous to cdk5rap2, a centrosomal protein essential for neural proliferation (bond et al . 2005; buchman et al . Two previous analyses report a significant association between duf1220 copy number and brain mass, cortical neuron number (dumas et al . 2012), cortical gray and white matter, surface area, and gyrification (keeney, davis, et al . First, duf1220 copy number was assessed across species using a blat / blast (blast - like alignment tool / basic local alignment search tool) analysis with a query sequence from humans, which introduces a bias that could partly explain the observed phylogenetic decay . Second, counts were not restricted to those domains occurring in functional exonic sequence and therefore many duf1220 domains found in human pseudogenes were included in the analyses . Third, the analyses were limited to a small number of species (48 primates), using parametric statistics that may not be suitable for count data, and which do not correct for phylogenetic nonindependence (felsenstein 1985). This is not a negligible issue, as it can result in the overestimation of statistical significance (carvalho et al . Finally, previous phenotypic associations have been reported for multiple cortical phenotypes all of which are strongly correlated with one another or are nonindependent . Therefore, to date, these studies have not provided evidence for a specific association with neocortex size, neither have they tested the strength of the association with different periods of brain development, which may provide new clues as to the functional relevance of duf1220 domain copy number . Here, we use nucleotide hidden markov models (hmms) (hmmer3; eddy 2011) to more accurately query the duf1220 domain number of distantly related genomes . After filtering these counts to limit the analysis to exonic sequence, we use phylogenetic comparative methods that correct for nonindependence to test whether duf1220 copy number is robustly associated with brain size, whether this is due to an association with pre- or postnatal brain development, and whether the association is specific to the neocortex . We confirm significant interspecific variation in duf1220 counts across primates (table 1, fig . 1). Phylogenetic generalized least square (pgls) regressions (pagel 1999) using square - root, or log10-transformed duf1220 counts support previous reports of an association with brain volume (sqrt: t10 = 3.165, p = 0.005, r = 0.455; log10: t10 = 4.770, p <0.001, r = 0.655). The same associations are also found after excluding homo sapiens from the analysis (sqrt: t9 = 3.810, p = 0.002, r = 0.569; log10: t9 = 3.952, p = 0.002, r = 0.586). However, these data transformations may not be appropriate for count data where models based on poisson distributions provide more accurate results (ohara and kotze 2010). 1. (a) phylogeny of ensembl primate genomes showing the number of duf1220 domains in functional, annotated genes with a cm promoter, and brain mass . (b) the relationship between square - root transformed duf1220 counts and log10(brain mass), and (c) the relationship between log10 transformed duf1220 counts and log10(brain mass). The regression lines are shown with (red) and without (gray) the inclusion of the h. sapiens data . In all cases, they are significant . (2012)whole genomefunctional exonic with cm promoterhomo sapiens272302262pan troglodytes12513832gorilla gorilla999732pongo abelii9210127nomascus leucogenys53596papio anubis7515chlorocebus sabaeus4816macaca mulatta357410callithrix jacchus31759tarsius syrichta472microcebus murinus241otolemur garnettii342 (a) phylogeny of ensembl primate genomes showing the number of duf1220 domains in functional, annotated genes with a cm promoter, and brain mass . (b) the relationship between square - root transformed duf1220 counts and log10(brain mass), and (c) the relationship between log10 transformed duf1220 counts and log10(brain mass). The regression lines are shown with (red) and without (gray) the inclusion of the h. sapiens data . In all cases, they are significant . Using a bayesian approach that corrects for phylogenetic nonindependence and fits a poisson distribution to the duf1220 count data (mcmcglmm; hadfield 2010), we again find evidence that cm - associated exonic duf1220 counts are associated with brain mass across primates (n = 12, posterior mean = 1.927, 95% confidence interval [ci] = 0.8003.040, pmcmc = 0.001). This association is robust to the exclusion of h. sapiens (posterior mean = 1.271, 95% ci = 0.4902.019, pmcmc = 0.003), and found when hominoids (n = 5, posterior mean = 3.679, 95% ci = 0.9666.258, pmcmc = 0.018) or anthropoids (n = 9, posterior mean = 2.019, 95% ci = 0.3523.684, pmcmc = 0.010) are analyzed alone, suggesting a consistent phylogenetic association . When body mass is included as a cofactor in the model, the positive association is restricted to brain mass (table 2a, fig . Table 2mcmcglmm results of multivariate modelsmodelposterior mean95% cipmcmc(a) brain mass and body mass 1 . Log(brain mass)4.1052.163 to 6.0000.001 + log(body mass)1.9863.544 to 3.9000.988(b) prenatal and postnatal growth 1 . Log(postnatal brain growth)2.9101.641 to 4.151<0.001 + log(postnatal body growth)1.2412.442 to 0.0520.977(c) brain regions 1 . Log(neocortex volume)6.0760.139 to 12.57120.025 + log(cerebellum volume)0.3699.5128 to 8.9610.526 + log(rob volume)5.49415.814 to 5.2880.872 mcmcglmm results of multivariate models separation of pre- and postnatal development specifically links duf12220 number to postnatal brain growth . Analyzed separately, the association with prenatal brain growth is weaker (n = 11, posterior mean = 1.758, 95% ci = 0.039 to 3.543, pmcmc = 0.023) than with postnatal brain growth (posterior mean = 1.839, 95% ci = 0.8952.808, pmcmc = 0.001). If both traits are included in the same model, only the positive association with postnatal brain growth remains (table 2b, fig . Multiple regression analysis also confirms that the association is specific to postnatal brain growth, rather than postnatal body growth (table 2b model 2). 2. (a) posterior means of the association between duf1220 count and brain mass (red) and body mass (black). (b) posterior means of the association between duf1220 count and postnatal brain growth (red) and prenatal brain growth (black). (c) posterior means of the association between duf1220 count and neocortex volume (red), cerebellum volume (solid black), and rest - of - brain volume (dashed black). (a) posterior means of the association between duf1220 count and brain mass (red) and body mass (black). (b) posterior means of the association between duf1220 count and postnatal brain growth (red) and prenatal brain growth (black). (c) posterior means of the association between duf1220 count and neocortex volume (red), cerebellum volume (solid black), and rest - of - brain volume (dashed black). Finally, we not only examined the hypothesized relationship with neocortex volume (e.g., keeny, davis, et al . 2014; keeny, dumas, et al . 2014), but also considered cerebellum volume, as this region coevolves with the neocortex (barton and harvey 2000), has expanded in apes (barton and venditti 2014), and shows high levels of nbpf expression (popesco et al . 2006). When the rest - of - the - brain (rob) is included as a cofactor, to account for variation in overall brain size, a positive association is found for neocortex volume but not cerebellum volume (table 2c models 1 - 3, fig . 2c). Our phylogenetic analyses substantiate the hypothesis that the increase in duf1220 number coevolves with brain mass (dumas et al . 2012; keeney, davis, et al . 2014), and may contribute to the proximate basis of primate brain evolution . We extend the results of previous studies by demonstrating specific associations with neocortex volume, and postnatal brain growth rather than prenatal brain growth . Together these results imply a role for duf1220 in evolutionary changes in the maturation and postnatal development of the neocortex . Previous hypotheses concerning the phenotypic relevance of duf1220 domain number have focused on their possible contribution to neurogenesis (dumas and sikela 2009; keeny, davis, et al . 2014; keeny, dumas, et al . 2014). This is supported by homology to genes with known functions in cell cycle dynamics (popesco et al . 2006; thornton and woods 2009), relevant spatial and temporal expression patterns (keeney, davis, et al . 2014), and an effect on the proliferation of neuroblastoma cell cultures (vandepoele et al . However, a direct effect of variation in duf1220 domain number on neural proliferation has not been demonstrated (keeney et al . 2015). If duf1220 domains do regulate neurogenesis, we would expect them to coevolve with prenatal brain growth, as cortical neurogenesis is restricted to prenatal development (bhardwaj et al . Existing data on duf1220 domain function suggest two potential roles that may explain this association: 1) a contribution to axonogenesis through initiating and stabilizing microtubule growth in dendrites; and 2) a potential role in apoptosis during brain maturation . Both hypotheses are consistent with the reported association between variation in duf1220 dosage and asd (davis et al . 2014). Indeed, an emphasis on postnatal brain growth is potentially more relevant for asd, which develops postnatally, accompanied by a period of accelerated brain growth in early postnatal development (courchesne et al . Microtubule assembly is essential for dendritic growth and axonogenesis (conde and cceres 2009). Pde4dip, which contains the ancestral duf1220 domain, has known functions in microtubule nucleation, growth, and cell migration (roubin et al . There is also evidence that nbpf1 interacts with a key regulator of wnt signaling (vandepoele et al . 2010) that has important roles in neuronal differentiation, dendritic growth, and plasticity (inestrosa and varela - nallar 2014). Consistent with this function, duf1220 domains are highly expressed in the cell bodies and dendrites of adult neurons (popesco et al . A role for duf1220 domains in synaptogenesis could potentially explain the association with asd severity (davis et al . 2002) and cortical white matter (hazlett et al . 2005; courchesne et al . 2011), both of which suggest a disruption of normal neuronal maturation (courchesne and pierce 2005; minshew and williams 2007). Alternatively, nbpf genes are also known to interact with nf-b (zhou et al . 2013), a transcription factor implicated in tumor progression, with a range of roles including apoptosis and inflammation (karin and lin 2002; perkins 2012). 2003; madden et al . 2007), including regulating neuronal density (sanno et al . 2010), and apoptotic genes may have been targeted by selection in relation to primate brain expansion (vallender and lahn 2006). Disruption of apoptosis causes microcephaly (poulton et al . 2011), potentially explaining the association between duf1220 dosage and head circumference (dumas et al . 2001) is also intriguing, given the growing evidence that the inflammatory response is linked to the risk and severity of asd (meyer et al . If duf1220 domain number does contribute to the evolution of postnatal brain growth, this contrasts with results of previously studied candidate genes with known roles in neurogenesis that coevolve with prenatal brain growth (montgomery et al . This suggests a two - component model of brain evolution where selection targets one set of genes to bring about an increase in neuron number (e.g., montgomery et al . 2011; montgomery and mundy 2012a, 2012b), and an independent set of genes to optimize neurite growth and connectivity (e.g., charrier et al . This two - component model is consistent with comparative analyses that indicate pre- and postnatal brain developments evolve independently, and must therefore be relatively free of reciprocal pleiotropic effects (barton and capellini 2011). Finally, these results add further evidence that many of the genetic changes that contribute to human evolution will be based on the continuation or exaggeration of conserved gene - phenotype associations that contribute to primate brain evolution more broadly (montgomery et al . 2011; scally et al . Understanding the commonalities between human and nonhuman primate brain evolution is therefore essential to understand the genetic differences that contribute the derived aspects of human evolution . Hmmer3.1b (eddy 2011) was used to build an hmm from the duf1220 (pf06758) seed alignment stored in the pfam database (finn et al . The longest isoforms for all proteomes of 12 primate genomes from ensembl v.78 (cunningham et al . 2015) (fig . 1a) were searched using the protein duf1220 hmm (hmmsearch, e value <1e-10) (supplementary table s1, supplementary material online). We extracted the corresponding cdna regions to build a duf1220 nucleotide profile hmm (nhmm) using a mafft sequence alignment, allowing for more sensitive analysis across a broad phylogenetic range . The duf1220 nhmm was used to search the complete genomic dna for all 12 species . These counts were filtered to remove any duf1220 domains not located in annotated exonic sequence, or located in known pseudogenes . We next filtered our counts to limit them to exonic sequence in close proximity to the nbpf - specific conserved - mammal (cm) promoter (obleness et al ., we built a nucleotide hmm for the cm promoter based on a mafft (katoh et al . 2002) alignment of the 900-bp cm region upstream of human genes nbpf4, nbpf6, and nbpf7 . Using this cm promoter nhmm, we searched 1,000-bp up- and downstream of genes containing duf1220 domains for significant cm promoter hits (nhmmer, e value <1e-10). This provided final counts for duf1220 domains within exonic regions and associated with the cm promoter (table 1). Supplementary material online, we compare our counts with previous estimates and discuss possible sources of error . All scripts and data used in the analysis are freely available from: https://github.com/qfma/duf1220 pgls regressions were performed using log - transformed phenotypic data and log- or square root - transformed duf1220 count data in bayestraits (pagel 1999). Phylogenetic multivariate generalized mixed models were implemented using a bayesian approach in mcmcglmm (hadfield 2010), to test for phylogenetically corrected associations between duf1220 counts and log - transformed phenotypic data (supplementary table s2, supplementary material online). All analyses were performed using a poisson distribution, as recommended for count data (ohara and kotze 2010), with uninformative, parameter expanded priors for the random effect (g: v = 1,n = 1, alpha. = 0, alpha.v = 1,000; r: v = 1, = 0.002) and default priors for the fixed effects . We report the posterior mean of the cofactor included in each model and its 95% cis, and the probability that the parameter value is greater than 0 (pmcmc) as we specifically hypothesize a positive association (dumas et al . Alternative data treatments lead to similar conclusions (supplementary information, supplementary material online). Hmmer3.1b (eddy 2011) was used to build an hmm from the duf1220 (pf06758) seed alignment stored in the pfam database (finn et al . The longest isoforms for all proteomes of 12 primate genomes from ensembl v.78 (cunningham et al . 2015) (fig . 1a) were searched using the protein duf1220 hmm (hmmsearch, e value <1e-10) (supplementary table s1, supplementary material online). We extracted the corresponding cdna regions to build a duf1220 nucleotide profile hmm (nhmm) using a mafft sequence alignment, allowing for more sensitive analysis across a broad phylogenetic range . The duf1220 nhmm was used to search the complete genomic dna for all 12 species . These counts were filtered to remove any duf1220 domains not located in annotated exonic sequence, or located in known pseudogenes . We next filtered our counts to limit them to exonic sequence in close proximity to the nbpf - specific conserved - mammal (cm) promoter (obleness et al ., we built a nucleotide hmm for the cm promoter based on a mafft (katoh et al . 2002) alignment of the 900-bp cm region upstream of human genes nbpf4, nbpf6, and nbpf7 . Using this cm promoter nhmm, we searched 1,000-bp up- and downstream of genes containing duf1220 domains for significant cm promoter hits (nhmmer, e value <1e-10). This provided final counts for duf1220 domains within exonic regions and associated with the cm promoter (table 1). Supplementary material online, we compare our counts with previous estimates and discuss possible sources of error . Pgls regressions were performed using log - transformed phenotypic data and log- or square root - transformed duf1220 count data in bayestraits (pagel 1999). Phylogenetic multivariate generalized mixed models were implemented using a bayesian approach in mcmcglmm (hadfield 2010), to test for phylogenetically corrected associations between duf1220 counts and log - transformed phenotypic data (supplementary table s2, supplementary material online). All analyses were performed using a poisson distribution, as recommended for count data (ohara and kotze 2010), with uninformative, parameter expanded priors for the random effect (g: v = 1,n = 1, alpha. = 0, alpha.v = 1,000; r: v = 1, = 0.002) and default priors for the fixed effects . We report the posterior mean of the cofactor included in each model and its 95% cis, and the probability that the parameter value is greater than 0 (pmcmc) as we specifically hypothesize a positive association (dumas et al . Alternative data treatments lead to similar conclusions (supplementary information, supplementary material online). Supplementary information, figures s1s3, and tables s1s3 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).
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The millennium development goal 5 (mdg5) of reducing the maternal mortality ratio by 75% between 1990 and 2015 (1) is unlikely to be achieved in africa, because there was no progress that has been made to achieve such goals . Most of the current strategies in the developing world are focusing on emergency obstetric care . In sudan, the mortality rate was 1,363 per 100,000 during 1995 - 1999 and sepsis was found to be the main cause of death in one third of cases . Furthermore, it was the leading cause of maternal mortality during 1985 to 2000 (2). Those who survive may develop serious complications as a result of puerperal sepsis such as infertility and chronic pelvic pain (3, 4). However, treatment based only on symptoms and clinical diagnosis can be misleading, and it can results in serious complications (5, 6). The present study, was conducted to determine the exact pathogenic infections in women with puerperal sepsis, and their susceptibility test to the currently use antimicrobial therapy in a rural hospital, in sudan . This prospective cross - sectional study was conducted at hussein mustafa hospital, department of obstetrics and gynecology in gadarif state, eastern sudan, during the period from june 2011 to june 2012 . Hundred and seventy patients who had puerperal sepsis (who definition) were included in this study . Maternal sepsis was defined as infection of the genital tract occurring at any time between the rupture of membranes or labor, and 42 days postpartum in which 2 or more of the following are present: a) pelvic pain; b) fever i.e. Oral temperature 38c or higher on any occasion; c) abnormal vaginal discharge, e.g. Presence of pus; d)abnormal smell / foul odor of discharge and delay in the rate of reduction of the size of uterus (<2cm / day during the first 8 days) (7). To ensure avoiding concomitant infections, twenty ml of venipuncture blood were drawn into two bottles of blood culture under aseptic condition, and it was immediately transferred to the laboratory for bacteriological examination ., uk) were incubated one aerobically and the other anaerobically at 37c for 24 - 48 hours . Presumptive turbidity growth in the blood culture bottles were sub cultured in aerobic and an anaerobic condition into blood agar and nutrient agar plates(plasmatic ltd ., uk), at 37c for 24 - 48 hours before discarded the pates after 3 day (8). The presumptive culture of the plate cultures were examined morphologically with the naked eye (size, pigment, edge, etc) and grams stain . Organisms that had pure growth were subjected to subsequent different biochemical tests for species identification, according to methods described previously by many workers (8, 9, 10). The isolated bacteria were subjected to antibiotics susceptibility test for numbers of antibiotics routinely uses in hospital which including; penicillin (10 g), ampicillin (10 g). Vancomycin (30 g) metronidazole (5 g), gentamicin (10 g), methicillin (10 g), ceftriaxone (30 g) by disc diffusion technique (kirby - bauer method). The result was reported by measured zone of the inhibition growth by mm around antibiotic discs according to the nccls for different zone diameter standards to determine sensitive, intermediate or resistant strains (8). Two to three colonies of the tested organism taken from the plate performed for antibiotic susceptibility test containing penicillin g and cephalosporin (11) (dominique, 2004). Change in color of the test strip from purple to yellow in the area of inoculation within 5 - 10 minutes considered a positive beta - lactamase test according to the manufacturer procedure (abtekbiologicals ltd ., methicillin - resistant staphylococcus aureus strips test: strips were used to confirm rapidly methicillin - resistant s.aureus (11). According to the manufacturer procedure (abtekbiologicals ltd ., liverpool). The statistical package for the social sciences (spss 15 for windows) was used for data recording and statistical analyses . Twenty ml of venipuncture blood were drawn into two bottles of blood culture under aseptic condition, and it was immediately transferred to the laboratory for bacteriological examination . Two bottles of blood culture (plasmatic ltd ., uk) were incubated one aerobically and the other anaerobically at 37c for 24 - 48 hours . Presumptive turbidity growth in the blood culture bottles were sub cultured in aerobic and an anaerobic condition into blood agar and nutrient agar plates(plasmatic ltd ., uk), at 37c for 24 - 48 hours before discarded the pates after 3 day (8). The presumptive culture of the plate cultures were examined morphologically with the naked eye (size, pigment, edge, etc) and grams stain . Organisms that had pure growth were subjected to subsequent different biochemical tests for species identification, according to methods described previously by many workers (8, 9, 10). The presumptive culture of the plate cultures were examined morphologically with the naked eye (size, pigment, edge, etc) and grams stain . Organisms that had pure growth were subjected to subsequent different biochemical tests for species identification, according to methods described previously by many workers (8, 9, 10). The isolated bacteria were subjected to antibiotics susceptibility test for numbers of antibiotics routinely uses in hospital which including; penicillin (10 g), ampicillin (10 g). Vancomycin (30 g) metronidazole (5 g), gentamicin (10 g), methicillin (10 g), ceftriaxone (30 g) by disc diffusion technique (kirby - bauer method). These antibiotics discs were commercially prepared (plasmatic ltd ., uk). The result was reported by measured zone of the inhibition growth by mm around antibiotic discs according to the nccls for different zone diameter standards to determine sensitive, intermediate or resistant strains (8). Two to three colonies of the tested organism taken from the plate performed for antibiotic susceptibility test containing penicillin g and cephalosporin (11) (dominique, 2004). Change in color of the test strip from purple to yellow in the area of inoculation within 5 - 10 minutes considered a positive beta - lactamase test according to the manufacturer procedure (abtekbiologicals ltd ., methicillin - resistant staphylococcus aureus strips test: strips were used to confirm rapidly methicillin - resistant s.aureus (11). According to the manufacturer procedure (abtekbiologicals ltd ., liverpool). The statistical package for the social sciences (spss 15 for windows) the total study population comprised 170 women with severe puerperal sepsis, diagnosed by the clinician according to the history, symptoms; signs . Patients were admitted to hussein mustafa hospital, sudan within the first two weeks after delivery . The mean maternal ages of patients were 25.48 years (ranged 17 to 35). Out of 170 admitted cases, 124(72.9%) patients had positive bacterial blood culture whereas; in the remaining patients, 46 (27.1%) no potential pathogens were isolated . The majority of these isolates were aerobic bacteria 77 (62.1%), and the anaerobic bacteria were isolated from34 (27.4%) patients . The age groups between 21 to 25 years had the highest rate of infection were 44(35.5%); followed by the age groups of 26 to 30 years were 31(25%); while those at the extreme of age in less than 20 years were 26(21%) and 31 years and more23 (18.5%) had the lowest rate of infection, (figure 1). Out of 124 of the isolates, staphylococcus aureus was the most prevalent organism 49 (39.5%) of which methicillin - resistant s. aureus (mrsa) was 41% (n=41/49); followed by c. perfringens, which constituted 34(27.4%), l monocytogenes showed prevalence of 21(16.9%); e. cloacae 13(10.5%); and staphylococcus epidermis was identified in 5.6% (n=7) of cases . The results showed that the highest rate of infection was among women who delivered vaginally 121 (97.6%) compared to those who delivered by caesarean section 3 (2.5%). Out of the 116 women who delivered vaginally and had a sepsis; 112 (96.6%) of them were home deliveries, whereas only 4 (3.4%) delivered vaginally at the hospital . It is also shown that the percentage sensitivity of the different isolates to various antimicrobial agents used in 124 women with puerperal sepsis . The isolated bacteria were subjected to in vitro antimicrobial susceptibility test using the disc diffusion technique (kirby - bauer method). The degree of sensitivity, described as sensitive, intermediate, and resistance of the isolated bacteria to antibiotics were recorded . In the past decade, maternal sepsis has been a common pregnancy - related event, which could eventually lead to fatal obstetric complications . This study was conducted in response to hospital records what showed an increasing rate of puerperal sepsis . In 2010, our findings do not represent maternal morbidity in gadarif district because it was a hospital - based study, only patients with severe disease reported to the hospital and most of the deliveries occurred at home due to unavailability of medical care, financial constrains, lack of transportation and cultural believes . In this study, the highest incidence of maternal sepsis was observed among young females, aged 21 - 25 years (35.5% . ), followed by 26 - 30 years (25%). It reasonable to expect such finding occurred in old women due to increase in several pregnancy complications including maternal sepsis (14, 15). We speculate that women at this age were likely to be primigravida with untested pelvises they resort to hospitals when labor became obstructed and infected . Strikingly, 72.9% (n=124) of our patients had positive bacterial blood culture and the majority 72.6% (n=90) of these isolates were aerobic bacteria, which were mainly s. aureus39.5%, of which methicillin - resistant s. aureus (mrsa) was the predominant isolates 41% (n=41/49). Traditionally, in the western countries, streptococcus pyrogen have been a major cause of maternal puerperal sepsis (16, 17). Recently, community associated mrsa (ca - mrsa) have become the predominant isolates, it has been described in patients with skin, soft tissue infections and pneumonia . Carrier may transmit the organism to another person via direct contact with infected hands (18). However, this high rate of mrsa, which may indicate community - acquired infection because the majority 96.6% of our patients delivered at home under unhygienic condition and most of these deliveries were conducted by traditional birth attendant . Such results necessitate urgent tracing of the risks factors by conducting further researches . In the present study, staphylococcus epidermidis was isolated in 5.6% of cases; earlier reports documented that s. epidermidis were the most common bacterial isolates in puerperal sepsis gerstner et al (19). Although, s. epidermidis is not usually pathogenic, but patients may acquire the infection when they have compromised immunity as in pregnancy . These findings may indicate regional variation in isolates as a cause of puerperal sepsis due to differences in geographical locations and immunity . In addition, since the majority of the studied population were home deliveries the source of infection might be exogenous where pathogens from nearby skin flora or contact with contaminated nonsterilized instruments, frequent vaginal examination with unwashed hands . In addition, the use of local herbal products for proper wound healing and treatment of established wound infections may contribute to increase of infection rate . In this study, 16.9% of the study population was found to have positive blood culture for l monocytogenes . Jos a. et (20) reported that pregnant women and immune - compromised patients are predominantly affected with l. monocytogenes . In the past, several outbreaks of food borne diseases were reported, farber and peterkin (21) reported in 1991 outbreaks with mortality of 24% . Our high reported cases of such infection are possible due outbreaks rather than sporadic cases, as there was 12% increase in the rate of sepsis which initiated the idea of this study . From the total patients who are infected with c. perfringens are not essentially developing gas gangrene; the disease can display a wide spectrum of clinical presentations (22, 23). Recently, it has become apparent that the presence of clostridium perfringens is unusual, but not rare, causes of tissue and bloodstream infections . Studies in which blood samples were obtained from patients in tertiary - care facilities have shown that c. perfringens were the most frequently identified pathogens accounted for 20% to 50% of isolates (24). In la crosse, wisconsin, a retrospective study carried from 1990 through 1997 to determine the incidence and clostridium species among the inpatients, the main clostridium species were perfringens with an incidence rate of 21.7% . The high rate in our study may be due to the fact that women in rural areas experiences unattended labor, during bearing down they soiled the perineum with fecal matter . These organisms may get access to the blood through fecally infected episiotomy and decircumcision; wounds leading to c. perfringens bacteremia . The present study showed differences in antibiotic susceptibility pattern of the isolates to antibiotics used for the treatment of puerperal sepsis . The isolates of s. aureus were in considerable variation in term of antibiotic susceptibility, in which 83.7% of the isolated strains were identified as mrsa . Similar study as reported by stone (26) which showed that 85.5% (55/47) of the isolates were mrsa . This variation could be ascribed to geographical variation and the difference in the immune response . Metronidazole was shown to have great affectivity against anaerobic bacteria, and it was ineffective to all aerobic bacteria . Metronidazole is regarded as the drug of choice for the treatment of anaerobic bacteria sepsis boyanova (29).s . Aureus; l. monocytogenes were 100% sensitive to vancomycin, while c. perfringens sensitivity to vancomycin was 94.1% . Ampicillin was 100% effective against l. monocytogenes, while it was 88.2% effective against c. perfringens . Penicillin was remarkably effective against l. monocytogenes (100%), but it was ineffective against s. epidermidis and e. cloacae . On the other hand, gentamicin was more effective against e. cloacae, l. monoctogenes (100%), while 94.1% of c. perfringens were resistant to gentamicin . A previous study demonstrated a similar finding (30) which showed that gentamicin is ineffective against anaerobic bacteria, primarily useful in aerobic gram negative bacterial infection such as enterobacter . The shortcomings of this report, it was a hospital based study which did not reflect puerperal infection in the whole community, and we did evaluated maternal fetal comes in the study population . In this study, the infection rate in this study was high, and the isolates were totally different from previous reports . These changes in microbial profile require a revision of the antimicrobial sensitivity pattern in patients with puerperal sepsis . Community - based studies will be more valuable and informative due variations in habits and cultures . To ensure achievement towards mdg5, active intervention by medical staff and government is required to achieve a 5.5% annual reduction rate as proposed by mdg5 . In this study, the rate of sepsis increased with home deliveries, in addition to younger age group . Improving accessibility to in - hospital care and midwife services will reduce morbidity associated with maternal sepsis in rural areas.
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The search for esthetic materials has led to advances in the study of dental materials, especially composite resins . The main advantages of resins are related to the material's esthetic properties, decrease of marginal leakage, increased resistance of the tooth remnant, and less need for removal of healthy tooth structure15 . In addition, the reduced polymerization contraction and improved wear resistance of resins permit their use not only in anterior but also in posterior teeth2 . Both esthetics and longevity of restorations strongly depend on the quality of the surface finishing and polishing . The presence of irregularities can influence appearance, plaque retention, surface discoloration, gingival inflammation4,5,16,19,20,26,29,30, solubility of the organic matrix due to the formation of acetic, propionic and lactic acids by adhered plaque, and the occurrence of secondary caries12 . In addition, the surface roughness of composites can reduce some mechanical properties such as hardness13,16,17 and increase the wear of restorations . Thus, polished and smooth composite resin restorations present a better esthetic appearance and greater longevity21 . Final polishing using extremely fine abrasives reduces the remaining roughness and is of special importance since rough surfaces accumulate more plaque and stains14 and may cause excessive enamel wear of the antagonistic tooth in areas of occlusal contact9 . Another important aspect is the need for removing the superficial resin layer that does not polymerize when in contact with oxygen18 . Studies have shown that a smoother surface is obtained when the resin is cured against a strip of appropriate matrix1,3,11 removal of this surface by the usually required finishing procedures will produce a harder, more resistant and esthetically acceptable surface24 . Thus, it is important to determine which finishing / polishing system will provide the smoothest surface for the different commercially available composites23,25 . Unfortunately, the use of these disks is not always possible because of the anatomic shape and difficult access to the restoration . Factors that can influence the surface roughness of composites include the type, size and quantity of load of the composite as well as the type, size and hardness of the abrasives and the finishing and polishing technique used10,13 . Various polishing protocols have been tested in vitro to evaluate their effects on the surface roughness of restorative materials . Several composite resins have been the subject of surface roughness studies, but few investigations are available comparing the surface roughness of microhybrid resins, as well as the use of a new microdiamond polishing system (pogo) recently launched on the market . Therefore, the objective of the present study was to evaluate the effect of three polishing systems on the surface roughness of five types of microhybrid composite resins . Five photoactivated microhybrid composites indicated for direct restorations were used in the present study (table 1). The specimens were prepared in acrylic resin plates (15 cm long x 5 cm wide), with one plate for each composite . Thirty - six specimens (circular cavity measuring 5 mm in diameter and 3 mm in depth) were prepared for each composite resin and divided into four groups (n=9): three polishing systems and one control . Information supplied by the manufacturer . The resins were inserted into the cavities in three increments (the first layer was cured for 20 s, the second layer was cured for 20 s and the last layer was cured for 60 s) and activated using an led curing unit (optilight ld ii, gnatus, ribeiro preto, sp, brazil) with intensity of about 5.2 w. the last layer was cured against a polyester matrix (tdv dental ltda ., pomerode, sc, brazil), with pressure being applied to the ends in order to produce extravasation and trim excess material . After preparation, the specimens were stored in distilled water at 37c for 24 h and then submitted to finishing and polishing and subsequent analysis of surface roughness . In the control group (group 1), the specimens were not submitted to any finishing or polishing procedure after curing against a polyester matrix . In groups 2, 3 and 4 the specimens were finished with fine grit diamond burs (gold 3168f, kg sorensen, barueri, sp, brazil), followed by extra - fine diamond burs (silver 3168ff, kg sorensen), each applied for 15 s with a high - speed handpiece under water cooling . After this step, the specimens were polished using the systems presented in table 2 according to the instructions of each manufacturer, for a total period of 60 s. information supplied by the manufacturer . In group 2, the specimens were polished with aluminum oxide - impregnated disks (sof - lex, 3m / espe) (dark blue, medium blue and light blue back, measuring 19.05 mm in diameter) at intermittent pressure and low speed for 20 s each . The specimens were washed with an air / water spray to remove debris, air dried and then polished with another disk of lower grit for the same period of time . In group 3, the specimens were polished with disk - shaped aluminum oxide - impregnated silicon points (enhance, dentsply) at low speed for 30 s, followed by treatment with the pogo diamond polishing system for an additional 30 s. in group 4, felt disks (diamond, fgv) in combination with diamond paste (excel diamond paste, fgv) were applied to the restoration surface at low speed for 60 s. average surface roughness (ra, in m) of the specimens was determined with a previously calibrated mechanical roughness tester (surftest 301, mitutoyo america corporation, suzano, sp, brazil) over a distance of 0.25 mm . Six measurements were made in the center of each specimen in two directions (three in the vertical and 3 in the horizontal direction), for a total of 54 measurements per group . The shapiro - wilk test was applied to determine whether the data showed a normal distribution or not . Surface roughness was compared between the control and treatment groups by two - way anova with repetition of the factorial design, with the level of significance set at 1% . When the difference was statistically significant (p<0.05), the tukey - kramer test was used for comparison between means . The interaction between the two main factors (composite resin and polishing system) was highly significant (p<0.01). Table 3 shows the ra values obtained for the different combinations of factors and table 4 shows the results of factorial anova . Analysis of the ra values obtained for the different polishing systems / composite resins showed that treatment of z250 with the felt disk+diamond paste system presented the best performance (ra = 0.1846 0.06). The highest ra (1.3663 0.32) was obtained for the sof - lex system applied to tph spectrum, with the difference being statistically significant from the other combinations . All ra values were higher than those observed for the control group (polyester matrix). The effectiveness of surface finishing and polishing procedures is of fundamental importance for any restoration11 . These procedures are commonly required after placement of direct composite resin restorations since they minimize the retention of plaque and stains and other problems resulting from the exposure of rough surfaces to the oral environment . Smoother composite surfaces are obtained when the material was cured against a polyester matrix1,4,11,16,23,27 . Even if care is taken in the placement of the matrix, removal of excess material and recontouring of restorations are frequently necessary . However, these procedures significantly increase surface roughness . The factors determining the micromorphology of the surface of composite resin restorations after finishing and polishing include composite characteristics such as size, hardness, type and amount of particles1 and factors related to the abrasive system such as flexibility of the material in which the abrasive is impregnated, hardness of the abrasive, and geometry, speed and form of application of the instruments used4,27 . In the studies of ozgnaltay et al.11 and yap, et al.28, the use of a polyester matrix resulted in the lowest ra, which differed significantly from all other finishing and polishing procedures (p=0.001). In addition, all procedures used for finishing and polishing of the restorations reduced the smoothness obtained with the matrix . A similar result was obtained in the present study, with the lowest ra values being obtained when the composite resins were cured against a polyester matrix and an increase in roughness being observed after surface treatment, except for z100 . In this case, a higher ra was observed for the control group than when finishing and polishing were performed with the felt disk+diamond paste system . This fact might be explained by bubble formation on the resin surface when pressed against the polyester matrix during curing . The sof - lex disk system yielded rougher surfaces and differed significantly from the other systems, irrespective of the composite resin used . This result disagrees with most studies comparing the sof - lex system with silicon points (enhance). However, in the present study the pogo microdiamond system was applied after the enhance system which resulted in better surface smoothness . Some investigators16,19,23 reported significant differences in ra between specimens polished with the sof - lex disk system and those polished with silicon points using the same resin, with smoother surfaces being obtained with the former and rougher surfaces with the latter . The capacity of disks impregnated with aluminum oxide particles to produce smooth surfaces is related to their ability of equally removing particles and organic matrix . The disks are difficult to produce, as is the finishing and polishing of the anatomic contours of the surfaces, especially in the posterior regions of the mouth3,11 . In contrast, the enhance polishing system consists of a rubber - like flexible material, a polymerized resin impregnated with an abrasive . This system may wear the resin matrix and only contour prominent surfaces, resulting in a higher surface roughness27 . Therefore, in the present study the enhance system was combined with pogo points in order to refine the previous polishing . Trkn and trkn24 observed no significant difference between surfaces polished with the pogo system and the control group (p=0.01), and concluded that among the polishing systems tested the pogo system produced the smoothest finishing for all composite resins . In the present study, the combination of diamond paste and felt disks was highly efficient, resembling a final polishing in view of the low grit diamond particles (2 - 4 m) used in composite restorations5 . The felt disk+diamond paste group produced low ra values for the composites tested, similar to the control and enhance+pogo groups, except for the tph resin / felt disk+diamond paste group . This combination presented the highest ra when compared to the other groups, demonstrating that each resin behaves according to the polishing system used . This finding agrees in part with the study of turssi et al.25, who observed a smoother surface when the specimens were polished with sof - lex followed by prisma gloss aluminum oxide paste, while the worst result was obtained when only enhance points were used . Similar to the present study, other investigators4,6 also showed that the effect of diamond - impregnated felt disks on the surface roughness of hybrid composites is superior to that of flexible disks . The lowest ra was observed after polishing with the felt disk+diamond paste for composite z250 (ra = 0.1846 0.06). The present results showed a significant change in the surface of the composites according to the polishing system used . A similar study reported that polishing z250 composite resin with micro - polisher disks (pogo) (0.51 0 .15) resulted in significantly lower surface roughness than the use of aluminum oxide (sof - lex) (1.12 0.27) and rubber polishing disks (identoflex) (1.53 1.70). In addition, no significant difference in surface roughness was found between unfinished materials (polyester matrix surface)22 . This study agrees with our findings . The structure of the composites (particle size, consistency and quantity of load, type of matrix and degree of reticulation) can also influence the results8 . In the present study, in contrast, z250 contains a smaller range of filler particles than the other composites tested and presents one of the smallest mean particle sizes, which may partially explain the lower roughness obtained with this composite resin . Reis, et al.13 (2003) reported that the smoothest surfaces were recorded for z250 microhybrid resin when compared to solitaire, surefil and alert (condensable) composites, and better polishing was obtained when diamond paste was applied . The good results observed for this composite might be explained by particle size (0.01 to 3.5 m) and arrangement . In addition, these authors concluded that z250 is more easily polished than condensable composites and presents low staining susceptibility, in agreement with the present study in which the smoothest surface was observed for z250, especially when polished with the felt disk+diamond paste system . With respect to particle size, the present findings agree with the manufacturer information that the mean size of the z250 particles is one of the smallest among the resins studied . On the other hand, although the highest ra was obtained for tph, z100 presented the largest particle size (4.5 m). The critical surface roughness threshold established for bacterial adhesion is 0.2 m . Whereas no reduction in bacterial accumulation is expected below this threshold, any increase in surface roughness above 0.2 m results in a simultaneous increase of plaque accumulation and of the risk of caries and periodontal inflammation, because can promise the esthetics and longevity of the restoration1 . Since all treated surfaces presented a ra higher than 0.2 m, the effect of the finishing / polishing systems on the finished surface of microhybrid composite resins is clinically relevant . Thus, the role of polishing is to produce a smooth glossy aspect on the restoration surface similar to that of enamel . This procedure is routinely used in daily dental practice and the absence of these characteristics may compromise the esthetics and longevity of restorations . The lowest surface roughness was observed for microhybrid composites submitted to finishing and polishing procedures with diskshaped aluminum oxide - impregnated silicon points and felt disks using diamond paste or felt disks plus diamond paste.
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In mammals, fertilization results in a zygote, the totipotent stem cell that can give rise to all cell types of a developing embryo and adult . A pre - implantation mouse embryo at the blastocyst stage consists of three cell lineages, inner cell mass (icm), trophectoderm (te) and primitive endoderm (pe). Te is the first morphologically distinct cell type of the trophoblast lineage, which becomes discernible at the morula stage and gives rise to the placental trophoblasts . Pe is the first morphologically distinct cell type of the extraembryonic endoderm, which becomes visible at the late blastocyst stage as a layer on the mural surface of the icm and gives rise to the yolk sac endoderm . Icm forms the epiblast, the first morphologically distinct cell type of the fetal lineage . The pre- and peri - implantation embryo in culture can produce stable cell lines that show characteristics of the three blastocyst lineages . The first prototypes of such cell lines consists of embryonic stem (es) cells from the mouse icm 2; 3, which closely resemble nascent epiblast 4 . Es cell lines have also been isolated from the epiblast in rat and mouse, which closely resemble the post - implantation epiblast and human es cells in gene expression profiles and transcription factor networks 5; 6 . Trophoblast stem (ts) cell lines have been isolated from blastocysts in the mouse 7 . Extraembryonic endoderm (xen) stem cell lines have been isolated from mouse blastocysts 8 . Thus, there are cell lines that represent the earliest stages of the fetal pathway in vitro and appear to be remarkably similar across mammalian species . Although these stem cells are capable of self - renewal and differentiation, none of them is totipotent, because they originate from the committed blastocyst lineages and maintain the respective properties of their origins . For example, these mammalian stem cells exhibits gene expression profiles characteristic of their sources 1; 7; 8 . More importantly, upon blastocyst transplantation and morula aggregation, mammalian es cells can contribute to the epiblast but not extraembryonic lineages 9, whereas ts and ps cells usually enter the te and pe of extraembryonic lineages but not the epiblast and its derivatives in the embryo proper 7; 8 . The attempt towards totipotent stem cell cultures from earlier developing embryos has not been available . The use of blastocyst stage embryos has conceptually excluded the possibility for totipotent cell derivation by origin . Recently, induced pluripotent stem (ips) cells have been obtained by forced expression of pluripotency transcription factors even in differentiated mammalian cells 10 . However, it is unclear whether this approach can lead to totipotent stem cell cultures . Recently, small molecules operating on several signaling pathways have been used to partially convert mouse epiblast stem cells to an earlier pluripotency state 11 . In addition, primary cultures from early embryos can produce a uniform population for transcriptome and/or proteome approaches to study early events such as early lineage restriction and cellular differentiation in a controllable conditions . For example, primary cultures of single zebrafish embryos at early stages has been used to study myogenesis 12 . This laboratory fish is well - suited for analyzing vertebrate development 13 . In this organism, we have previously established a feeder - free culture system that allowed for derivation of diploid es cells 14 - 16 from fertilization midblastula embryos as the equivalent of mammalian blastocysts, male germ stem cells from the adult testis 17 and even haploid es cells from gynogenetic embryos18; 19 . We are interested in the possibility towards totipotent stem cell derivation . As a first step, this study was aimed at determining the suitability of earlier embryos prior to the midblastula stage for cell isolation and culture . We show that single cells from medaka embryos ranging from the 32-cell early morula stage to the 1000-cell early blastula stage can survive and proliferate in culture on gelatin - coated multiwell plates . More importantly, the cells from these earlier embryos can form a monolayer during 6 days of culture and display phenotypes similar to es cells . These data suggest the possibility for stem cell derivation from these early embryos in this organism . Fish . Work with fish followed the guidelines on the care and use of animals for scientific purposes of the national advisory committee for laboratory animal research in singapore (permit number 27/09). Medaka was maintained under an artificial photoperiod of 14-h light to 10-h darkness at 26c 20 . Medaka strains hb32c, sok and i were used for cell culture in this study . Hb32c is a wild - type pigmentation strain from which diploid es cell lines were derived14 - 16, whereas i is an albino strain from which haploid es cells generated 18; 19 . Embryo manipulation, cell isolation and cultures were done essentially as described 14 - 16 . Briefly, fertilization embryos were treated with proteinase k (10 mg / ml) for 60 min at 28c to remove the attachment filaments, rinsed twice in phosphate - buffered saline (pbs) and sterilized in pbs-0.1% bleach for 2 min, and rinsed 5 times in pbs . Embryos were incubated in pbs and monitored for developmental stages under a stereo microscope at aseptic conditions . The chorion was manually torn with a pair of fine forceps through the yolk sac at the vegetal half . Cells were dissociated by gentle pipetting, rinsed 5 times by partial pbs changes and seeded into gelatin - coated 96-well plates containing 150 l of esm2 . Cell growth, attachment, proliferation and differentiation were monitored at regular intervals of culture at 28c in air . Observation and photography on leica mzfiii stereo microscope, zeiss axiovertinvert 2 invert microscope and axiovert 200 upright microscope with a zeiss axiocam m5rc digital camera (zeiss corp ., germany) were as described previously 19 . In medaka, early embryos undergo rapid synchronous cell divisions until the midblastula stage with ~2000 cells . Medaka es cells have previously been derived from the midblastula embryos . In this study, we chose 6 earlier stages to test their possibility for cell culture derivation ., the early blastula stage has 1000 cells of three lineages, pluripotent deep cells that produce the future embryo proper, and two extraembryonic lineages called the envelop layer (evl) and yolk syncytial layer (ysl) 21 . The deep cells, evl and ysl are equivalent to the inner cell mass or epiblast, trophectoderm and primitive endoderm of the mouse blastocyst embryo 1 . Medaka morula embryos have 128, 256 and 512 cells, which form deep cells and evl . Medaka cleavage embryos have 32 and 64 cells, which are indeterminate in lineage commitment and thus have a promise to generate totipotent stem cell cultures . These 6 stages thus appear to represent a hierarchy of restriction in totipotency / pluripotency . We found that embryos from 128-cell stage onwards allowed for robust dissociation of individual cells, whereas cell isolation from 32- and 64-cell embryos was more demanding for skills . Upon isolation and seeding into culture plates, individual cells remained intact and viable as evidenced by pseudopodia (fig . Isolated cells even from the same embryos exhibited varying sizes, and some of them were connected by intercellular bridges . Importantly, they continued active divisions and formed clusters of smaller daughter cells until 4 h of culture (fig . 2), when cell death were apparent in certain cells without division in cultures from 64- and 128-cell embryos (fig . 2a and b) and with one or two divisions in cultures from 256-cell embryos (fig.2c). 3), as has been observed for cell culture initiation from diploid 14 and haploid midblastula embryos 19 . With regular medium changes, cell cultures from all the 6 stages survived and proliferated over the entire period of observation for up to 2 weeks . These data demonstrate the suitability of early medaka embryos prior to the midblastula stage for cell isolation and cell culture initiation . Like es cell derivation from midblastula embryos (hong and schartl ., 1996), cells from all the 6 stages continued active proliferation and at 6 days of culture, formed monolayers (fig . 3a - f), which consisted of cells phenotypically resembling medaka es cells 15; 19 . In addition, in cultures from 32-cell embryos, we observed ysl cells . These were giant cells with multiple nuclei at the periphery and typical syncytial cytoplasm at the center; and the cytoplasm exhibited motility and changed its shape by pseudopodial formation (fig . Dissociated cells from embryos at 32- to 1024-cell stages can produce es - like cell cultures and ysl cells, pointing to the possibility to derive pluripotent and even totipotent stem cell cultures from early medaka embryos . In medaka, early embryos undergo rapid synchronous cell divisions until the midblastula stage with ~2000 cells . Medaka es cells have previously been derived from the midblastula embryos . In this study, we chose 6 earlier stages to test their possibility for cell culture derivation ., the early blastula stage has 1000 cells of three lineages, pluripotent deep cells that produce the future embryo proper, and two extraembryonic lineages called the envelop layer (evl) and yolk syncytial layer (ysl) 21 . The deep cells, evl and ysl are equivalent to the inner cell mass or epiblast, trophectoderm and primitive endoderm of the mouse blastocyst embryo 1 . Medaka morula embryos have 128, 256 and 512 cells, which form deep cells and evl . Medaka cleavage embryos have 32 and 64 cells, which are indeterminate in lineage commitment and thus have a promise to generate totipotent stem cell cultures . These 6 stages thus appear to represent a hierarchy of restriction in totipotency / pluripotency . We found that embryos from 128-cell stage onwards allowed for robust dissociation of individual cells, whereas cell isolation from 32- and 64-cell embryos was more demanding for skills . Upon isolation and seeding into culture plates, individual cells remained intact and viable as evidenced by pseudopodia (fig . Isolated cells even from the same embryos exhibited varying sizes, and some of them were connected by intercellular bridges . Importantly, they continued active divisions and formed clusters of smaller daughter cells until 4 h of culture (fig . 2), when cell death were apparent in certain cells without division in cultures from 64- and 128-cell embryos (fig . 2a and b) and with one or two divisions in cultures from 256-cell embryos (fig.2c). Until one day of culture, cell attachment was seen for all the 6 stages of embryos (fig . 3), as has been observed for cell culture initiation from diploid 14 and haploid midblastula embryos 19 . With regular medium changes, cell cultures from all the 6 stages survived and proliferated over the entire period of observation for up to 2 weeks . These data demonstrate the suitability of early medaka embryos prior to the midblastula stage for cell isolation and cell culture initiation . Like es cell derivation from midblastula embryos (hong and schartl ., 1996), cells from all the 6 stages continued active proliferation and at 6 days of culture, formed monolayers (fig . 3a - f), which consisted of cells phenotypically resembling medaka es cells 15; 19 . In addition, in cultures from 32-cell embryos, we observed ysl cells . These were giant cells with multiple nuclei at the periphery and typical syncytial cytoplasm at the center; and the cytoplasm exhibited motility and changed its shape by pseudopodial formation (fig . Dissociated cells from embryos at 32- to 1024-cell stages can produce es - like cell cultures and ysl cells, pointing to the possibility to derive pluripotent and even totipotent stem cell cultures from early medaka embryos . In this study we report the ability to isolate and culture early medaka embryonic cells as a step towards stem cell derivation from embryos before and after major lineage restriction . We show that medaka embryos as early as 32-cell stage have the accessibility for the isolation of viable cells capable of generating cell cultures . Importantly, embryonic cell cultures derived from earlier stages consist of cells exhibiting continuous growth and phenotypes that closely resemble well - characterized medaka es cells derived from midblastula embryos 14; 15; 19 suggesting that the competence for es cell derivation is established at the 32-cell stage for stem cell derivation . In fish, no lineage restriction has been reported at this stage, which represents the earliest stage for cell culture derivation in vertebrates . In mammals, the blastocyst embryo after one or two events of lineage restriction leading to the inner cell mass, trophectoderm and primitive endoderm has successfully been used for cell culture, resulting in the derivation of es cells 2; 23, trophoblasts stem cells 7 and extraembryonic endoderm stem cells 8 . It is unknown whether mammalian embryos at the 32-cell stage can produce cell cultures . Interestingly, we have demonstrated that dispersed cells from embryos at all the 6 stages tested including the 32-cell stage can generate yolk syncytial layer cells of the primitive endoderm origin, one of the earliest extraembryonic lineages . It remains to be determined whether the ysl fate is predetermined at the 32-cell stage or specified under culture conditions . In medaka, the ysl originates from marginal blastomeres and its nuclei undergo endomitosis 21 . In zebrafish, ysl formation has been studied in more detail, where some marginal blastomeres are confluent with the yolk cell cytoplasm resulting from incomplete division collapse and deposit their nuclei and cytoplasm into the cytoplasmic cortex of the yolk cell, thereby forming the ysl, whose nuclei then undergo three to five rounds of endomitosis without cytokinesis 24 . The ysl is elusive for study due to difficulties in interfering specifically with ysl formation and morphogenesis 24 . Our finding that ysl cells can form and survive in cultures even from dissociated blastomeres of 32-cell embryos makes medaka an excellent model to study ysl formation in vitro . Among the 6 tested stages, more advanced embryos are better in terms of ease and efficiency for cell isolation and culture . A larger cell size of earlier embryos is prone to mechanical damages during embryo manipulations, cell dissociation and seeding . For the possible derivation of totipotent stem cell cultures, however, the embryo even prior to the 32-cell stage is preferred . At the 32-cell stage, there are already two layers of cells that display differences in size and position, which may indicate the presence or onset of cell - cell interactions and different developmental pathways in differently positioned cells . Future work will determine whether 16- and even 8-cell embryos are amenable for cell isolation and culture . In this study, our primary attention was on the possibility to isolate and cultivate early embryonic cells . We observed similar growth and proliferation during the entire 14-day period of culture between the 32-cell embryos obtained in this study and the midblastula embryos that eventually led to stable es cell lines in our previous studies 14; 15; 19; 25 . Therefore, our results are not against the possibility that early embryonic cells can develop into stable cultures and even cell lines . In this regard, our detailed analyses of differential rna expression of seven pluripotency genes and the telomerase reverse transcriptase in companion papers 26, 27 provide valuable information for characterizing putative stem cells at the molecular level.
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Traditionally, implant treatment is based on a 2-stage protocol with a healing period of 3 - 6 months during which the implants are submerged to achieve osseointegration.1 recently, this clinical suggestion has been challenged . Numerous practitioners now advocate immediate or early loading of implants.2 the advantages of immediately loaded implants are clear: they require shorter treatment periods and allow immediate recovery of function and esthetics.3 high success rate of immediately loaded implants in humans was first documented in the middle 1980s . The 88% cumulative success rate on 1739 immediately loading implants was suggested.4 the clinical performance and prognosis of the single - stage surgical protocol are known to be comparable to the traditional 2-stage method.5 there are some articles reporting a cumulative survival rate of 95%, which investigated immediately loaded single implants.6,7 results from these studies suggest that immediate loading could achieve equal success rates as those found in delayed loading . It is also known as a common claim that treatment with immediate loading improved patient satisfaction and was cost effective although no scientific evidence was presented to support.8 however, advantages of early or immediate loading as mentioned may be offset by an increased risk of implant failure . It was reported that immediately loaded implants were approximately 3 times more likely to fail within 1 year of placement.3 furthermore, there have been few clinical studies investigating the success or failure rates of immediate loading based on korean implant systems . The aim of this preliminary prospective study was to evaluate the outcome of immediate functional loading in partial edentulism, using sinusquick eb (neobiotech co., seoul, korea) implant system . Four subjects (2 smokers and 2 non - smokers) recruited from a population of patients under routine care at seoul national bundang hospital were enrolled in the study . The patients were selected according to the following inclusion criteria: they were in normal general health with sufficient bone to allow the placement of implants at least 7 mm length . The mean age of the subjects was 50.3 years (range from 39 to 65 years), with a gender distribution of 100% men . Informed written consent was obtained from all subjects following approved institutional review board guidelines for clinical research . Surgery was performed under local anesthesia (1: 100,000 epinephrine) or conscious intravenous sedation with 1% propofol solution and midazolam . Implants were inserted according to the procedures recommended by manufacturers . During the period from april to october 2009 (range from 2 to 6 months) immediate loading was applied to the implants showing implant stability quotient (isq) and insertion torque values that were more than 60 and 35 ncm, respectively . Immediate loading is defined as provisional or final implant - supported restoration delivered within 2 weeks.9 therefore, provisional implant - supported fixed partial dentures were delivered within 2 weeks (fig . Periapical radiographs were taken using commercially available film holders and a paralleling imaging technique during the investigating period . In each patient, peri - implant marginal bone level was evaluated by impax (agfa co., mortsel, belgium) system of periapical radiographs . Measurements were recorded at the time of surgery, immediate loading, after 3-months of continued loading, and at the last follow - up (fig . Marginal bone height was determined on these images by measuring the distance from a reference point, defined as the platform of the implant (fig . 2), to the most coronal point of bone - to - implant contact on both the mesial and distal sides of the implant . A single value for marginal bone height was then calculated by obtaining the mean of these two measurements for each implant . The definition of implant success was based on the following clinical and radiologic criteria: 1) absence of clinically detectable implant mobility, 2) absence of pain or any subjective sensation, 3) absence of recurrent peri - implant infection, and 4) absence of continuous radiolucency around the implant.10 marked variability was noted in the implant sizes selected for placement, although implants 11.5 mm length and 5.0 mm diameter were most commonly used . The mean follow - up period was 4.8 months (range, 2 to 6 months). Mean marginal bone loss from implant surgery to immediate loading, 3-months follow - up and last follow - up was found to be 0.03 mm, 0.16 mm and 0.29 mm respectively (table ii). No implant failed up to 6 months after insertion, resulting in a 100% survival rate . All the inserted implants showed successful integration and stable peri - implant condition up to six months . Primary stability was reported to be the most important determining factor on immediate implant loading.6 micromovements of more than 100 m were sufficient to jeopardize healing with direct bone - to - implant contact.6 szmukler - moncler et al . Indicated that micromotions at the bone - implant interface beyond 150 m resulted in fibrous encapsulation instead of osseointegration.11 if the primary implant stability could not be achieved or was questionable, it was strongly recommended to follow a conventional treatment protocol.6 most agreed that an insertion torque of at least 32 nm and a resonance frequency analysis of at least 60 isq was required to achieve a high level of stability.12 in this study, mean isq of 15 early loaded implants was 64.9 4.9 . Generally, clinicians agreed that the quality of bone was significant for success in immediate loading . The initial stability of the implant reduces in the first 3 - 6 weeks after placement due to remodeling and an increased ratio of woven to lamellar bone.12 barewal et al . Indicated that implants placed in areas of high bone quality are relatively stable over the early healing periods.13 however, we reported that both maxillary and mandibular arches showed no failure of implants although the sample size was too small to analyze the data . Horiuchi et al . Also reported about no difference in the success rate between arches in immediate loading.14 further studies are required about the relationship between bone quality and the success rate of immediate loading . It has been established that there are no absolute contraindications to implant placement although a number of conditions exist, which are associated with an increased risk of failure.12 tobacco was reported to be only a risk factor for the implant failure.3 however, the results of this investigation showed there was no implant failure in the participating patients who were smokers . Further controlled clinical studies are needed to evaluate the long - term success of early loaded implants . Within the limitation of this clinical study the preliminary results indicate that immediate loading of the implants in partial edentulism, based on sinusquick eb implant system, may be successful for short period up to six months.
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Ammonium dichromate is an odorless, bright orange to red, crystalline powder used in pyrotechnics, fireworks, photography, and in dyes used for screen and color printing . Skin manifestations after industrial exposure of chromate salts are well - known, but little is known about systemic toxicity and its treatment . A 25-year - old female patient presented with history of vomiting and oliguria for 10 days . Patient had taken a handful of orange red crystals with suicidal intent 10 days earlier while working in a printing press [figure 1]. She was taken to a local hospital where stomach wash was given and managed conservatively . Ammonium dichromate crystals on examination, patient was dehydrated with blood pressure of 130/80 mm of hg and pulse of 98/min . Her investigations showed hemoglobin 7.2 g / dl, urea 650 mg / dl, creatinine 19 mg / dl, sodium 122 meq / l, potassium 7.9 meq / l, calcium 6.7 mg / dl, phosphorus 5.6 mg / dl, bilirubin 0.8 mg / dl, albumin 4.1 g / dl, total protein 7.2 g / dl, alanine transaminase 28 iu / l, aspartate transaminase 106 iu / l, alkaline phosphatase 66 iu / l, lactate dehydrogenase 124 iu / l, and creatine kinase 67 her arterial blood gas analysis showed severe metabolic acidosis with ph of 7.01, pco2of 23 mm of hg and bicarbonate of 6 meq / l . Her electrocardiogram showed tall peaked t waves with prolongation of pr interval and qrs duration . Patient urine output improved gradually and she was discharged with creatinine of 1.3 mg / dl . Her serum creatinine during the last follow - up was 1.1 mg / dl . Hexavalent chromium salts have been recognized as occupational health hazards for more than two centuries . Chromium compounds can exist in a variety of valence states, of which chromium (iii) appears to be the most stable and important form . Chromium (iii) will form stable organic complexes with proteins, and amino acids . Being a strong corrosive agent, dermal contact with chromium (vi) compounds can cause allergic dermatitis or sensitization . After ingestion, ammonium dichromate causes corrosive injury to the oral mucosa, esophagus, and stomach . The systemic manifestations include intravascular hemolysis, acute respiratory distress syndrome, toxic hepatitis, acute kidney injury, thrombocytopenia, and encephalopathy . Chemical analysis of the stomach aspirate is often not useful as ammonium dichromate is highly dissociable compound . Various treatment modalities tried are exchange transfusion, ascorbic acid, n - acetylcysteine, chelation with dimercaprol and dialysis . Ascorbic acid has reducing property and in large doses accelerates the rate of reduction of chromium vi to chromium iii leading to the formation of chromium protein complex, which is nontoxic and excreted in the urine . Though exact dose and duration of ascorbic acid is not known, large doses of ascorbic acid (1 g) has been successfully used to prevent renal failure . Hemodialysis can effectively remove chromium (vi) though the reports on its efficacy in clinical settings are conflicting . Meert et al ., report the death of a child following a 1 g ingestion of ammonium dichromate . Hassan reported a case of 24-year - old female who presented with multiorgan failure after ammonium dichromate ingestion . She was treated with hemodialysis and ascorbic acid (500 mg / day) after which she improved . The cause of renal failure in our patient is probably acute tubular necrosis secondary to both emesis related hypovolemia and direct tubular toxicity of chromium compound . Renal biopsy was not done as her urine output started improving during 2 week of admission . Our patient presented 10 days after the ingestion with severe renal failure without features of any other organ involvement . Since she presented very late, we did not treat her with ascorbic acid and chelating agents . Screen - printing and coloring work is becoming more and more popular in india and the availability of these compounds is much easier now . The health care professionals should be aware of the manifestations of chromate poisoning and benefits of early management, especially with large doses of ascorbic acid.
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Snakes are geographically distributed around the world except for the frozen polar - ice zones and high mountainous altitudes . The majority of species are terrestrial, but others inhabit shorelines, rivers, swamps, and estuaries, and are not found far away from terrestrial environments and habitats . A few aquatic species inhabit the pacific and indo - pacific oceans and associated waters, and a few are pelagic in nature . Approximately, 10% of the approximate 3,500 species of snakes worldwide are considered to be potentially dangerous to humans . Recent estimations of the global toll to humans as a result of venomous snakebite envenomation suggest that 421,000 snake envenomations, and 20,000 deaths occur annually, compared with historical estimates of 5,000,000 snakebites having occurred each year, with greater than 100,000 of them leading to death . Regardless of these reported numbers, it is likely that the reported data represent an under - estimation of the true magnatude of the global snakebite problem . Kasturante postulates that given the statistic that envenomation is said to result from one out of every four snakebites, there may be 1.2 - 5.5 million snakebites occurring annually around the world . Geographically, most cases of venomous snakebite deaths occur in south and southeast asia and sub - saharan africa . In the country of iran, 69 indigenous snake species have been identified, of which 36 are non - venomous, 25 are venomous, and 8 are considered semi - venomous . The recorded number of snakebites from 2001 to 2009, were approximately 5,000 - 7,000 per year, of which, approximately seven deaths were reported each year . Snake envenomation patterns, depending on the species, can vary among the four different families common in iran namely; colubridae, elapidae, viperidae and hydrophidae . Envenomation can cause a range of symptoms and severity, ranging from mild envenomation where a bite is evidenced by skin punctures and mild swelling to severe symptoms of neurotoxicity, vasculotoxicity, hematological toxicity, and myotoxicity . Iran's venomous snakes have a broad spatial geographic distribution, especially those stretching across great expanses of desert . Venomous snake species within the country are represented by both the elapidae family (oxus cobra, naja oxiana) and viperidae family (haly's viper, gloydius halys; saw - scaled viper, echis carinatus; levantine viper, m. l. obtusa; field's horned viper, pseudocerastes fieldi; persian horned viper, pseudocerastes persicus; iranian mountain viper, vipera albicornuta). Species that inhabit desert ecosystems are m. l. obtusa, e. carinatus, p. fieldi, malpolon monspessulanus (montpelier snake), and psammophis schokari (sand racer). P. persicus and p. fieldi inhabit rocky, semi - desert areas mixed with vegetation at elevations up to 2,000 m, and are not found in soft shifting sand areas . Given the varying habitat distribuion of the different species, it is thus apparent that, historically, iranian people have always been exposed to the potential risk of venomous snakebites in any area of the country in which they lived . Snakes in the viperidae family typically possess curved, tubular - shaped canine teeth that are retractable and canalized located in the upper maxillary region, of the mouth . Species such as p. fieldi and p. persicus move in a side - winding motion . The family viperidae is subdivided into two subfamilies, viperinae (true vipers) and crotalinae (pit vipers). These two subfamilies are differentiated by the presence of a heat - sensing foramen or pit bilaterally located between each eye and nostril, this anatomical feature is not present in the vipers native to middle east geographic regions, as true viper species inhabit the region . The dorsal cranial portions of their viper heads have small and non - symmetrical scales, but in a few species there are symmetrical scales . A unique anatomical feature of p. persicus and p. fieldi is the presence of scales that form horns above the eye (suprocular shields). However, knowledge of the unique habitat characteristics, which are specific for each snake species, is an important factor aiding in correct species identification, and minimizing confusion with non - venomous species in the identification process . Thus, the anatomical features, geographical distribution of different snake species, their preferred habitat and associated vegetation types, show unique characterisitics for each species . Severity of envenomation falls into three general categories of mild, moderate, and severe . In mild envenomation, symptoms include swelling, pain, and tenderness . A moderate envenomation manifests with swelling, pain, tenderness, and systemic effects such as nausea, vomiting, tremor, mild hypotension with evidence of coagulopathy, but without clinical bleeding . In severe envenomation, the complications that develop are shock, profound bradycardia, tachypnea, or respiratory failure, and coagulation disorders characterized by bleeding, and other related manifestations . The mainstay in the treatment of envenomation is antivenin . In iran, of three types of antivenin, including mono-, tetra-, and polyvalent, up to now polyvalent are produced by the razi vaccine and serum research institute and have been specifically developed for the treatment of snakebite patients . In general, treatment of mild, moderate, and severe cases require, 2 - 5 vials, 5 - 10 vials, and 10 - 20 vials of antivenin, respectively . Kashan city is located in the north of isfahan province with an approximate distance of 220 km from isfahan city . It is in the vicinity of semnan and qom province from north, markazi province from east, and the central plain from west . The plains are of a typical desert climate with very hot dry summers and mild winters, while the mountainous area has a semi - arid to arid climate with mild, dry summers and cold winters . Rainfall in both areas is minimal and the desert climate is more prominent with very low rainfall and increasing temperature in the summer being significant complications . Overall, intense dry weather conditions are the outstanding climatic characteristics for the region . In a previous study performed in kashan city, the incidence of snakebite was reported to be 2.5 cases per 100,000, which is lower than the average number of snakebites reported for the country of iran as a whole (6.9 cases per 100000). The majoritiy of snakebite patients were male (96%), and 68% of snakebites occured in rural areas of kashan . The greatest rate of snakebite occurred in 15 - 24-year - old people, an age range that also represented the largest working group . Management of venomous snakebite patients has always been a major health - problem within the country . The diversity of envenomation profiles resulting from bites by snakes with this wide geographic distribution, ranging from the southern island in the persian gulf to the northern area of iran, presents a medical problem that requires caution and pause for medical personnel when considering treatment of the envenomated patient . Reporting the specific identity of animal species, especially venomous snake species is therefore, important and allows critical evaluation as to which are the more medically important species, leading to more enhanced medical care of patients envenomated by certain snake species . This study was performed in kashan city, in the isfahan province of central iran, and the study's aim was to determine the species of snakes responsible for snakebite in this region of iran, and those impacting human health . We also aimed to provide general diagnostic and treatment guidelines to treat the envenomation by these snakes . This was a cross - sectional study of snakebite reports from kashan city of the isfahan province in central iran for the period from 2004 to 2010 . All snake specimens, living or non - living, were collected in both active and passive ways for evaluation . Non - living specimens were preserved in ethanol, labeled with date, geographic locality, and specific place of collection documented . All specimens were sent to kashan city, department of environmental health, kashan city, and university of medical sciences, laboratories, iran . Rohollah dehghani, and classified taxonomically based on specific morphological features, and anatomical characteristics with the use of taxonomic identification keys for assignment to proper genus and species, and classified as non - venomous, semi - venomous, or venomous . Uniform questionnaires were used for data collection and filled out by individuals who collected the snake specimens . All questionnaires were reviewed and relevant data extracted for determination of: (1) where snakes were collected geographically (urban, suburban, or rural), (2) type of habitat environment where collected (desert, mountain, forest), (3) collection site (indoors, outdoors, abandoned homes and factories), (4) date and time of collection (day, night, hour), (5) specimen disposition (alive, dead, preserved, killed), (6) snake morphological features (body length, triangular head shape, vent to tip of tail length, body color and pattern, (7) classification to family, genus and species, and (8) species confirmed as venomous, semi - venomous, or non - venomous . Individual snake collector status with respect to type of career work was documented, and whether they were bitten or unharmed during the specimen / snake collection process was also recorded . Medical details of envenomation were not collected . A total of 46 snakes were collected and presented to the laboratory (32 presented alive), for evaluation . The majority of specimens, 32 snakes (70%) were collected from rural areas, none were collected in suburban areas, and 14 snakes, (30%) were from urban areas . Non - venomous specimens were the most common and accounted for 37 (80%) of the total number of snakes evaluated . Seven (15%) specimens were confirmed as venomous species of the viperidae family: m. l. obtusa (n = 3), p. persicus (n = 2), p. fieldi (n = 1), and e. carinatus (n = 1). Two specimens (4%), malpolon monspessulanus insignitus and p. schokari were classified as semi - venomous . M. l. obtusa, was the most frequently collected venomous snake species, and specimans were collected from armack, naragh, and niasarsar [figure 1]. The single p. fieldi specimen represents the first such speciman to be collected, reported, and documented in central iran [figures 3a, b]. M. m. insignitus was collected in the rural area between mashkan and khoragh, in the vicinity of a milk pasteurizing factory [figure 5]. Non - venomous snakes collected were represented by eleven different species, primarily of the genus coluber . Macorvipera lebetina obtusa (picture by dehghani r) pseudocerastes persicus (picture by dehghani r) pseudocerastes persicus (picture by dehghani r) pseudocerastes fieldi (picture by dehghani r) echis carinatus (picture by keyler d) malpolon monspessulanus insignitus occurrence of snakes in urban and rural kashan city, isfahan province, iran represent the total number of snakes collected in urban and rural environments map of kashan and study areas the significance of this study's results relate to the confirmation that four species of venomous snakes, and two species of semi - venomous snakes, belonging to the viperidae and colubridae families, respectively, are currently found in the kashan city in isfahan province, iran . Additionally, there are eight other non - venomous species present in the region . Classification of venomous, semi - venomous, and non - venomous snakes can be performed in several ways . The most confirmative method to distinguish the differences between non - venomous, semi - venomous, or venomous species, involves the inspection of a snakes dentition (anatomical fang structures or lack of fang structures). Non - venomous snakes are aglyphous in that they have simple rows of shorter, conical - shaped teeth, which are not canaliculated or grooved, and are not connected to a secretory or venom gland . The semi - venomous snakes m. m. insignitus and p schokeri are opisthoglyphous, possessing posterior maxillary teeth, which are larger and more prominent than the anterior teeth, and connected to glands that are of a lesser defined anatomical structure . Viperid snakes are are solenoglyphous, possessing retractile fangs that are canaliculated (having a hollow lumen). Climatic conditions and weather patterns occurring in different parts of the mountane and dessert regions of this defined geographic kashan city area are highly variable, providing favorable conditions and habitats for multiple different snake species . In this study, the most abundant venomous species collected was m. l. obtusa, and it is a speices known to tolerate a spectrum of climatic weather conditions from the northern to southern and eastern to western parts of its range . Have previously reported that the bite of this species can be dangerous to humans, and of significant medical importance . P. persicus was the second most frequently collected venomous snake species in this study . Surveying for collection of this species involved unique habitat search strategies in rural country involving cemetery grave - stones, hedges, fence lines, and wall cracks / spaces of abandoned old thatch cottages . The distribution of this species has been previously reported in khorasan, sistan, yazd, esfahan, fars, semnan, markazi, khuzestan, and zanjan, provinces . E. carinatus was responsible for a single bite in this study, and has been previously reported from semnan, khorasan, sistan, kerman, fars, hormozgan, khuzestan, and mazandaran provinces . More recent confirmation of this viperid specie's presence in iran has been reported by stmpel et al . It is interesting; however, that prior to this study, there were no reports confirming the biological activity or existence of e. carinatus in isfahan province . Of all the specimens collected, p. fieldi was the most surprising, for despite this species having been reported from kermanshah city, and fars province . Thus, the confirmation of p. fieldi here represents the first reporting of the viperid species in isfahan province, and suggests that the species has a quite limited geographic distribution in iran . M. m. insignitus represents the only semi - venomous colubrid species responsible for snakebite to a human in this case series . It does have enlarged rear fangs, a functional secretory gland, and an aggressive behavior . Although this subspecies is reported as semi - venomous, a bite in france by m monspessulanus has been reported to cause significant neurological symptoms following envenomation . Distribution of this species has been reported in eastern and western azarbaijan, and ardabil, markazi, tehran, khorasan, hamedan, kermanshah, hormozgan, khuzestan, mazandaran and ghazvin provinces . Furthermore, we have previously reported this species from isfahan province, and the documentation represents current confirmation of the species in the province . P. schokari, inhabits the arid kavir desert, the great salt desert of central iran, and is a fast moving snake . Although it possesses enlarged posterior maxillary teeth and is considered semi - venomous, the consequences of its bite are not known to be documented . Clinical signs and symptoms associated with bites from the less venomous snakes of the colubridae family include local swelling, bleeding from the fang marks and sometimes fainting, and ecchymosis in rare instances . Envenomation following bites by the dangerous colubrids of africa (dispholydus typus and thelotornis spp .) Can result in delayed vomiting, colicky abdominal pain and headache, widespread systemic bleeding with extensive ecchymoses, incoagulable blood, intravascular haemolysis, and kidney failure . Viperidae (sub - families crotalinae and viperinae) envenomation can be evidenced by severe local signs and symptoms . Swelling may become detectable within 15 min, but in unusual circumstances my be delayed for several hours . Generally, swelling spreads rapidly and may involve the whole limb and adjacent trunk, with pain and tenderness in regional lymph nodes exist, bruising, blistering, and necrosis developing during the next few days . Spontaneous systemic haemorrhage is most often detected in the gums, but may also be seen as epistaxis, haematemesis, cutaneous ecchymoses, haemoptysis, sub - conjunctival, retroperitoneal, and intracranial haemorrhages . Direct involvement of the heart muscle is suggested by an abnormal electro cardiogram (ecg) or cardiac arrhythmia . Laboratory values indicating signs of snake envenomation include raised peripheral neutrophil count (up to 20,000 cells/l). Initial hemo - concentration resulting from extravasation of plasma, followed by anemia due to bleeding or haemolysis, and thrombocytopenia . A useful test for venom - induced defibrin (ogen) a few millilitres of venous blood is placed in a new, clean, dry, glass test tube, left undisturbed for 20 min at ambient temperature, and then tipped to see if it has clotted . Patients with generalized rhabdomyolysis show a rise in serum creatine kinase, myoglobin, and potassium . Urine should be examined for blood / haemoglobin, myoglobin and protein and for microscopic haematuria and red cell casts . While removing the patients to the nearest medical facility, movements of the bitten limb should be avoided by a splint or sling . Local incisions and suction, vacuum extractors, potassium permanganate and cryotherapy, tourniquets, and compression bands should also be avoided . In cases of elapid envenoming (such as cobra), the pressure immobilization method (firm but not tight bandaging of the entire bitten limb with a crepe bandage 4 - 5 m long by 10 cm wide starting over the site of the bite and incorporating a splint) may be useful . This method is not warranted in viperid snakebites where local swelling, necrosis, and coagulopathy are the main venom - induced features (e.g., m. l. obtusa). Patients being transported to the hospital should be laid on their side to prevent aspiration of vomit or excessive salivary secretions . Syncope, shock, angio - oedema and other anaphylactic symptoms are treated with 0.1% adrenaline by subcutaneous injection (0.5 ml for adults, 0.01 ml / kg body weight for children), and an antihistamine such as chlorpheniramine (or diphenhydramine 50 mg) is given by slow intravenous injection (10 mg for adults, 0.2 mg / kg body weight for children). General indications for antivenom include haemostatic abnormalities such as spontaneous systemic bleeding and profound thrombocytopenia, neurotoxicity, hypotension and shock and/or abnormal ecg, impaired consciousness, and generalized rhabdomyolysis . Local swelling involving more than half the bitten limb, extensive blistering or bruising, bites on digits, and rapid progression of swelling are other indications for antivenom . It is important in the decision process for antivenom administration to consider the prognosis in the absence of antivenom therapy, as a therapeutic goal with antivenom use is to neutralize venom toxins, and prevent the worsening of venom - induced toxicological problems . Mono - specific (monovalent) antivenom (antivenom containing antibodies against the venom of a single snake species) is ideal for treatment if the biting species is known . However, poly - specific or polyvalent antivenoms (antivenom containing antibodies against the venom toxins of multiple snake species) are generally used and advantageous because identification of the snake responsible for a bite may be difficult and uncertain . Antivenom treatment is indicated as long as signs of systemic envenoming persist; however, it is most effective when given as soon as signs of envenomation appear . Intravenous infusion of antivenom diluted in approximately 5 ml of isotonic fluid / kg body weight allows for a more easily regulated controlled delivery, and it is more safe than intravenous push injection of undiluted antivenom . Furthermore, controlled administration of diluted antivenom, usually reduces the risk of adverse allergic reactions . In most countries the dosing of antivenom is empirical . Use of antivenin is usually recommended in patients suffering severe envenomation, but in some patients it's use may cause life - threatening hypersensitivity reactions . Marked symptomatic improvement may be seen soon after antivenom infusion has been completed . In patients suffering shock, spontaneous systemic bleeding usually stops within 15 - 30 min, and blood coagulability is restored within 6 h of antivenom . More antivenom should be given if severe signs of envenoming persist after 1 - 2 h or if blood coagulability is not restored within about 6 h. systemic envenoming may recur hours or days after an initially good response to antivenom . This is explained by continuing redistribution of venom from tissues following its initial distribution, resdidual venom distributon from the injection site, and the pharmacokinetic differences in venom and antivenom clearance from the blood . Envenomed patients should therefore, be assessed daily for at least 3 or 4 days, and followed with outpatient visits for 2 - 3 weeks . If signs of necrosis appear, surgical debridement, and broad - spectrum antimicrobial coverage provided . In rare instances split skin grafting may be required . Once specific antivenom has been given, and neutralization of venom procoagulants, restoration of coagulability and platelet function established, fresh whole blood, fresh frozen plasma, cryoprecipitates or platelet concentrates further correct insufficiencies . The majority of snakebites occurring in the urban areas of kashan are non - venomous . In contrast, patients presenting to clinics bitten in rural areas have a greater probability of suffering from a venomous snakebite . Knowledge of the characteristics, geographic distribution, and environmental habitats of venomous snakes is of value to physicians for rapidly recognizing species potentially responsible for the snakebite in a specific geographic region; thus, allowing for the timely and correct treatment based on identification with respect to the presence or absence of a given snake species in the specific region . Medical personnel have a significant role, by their permanent presence and medical practice in regions of high snakebite risk . Although they can initiate treatment following a bite, providing knowledge to medical professionals for explaining general information about venomous snakes to the locally native people would potentially decrease the risk of snakebite with consequent reduction in morbidity and mortality associated with venomous snakebite . Education of native people and their children to increase awareness about the possibility of venomous snakes being present where they live, and that the risk of snakebite is greater during the warm months of the year is important . The four venomous viperid species in the kashan city area of the isfahan province (p. fieldi, m. l. obtuse, p. persicus, and e. carinatus) possess venom toxins that may cause severe hematological complications following envenomation . Finally, general awareness for those who intend to hike or camp in areas known to be inhabited by venomous snake species is a potentially important preventive measure for reducing the risk of a venomous snakebite.
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Telemedicine can be considered as the application of telecommunications technology to assist in delivering health services at a distance and is not new.1 teleophthalmology was long considered as one of telemedicine s most challenging applications.2 spectral domain ocular coherence tomography (sd oct) imaging is being used in optometric primary care with referral of sd oct images to hospital eye services (he s). Collaboration between primary and secondary care in the application of retinal imaging technology in this way, while innovative, is not unique.3,4 however no such previous publications included oct imaging . The primary care to specialist interface is a key organizational feature of many health care systems . Patients are referred to specialist care when investigations or therapeutic options are exhausted and more specialized care is needed, or thought to be needed . In united kingdom ophthalmology practice, the community optometrist (co) is usually the source of most referrals . There is evidence that referral processes can be improved.5,6 currently, patients attending a co in the authors area, following general practitioner (gp) referral or self - referral, can expect waiting times of up to 2 days in the community . Those community optometric patients requiring forward referral by the gp for specialist ophthalmic opinion or treatment may, depending on the efficiency and work pressures at the gp s practice, perhaps wait several days for a referral to be made by the gp to an ophthalmologist . At present there is no reliable method for the co to check that such referral has occurred or has been received by the he s . Critically there is a clinical risk of patient referrals being lost or delayed as a result of the multiple steps in referral . Furthermore, in the authors experience, some gps add little additional clinical information to the optometric referral . There is then often a possible additional waiting time of 18 weeks in the english national health service (nhs) for nonurgent cases . Further pathway delay can occur, even in urgent cases, as retinal patients are frequently first seen in the general clinic or eye casualty in the he s and only then often referred to a medical retina consultant . Several retinal conditions are time sensitive and require prompt treatment if good clinical outcomes are to be achieved . Timely access to nhs services for macular patients is under pressure and follow - up appointment capacity is problematic and is a patient safety concern.7 a recent study from scotland highlighted further patient safety concern that optometrists find it difficult to accurately elicit the signs of macular disease.8 traditional methods of retinal image transfer in the nhs are often unsatisfactory and described in table 1 . Optometrists undertake retinal examinations in the community and generally inform the patient s gp of any detected eye disease . It is usually at the discretion of the gp (acting as a gatekeeper) to make a further referral (acting as a mailbox) to an ophthalmologist for further specialist review . Many gps in the united kingdom see merit in direct referral by cos as many gps lack instruments for, or advanced training in, assessment of eye disorders.9,10 many retinal conditions can be better diagnosed with sd oct imaging . The objective in this report was to evaluate the novel service innovation of attaching sd oct images to emailed clinical correspondence and consider implications for telemedicine ophthalmology consultation (teleophthalmology) involving patients with suspected macular disease . The authors were keen to improve triage of age - related macular degeneration (amd) patients from optometry . This manuscript is an evaluation of 50 retinal e - consultations undertaken from june 2010 to august 2011 based on sd oct imaging in the community . In contrast to many telemedicine reports involving rural and or remote areas, these locations are within 6 miles of each other, in greater manchester, united kingdom . The patients referred were examined at the co practice and all had, or were suspected of having, macular disorders . Patients attending for diabetic retinopathy screening were not included as separate local pathways exist for such patients . The co forwarded referrals with sd oct images captured in primary care to an ophthalmologist for consideration, triage, advice, and diagnosis . A topcon 3d oct-2000 instrument (topcon corporation, tokyo, japan) was used by the co. a color fundus image is included with oct images captured from this instrument . Oct images were stored onsite at the optometrist s facility in the oct instrument and offsite backup was made . The images were forwarded by secure email (nhsmail) and viewed by the ophthalmologist at the hospital or on a secure personal laptop computer . Images and text from the referrals were stored in a personal folder on the hospital nhs foundation trust s network in accordance with the trust s data protection guidance . The co also printed out the email correspondence and provided it on paper to the patients gps . Trainee ophthalmologists were also able to participate in the e - consultation using secured smartphones . Sd oct imaging was conducted by the co with the patient s permission and at an additional charge to the sight test fee . If patients could not afford oct imaging, the cost was born by the optometry practice . It is at the discretion of the co to decide what further examinations are appropriate.11 an email assessment of the patients consultation was sent by the co if an ophthalmic opinion on suspected macular disease was required . Where appropriate, other clinical data such as previous color fundus images captured by the co or descriptions of visual fields or amsler testing were included . Patients who subsequently attended hospital had oct imaging by nurses on the zeiss cirrus sd oct (carl zeiss meditec ag, jena, germany) instrument and other investigations as required . Specifically, in 96% of cases, analysis of the referrals and a working diagnosis / care pathway was provided by the ophthalmologist to the optometrist within the next calendar day (tables 2 and 3). The patient pathway is outlined in figure 1 . Where the teleophthalmology consultation recommended referral to the he s, the optometrist made recommendations to the gp for referral for further ophthalmic care . On occasion the telemedicine ophthalmologist advised direct urgent referral to he s based on the e - referral . This was either direct referral for face - to - face examination at the local he s in bolton (n = 30) or tertiary he s retinal care (n = 2) in manchester or by the gp (n = 1). If needed, the ophthalmologist contacted patients by telephone to arrange direct urgent attendance at the next day s ophthalmic clinic (n = 5). Seventeen cases (34%) did not, in the consensus opinion of the optometrist and ophthalmologist, require medical review and were further managed in primary optometric care . In all cases the community sd oct image quality was considered by the ophthalmologist to be as good as, or better than, the sd oct images captured by the hospital nursing staff . In two cases the community sd oct image was superior to that captured in hospital clinic on initial attempt . Figure 2 depicts an example of a wet amd case imaged at the co s practice and at the hospital clinic on successive days . Patient satisfaction was not formally accessed, but it was noted that most patients spontaneously remarked on how satisfied they were . E - health and telemedicine has the potential for significant improvement in the quality and productivity of patient care compared to traditional methods . Service evaluation is conducted to judge current care and thus differs from clinical research or clinical audit inter alia in that no randomization is undertaken.12 a recent service evaluation showed e - consultation with hospital nephrologists promotes effective management of patients with mild - to - moderate chronic kidney disease in primary care.13 similarly in the current service evaluation retinal referrals from optometry to hospital ophthalmologists were reduced by e - consultation . This reduction allowed swifter attention to referred cases and, importantly, urgent attention to clinically urgent retinal cases . Benefits also included more efficient triage and prioritization of referrals and appointments at the hospital . The benefits in the community included ease of access to oct scanning at one - stop visits, enhanced education of optometrists in the care of retinal patients, and improved interdisciplinary professional working . This innovation has the potential to support the quality, innovation, productivity and prevention scheme.14 with respect to suspected wet amd patients, direct faxed referral from the community to the he s is endorsed by relevant stakeholders.15 email is faster and superior in quality to such faxed and often handwritten optometric or gp urgent referral documents . Specifically, typed text is clearer than handwritten text and the author and date of writing are easy to determine . In the authors opinion, secondly, it is also often at the discretion of the gp and or ophthalmologist to inform the referring optometrist with the outcome if referred to he s . Importantly, despite written patient consent, such feedback to cos is often lacking.16 such disconnect has the potential to lead to breakdown in continuity of care at the patient s next visit to the co and may lead to further referrals . Waste in the healthcare system and a lost opportunity for multidisciplinary education and audit . Furthermore as well as providing savings to the nhs by reduced hospital and gp clinic visits, this service development also benefits the economy and ecology by reduced patient travel and importantly reduces patient concern by swift response to consultation . It is not intended that teleophthalmology would replace face - to - face consultation; rather, electronic referral of oct images is, in the authors opinion, a useful tool to assist, prioritize, and refine referral of retinal patients in the digital age . The proportion of referrals from optometrists compared to those from gps is increasing.17 currently, there are fewer ophthalmologists in the united kingdom, pro rata, than in any other european union nation . Together with a high level of undetected, yet treatable visual morbidity and an increasing elderly and diabetic population, improvements in primary eye care are also needed . Treatment of wet amd patients requires regular intravitreal injections and is an area of high volume care under pressure in england and wales.7 in the authors opinion, teleophthalmology may assist with such pressures . A recent report described a pilot teleconsultation network of general and specialist ophthalmologists in italy for retinal cases.18 others have used teleophthalmology in glaucoma, acute eye conditions, retinopathy of prematurity, and strabismus.1922 there is merit of teleophthalmology in challenging locations, such as in rural areas,3,20 prisons,23 and in military settings.24 this project did not include diabetic patients, as separate pathways for such patients exist locally within diabetic retinal screening services . The potential for teleophthalmology with oct in such diabetic retinal screening services, and which are now embedded in the nhs, requires further analysis . The present evaluation of a pilot scheme highlights the potential wider application of innovation in clinical imaging and information technology transfer such as has occurred within picture archiving and communication systems . Access to nhsmail by optometrists is available from the nhs on request and this requires publicity and adoption within the optometry profession . Optometry connection to the nhs n3 network for picture archiving and communication systems transfer and or use of image exchange portals are further quality improvements that could be rolled out . If teleophthalmology services do develop, it is important that they are compliant with data protection and patient safety aspirations . The comprehensive guidelines on teleradiology services developed by the royal college of radiologists may be of merit to those seeking to roll out teleophthalmology services.25 teleophthalmology consultation with community optometry enables ophthalmologists to focus on macular patients with significant / urgent disease and, if applied more widely, could reduce referrals, costs, and waiting times for such services in the united kingdom . A limitation is that a 24/7/365 rapid response teleophthalmology service is unlikely to be sustained unless motivation or remuneration is provided to those ophthalmologists, optometrists, and providers involved . This innovation would therefore need to be recognized in service planning or commissioning . In conclusion, this service evaluation indicates the value of ophthalmic images transfer between primary and secondary care in the better management of patients with macular conditions . Telemedicine is of known benefit in image dependent specialties such as radiology and ophthalmology . In england, remote review of ocular fundus images is in use to underpin nhs diabetic retinopathy screening services . The merits of teamwork in community optometric and hospital opthalmic practice for patients using innovative technology . Telemedicine is of known benefit in image dependent specialties such as radiology and ophthalmology . In england, remote review of ocular fundus images is in use to underpin nhs diabetic retinopathy screening services . The merits of teamwork in community optometric and hospital opthalmic practice for patients using innovative technology.
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Colorectal cancer (crc) occupies an important place among cancers that affect both men and women; it ranks third among all cancers in terms of its incidence among both men and women, as well as being the third most common cause of death from cancer in the usa . The estimated number of new cases and of deaths from crc in the usa was reported to be 136,830 and 50,310, respectively . Changes in lifestyle have led to a rapid increase in crc incidence in developing countries . Many adenomas are polyps which advance from small to large (> 1 cm) size, and then to dysplasia and then cancer . Gross appearance cannot be used to distinguish these forms reliably, and diagnosis requires a biopsy and pathology evaluation . If adenoma advances to carcinoma, the process can take at least 10 years, on average . A number of inflammatory tests, including crp and glasgow prognostic score, have been found to correlate with poor prognosis of cancer patients . As a reflection of systemic inflammation, the neutrophil - to - lymphocyte ratio (nlr) was initially established as a prognostic factor among patients in intensive care, then found also to be associated with poor prognosis in various types of cancer, including colon cancer [710]. Crc has a low morbidity and mortality rate when diagnosed at early stages, and it is possible to treat crc through surgery in these early cancer stages . Since hematological testing is a routine procedure for most patients, nlr constitutes a simple, reliable, and easy - to - use indicator for the inflammatory response . The aim of the present study was to conduct research into the relationship between nlr and the colorectal adenoma the study sample included 397 patients who had colonoscopic polypectomy between january 2010 and december 2014 at the endoscopy unit of the faculty of medicine, dicle university . This study was approved by the ethics board of the faculty of medicine of dicle university . Patient pre - operative demographic information and laboratory results of these patients was obtained retrospectively through the hospital s automated record system . The study excluded patients with active infectious diseases, hematological or solid organ tumors, obstructive or ulcerated lesions identified distinctly in colonoscopy, more than 15 polyps, use of cyclooxygenase-2 (cox-2) inhibitors, and a history of surgery for colon tumors . The patients were divided into four groups: patients with hyperplastic polyps (group a), patients with adenomatous polyps (group b), patients with adenomatous polyps indicating dysplasia (group c), and patients with polyps diagnosed as adenocarcinoma (group d). Hematological parameters were measured using an automated hematology analyzing system (abbott cell - dyn 3700; abbott laboratory, illinois, usa). The nlr was calculated by the division of the neutrophil count by the lymphocyte count . Statistical analyses were performed using the spss 18.0 for windows software package (spss inc ., descriptive statistics pertaining to continuous variables were indicated as average and standard deviation (sd) values . Variables that lacked a normal distribution were expressed as mean (min max range) values . Variance analysis (anova) the kruskall - wallis test was used for variables that lacked normal distributions during the comparison of three groups, and the mann - whitney u test was used for subgroup comparisons . The threshold value of nlr in the prediction of polyps indicating dysplasia - cancer was identified by receiver operating characteristic (roc) curve analysis . Statistical analyses were performed using the spss 18.0 for windows software package (spss inc ., descriptive statistics pertaining to continuous variables were indicated as average and standard deviation (sd) values . Variables that lacked a normal distribution were expressed as mean (min max range) values . Variance analysis (anova) was used for variables with normal distributions during the comparison of three groups . The kruskall - wallis test was used for variables that lacked normal distributions during the comparison of three groups, and the mann - whitney u test was used for subgroup comparisons . The threshold value of nlr in the prediction of polyps indicating dysplasia - cancer was identified by receiver operating characteristic (roc) curve analysis . The age range of the patients was 1790 years and the mean age was 57.715.7 years . Of the 379 patients, 240 (60.4%) were male . In all, 31.5% of the patients had hyperplastic polyps (group a, n=125); 54.7% had adenomatous polyps (group b, n=217: 185 tubular, 37 tubulovillous and 5 villous); 5.0% of patients had polyps indicating dysplasia (group c, n=20), and 8.8% had polyps indicating cancer (group d, n=35). The ratio of smoking was 26.7% among men and 8.6% among women (p<0.001). The rates of smoking for patients in groups a, b, c, and d were 41%, 34%, 25%, and 33%, respectively (p=0.49). No correlation was identified between dysplasia, cancer, and smoking . Table 1 presents the median (min max range) for white blood cells, neutrophils, lymphocytes, and nlr values of the groups . The comparison of the groups showed significant differences only in the lymphocyte count and nlr (p values were 0.002 and 0.007, respectively). Nlr values increased in accordance with the adenomatous polyp dysplasia - cancer sequence (2.05 (0.2710), 2.34 (0.8314.70) and 3.25 (0.8110.0), respectively). When the groups were compared, the lymphocyte count was significantly lower among cancer patients than among the groups with hyperplastic and adenomatous polyps (p values were 0.011 and 0.003, respectively). The lymphocyte count was significantly lower among patients with dysplasia only when compared to the adenomatous group (p=0.009). Nlr comparison included age, sex, polyp size, and polyp location (table 2). Nlr median value was found to be significantly higher among those with polyps larger than 10 mm [2.71 (0.9014.70)] compared to those with polyps smaller than 10 mm [2.28 (0.2711.67)] (p<0.001). The relationship between nlr and cancerous polyps was evaluated by roc curve analysis . With the nlr threshold value set at 2.20, it was possible to predict the diagnosis of cancerous polyps with a sensitivity of 71.4% and a specificity of 52.5% (auc: 0.665, 95% ci: 0.5590.772, p=0.001), (figure 1). A limited number of studies have evaluated the relationship between polyps and nlr, and these studies addressed patients with neoplastic polyps and crc patients separately . To the best of our knowledge, the present study is the first study that approaches non - neoplastic polyps and all lesions in the adenoma - dysplasia - cancer sequence of the colon together . The present study found a gradual increase in nlr as the patient approaches cancer diagnosis in line with the adenoma - dysplasia - cancer sequence . Nlr was found to be significantly higher among cancer patients than among patients with neoplastic or non - neoplastic polyps . Again, with the nlr threshold set at 2.20, we found that cancerous polyps could be predicted with a sensitivity of 71.4% and a specificity of 52.5% . Furthermore, a significant relationship was established between polyp size, which represents an important factor in the progression from neoplastic polyps to cancer, and nlr . The relationship between cancer and inflammation was reported by virchow in the nineteenth century, and ample evidence has been gathered since then to support this relationship . Levels of white blood cells, neutrophils, lymphocytes, and platelets in serum, and acute - phase proteins, including c - reactive protein (crp) and albumin, are known indicators of a response to inflammation . These simple and easy - to - measure parameters are widely used as standard assays . Nlr can be used as a tool to indicate the balance between the activation of the pro - tumor inflammatory pathway and the anti - tumor function of the immune system . Current theories focus on the relative neutrophilia and lymphocytopenia that emerges as part of the cancer - induced systemic response to inflammation . In malignant tumors, systematic inflammation is considered to be created by tumor hypoxia or necrosis and the relevant anti - apoptosis signaling pathway motivation . Mechanisms proposed to explain neutrophilia include the release of g - csf from tumor cells and inflammation in cancer through the release of il-1 and tnf - alpha . Relative neutrophilia leads to an increase in the number of inflammatory markers that incorporate pro - angiogenic factors (vegf), growth factors (cxcl8), proteases (metalloproteinase tissue inhibitors), and anti - apoptotic markers (nf-b) that extend support to tumor growth and progression [1618]. A number of studies have established that a decrease in the t cell activity in tumors is correlated with the progression of the primary tumor . It has been argued for some time that anti - tumor activity is primarily realized through the mediation provided by cellular immune reactions dependent on lymphocytes . Lymphopenia has been shown to be associated with disease severity and immunological escape of tumor cells from infiltrating lymphocytes [2023]. The present study established a significant difference among the patient groups in terms of lymphocyte count, despite the absence of any significant difference identified among the groups in terms of the neutrophil count . According to inter - group comparisons, lymphocyte count was determined to be significantly lower among patients with cancer and dysplasia when compared to patients with non - dysplastic adenomatous polyps . This, in turn, signifies that the increase in nlr correlates with the decrease in lymphocyte count, as has been emphasized in the literature . An increased count of lymphocytes infiltrating tumors has been acknowledged as a factor predicting good prognosis . It is possible to utilize nlr as a means to indicate the balance between the activation of the pro - tumor inflammatory pathway and the immunological anti - tumor function . Neutrophils in tumor tissue have been shown in histopathology studies to be a factor for poor prognosis in patients with crc . In the present study, the high level of intratumoral cd66b neutrophils was positively correlated with pathologic and clinical stages . Neutrophils in the blood have also been found to be an independent prognostic marker for poor survival in crc exhibiting metastasis . One study found that for patients with advanced crc who receive oxaliplatin - based chemotherapy, increased nlr predicted poor prognosis as an independent factor . An increase in nlr (> 5) also provided an independent prediction of poor prognosis for colorectal metastasis in the liver following percutaneous radiofrequency ablation . A recent study identified the association of systemic inflammation before the operation (glasgow prognostic score criteria), peritumoral inflammatory infiltrate, and cancer - specific survival among patients who had undergone resection with a remedial potential for colorectal cancer . In addition, the aforementioned study demonstrated that a low - grade peritumoral infiltrate was associated with an increase in the dukes stage and in the total white cell count and neutrophil count . Another recent study established a positive correlation between crp and metastatic lymph node ratio, an independent indicator of prognosis among patients with stage iii colon cancer . The seemingly inverse proportion between markers of the response to systemic inflammation and the response to local inflammation most probably reflects the imbalances in the innate and adaptive immune systems that disrupt effective host - tumor immune responses . In one study, the presence of increased inflammation was identified by histopathology in adenomatous and hyperplastic polyps; however, both acute and chronic inflammation was found to be prominent only when compared to both normal tissue and hyperplastic polyps among adenomatous polyps with neoplastic potential . Cox-2 proteins, which are negative in normal colon epithelia, increase in both adenomatous polyps and crc cells . Clinical and experimental studies have established that selective and nonselective nonsteroidal anti - inflammatory (nsaids) prevent the development of cancer from neoplastic polyps . Studies that showed cox-2 to be a significant factor that regulates apoptosis, angiogenesis (vegf), and invasiveness in tumor cells also provided mechanistic insights into the position of cox-2 in intestinal tumorigenesis . In addition, studies have indicated that nsaids inhibit cell proliferation and induce apoptosis in colon and other tumor cell lines in culture . Reported that nlr was able to distinguish neoplastic polyps from non - neoplastic ones . In the present study, nlr values> 1.9 could predict neoplastic polyps with a sensitivity of 71% and a specificity of 50% . However, we could not establish a significant difference in the nlr between these two groups . This situation may be associated with the low villous adenoma ratio (7.4% and 2.3%) despite the relatively high total number of patients included in the study . Another study looking at the polyp - nlr relationship found that nlr was able to distinguish crc from neoplastic polyps . In the present study, nlr was found to be significantly higher in the crc group than in both the group with neoplastic polyps and the healthy group, whereas no difference was identified between the nlr values of patients with neoplastic polyps compared to healthy controls . The earlier study established nlr values> 2.28 as able to predict crc with a sensitivity of 68% and a specificity of 42% . These values correspond to the results of our study . In histopathologic terms, a correlation was determined between polyp size and both acute and chronic inflammation . The present study identified significantly higher nlrs in polyps larger than 10 mm and demonstrated the same finding in systemic terms . The first, most important limitation of the present study is its concentric and retrospective nature . Second, the study could not assess the effects on the immune system fully due to its retrospective design . Another weakness of the study was the low number of patients with cancerous and dysplastic polyps compared to the number of patients with hyperplastic and adenomatous polyps . Furthermore, nlr remained insufficient in predicting dysplastic polyps and could only predict cancer in the adenoma - dysplasia - cancer sequence . This may have been the result of the small number of patients in the dysplasia group, as well as the low number of high - grade dysplastic patients in this group (n=3). These results provide preliminary information for multi - centered, prospective and long - term studies to be undertaken in the future . The present study established that nlr increased gradually in the pathway extending from adenoma to cancer . We detected that nlr was significantly higher in cancerous polyps when compared to non - neoplastic polyps and adenomatous polyps . Furthermore, with the nlr cutoff value set at 2.2, we were able to predict patients with cancerous polyps from amongst all patients exhibiting polyps with a medium level of sensitivity and specificity . Nlr is a cheap, universally available, simple, and reliable test that can predict cancerous polyps . Large - scale prospective studies to be conducted in the future may enable nlr to be utilized as a useful biomarker in the follow - up of patients with polyps and the selection of patients for such modes of treatment as cox-2 inhibitors that inhibit the progression of crc.
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Many children with acute glomerulonephritis (agn) are first seen in their primary physicians' offices . This initial contact may be crucial in determining the child's most appropriate disposition as well as identifying any immediate threats to life . This paper will review the office approach to agn in children on the basis of upon a firm grounding in pathophysiology . It will begin with an overview of the pathology and pathophysiology of glomerulonephritis and then present a practical outline of the important aspects of the history and physical examination pertinent to a child with suspected agn . It will then provide guidance in choosing and interpreting appropriate laboratory studies for the initial evaluation . Finally, some guidance will be provided on referral of children with agn, including a discussion of some situations in which management by the primary caretaker may be appropriate . Agn is a complex of findings which is marked histologically by a generalized glomerular inflammation . Frequently, renal biopsy is not available, but agn can usually be recognized by the clinical picture of hematuria, fluid overload (edema and hypertension), and some evidence of renal insufficiency (elevation of bun and creatinine). In most circumstances, glomerular inflammation begins with an antigen - antibody reaction, either direct antibody binding to an antigen expressed or trapped in the glomerulus, or the localization of a circulating complex in the kidney . This incites injury by activating one or more systems of inflammatory mediators: the complement cascade, coagulation factors, cytokines, growth factors, and others . The inflammation is marked by proliferation of resident glomerular cells and infiltration by lymphocytes or neutrophils . The glomerular inflammation and expansion impairs the microcirculation, reducing the glomerular filtration rate (gfr) and usually resulting in an increase in bun and creatinine . This reduction in gfr, in turn, leads to the retention of salt and water, causing fluid overload . Indeed, hypertensive encephalopathy may be the presenting complaint in some children with agn . In some situations, agn is a primary process, and virtually, all of the clinical findings are a consequence of the renal lesion . The agn is but one manifestation of a systemic illness which has targeted multiple organs, each of which may be independently injured . In children, fortunately, most cases of agn in children are either self - limited or amenable to therapy although there may be devastating complications of the illness during the acute phase . Less commonly, what begins as an apparent agn may presage the development of a chronic process, which ultimately may progress into irreversible end - stage renal disease (esrd). Most typically, the child with agn will be seen because of the sudden development of change in urine color . On occasion, however, the presenting complaint may relate to a complication of the disease: hypertensive seizures, edema, and so forth . True bright red blood in the urine is more likely a consequence of anatomic problems such as urolithiasis than glomerulonephritis . The gross hematuria of agn is virtually always painless; dysuria accompanying gross hematuria points to acute hemorrhagic cystitis rather than renal disease . A history of previous such episodes would point to an exacerbation of a chronic process such as iga nephropathy . Although a history of a recent documented streptococcal infection would be consistent with poststreptococcal agn, such a history is frequently unavailable . These might include shortness of breath or exercise intolerance from fluid overload or headaches, visual disturbances, or alteration in mental status from hypertension . Since agn may be the presenting complaint of a multisystem illness, a complete review of systems is vital . Particular attention should be paid to rash, joint discomfort, recent weight change, fatigue, appetite changes, respiratory complaints, and recent medication exposure . The family history should address the presence of any family members with autoimmune disorders, as children with both sle and membranoproliferative glomerulonephritis (mpgn) may have such relatives . A family history of renal failure (specifically asking about dialysis and kidney transplantation) may be the first clue to a process such as alport syndrome, which may initially present with an agn picture . The physical examination begins with a careful assessment of vital signs, particularly blood pressure . Blood pressures 5 mm above the 99th percentile for the child's age, sex, and height, especially if accompanied by any alteration in mental status, demand prompt attention . Tachycardia and tachypnea point toward symptomatic fluid overload . Careful examination of the nose and throat may provide evidence of bleeding, suggesting the possibility of one of the anca - positive vasculitides such as wegner's granulomatosis . The cardiopulmonary examination will provide evidence of fluid overload or the pulmonary involvement characterizing the rare kidney - lung syndromes . Scrotal edema may occur in nephrotic syndrome as well, and orchitis is an occasional finding in hsp . The rash of hsp, while characteristic when florid, may initially be subtle and limited to the buttocks or the dorsa of the feet . Some peripheral edema from salt and water retention is seen in agn, but this tends to be a more subtle brawny edema than the pitting edema characteristic of nephrotic syndrome . Small joint (e.g., fingers) is more typical of sle, while or knee involvement is seen with hsp . Obviously, a good urinalysis is the first order of business in assessing a child with suspected agn . The presence of red blood cell casts, while not invariably seen, is diagnostic of glomerulonephritis if present . Agn is an inflammatory process, so it is not at all unusual to see white blood cells in nephritic urine . Proteinuria is also nearly invariant in agn although any cause of gross hematuria can lead to some urinary protein . If the urine is not grossly bloody, however, the combined presence of hematuria and proteinuria virtually always means glomerulonephritis . The initial blood work required in suspected agn is actually limited; more sophisticated immunologic investigations, for example, are really second tier studies after the initial results are known . Obviously, assessing renal function and electrolytes is an important first step, as is obtaining a hemogram . A mild degree of anemia is frequently seen with agn and likely is dilutional; more significant anemia would be evidence that the process may be more chronic . There are typically no important changes in the white blood cell count or platelet count in most causes of agn . A normal platelet count in the presence of petechiae and purpura is the usual finding in hsp . Beyond these basic tests, a serum albumin is usually included; a slight degree of hypoalbuminemia is typical of many inflammatory processes such as hsp, but values <2.0 gm / dl are quite unusual in straightforward agn and point to a process with a nephrotic syndrome component . By far, the most important (and frequently forgotten) test to obtain initially is an assessment of the complement system . This generally means obtaining a serum c3 and c4; the total hemolytic complement (ch50) is generally of only historical interest . Poststreptococcal agn is characterized by a very low c3, sometimes with minimal decreases in c4 . The hypocomplementemia of poststreptococcal agn is evanescent, typically normalizing in six to eight weeks . On the other hand thus, if a child with a few weeks' of abnormal urine has not had a c3 measurement earlier, it may be impossible to make a diagnosis of poststreptococcal agn with certainty without a kidney biopsy . All of these tests should be easily obtained in the primary care setting and will usually identify the child for whom referral is going to be necessary . The child with severe hypertension (more than 5 mm above the 99th percentile), especially if accompanied by any neurologic complaints, must be referred immediately . Similarly, children with significant renal insufficiency should be assessed by a specialist . When agn is accompanied by a nephrotic syndrome, the additional diagnostic and therapeutic interventions are also beyond the typical primary care practice . Beyond these situations, however, many such children can be reasonably managed in the primary care setting . The child with agn in the setting of hsp, for example, who is normotensive, has normal renal function, and who is not nephrotic requires little more than careful serial observation . Although the urinary abnormalities may persist for some time after the rest of the disease has resolved, these children have little if any risk of permanent kidney injury . Many children with poststreptococcal agn may also be followed in the primary care setting, but this will entail a commitment to serial examination . The major threat to such children is hypertension and its complications, and this may evolve over a few days . In otherwise typical poststreptococcal agn with minimal hypertension (e.g., blood pressure between the 95th and 99th percentiles) and no renal failure, the urinary abnormalities in poststreptococcal agn may persist for a long time, even a year . The best indicator of resolution of the disease is the return of the c3 level to normal . Persistent decrease in c3 by this time merits referral, as this could be an indicator that the agn
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Elisa kits for the determination of insulin were purchased from alpco diagnostics, and leptin kits were purchased from millipore . [p]-atp was purchased from perkin - elmer, sn-1,2-dioleylglycerol was from avanti, and most other reagents and chemicals were purchased from sigma - aldrich . The generation of the hypoxia - induced rat model of iugr has been described in detail previously (2325). At birth, litters were reduced to eight male pups and litters that contained fewer than eight viable male pups were reduced to eight offspring with as many males as possible . At 3 weeks of age, female offspring were killed and male pups were weaned and housed two per cage . Immediately after weaning, all offspring exposed to different prenatal interventions (control, n = 36 from seven litters; and iugr, n = 36 from eight litters) started receiving an hf diet (45% fat; research diets d12451). In addition, half of the animals from each litter were randomly allocated to receive additional supplementation with resv in the diet (4 g / kg of diet). Therefore, four experimental groups were created: control offspring receiving hf diet (control hf - c; n = 18 from six litters), control offspring receiving hf diet plus resv (control hf - r; n = 18 from six litters), iugr offspring receiving hf diet (iugr hf - c; n = 18 from six litters), and iugr offspring receiving hf diet plus resv (iugr hf - r; n = 18 from six litters). The bioavailability of resv administered in the diet is low (0.1%), mainly because of intestinal breakdown, absorption, and liver metabolism (14). Therefore, the final plasma concentration of this molecule achieved with the dose administered in this study was within expected therapeutic values (1020 mol / l range) (26). To determine which measurements were performed on each animal, rats born from the same dam within each experimental group were randomly assigned to three possible subgroups (designated a, b, or c); see supplementary fig . 1 for details . Because all rats included in each subgroup belonged to a different litter, we used the offspring as the unit of analysis . After 9 weeks of nutritional intervention, body composition was determined using a whole body composition analyzer based on time - domain nuclear magnetic resonance technology (echomri 4-in-1/1000; echo medical systems). For the determination of abdominal fat in vivo, rats were anesthetized using inhaled isoflurane (2% in compressed air) and placed into a micro single - photon emission computed tomography (spect) scanner flex pre - clinical platform xo - xpet - xspet instrument (-medica ideas). The -camera was programmed to scan 512 projections (sum of frames = 4, voltage 60 kdp and 390 ua). The observation window was set between the diaphragmatic membrane and the acetabulum for each animal (91.78 mm), and the magnification factor on the camera was set to 1.29 . The intra - abdominal fat volume was calculated using the software gmi - amira 3.1.1 . Threshold density was adjusted by internal volume so that internal organs and large vessels were not counted as intra - abdominal fat tissue and were excluded from calculations . For additional measurements of intra - abdominal fat content, mechanical extraction and weight of different abdominal fat depots (retroperitoneal, perirenal, mesenteric, epiploic, and subdiaphragmatic) was performed after dissection . The pearson correlation between methods used to determine the intra - abdominal fat content (mechanical extraction and micro ct scan) was r = 0.65, p = 0.002 . The bland and altman analysis demonstrated a bias between the techniques of 0.14 14.7 g, which demonstrates that both techniques were highly correlated and that the average variability in the determinations made with these techniques was less than 1 g (<2% of the average value). Intra - abdominal organs including liver, spleen, pancreas, and kidneys were dissected and weighed before being frozen . In a subset of rats and after 2 h of fasting, rats were anesthetized using inhaled isoflurane, followed by cervical dislocation, collection of a sample of blood by cardiac puncture, and for the analysis of ampk activity, tissues were dissected and immediately frozen in liquid nitrogen, as described (27). In a separate set of animals, rats were fasted 2 h before intraperitoneal injection with insulin (1 mu / kg) and killed as above 15 min after injection . The length of the right tibia bone was measured in all animals . For immunoblotting, homogenates were prepared in ice - cold sucrose homogenization buffer as described (27). Intra - abdominal fat was extracted and weighed, and samples of omental adipose tissue were fixed in 10% formalin for 48 h and then in 10% methanol . Histological sections and hematoxylin / eosin staining were performed at the alberta diabetes institute histology core (edmonton, ab, canada) following standardized protocols . Digital images of three representative fields were taken using a digital camera mounted on a light microscope at 40 magnification . All images were analyzed with imagej software (ver 1.43u; national institutes of health). Lipids were extracted from 200 l of plasma, and tg, cholesterol ester, and free fatty acids were separated by fast - protein liquid chromatography as described (28). Liver and skeletal muscle tissues were homogenized, and tg, cholesterol ester, and ceramide content was determined by fast - protein liquid chromatography (27). Diacylglycerol content was determined by a diacylglycerol kinase assay, according to a well - established procedure (29). Indirect calorimetry was performed using the comprehensive laboratory animal monitoring system (oxymax / clams; columbus instruments). After an initial 24-h acclimatization period, rats were monitored every 13 min for 24 h to complete a 12-h dark (active)/12-h light (inactive) cycle . The respiratory exchange ratio (rer), vo2, vco2, heat production, and physical activity were measured . After a 5-h fast, rats were injected intraperitoneally with a 50% glucose solution (2 g / kg) for the glucose tolerance test (gtt). Blood glucose concentrations were determined using an accu - chek advantage glucometer (roche diagnostics) using blood from the tail at baseline and after glucose injection (15, 30, 60, 90, and 120 min). For the insulin tolerance test (itt), rats were injected intraperitoneally with insulin (1 mu / kg) after a 2-h fast and the blood glucose concentration was measured from the tail at baseline and after insulin injection (15, 30, 60, 90, and 120 min). Differences in measurements performed among four groups were analyzed using two - way anova and a bonferroni post hoc test with both iugr and administration of resv as sources of variation . Measurements of body weight and food consumption over time, as well as gtt and itt, were analyzed using a two - way anova with both time and group as sources of variation . When interaction between sources of variation included in the two - way anova was detected, interpretation of the overall anova was dictated by the significance observed in the post hoc analyses . Elisa kits for the determination of insulin were purchased from alpco diagnostics, and leptin kits were purchased from millipore . [p]-atp was purchased from perkin - elmer, sn-1,2-dioleylglycerol was from avanti, and most other reagents and chemicals were purchased from sigma - aldrich . The generation of the hypoxia - induced rat model of iugr has been described in detail previously (2325). At birth, litters were reduced to eight male pups and litters that contained fewer than eight viable male pups were reduced to eight offspring with as many males as possible . At 3 weeks of age, female offspring were killed and male pups were weaned and housed two per cage . Immediately after weaning, all offspring exposed to different prenatal interventions (control, n = 36 from seven litters; and iugr, n = 36 from eight litters) started receiving an hf diet (45% fat; research diets d12451). In addition, half of the animals from each litter were randomly allocated to receive additional supplementation with resv in the diet (4 g / kg of diet). Therefore, four experimental groups were created: control offspring receiving hf diet (control hf - c; n = 18 from six litters), control offspring receiving hf diet plus resv (control hf - r; n = 18 from six litters), iugr offspring receiving hf diet (iugr hf - c; n = 18 from six litters), and iugr offspring receiving hf diet plus resv (iugr hf - r; n = 18 from six litters). The bioavailability of resv administered in the diet is low (0.1%), mainly because of intestinal breakdown, absorption, and liver metabolism (14). Therefore, the final plasma concentration of this molecule achieved with the dose administered in this study was within expected therapeutic values (1020 mol / l range) (26). To determine which measurements were performed on each animal, rats born from the same dam within each experimental group were randomly assigned to three possible subgroups (designated a, b, or c); see supplementary fig . 1 for details . Because all rats included in each subgroup belonged to a different litter, we used the offspring as the unit of analysis . Body weight and food intake was measured weekly from birth . After 9 weeks of nutritional intervention, body composition was determined using a whole body composition analyzer based on time - domain nuclear magnetic resonance technology (echomri 4-in-1/1000; echo medical systems). For the determination of abdominal fat in vivo, rats were anesthetized using inhaled isoflurane (2% in compressed air) and placed into a micro single - photon emission computed tomography (spect) scanner flex pre - clinical platform xo - xpet - xspet instrument (-medica ideas). The -camera was programmed to scan 512 projections (sum of frames = 4, voltage 60 kdp and 390 ua). The observation window was set between the diaphragmatic membrane and the acetabulum for each animal (91.78 mm), and the magnification factor on the camera was set to 1.29 . The intra - abdominal fat volume was calculated using the software gmi - amira 3.1.1 . Threshold density was adjusted by internal volume so that internal organs and large vessels were not counted as intra - abdominal fat tissue and were excluded from calculations . For additional measurements of intra - abdominal fat content, mechanical extraction and weight of different abdominal fat depots (retroperitoneal, perirenal, mesenteric, epiploic, and subdiaphragmatic) was performed after dissection . The pearson correlation between methods used to determine the intra - abdominal fat content (mechanical extraction and micro ct scan) was r = 0.65, p = 0.002 . The bland and altman analysis demonstrated a bias between the techniques of 0.14 14.7 g, which demonstrates that both techniques were highly correlated and that the average variability in the determinations made with these techniques was less than 1 g (<2% of the average value). Intra - abdominal organs including liver, spleen, pancreas, and kidneys were dissected and weighed before being frozen . In a subset of rats and after 2 h of fasting, rats were anesthetized using inhaled isoflurane, followed by cervical dislocation, collection of a sample of blood by cardiac puncture, and for the analysis of ampk activity, tissues were dissected and immediately frozen in liquid nitrogen, as described (27). In a separate set of animals, rats were fasted 2 h before intraperitoneal injection with insulin (1 mu / kg) and killed as above 15 min after injection . The length of the right tibia bone was measured in all animals . For immunoblotting, homogenates were prepared in ice - cold sucrose homogenization buffer as described (27). Intra - abdominal fat was extracted and weighed, and samples of omental adipose tissue were fixed in 10% formalin for 48 h and then in 10% methanol . Histological sections and hematoxylin / eosin staining were performed at the alberta diabetes institute histology core (edmonton, ab, canada) following standardized protocols . Digital images of three representative fields were taken using a digital camera mounted on a light microscope at 40 magnification . All images were analyzed with imagej software (ver 1.43u; national institutes of health). Lipids were extracted from 200 l of plasma, and tg, cholesterol ester, and free fatty acids were separated by fast - protein liquid chromatography as described (28). Liver and skeletal muscle tissues were homogenized, and tg, cholesterol ester, and ceramide content was determined by fast - protein liquid chromatography (27). Diacylglycerol content was determined by a diacylglycerol kinase assay, according to a well - established procedure (29). Indirect calorimetry was performed using the comprehensive laboratory animal monitoring system (oxymax / clams; columbus instruments). After an initial 24-h acclimatization period, rats were monitored every 13 min for 24 h to complete a 12-h dark (active)/12-h light (inactive) cycle . The respiratory exchange ratio (rer), vo2, vco2, heat production, and physical activity were measured . After a 5-h fast, rats were injected intraperitoneally with a 50% glucose solution (2 g / kg) for the glucose tolerance test (gtt). Blood glucose concentrations were determined using an accu - chek advantage glucometer (roche diagnostics) using blood from the tail at baseline and after glucose injection (15, 30, 60, 90, and 120 min). For the insulin tolerance test (itt), rats were injected intraperitoneally with insulin (1 mu / kg) after a 2-h fast and the blood glucose concentration was measured from the tail at baseline and after insulin injection (15, 30, 60, 90, and 120 min). Differences in measurements performed among four groups were analyzed using two - way anova and a bonferroni post hoc test with both iugr and administration of resv as sources of variation . Measurements of body weight and food consumption over time, as well as gtt and itt, were analyzed using a two - way anova with both time and group as sources of variation . When interaction between sources of variation included in the two - way anova was detected, interpretation of the overall anova was dictated by the significance observed in the post hoc analyses . Consistent with our previous report (9), exposure of rats to prenatal hypoxia had no effect on body weight gain (fig . Food intake was decreased in iugr offspring compared with control rats independently of the administration of resv, despite similar body weights (fig . These data suggest that additional mechanisms must be involved in maintaining body weight in iugr offspring to compensate for the decrease in food consumption . Consistent with this finding, physical activity was significantly reduced in iugr offspring compared with control offspring (fig . Although the mechanisms involved in maintaining body weight in control rats and iugr rats may differ, neither iugr nor resv administration affected whole body lean and fat tissue composition (fig . After exposure to a normoxic (21% o2) or a hypoxic (11.5% o2) prenatal environment that caused iugr, we examined the effect of postnatal feeding an hf (hf - c) diet or an hf diet supplemented with resv (hf - r) to rat offspring for 9 weeks d: total physical activity in 24 h. e: total body composition estimated by spect . Resv is 4 g / kg of diet . * p <0.05 for the respective sources of variation (iugr and resv administration) using two - way anova; p <0.05 vs. controls after a bonferroni post hoc test comparing iugr and control offspring receiving the same diet (n = 6 per group). As expected, rats fed an hf diet exhibited a low rer as a result of increased availability of fat as an energy source (table 1). Changes in the rer were not observed in control offspring compared with offspring born iugr . However, iugr rats exhibited a reduction in vo2, vco2, and heat production during both light and dark cycles relative to controls regardless of whether they were receiving resv in their diets (table 1). Gas exchange ratio and heat production in control and iugr rats fed hf diet with or without resv experiments were performed after 9 weeks of nutritional intervention (int). * values of p <0.05 for the respective sources of variation (iugr and resv) using two - way anova (n = 6 per group). Despite similar body weight and total body composition, offspring born iugr and exposed to an hf diet exhibited increased total and relative (adjusted by total body fat) intra - abdominal fat distribution compared with control rats, whereas resv reduced the abdominal fat content as determined by both abdominal tomographic imaging and surgical dissection of fat depots (fig . The effect of iugr and administration of resv on fat distribution among the different intra - abdominal fat depots is reported in supplementary table 1 . Rats receiving resv displayed a reduction in the absolute abdominal fat content regardless of whether the rats were born iugr (fig . Although the administration of resv to control offspring did not affect the content of abdominal fat as a percentage of body weight, resv reduced this parameter in iugr offspring to levels comparable with those observed in controls (fig . 2d), suggesting that resv causes a redistribution of fat to depots other than the abdomen in iugr rats . Consistent with our previous results (9), iugr offspring fed an hf diet had larger adipocyte diameters than control offspring receiving the same nutritional intervention (fig . 2f and g), whereas resv caused a comparable decrease in the relative adipocyte diameter in both iugr and control offspring (fig . 2f and g). In agreement with greater abdominal fat mass, circulating leptin levels were higher in hf - fed iugr rats compared with control hf - fed offspring (fig . More importantly, resv decreased the plasma levels of leptin in offspring born iugr but had little or no effect on controls (fig . Measurements were made after 9 weeks of an hf - c or an hf - r (resv is 4 g / kg of diet). A: representative axial views of the abdominal cavity obtained by x - ray computed tomography and (b) subsequent three - dimensional reconstructions of intra - abdominal fat deposits . Total (c) and relative (d) intra - abdominal fat adjusted by total body fat determined by spect is shown . F: representative pictures of omental fat tissue histological preparations (hematoxylin - eosin; scale bar, 50 m). G: average intra - abdominal adipocyte diameter . * p <0.05 for the respective sources of variation (iugr or resv) using two - way anova; p <0.05 vs. controls after a bonferroni post hoc test comparing iugr and control offspring receiving the same diet (n = 6 per group). (a high - quality digital representation of this figure is available in the online issue .) Interestingly, although administration of resv had no effect on control offspring, resv reduced circulating levels of tg and free fatty acids in offspring born iugr (table 2). In addition, liver and skeletal muscle tg, diacylglycerol, and ceramide levels were elevated in iugr offspring compared with control offspring, and these values were significantly lower in resv - treated rats (table 2), demonstrating a plasma lipid lowering effect of resv as well as the prevention of accelerated peripheral organ steatosis during the consumption of an hf diet . Circulating and tissue lipid concentrations of control and iugr rats fed hf diet with or without resv measurements were made after 9 weeks of hf diet with or without resv 4 g / kg of diet . Ffa, free fatty acids . * p <0.05 for the respective source of variation such as prenatal hypoxia (iugr), resv, or int using two - way anova; p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). Because the major effect of high levels of circulating lipids and their accumulation in tissues is the development of insulin resistance and impaired glucose handling, we used gtt and itt to investigate whole body glucose homeostasis . Although iugr and resv had no effect on fasting blood glucose levels after 9 weeks of hf diet (fig . 3a), resv prevented the development of hyperinsulinemia and elevated the homeostasis model assessment index in iugr offspring (fig . 3 g and i) compared with control offspring, and resv improved both glucose disposal (fig . Together, these results indicate that resv administration prevents iugr rats from developing hf - induced insulin resistance . Measurements were made after 9 weeks of hf - c or hf - r (resv is 4 g / kg of diet) on: fasting blood glucose levels (a), fasting plasma levels of insulin (b), and homeostasis model assessment (homa) index (c). Hf - c rats gtt (d), hf - r fed rats gtt (e), and gtt summary information (f) are presented as area under the curve (auc). Hf - c rats itt (g), hf - r fed rats itt (h), and itt summary information (i) are presented as auc . Ip, intraperitoneal . * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs) or a repeated - measures anova (gtt and itt); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). The insulin signaling cascade regulates both hepatic glucose output as well as glucose transport into skeletal muscle; therefore, we determined the phosphorylation status of protein kinases in the insulin signaling cascade in the tissues of rats isolated 15 min after insulin injection . Feeding an hf diet to iugr rats impaired insulin - stimulated akt phosphorylation at ser-473 (activating site) in both liver and gastrocnemius muscle (fig . 4a). In agreement with the interpretation of data from gtt and itt, the addition of resv to the hf diet of iugr rats prevented a decrease in insulin - stimulated akt phosphorylation in the liver and skeletal muscle . Interestingly, insulin - stimulated akt phosphorylation was higher in the muscles of both control and iugr offspring fed an hf diet supplemented with resv compared with those rats receiving an hf diet alone, demonstrating that the muscle insulin sensitizing effects of resv were not confined to only iugr offspring . To further characterize the source of the signaling defect, we measured the phosphorylation of upstream regulators of akt . Phosphorylation of insulin receptor substrate (irs)-1 at ser-1101 (inhibitory site) was higher in liver and skeletal muscle of iugr offspring compared with controls (fig . Moreover, the inhibitory phosphorylation of irs-1 was reversed by resv treatment in both organs (fig . 4b). Because irs-1 is directly phosphorylated by protein kinase c (pkc) at ser-1101 and pkc is activated in tissues where lipids, especially diacylglycerols, accumulate, we measured the phosphorylation of pkc at thr-538 (activating site). During hf - diet feeding, liver and skeletal muscle pkc activity was increased in iugr rats compared with controls (fig . 4c). More importantly, and consistent with the reduction of lipid accumulation in the tissues (table 1) and prevention of glucose intolerance and insulin resistance (fig . 3), pkc phosphorylation was reduced by resv in the iugr offspring (fig . 4c). Together, these data indicate that resv prevented molecular signaling defects observed in iugr rats fed an hf diet, and these improvements contributed to the prevention of hf - induced insulin resistance in iugr rats . Measurements were made in the liver and gastrocnemius tissues of rats after 9 weeks of hf - c or hf - r . Phosphorylation of akt (p - akt to akt ratio; a), phosphorylation of the irs-1 (p - irs to irs ratio; b), and phosphorylation of pkc (p - pkc/tubulin; c) are shown . * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). To further characterize the mechanisms for the metabolic benefits of supplementing the diets of iugr rats with resv, we investigated ampk signaling . 5a), resv clearly activated ampk as determined by its phosphorylation at its activating site (thr-172). Consistent with the increased ampk activity, resv increased acc phosphorylation at ser-79 (inhibitory site) in rat liver and skeletal muscle (fig . 5b). Given the ability of activated hepatic ampk to decrease fatty acid and tg synthesis (19,20) and increased ampk in skeletal muscle to promote glucose disposal (22), these data indicate that resv - mediated activation of ampk contributes to the prevention of hf - induced insulin resistance in iugr rats . Measurements were made in the liver and gastrocnemius muscle tissues of rats after 9 weeks of hf - c or hf - r . A: phosphorylation of ampk (p - ampk to ampk ratio). B: phosphorylation of acc (p - acc to acc ratio). * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). Consistent with our previous report (9), exposure of rats to prenatal hypoxia had no effect on body weight gain (fig . Food intake was decreased in iugr offspring compared with control rats independently of the administration of resv, despite similar body weights (fig . These data suggest that additional mechanisms must be involved in maintaining body weight in iugr offspring to compensate for the decrease in food consumption . Consistent with this finding, physical activity was significantly reduced in iugr offspring compared with control offspring (fig . Although the mechanisms involved in maintaining body weight in control rats and iugr rats may differ, neither iugr nor resv administration affected whole body lean and fat tissue composition (fig . After exposure to a normoxic (21% o2) or a hypoxic (11.5% o2) prenatal environment that caused iugr, we examined the effect of postnatal feeding an hf (hf - c) diet or an hf diet supplemented with resv (hf - r) to rat offspring for 9 weeks d: total physical activity in 24 h. e: total body composition estimated by spect . Resv is 4 g / kg of diet . * p <0.05 for the respective sources of variation (iugr and resv administration) using two - way anova; p <0.05 vs. controls after a bonferroni post hoc test comparing iugr and control offspring receiving the same diet (n = 6 per group). As expected, rats fed an hf diet exhibited a low rer as a result of increased availability of fat as an energy source (table 1). Changes in the rer were not observed in control offspring compared with offspring born iugr . However, iugr rats exhibited a reduction in vo2, vco2, and heat production during both light and dark cycles relative to controls regardless of whether they were receiving resv in their diets (table 1). Gas exchange ratio and heat production in control and iugr rats fed hf diet with or without resv experiments were performed after 9 weeks of nutritional intervention (int). * values of p <0.05 for the respective sources of variation (iugr and resv) using two - way anova (n = 6 per group). Despite similar body weight and total body composition, offspring born iugr and exposed to an hf diet exhibited increased total and relative (adjusted by total body fat) intra - abdominal fat distribution compared with control rats, whereas resv reduced the abdominal fat content as determined by both abdominal tomographic imaging and surgical dissection of fat depots (fig . The effect of iugr and administration of resv on fat distribution among the different intra - abdominal fat depots is reported in supplementary table 1 . Rats receiving resv displayed a reduction in the absolute abdominal fat content regardless of whether the rats were born iugr (fig . Although the administration of resv to control offspring did not affect the content of abdominal fat as a percentage of body weight, resv reduced this parameter in iugr offspring to levels comparable with those observed in controls (fig . 2d), suggesting that resv causes a redistribution of fat to depots other than the abdomen in iugr rats . Consistent with our previous results (9), iugr offspring fed an hf diet had larger adipocyte diameters than control offspring receiving the same nutritional intervention (fig . 2f and g), whereas resv caused a comparable decrease in the relative adipocyte diameter in both iugr and control offspring (fig . 2f and g). In agreement with greater abdominal fat mass, circulating leptin levels were higher in hf - fed iugr rats compared with control hf - fed offspring (fig . 2e). More importantly, resv decreased the plasma levels of leptin in offspring born iugr but had little or no effect on controls (fig . Measurements were made after 9 weeks of an hf - c or an hf - r (resv is 4 g / kg of diet). A: representative axial views of the abdominal cavity obtained by x - ray computed tomography and (b) subsequent three - dimensional reconstructions of intra - abdominal fat deposits . Total (c) and relative (d) intra - abdominal fat adjusted by total body fat determined by spect is shown . F: representative pictures of omental fat tissue histological preparations (hematoxylin - eosin; scale bar, 50 m). G: average intra - abdominal adipocyte diameter . * p <0.05 for the respective sources of variation (iugr or resv) using two - way anova; p <0.05 vs. controls after a bonferroni post hoc test comparing iugr and control offspring receiving the same diet (n = 6 per group). (a high - quality digital representation of this figure is available in the online issue .) Interestingly, although administration of resv had no effect on control offspring, resv reduced circulating levels of tg and free fatty acids in offspring born iugr (table 2). In addition, liver and skeletal muscle tg, diacylglycerol, and ceramide levels were elevated in iugr offspring compared with control offspring, and these values were significantly lower in resv - treated rats (table 2), demonstrating a plasma lipid lowering effect of resv as well as the prevention of accelerated peripheral organ steatosis during the consumption of an hf diet . Circulating and tissue lipid concentrations of control and iugr rats fed hf diet with or without resv measurements were made after 9 weeks of hf diet with or without resv 4 g / kg of diet . * p <0.05 for the respective source of variation such as prenatal hypoxia (iugr), resv, or int using two - way anova; p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). Because the major effect of high levels of circulating lipids and their accumulation in tissues is the development of insulin resistance and impaired glucose handling, we used gtt and itt to investigate whole body glucose homeostasis . Although iugr and resv had no effect on fasting blood glucose levels after 9 weeks of hf diet (fig . 3a), resv prevented the development of hyperinsulinemia and elevated the homeostasis model assessment index in iugr offspring (fig . 3 g and i) compared with control offspring, and resv improved both glucose disposal (fig ., these results indicate that resv administration prevents iugr rats from developing hf - induced insulin resistance . Measurements were made after 9 weeks of hf - c or hf - r (resv is 4 g / kg of diet) on: fasting blood glucose levels (a), fasting plasma levels of insulin (b), and homeostasis model assessment (homa) index (c). Fed rats gtt (e), and gtt summary information (f) are presented as area under the curve (auc). Hf - c rats itt (g), hf - r fed rats itt (h), and itt summary information (i) are presented as auc . * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs) or a repeated - measures anova (gtt and itt); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). The insulin signaling cascade regulates both hepatic glucose output as well as glucose transport into skeletal muscle; therefore, we determined the phosphorylation status of protein kinases in the insulin signaling cascade in the tissues of rats isolated 15 min after insulin injection . Feeding an hf diet to iugr rats impaired insulin - stimulated akt phosphorylation at ser-473 (activating site) in both liver and gastrocnemius muscle (fig . 4a). In agreement with the interpretation of data from gtt and itt, the addition of resv to the hf diet of iugr rats prevented a decrease in insulin - stimulated akt phosphorylation in the liver and skeletal muscle . Interestingly, insulin - stimulated akt phosphorylation was higher in the muscles of both control and iugr offspring fed an hf diet supplemented with resv compared with those rats receiving an hf diet alone, demonstrating that the muscle insulin sensitizing effects of resv were not confined to only iugr offspring . To further characterize the source of the signaling defect, we measured the phosphorylation of upstream regulators of akt . Phosphorylation of insulin receptor substrate (irs)-1 at ser-1101 (inhibitory site) was higher in liver and skeletal muscle of iugr offspring compared with controls (fig . Moreover, the inhibitory phosphorylation of irs-1 was reversed by resv treatment in both organs (fig . Because irs-1 is directly phosphorylated by protein kinase c (pkc) at ser-1101 and pkc is activated in tissues where lipids, especially diacylglycerols, accumulate, we measured the phosphorylation of pkc at thr-538 (activating site). During hf - diet feeding, liver and skeletal muscle pkc activity was increased in iugr rats compared with controls (fig . 4c). More importantly, and consistent with the reduction of lipid accumulation in the tissues (table 1) and prevention of glucose intolerance and insulin resistance (fig . 3), pkc phosphorylation was reduced by resv in the iugr offspring (fig . 4c). Together, these data indicate that resv prevented molecular signaling defects observed in iugr rats fed an hf diet, and these improvements contributed to the prevention of hf - induced insulin resistance in iugr rats . Measurements were made in the liver and gastrocnemius tissues of rats after 9 weeks of hf - c or hf - r . Phosphorylation of akt (p - akt to akt ratio; a), phosphorylation of the irs-1 (p - irs to irs ratio; b), and phosphorylation of pkc (p - pkc/tubulin; c) are shown . * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). To further characterize the mechanisms for the metabolic benefits of supplementing the diets of iugr rats with resv, we investigated ampk signaling . 5a), resv clearly activated ampk as determined by its phosphorylation at its activating site (thr-172). Consistent with the increased ampk activity, resv increased acc phosphorylation at ser-79 (inhibitory site) in rat liver and skeletal muscle (fig . 5b). Given the ability of activated hepatic ampk to decrease fatty acid and tg synthesis (19,20) and increased ampk in skeletal muscle to promote glucose disposal (22), these data indicate that resv - mediated activation of ampk contributes to the prevention of hf - induced insulin resistance in iugr rats . Measurements were made in the liver and gastrocnemius muscle tissues of rats after 9 weeks of hf - c or hf - r . A: phosphorylation of ampk (p - ampk to ampk ratio). B: phosphorylation of acc (p - acc to acc ratio). * p <0.05 for the respective source of variation (iugr or resv) using two - way anova (bar graphs); p <0.05 vs. controls receiving the same diet after a bonferroni post hoc test (n = 6 per group). Our previous work established that iugr offspring subjected to an hf diet are more susceptible to developing several components of the mets than control offspring fed the same diet (9). Because resv has been demonstrated to be an insulin - sensitizing molecule (13,14,20,30), we tested the efficacy of resv in alleviating the development of hf - mediated pathologic metabolic phenotypes in offspring born from pregnancies complicated with iugr . Interestingly, even in the absence of increased body weight, iugr offspring exposed to an hf diet exhibited increased abdominal fat, elevated abdominal adipocyte size, hyperlipidemia, insulin resistance, and impaired glucose disposal . Importantly, these significant pathophysiological alterations were alleviated when the hf diet was supplemented with resv . Although resv has been shown to improve insulin resistance and glucose disposal in other rodent models (13,14,20,30), to our knowledge this is the first study demonstrating that early postnatal administration of resv in the diet of young rats improved the metabolic profile of hf - fed offspring born iugr secondary to a prenatal hypoxic insult . An interesting phenomenon observed with our iugr model is that although prenatal hypoxia had no effect on total body composition after an hf diet, iugr was associated with increased absolute and relative intra - abdominal fat content compared with control offspring . Although we have yet to determine the mechanism(s) responsible for this, we do show that resv decreases the amount of fat located in the abdominal cavity and reduces adipocyte diameter . This observation is important given the relatively short length of the intervention (only 9 weeks) compared with other studies showing the beneficial effects of resv on fat distribution when administered for up to 60 weeks (3133). Because we also show that administration of resv prevented the diet - induced increase in plasma lipids that is observed in iugr offspring, it is likely that reduced circulating levels of lipids contribute to the reduced lipid storage by adipocytes . However, we cannot explain why resv specifically reduces visceral fat without altering whole body fat content . With that said, previous work has shown that incubation of isolated adipocytes with resv reduced lipogenesis and increased lipolytic rates (34). Based on this, we speculate that resv preferentially targets abdominal adipocytes to increase lipolysis and reduce the mass of this fat depot . Because intra - abdominal fat is a major source of adipokines and cytokines associated with metabolic disorders (35,36), the reduced abdominal fat mass in iugr offspring fed an hf diet supplemented with resv corresponds with a lower level of leptin observed in these rats . These results are consistent with evidence showing that supplementation of hf diets with resv can prevent diet - induced obesity in rodents (13,14,20), in part, by diminishing visceral adipose tissue (14,37), although this is the first evidence that resv reduces adiposity in young rodents . As mentioned, resv reduced the hf diet - induced hyperlipidemia that is observed in iugr offspring . Resv inhibited fatty acid and tg synthesis by isolated hepatocytes (37,38), suggesting that the lipid - lowering effects of resv could be attributed to direct effects of this molecule on hepatic function . Consistent with this, we provide evidence that the lipid - lowering effects of resv may be attributed to activation of ampk and reduced activity of acc in the liver, thus resulting in the inhibition of fatty acid synthesis and decreased accumulation of tg in the liver . Because increased fatty acid utilization by skeletal muscle also contributes to lowering plasma lipid levels (39), we also show that activation of the ampk / acc axis occurs in the skeletal muscle as well . Because activation of the ampk / acc signaling axis in muscle increases fatty acid oxidation (18), this may also contribute to lowering plasma lipid levels in iugr offspring when hf diets are supplemented with resv . Therefore, the lipid - lowering effects of resv in iugr rats could be a result of ampk - mediated reduction of hepatic lipid synthesis (17) and increased oxidation of fatty acids by the skeletal muscle (18). Our finding that resv activates ampk in young rodents fed an hf diet is consistent with previous reports indicating that ampk activation is the primary mechanism by which resv mediates these beneficial effects (1517). Recent findings using hf - fed ampk-1 and -2deficient mice demonstrated that the effects of resv on insulin sensitivity required the presence of either ampk isoform (16). Moreover, activation of ampk in skeletal muscle increases glucose utilization in glucose - intolerant mice in an insulin - independent fashion (40). Although our data implicate ampk as being involved in the beneficial effects of resv, we also investigated whether additional molecular signaling mechanisms may contribute . For example, the pkc / irs / akt signaling pathway is centrally involved in insulin sensitivity in the liver and skeletal muscle (41) and subsequently regulates glucose uptake in muscle (42). In agreement with this, previous work suggested that impaired signaling through this pathway contributes to insulin resistance and decreased glucose disposal in association with the accumulation of lipotoxic lipid intermediates (43). Consistent with this, our data show that the pkc / irs / akt signaling pathway in the liver and skeletal muscle of iugr offspring fed an hf diet is significantly impaired, whereas resv restores this pathway and tissue lipids to levels observed in control offspring fed an hf diet . Thus we conclude that the ability of resv to prevent insulin resistance and glucose intolerance in iugr offspring exposed to an hf diet involves activation of ampk and the prevention of impaired akt signaling in liver and skeletal muscle . In conclusion, our data suggest that the consumption of hf diets by iugr offspring predisposes young rats for the development of glucose intolerance and peripheral insulin resistance without affecting insulin secretion . Most importantly, this is the first study demonstrating that postnatal administration of resv in the diet of young rats can improve the metabolic profile of hf - fed offspring born from pregnancies complicated by iugr . Specifically, we show that supplementation with resv is well tolerated and can reduce the susceptibility to hf diet induced metabolic alterations in fat distribution, adipocyte size, hyperlipidemia, glucose disposal, and insulin resistance . Moreover, we also provide molecular evidence that the improvement of glucose homeostasis by resv may be attributed to insulin sensitization of the peripheral tissues by preventing impaired akt signaling as well as by activation of ampk and potentially improving glucose utilization via an insulin - independent mechanism . Based on these data, we suggest that resv should be considered for further testing as a therapy for the pediatric population born iugr . Indeed, postnatal therapies for the metabolic defects associated with being born iugr are currently lacking, and resv represents the first potential pharmacological treatment that, if administered to vulnerable pediatric patient populations consuming high - calorie diets, could reduce the susceptibility for an adverse metabolic profile.
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Vulvar hematomas are usually seen in the obstetric population following repair of episiotomies and birthrelated soft tissue injury . However, traumatic nonobstetric vulva hematomas are rare . They may arise secondary to blunt trauma sustained during a fall from height 1, 2, 3, sexual assault, foreign body insertion, and coitus 4, 5, 6, 7 . Although most are small and pose little threat to the patient, nonobstetric hematomas may become large enough to cause hemodynamic instability . While conservative management is often the mainstay of treatment we detail the presentation and management of a young woman with a significant vulva hematoma following accidental blunt trauma . A healthy 16yearold female, with no known comorbidities, presented to the outpatient department with severe perineal pain . The mechanism of injury was a fall from height in which the patient sustained blunt trauma to the perineum . The patient reported immediate onset of severe pain associated with a progressive swelling of the vulva and the presence of blood . This was associated with the gradual onset of headache and symptoms of presyncope, including generalized weakness, nausea, and lightheadedness . The past medical history was noncontributory and the patient reported no known allergies or current medications . On physical examination the heart rate was 115 bpm with a manual blood pressure of 100/60 mmhg and an oral temperature of 37.1c . Examination of the perineum revealed a 10 8 cm swelling of the left vulva with right deviation of the right labia and clotted blood over the left labia minora (fig . Basic laboratory investigations revealed a hb of 10.2 g / dl, with a normal wbc and platelet count . Initial resuscitation consisted of 1500 ml iv bolus of crystalloid solution . Despite the infusion, the patient remained hypotensive and tachycardic . Given the degree of swelling and accompanying hemodynamic instability, the patient was consented for surgical intervention . Intraoperative, a large hematoma was evacuated through a medial incision of the left labia majora . A single, actively bleeding vessel was identified and suture ligated . The cavity was irrigated and inspected for hemostasis, which was subsequently closed with 10 chromic catgut (fig . The patient had an uneventful postoperative course with prompt removal of urinary catheter and discharge on postoperative day three . The patient was seen in followup at 4 weeks and the vulva appeared symmetric with the swelling completely resolved . Preoperative: swollen vulva with a clotted blood mass on left labia minora and lateral deviation of right labia majora . The vulva consist mostly of loose connective tissue and smooth muscle that is richly supplied by branches of the pudendal artery; a significant branch of the internal iliac artery 8 . The venous drainage consists of labial veins, which are tributaries of the internal pudendal vein and venae comitantes . The injury to labial branches of the internal pudendal artery, which is located in the superficial fascia of the anterior and posterior pelvic triangle, can cause significant vulvar hematomas 9 . Blunt trauma to the vulva may occur secondary to reflex adduction of the thighs immediately preceding trauma . Its anatomical position and rich vascular supply make it susceptible to hematoma formation during perineal injury . In the case of a fall, a patient falls astride an object compressing the soft tissue of the vulva on the underlying pelvic girdle, which leads to laceration and hematoma formation . It is imperative to elicit the nature of the trauma in order to provide the most appropriate pre and postoperative care . Social and cultural barriers to a thorough sexual history must be addressed to provide appropriate management . The true incidence of nonobstetric vulvar hematoma is unknown but is likely underreported in the available literature . There are no consensus statements or best practice guidelines for the necessity or timing of surgical intervention . 10 observed that in the absence of acute hematoma expansion, nonoperative conservative management can yield good results . However, benrubi et al . 11 found that conservative management of hematomas was associated with longer stays in hospital, an increased need for antibiotics and blood transfusion and greater subsequent operative intervention . . This may be particularly useful in the highrisk operative candidate with prohibitive comorbidities, as the procedure does not require a general anesthetic . It may also be advantageous in the situation where the degree of soft tissue trauma makes careful delineation of the anatomy difficult 2, 14, 15, 16 . However, this approach requires unique institutional resources that are often not available at the community or district level facility . Reported complications include groin hematoma, muscle pain, puncture site infection, guide wire perforation, and vagina fistula 16 . In the case presented, early surgical intervention is offered because of the enlarging hematoma and associated hemodynamic instability . For small, nonexpanding hematomas a conservative approach consisting of appropriate analgesia, cold compression, and close observation is appropriate . Surgical intervention is essential in the patient with a rapidly expanding hematoma or associated hemodynamic instability that does not immediately respond to crystalloid infusion . In cases of severe nonobstetric blunt trauma to the perineum, hematoma of the vulva can represent a potentially lifethreatening hemorrhage that requires urgent surgical intervention . While conservative management plays an important role in minor perineal injuries, surgical hematoma evacuation and hemostasis is often desirable to minimize hospital stay and diminish the impact of longterm complications . Written informed consent was obtained from the patient for publication of this case report and any accompanying images . A copy of the written consent is available for review by the editor of this journal on request.
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American ginseng (panax quinquefolium) is an important medicinal plant belonging to the araliaceae family . It is native to the hardwood - forested regions of the eastern part of north america, but it has been successfully cultivated in new zealand, china, australia, holland, france, and poland (koodziej et al . 2006). However, ginseng is a slow - growing plant, difficult to cultivate in the field, and prone to disease . Saponins, a type of ginsenoside, the pharmacologically active ingredient, can be obtained only after 47 yr of cultivation . Therefore, hairy root in vitro cultures of p. quinquefolium provide an attractive alternative for obtaining the biologically active compounds . The two major groups of ginsenosides are rb and rg groups, derived from the 20(s) protopanaxadiol and 20(s) protopanaxartiol structures, respectively . The main compounds are the ginsenosides rb1 (20(s) protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-o--d - glucopyranosyl(16)--d - glucopyranoside), rb2 (20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l-2)--d - glucopyranoside]-20-[o--l - arabinopyranosyl(l6)--d - glucopyranoside]), rc (20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-o--l - arabinofuranosyl (l6)--d - glucopyranoside), and rd (20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-(o--d - glucopyranoside) from the rb group, and re (re-20(s)-protopanaxatriol-6-[o--l - rhamnopyranosyl(l2)--d - glucopyranoside]-20-o--d - glucopyranoside), rg 1 (rg120(s)-protopanaxatriol-6,20-di - o--d - glucoside) from the rg group (figs . 1 and 2; table 1). Ginsenosides are responsible for most of the therapeutic action of ginseng (yuan et al . 2010).figure 1.chemical structure of protpanaxadiol and protopanaxartiol.figure 2.biosynthetic pathway of ginsenosides from squalene in p. ginseng . Triterpene undergoes oxidation, glycosylation and is finally converted into triterpene saponins (ginsenosides), according to kim et al (2009).table 1.sugar molecules in the structures of ginsenosides examined in this studymetaboliter1r220(s)-protopanaxadiolh h rb1glc triterpene undergoes oxidation, glycosylation and is finally converted into triterpene saponins (ginsenosides), according to kim et al (2009). Sugar molecules in the structures of ginsenosides examined in this study experimental research has identified a range of pharmaceutical activities possessed by many secondary plant metabolites including polysaccharides, flavonoids, coumarins, glycosides, phenolic acids, saponins, and also essential oils . Many have strong antibacterial, antifungal, antiviral, anti - inflammatory, antioxidant, and also anticancerogenic properties (sparg et al . 2004; chanwitheesuk et al . 2005; ahn et al . 2006; sienkiewicz et al ., enterococcus spp ., and gram - negative bacteria, mainly of the klebsiella spp . In addition, the broad and complex activity of plant metabolites, as well as their synergy of action, can make them a valued weapon against multidrug resistant bacterial strains (tan and vanitha 2004; mahesh et al . Currently, there is no literature concerning the antibacterial and antifungal activity of the extracts obtained from p. quinquefolium hairy root cultures . In this study, we investigated antimicrobial properties associated with ginseng hairy root extracts . P. quinquefolium l. hairy root cultures were established from seedlings (obtained from field cultivation in the agriculture university of lublin, lodz, poland) after infection with an agropine - type strain of agrobacterium rhizogenes atcc 15834 . After 68 wk, the roots emerged from the site of infection . When the roots were 1.52 cm long, they were individually excised and transferred into a hormone - free b-5 liquid medium with addition of 500 mg / l ampicillin . An aseptic culture of hairy roots was obtained, which grew rapidly on the same medium without antibiotic supplementation . Three clones, a, b, and g, were obtained and transformation was verified by pcr analysis (kochan et al . Hairy root cultures were grown in 300 ml shaken erlenmeyer flasks with 80 ml of hormone - free liquid b-5 medium (gamborg et al . The average inoculum size was about 300 mg fresh weight and 0.30 mg dry weight (dw). The cultures were maintained in the dark at 26c on a rotary shaker (100 rpm) and subcultured every 28 d. fresh roots, after drying on absorbent paper, were dried at room temperature and were processed for ginsenoside extraction and hplc analysis as previously described (kochan et al . Five standard strains of gram - positive bacteria were used: staphylococcus aureus atcc 433000, s. aureus atcc mr3, enterococcus faecalis van b atcc 51299, e. faecalis, vancomicin - sensitive atcc 29212, enterococcus faecium, and vancomicin - sensitive atcc 35667 . In addition, four gram - negative bacteria were used: escherichia coli atcc 35218, e. coli atcc 25922, pseudomonas aeruginosa atcc 27853, and the yeast strain candida albicans atcc 10231 . These standard bacterial and yeast strains used in the agar dilution method came from the collection of the medical and sanitary microbiology department, medical university of lodz, poland . Three antimicrobial agents were used: 5 g / ml ciprofloxacin (graso), a mixture of 20 g / ml amoxicillin 10 g / ml clavulanic acid (graso); and 25 g / ml fluconazole (graso). The bacteriological media were columbia agar (biomerieux), mueller hinton ii agar (emapol), and sabouroud dextrose agar (graso). The standard strains were cultivated in columbia agar medium, incubated at 37c for 48 h (bacteria) and in sabouroud dextrose agar at 28c for 48 h (yeast) in aerobic conditions . Bacterial and yeast suspensions were prepared with an optical density of 0.5 on the mcfarland scale using a bio merieux densitometer . Susceptibility testing was carried out using the disk diffusion method (jorgensen and turnidge 2007). Cultures were incubated at 37c for 1618 h (bacterial strains) and 28c for 24 h (yeast) in aerobic conditions . The results were interpreted according to clinical and laboratory standard institute guidelines (clsi 2009). For antimicrobial testing, a1, a2, b1, b2, g1, g2 independent weighted samples each of the extracts from the hairy root clones was weighed, diluted in ethanol a concentration of 97% w / v of extracts and used as a stock solution . An appropriate amount of this solution was mixed with columbia agar medium (bacteria) and sabouroud dextrose agar (yeast) to obtain concentrations from 0.8 to 1.4 mg / ml, and these were dispensed into petri dishes . An inoculum containing 1.510 cfu (0.1 ml) per spot was seeded either upon the surface of an agar plate with an extract from the hairy root clones (at various concentrations), or a plate with no extracts added (strains growth control). The minimal inhibitory concentration (mic) was determined after 24 h of incubation at 37c for bacteria and at 28c for yeast in aerobic conditions . The analyses of the antibacterial and antifungal activity of the extracts were performed independently three times . Control media containing only alcohol (at concentrations used in the dilutions of extracts) did not inhibit the growth of any of the bacterial or yeast strains . Three independently generated hairy root in vitro cultures were established from sterile seedlings of p. quinquefolium that had be transformed with a. rhizogenes . For the 28-d culture period, the highest increase of dry biomass (above eightfold) was recorded for line a of p. quinquefolium hairy root culture, with a slightly lower increase for line g (sevenfold), and the lowest for line b (fivefold). The obtained root cultures synthesized six identifiable saponins: rb1, rb2, rc, rd, (derivatives of protopanaxadiol), and rg1 and re (derivatives of protopanaxatriol). Ginsenoside production (expressed in mg / g dw) after 28 d of culture is shown in table 2 and fig . 3.table 2.ginsenoside content in three hairy root culture lines of p. quinquefoliumlines of hairy rootsginsenoside [mg / g dw]rb1rb2rcrdrg1retotala3.934 0.080.296 0.0190.9477 0.110.066 0.0211.216 0.143.662 0.3610.12 0.52b1.933 0.080.346 0.0380.6133 0.030.037 0.010.785 0.072.384 0.366.097 0.44g3.169 0.350.073 0.0150.7553 0.070.085 0.0360.992 0.073.006 0.098.079 0.34each value is a mean of six replicates sdfigure 3.ginsenoside contents of rb group and rg group in three lines of p. quinquefolium hairy roots . Ginsenoside content in three hairy root culture lines of p. quinquefolium each value is a mean of six replicates sd ginsenoside contents of rb group and rg group in three lines of p. quinquefolium hairy roots . Clone a of the hairy roots achieved the highest total ginsenoside level (10 mg / g dw) followed by clones g and b. the content of individual metabolites in clones a, b, and g differed . The quantitative level of rb1, rb2, rc, rd, re, and rg1 saponins shows the pattern rb1> re> rg1> rc> rb2> rd for clone a, with rb1 being the most abundant and rd the least . Clone g has similar pattern, but the content of ginsenoside rd was slightly higher than rb2 . Clone b followed the same sequence as clone a, but the main ginsenoside was the metabolite re (table 2). Metabolite rb was dominant among the protopanaxadiol derivatives, and metabolite re was dominant among the protopanaxatriol derivatives in all examined lines . The sum of the main components (rb1, re) was more than 70% of total ginsenosides . The mics of the hairy root extracts (a1, a2, b1, b2, g1, and g2) against the tested microorganisms and susceptibility to standard strains of bacteria and yeast are shown in table 3.table 3.mic (milligrams per milliliter) for extracts of the hairy root of p. quinquefoliumstandard strainmic (mg / ml) of hairy root clone extractcontrol of standard strains, susceptibility to antibiotics (mean dimeter zone of inhibiton (mm))ethanola1a2b1b2g1g2cipamcflus . Albicans atcc 102311.01.01.41.31.21.2ndnd28nicip ciprofloxacin (5 g) (r 15, 16 i 20, s 21), amc amoxicillin / clavulanic acid (20 g/10 g) (r 13, 14 i 17, s 18), flu fluconazole (25 g / ml) (r 14, 14 i 18, s> 18), nd not determined, ni not inhibited mic (milligrams per milliliter) for extracts of the hairy root of p. quinquefolium cip ciprofloxacin (5 g) (r 15, 16 i 20, s 21), amc amoxicillin / clavulanic acid (20 g/10 g) (r 13, 14 i 17, s 18), flu fluconazole (25 g / ml) (r 14, 14 i 18, s> 18), nd not determined, ni not inhibited the tested hairy root extracts were the most active against gram - negative standard strains: e. coli atcc 35218 and e. coli atcc 25922 . The mic values were between 0.8 and 1.3 mg / ml . The extracts from clones a were found to be very effective against the standard e. coli strains (mic 0.80.9 mg / ml). However, the mic values of the growth inhibition factors in the extracts from all the clones were higher against p. aeruginosa (1.01.4 mg / ml). The highest concentrations of extracts, between 1.1 and 1.3 mg / ml for a clones and 1.4 mg / ml for b and g hairy root clones, were effective against the vancomycin - resistant standard strain, e. faecalis van b atcc 51299 . However, the mic values for standard strain s. aureus atcc 4330 were between 0.9 and 1.2 mg / ml and for s. aureus atcc mr3 were between 0.8 and 1.4 mg / ml for the all clones . The mic of the hairy root extracts against c. albicans atcc 10231 was 1.0 mg / ml for the a clones, 1.2 mg / ml for the g clones and 1.31.4 overall, the strongest activity was observed by the a clones of the hairy root extracts . The total ginsenoside level in lines a, g, and b of the hairy roots was found to be about 10, 8, and 6 mg / g dw, respectively . A lower level of total ginsenoside (2.585.44 mg / g dw) was reported by mallol et al . (2001) in different phenotypes of hairy roots of p. ginseng . In the present study, ginsenosides rb1 and re were seen be the major component . Mathur et al . (2010) demonstrated that extracts of p. quinquefolium hairy roots also contained the highest level of metabolite rb1 having protopanaxadiol as sapogenin and re having protopanaxatriol as sapogenin after 4 wk of culture . The same metabolites, rb1 and re, dominated all 18 lines of p. ginseng hairy roots studied by woo et al . Hairy roots of hybrid p. ginseng and p. quinquefolium growing on b-5 medium also synthesized six of the ginsenosides examined in this study, but only metabolite rb1 significantly exceeded the level of the other saponins (washida et al . The lowest level of saponin rd, found in this study, was similar to that observed by washida et al (1998, 2004) and woo et al . (2004). In our tests, standard strains of s. aureus (n = 2), enterococcus spp . (n = 2), p. aeruginosa (n = 1), and c. albicans (n = 1) were sensitive to hairy root extracts at 0.81.4 mg / ml concentrations . Methanolic extracts from the leaves of cussonia sp . From the araliaceae family were active against p. aeruginosa (mic of 1.01.5 mg / ml) and s. aureus (mic of 1.8 mg / ml). In accordance with the literature, extracts from hairy root and untransformed roots of maytenus senegalensis showed an inhibitory effect against the growth of s. aureus at concentrations in the range of 0.651.25 mg / ml . However, the root extracts of both untransformed and transformed root cultures were active against gram - positive bacterial strains only (jain et al . Glycyrrhiza glabra root extracts were found to demonstrate significant antibacterial activity against two gram - positive (bacillus subtilis and s. aureus) and two gram - negative (e. coli and p. aeruginosa) bacterial strains (nitalikar et al . Studies on the antimicrobial properties of the essential oils obtained from the cultured hairy roots of salvia miltiorrhiza bunge containing four diterpenoid tanshinones and three phenolic acids revealed that the compounds have very strong activity against gram - positive and gram - negative bacteria, and one fungal species (zhao et al . Additionally, studies on the antimicrobial properties of korean red ginseng confirmed that ginsenosides possess antibacterial activities toward pathogenic gram - positive and gram - negative bacteria . In our tests, the highest antimicrobial activity was demonstrated by clone a hairy root culture, which was specifically correlated with the high content of ginsenosides . The literature reports confirm not only the antimicrobial properties of ginsenosides, but also their synergy of action with commercial antibiotics, such as kanamycin and cefotaxime, on antibacterial activity against methicillin - resistant s. aureus strains (sung and lee 2008). Three studied lines of hairy root cultures of p. quinquefolium a, b, g synthesize six types of known ginsenosides . The highest level of saponins is reported for line a.the tested extracts from hairy root clones inhibit the growth of standard bacteria and yeast strains . These have the potential to be used in combination with antibiotics in fighting infectious diseases . Three studied lines of hairy root cultures of p. quinquefolium a, b, g synthesize six types of known ginsenosides . The highest level of saponins is reported for line a. the tested extracts from hairy root clones inhibit the growth of standard bacteria and yeast strains . These have the potential to be used in combination with antibiotics in fighting infectious diseases.
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The use of the modern mri imaging techniques has changed the prospects for the treatment of cervical carcinoma and has become a valuable tool in the brachytherapy (bt) component [13]. The american brachytherapy society (abs) and the groupe europen de curiethrapie and the european society for radiotherapy and oncology (gec - estro) have recommended mri as the preferred image modality for image guided brachytherapy (igbt) [46]. In locally advanced cervical carcinoma with moderate extension to the parametrium, combined endocavitary and interstitial applicators (vienna or utrecht applicators, nucletron, an elekta company, elekta ab, however, these have coverage limitations in patients with more advanced disease or in those who respond poorly to external beam radiotherapy (ebrt). In such cases, interstitial templates such as the mupit (martinez universal perineal interstitial template, nucletron, an elekta company, elekta ab, stockholm, sweden) or the syed template (best medical international, inc . The current commercially available interstitial templates have inherent disadvantages: a) some do not allow addition of an intracavitary component, and b) they are incompatible with mri and therefore require use of ct dosimetry . In an attempt to combine the technical advantages of mupit and utrecht applicator and mri - based planning, we have developed a new intracavitary / interstitial applicator compatible with mri . In contrast to the existing multi - interstitial templates (mupit and syed), the benidorm template is completely compatible with mri, thus avoiding the artifacts and image distortion produced by stainless components and improving the mri image for treatment volume design and dosimetry . Use of this device facilitates adherence to the recommendations of abs / gec estro for igbt . The purpose of this work is to present the design of this new applicator and to describe clinical indications, the clinical procedure, and the advantages of mri dosimetry . The benidorm template (lorca marn s.a, murcia, spain) uses currently existing nucletron - elekta mri - compatible intrauterine tubes; these 15, 30, and 45 grade tubes are 4, 6, or 8 cm long and allow delivery of a large central dose . This device can also use 20 or 24 cm long titanium needles to cover the disease in all directions . The intracavitary component (ic) helps to straighten the uterus and to maintain the geometry of the needles . The benidorm template has 12 rows 1.1 cm apart (measured from center to center of each needle) to introduce straight and angled titanium needles . It has 7 rows, in which there are 35 straight holes for parallel needle placement and 5 rows, in which there are 16 angled holes (7) (figure 1a). This template allows coverage of the distal parametrium (up to 4.5 cm from the middle of the ic tube) and the entire vagina (figure 1b). It has also the possibility to include a vaginal cylinder 2.4 cm in diameter; these cylinders are available in lengths from 4 - 12 cm to accommodate different vaginal lengths, with 8 positions of needles in its surface (figure 1c and d). Plastic obturator tracks fasten the needles avoid displacement (figure 1e). A second plate of the exactly same design covers the first in order to achieve adequate docking and to prevent displacement of the components of the applicator (figure 1f). This second plate also can hold a central needle when the introduction of the intrauterine tube is not possible (figure 1 g and h). B) straight needles and angled needles (up to 4.5 cm from the middle of the ic tube). H) second plate with central needle this new template takes the well - known advantages of the mupit and complements them with an intrauterine tube, additional needle holes, and compatibility with mri for ctv - oar delineation . This novel template is specially indicated in locally advanced cervical carcinomas with bulky parametrial extension (medial or distal), bulky primary disease or disease with poor response to ebrt, extensive paravaginal involvement (tumor thickness greater than 0.5 cm) extending to middle and lower third of the vagina, or disease that has invaded the bladder or rectum (stage iva). In addition, this applicator can be used in patients who are not candidates for an intrauterine component because of unfavorable topography after ebrt and chemotherapy, narrow vaginas, primary tumors of the vagina, or post - hysterectomy recurrences or recurrences in a previously irradiated area . A pre - implant mri with the vaginal obturator is done one week before the bt procedure . T2 weighted mri images in the axial, coronal, and sagittal planes are taken (figure 2). Using these images, the number, position, and depths of the needles are defined, always taking into account that an offset of 1 cm from the tip is given to define the first dwell position . This means that the depth is considered appropriate if the ctv is covered in its entirety with up to a 1 cm superior margin . A foley tri - lumen catheter is placed in the bladder and inflated with 7 cc of saline solution . The obturator (vaginal cylinder) of the template assembly is placed in the vagina, so that it fits against the cervix and maintains that position . C) coronal plane different lengths of vaginal cylinders are available to match different anatomies . The plate is positioned against the perineum, and the template is fixed to the perineal skin by four or six stitches . With the obturator in the vagina and the template fixed to the perineum, titanium needles (in our case 20 cm long) are inserted through the holes of the template and through the perineum into the desired target; the obturator tracks exactly the depth previously defined with the pre - implant mri . If the lateral dimension must be increased, the oblique holes are used . After the implant, a second plate is placed covering the one fixed to the perineum to prevent longitudinal displacement of the needles . In the mri (general electric 1,5 t, milwaukee, wi, usa) we use a t2 sequence . To visualize the applicator and titanium needles, a t1 3d radio - frequency spoiled gradient recalled echo (spgr) sequence is also used (figure 3). Normal saline solution (50 cc) is injected into the bladder to help in volume definition . C) isodoses: 150% (magenta), 100% (yellow), 80% (green) due to the dose characteristics involved in using a single interstitial implant and a single prescription dose over the ctv, we delineate the oars (rectum and bladder) and the ctv . The ctv is defined taking into account bright regions of tumor seen in t2 sequences, initial image mri studies, clinical examination at the first consultation, and by examination under anesthesia at the time of the implant [13, 14]. Intrauterine tandem and needle reconstruction has been object of another work dedicated to the medical physics aspects of the benidorm template . Briefly, the reconstruction was determined by the relationship of the free length of the needles to mri markers specific to the benidorm template . For the intrauterine tandem dosimetry with this template is calculated in the same manner as with mupit - based ct planning . The advantage in using mri instead of ct is the more adequate ctv definition that typically results in a lower ctv volume than that derived using ct . The optimization was done first geometrically to minimize overdose volumes, and then the result was modified manually to maximize bladder and rectum sparing . Independent calculation verification was performed importing the points, dwell positions, and dwell times from the treatment planning system to a home - made spreadsheet . For these patients, the radiation oncology treatment strategy included an ebrt component that is delivered before bt to reduce tumor volume (46 - 50.4 gy to the pelvis with conventional fractionation). We tried to limit the overall treatment time to less than 8 weeks . The customary bt approach adopted by our department was used: six 4 gy fractions prescribed to ctv and given in four days, six hours apart . The bladder was filled with 50 cc of saline solution before each fraction to reproduce the planned treatment defined in the mri . Total doses were normalized to 2 gy per fraction equivalent doses (eqd2), assuming / of 10 for ctv and 3 for oar as recommended [5, 6]. This fraction size was chosen to decrease the risk of late toxicity that might result from the biologic effect of a larger fractional dose and the fact that two fractions were administered daily [18, 19]. We performed a single application implantation with multifractionated hdr irradiation . The planning aim was to deliver at least 75 - 85 gy eqd2(/=10) to de ctv d90 . Dose constraints applied to the oars were based on the embrace protocol for cervix, using a maximum dose to d2cc rectum of 70 - 75 gy eqd2(/=3) and 90 gy eqd2(/=3) to the d2cc of the bladder . Routine antibiotics, continuous intravenous analgesia, and anti - embolism prophylaxis were prescribed during the treatment period . The patients were provided with antibiotics and analgesics, and discharged the same day that the implant was removed . The benidorm template (lorca marn s.a, murcia, spain) uses currently existing nucletron - elekta mri - compatible intrauterine tubes; these 15, 30, and 45 grade tubes are 4, 6, or 8 cm long and allow delivery of a large central dose . This device can also use 20 or 24 cm long titanium needles to cover the disease in all directions . The intracavitary component (ic) helps to straighten the uterus and to maintain the geometry of the needles . The benidorm template has 12 rows 1.1 cm apart (measured from center to center of each needle) to introduce straight and angled titanium needles . It has 7 rows, in which there are 35 straight holes for parallel needle placement and 5 rows, in which there are 16 angled holes (7) (figure 1a). This template allows coverage of the distal parametrium (up to 4.5 cm from the middle of the ic tube) and the entire vagina (figure 1b). It has also the possibility to include a vaginal cylinder 2.4 cm in diameter; these cylinders are available in lengths from 4 - 12 cm to accommodate different vaginal lengths, with 8 positions of needles in its surface (figure 1c and d). Plastic obturator tracks fasten the needles avoid displacement (figure 1e). A second plate of the exactly same design covers the first in order to achieve adequate docking and to prevent displacement of the components of the applicator (figure 1f). This second plate also can hold a central needle when the introduction of the intrauterine tube is not possible (figure 1 g and h). B) straight needles and angled needles (up to 4.5 cm from the middle of the ic tube). H) second plate with central needle this new template takes the well - known advantages of the mupit and complements them with an intrauterine tube, additional needle holes, and compatibility with mri for ctv - oar delineation . This novel template is specially indicated in locally advanced cervical carcinomas with bulky parametrial extension (medial or distal), bulky primary disease or disease with poor response to ebrt, extensive paravaginal involvement (tumor thickness greater than 0.5 cm) extending to middle and lower third of the vagina, or disease that has invaded the bladder or rectum (stage iva). In addition, this applicator can be used in patients who are not candidates for an intrauterine component because of unfavorable topography after ebrt and chemotherapy, narrow vaginas, primary tumors of the vagina, or post - hysterectomy recurrences or recurrences in a previously irradiated area . A pre - implant mri with the vaginal obturator is done one week before the bt procedure . T2 weighted mri images in the axial, coronal, and sagittal planes are taken (figure 2). Using these images, the number, position, and depths of the needles are defined, always taking into account that an offset of 1 cm from the tip is given to define the first dwell position . This means that the depth is considered appropriate if the ctv is covered in its entirety with up to a 1 cm superior margin . A foley tri - lumen catheter is placed in the bladder and inflated with 7 cc of saline solution . The obturator (vaginal cylinder) of the template assembly is placed in the vagina, so that it fits against the cervix and maintains that position . C) coronal plane different lengths of vaginal cylinders are available to match different anatomies . The plate is positioned against the perineum, and the template is fixed to the perineal skin by four or six stitches . With the obturator in the vagina and the template fixed to the perineum, titanium needles (in our case 20 cm long) are inserted through the holes of the template and through the perineum into the desired target; the obturator tracks exactly the depth previously defined with the pre - implant mri . After the implant, a second plate is placed covering the one fixed to the perineum to prevent longitudinal displacement of the needles . In the mri (general electric 1,5 t, milwaukee, wi, usa) we use a t2 sequence . To visualize the applicator and titanium needles, a t1 3d radio - frequency spoiled gradient recalled echo (spgr) sequence normal saline solution (50 cc) is injected into the bladder to help in volume definition . C) isodoses: 150% (magenta), 100% (yellow), 80% (green) due to the dose characteristics involved in using a single interstitial implant and a single prescription dose over the ctv, we delineate the oars (rectum and bladder) and the ctv . The ctv is defined taking into account bright regions of tumor seen in t2 sequences, initial image mri studies, clinical examination at the first consultation, and by examination under anesthesia at the time of the implant [13, 14]. Intrauterine tandem and needle reconstruction has been object of another work dedicated to the medical physics aspects of the benidorm template . Briefly, the reconstruction was determined by the relationship of the free length of the needles to mri markers specific to the benidorm template . For the intrauterine tandem dosimetry with this template is calculated in the same manner as with mupit - based ct planning . The advantage in using mri instead of ct is the more adequate ctv definition that typically results in a lower ctv volume than that derived using ct . The optimization was done first geometrically to minimize overdose volumes, and then the result was modified manually to maximize bladder and rectum sparing . Independent calculation verification was performed importing the points, dwell positions, and dwell times from the treatment planning system to a home - made spreadsheet . For these patients, the radiation oncology treatment strategy included an ebrt component that is delivered before bt to reduce tumor volume (46 - 50.4 gy to the pelvis with conventional fractionation). We tried to limit the overall treatment time to less than 8 weeks . The customary bt approach adopted by our department was used: six 4 gy fractions prescribed to ctv and given in four days, six hours apart . The bladder was filled with 50 cc of saline solution before each fraction to reproduce the planned treatment defined in the mri . Total doses were normalized to 2 gy per fraction equivalent doses (eqd2), assuming / of 10 for ctv and 3 for oar as recommended [5, 6]. This fraction size was chosen to decrease the risk of late toxicity that might result from the biologic effect of a larger fractional dose and the fact that two fractions were administered daily [18, 19]. The planning aim was to deliver at least 75 - 85 gy eqd2(/=10) to de ctv d90 . Dose constraints applied to the oars were based on the embrace protocol for cervix, using a maximum dose to d2cc rectum of 70 - 75 gy eqd2(/=3) and 90 gy eqd2(/=3) to the d2cc of the bladder . Routine antibiotics, continuous intravenous analgesia, and anti - embolism prophylaxis were prescribed during the treatment period . The patients were provided with antibiotics and analgesics, and discharged the same day that the implant was removed . The objective of this manuscript is to describe the ctv coverage achieved with the ic / interstitial benidorm template applicator and to demonstrate the improvement in isodoses: 150% (magent), 100% (yellow), 90% (green) volume definition that was possible through the use of mri . In a future phase of this project, the clinical follow - up and tolerance results will be analyzed and compared with our previous results with ct - based dosimetry using the mupit applicator . Between april 2013 until december 2014, we used the benidorm template to place 15 implants in patients with locally advanced cervical carcinoma (8 stage iib, 3 stage iii, and 4 stage iva). Tumor characteristics, ctv d90, and bladder and rectum d2cc of these patients are listed in table 1 . Median d90 for ctv was 79.8 gy (71.5 - 89.9 gy eqd2(/=10)); median d2cc for bladder was 77.6 gy (69.8 - 90.8 gy eqd2(/=3)), and median d2cc for rectum was 71.9 gy (58.3 - 83.7 gy eqd2(/=3)). Eqd2 values includes both external beam radiotherapy and brachytherapy ebrt external beam radiation therapy, eqd2 equivalent dose at 2 gy, / alpha / beta ratio, d90 percent of the prescription dose covering 90% of the ctv, d2cc minimum dose to the most exposed 2 cm, mri magnetic resonance imaging a typical example of a dose plane distribution is shown on figure 3 . Despite medical advances, the treatment of cervix malignancies, mainly locally advanced tumors, remains one of the main clinical challenges for the radiation oncologist . External beam radiotherapy with concomitant chemotherapy and intracavitary bt represent the gold standard for these patients . In contrast with the old point - based techniques, the incorporation of ct - based tridimensional imaging for bt planning has been a very important and well - known advance . It allows volume definition providing dose - volume histograms to predict potential control and toxicity . Several studies have reported that mri can also be an extremely useful tool for diagnosis and prediction of response in cervical malignances [2225], and this modality has been recommended as superior to ct [1, 26]. The ability of mri to provide precise knowledge of disease extension has facilitated the tailoring of treatment to individual circumstances at different stages of the disease . Clearly, this characteristic of mri - based diagnosis aids in selection of the best radiation source placement for the bt procedure and the bt - applicator that will be able to cover the entire ctv [10, 2729]. In 2004, the image - guided brachytherapy working group of the abs published their guidelines for image - based bt for cervical carcinoma for north america . At almost the same time, several european specialist groups published reports with the gec - estro recommendations [5, 6], combining the work of both societies to facilitate inter - institutional standardization . The gec - estro recommendations defined different bt volumes (gtv, high risk ctv, and intermediate risk ctv), based on the disease and risk of recurrence because of microscopic tumor at the diagnosis and at the time of the bt procedure, i.e., clinical, and as described by mri . The gec - estro recommendations have introduced the term image - guided brachytherapy to indicate this stage of the treatment process . Application of these guidelines is intended to reduce toxicity and increase local control and survival; several different groups and institutions have reported on the efficacy of this approach [3234]. Despite these efforts, inadequate dose coverage remains the main reason for local failure . It is well known that higher doses in these tumors have increased local control and survival, producing control rates near 90 - 95% when new imaging techniques were used [2, 3537]. Brachytherapy continues to be the best treatment to increase dose in locally advanced cervical carcinoma [3840]. Mri findings have shown that there is a group of patients, in which the areas that should be treated are excluded from the classical treatments and prescriptions; these results have revealed the limitations of exclusive use of intracavitary applicators . To our knowledge, there is no established approach to treat these extended lesions using brachytherapy alone . An alternative is to combine brachytherapy with ebrt using imrt, and then control of image registration and dose combination are the critical points . Attempts to apply this kind of combined treatment have led to the development of new applicators . An interstitial component has been added to the basic ic applicators using metallic or plastic needles compatible with mri . The two manufactured devices that apply this solution are the classic ring (vienna applicator) and ovoids (utrecht applicator) [7, 8, 42]. When the extension to parametrium is moderate, these combined endocavitary and interstitial mri applicators extend the ctv coverage using a lateral line of needles . This type of coverage is inadequate in patients with more advanced disease, such as bulky parametrial extension, bulky primary disease, extensive paravaginal extension to middle or lower third of the vagina, or disease invading the bladder or rectum . In these situations there are manufactured templates for interstitial implants, such as mupit with angled openings or the syed template . However, the mupit lacks a central intracavitary component that can reach deep into the cervix and neither template is mri compatible . Plastic needles can be used but fibrosis tissue, tumors, or bones could divert these and prevent a uniform and homogenous implant . The few published series show the use of ultrasound [44, 45, mri [13, 46], or ct [47, 48] for needle insertion . All of these series have used plastic or stainless steel needles for ct planning (with or without fusion with mri imaging done without the applicator and the needles). It is well known that the unavoidable time difference between the mri and ct scans increases the overall uncertainty and can cause discomfort for patients . In our experience, these disadvantages can be resolved through the use of the benidorm template, which allows all planning procedures to be based solely on mri . The gec - estro recommends that, when using mr imaging for image - based adaptive cervical carcinoma brachytherapy, the contouring should be done in para - axial planes . In our current image protocol, we are using axial planes for contouring, however, there are some 3d protocols in progress to allow the application of these recommendations . One problem that we found in using this applicator with both the intracavitary and the interstitial component and mri planning was that following gec - estro volume definitions for prescription became more difficult . As mentioned earlier, different doses for the hr - ctv and the ir - ctv the most typical strategy in interstitial bt involves including both the gtv at diagnosis and the gtv at the time of the brachytherapy in a single ctv . In the literature, although the concept is basically the same, the specific descriptions vary greatly . Define a ctv that includes the clinical and radiological tumor at diagnostic and at the moment of the bt . Lee et al ., in reporting on a huge experience in interstitial implants and mri imaging, define the ctv as any regions thought by the physician to harbor microscopic disease spread based on the localization of the tumor or ctv-1 as the clinical evident disease by ct imaging and clinical examination, facilitated by clinical drawings, fiducial markers, and pre - brachytherapy mri when available and ctv-2 as any adjacent region with considerable risk of microscopic disease extension . Describe the ctv as the line joining all the peripheral needles visible in ct between the cranial and caudal extent of the disease . Also, some groups do not describe treatment volumes at all and employ ct only to define dose points in relation to catheter reconstruction . According to the literature and others institutions experience, we chose to base prescription on a single ctv without distinguishing between volumes and to consider the hr - ctv to be contained within it . The resulting dose distribution was different and tended to require a lower eqd2 than is described in embrace for cervix cases . However, it must be remembered that what appears to be a relatively low median eqd2 to the ctv does not take into account the effect of delivering 6 fractions two times a day in a short overall treatment time . Our median eqd2 dose to ctv reached 79.8 gy (71.5 - 89.9 gy); this is in the range described by others (eqd2 61.6 - 82 gy [13, 14, 18, 47, 50, 52, 54]. For oar evaluation, we followed the gec - estro recommendations on d2cc and the embrace protocol using dose constraints based on the eqd2 . From january 2006 to november 2013, 89 interstitial treatments using the mupit template were done at our department, 65 of these as a primary treatment for locally advanced cervical cancer . In 2006 we began to employ mri following the recommendations of gec - estro / abs in bt procedures for early stage cervical carcinoma; in 2009 we incorporated use of the utrecht applicator for more advanced disease . Drawing on our experience in perineal interstitial treatments, utrecht applicators and mri bt, we designed the benidorm template to combine the advantages of these approaches . To our knowledge, this is the first report of the use of a completely mri - compatible template with a mixed intracavitary and interstitial component that allows exclusive mri based planning . The design of this template addresses the disadvantages of currently commercially available templates: the inability of the intracavitary component to reach deep into the cervix (mupit), and the mri - incompatibility of these templates (mupit and syed), which necessitates use of ct imaging for the dosimetry . The use of this new mri - compatible template is practical and efficient, allowing improved contouring and ctv conformation and planning procedure based solely on mri.
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Maraviroc (mvc) is a first - in - class selective chemokine coreceptor type-5 (ccr5) antagonist indicated for the treatment of ccr5-tropic (r5) hiv-1 infection in both treatment - naive and treatment - experienced patients in the united states,1 and in treatment - experienced patients in the european union.2 mvc is primarily metabolized by hepatic cytochrome p450 (cyp) 3a enzymes, with negligible metabolic activity for other cyp enzymes and is also a substrate for the efflux transport p - glycoprotein (p - gp).3 mvc exposures have been shown to increase significantly when coadministered with potent cyp3a / p - gp inhibitors.3 as such, the recommended mvc dose in the presence of potent cyp3a / p - gp inhibitors is 150 mg twice daily (bid).1,2 patients with hiv-1 infection are disproportionately affected by viral hepatitis, specifically hepatitis c virus (hcv), which can lead to life - threatening complications.4 approximately 25% of hiv - infected patients in the united states and europe are coinfected with hcv, accounting for> 75% of liver - related deaths in hiv - infected patients.5 boceprevir (boc) and telaprevir (tvr) are newly approved hcv protease inhibitors indicated (in combination with pegylated interferon alpha and ribavirin) for the treatment of genotype 1 chronic hcv in adult patients with compensated liver disease.69 boc is a potent inhibitor of cyp3a, and tvr is also a potent inhibitor of cyp3a and an inhibitor of p - gp69; however, some unexpected drug interactions with hiv protease inhibitors have led to recommendations against the coadministration of boc with darunavir / ritonavir, atazanavir / ritonavir, lopinavir / ritonavir, and fosamprenavir / ritonavir, and against the coadministration of tvr with darunavir / ritonavir, lopinavir / ritonavir, and fosamprenavir / ritonavir.69 given the limited treatment options for hiv-1 and hcv coinfected patients, it is therefore important to investigate potential drug interactions of boc and tvr with mvc . This study was conducted to estimate the effect of boc 800 mg 3 times daily (tid) and tvr 750 mg tid on the pharmacokinetics (pk) of mvc, and to describe the pk of boc and tvr when dosed in combination with mvc 150 mg bid . The safety and tolerability of mvc in combination with boc and tvr was also assessed . Eligible volunteers were healthy adults (aged 1855 years) who had a body mass index of 17.530.5 kg / m, and a body weight of more than 50 kg . Volunteers who had used prescription or nonprescription drugs or dietary supplements within 7 days or 5 half - lives (whichever was longer) before the start of study treatment were not permitted to take part in the study . Volunteers who had used herbal supplements or hormonal methods of contraception within 28 days (6 months for depo - provera) were also not permitted to take part in the study . Volunteers with positive results for hiv-1, hiv-2, hepatitis b serology (hepatitis b surface antigen, hepatitis b core antibody), or anti - hcv serology (as determined by multi - antigen enzyme immunoassay), or who had a history of hypersensitivity to the study drugs, were excluded . This was an open - label, fixed - sequence, phase i study (nct01597895) conducted at a single site (pfizer clinical research unit, brussels, belgium). After a screening visit within 28 days before the start of treatment, volunteers received mvc 150 mg bid (every 12 hours) for 5 days (treatment period 1), followed by mvc 150 mg bid plus boc 800 mg tid (every 8 hours) for 10 days (treatment period 2), then mvc 150 mg bid plus tvr 750 mg tid (every 8 hours) for 10 days (treatment period 3), with a 10-day wash - out between periods 2 and 3 (fig . Study treatment was administered with 240 ml water at ambient temperature 30 minutes after a standard fat meal / snack (approximately 20 g fat and 500 calories). On pk assessment days (day 5 of treatment period 1 and day 10 of treatment periods 2 and 3), volunteers ate a standardized breakfast containing approximately 20 g fat and 8001000 calories and received only a single (morning) dose of mvc, as well as only the morning and afternoon doses of boc and tvr . Study personnel conducted mouth checks to ensure treatment compliance . To standardize conditions on pk sampling days, all volunteers were required to refrain from lying down [except when required for vital signs and electrocardiogram (ecg) assessments] and eating and drinking beverages other than water during the first 4 hours after dosing . Volunteers could be discontinued from the study at any time at their own request, or on the grounds of safety concerns, behavioral reasons, or inability to comply with the study activity or procedures, at the investigators' discretion . This study was conducted in compliance with the declaration of helsinki and good clinical practice guidelines established by the international conference on harmonization . The final protocol, amendments, and informed consent documentation were reviewed and approved by the study center institutional review board . Blood samples for mvc pk analysis were collected predose and at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours postdose on day 5 of treatment period 1 (mvc) and on day 10 of treatment periods 2 (mvc and boc) and 3 (mvc and tvr). Blood samples for boc and tvr pk analysis were collected predose and at 0.5, 1, 2, 3, 4, 6, and 8 hours postdose on day 10 of treatment periods 2 and 3, respectively . Samples of 4 ml were taken to provide a minimum volume of 1.5 ml plasma for pk analysis and were transferred into appropriately labeled tubes containing sodium heparin (mvc), dipotassium ethylenediaminetetra - acetic acid (k2edta) (boc), or tripotassium ethylenediaminetetra - acetic acid (k3edta) (tvr). All samples were centrifuged at approximately 1700 g for approximately 10 minutes at 4c . For mvc and boc, plasma was extracted and stored in appropriately labeled screw - capped polypropylene tubes at approximately 20c (mvc) or 70c (boc) within 1 hour of collection . Plasma extraction for tvr followed the same process, although after centrifugation approximately 1 ml plasma was transferred to an appropriately labeled screw - capped polypropylene tube containing 0.05 ml of 10% formic acid, before mixing thoroughly and being stored at approximately 70c . Formic acid solution was added to plasma to allow for accurate quantification of tvr by preventing tvr epimerization . Plasma samples were analyzed for mvc (tandem labs, west trenton, nj),10 and for boc and tvr (covance bioanalytical services, shanghai, china), using a solid - phase extraction and a validated high - performance liquid chromatography / dual mass spectrometry assay . Noncompartmental analyses were performed using standard methods with enca version 2.2 (pfizer, inc, new york, ny). Area under the plasma concentration time curve (auc) from predose (0 hours) to 12 hours (auc12; mvc) or 8 hours postdose (auc8; boc and tvr) was determined by the linear / log trapezoidal method, whereas plasma concentration at 12 hours (c12; mvc) or 8 hours postdose (c8; boc and tvr), maximum plasma concentration (cmax), and time to cmax (tmax) were determined by direct observation . All observed and volunteered adverse events (aes) were recorded and assessed by the investigator for severity and relationship to study treatment . Additional safety assessments included standard hematology, urinalysis, and chemistry laboratory assessments, physical examinations, vital signs (blood pressure and pulse) measurements, and ecgs . Orthostatic hypotension, a concentration - dependent ae observed with mvc,11 was defined as a decrease of 20 mm hg for systolic blood pressure or 10 mm hg for diastolic blood pressure 2 minutes after standing from a supine position, and may have been symptomatic or asymptomatic . A minimum sample size of 12 volunteers was required to provide 90% confidence intervals (cis) for the difference between treatments on the natural logarithmic scale of 0.1536 for mvc auc12, 0.2745 for mvc cmax, and 0.1493 for mvc c12, with 80% coverage probability . To allow for any volunteers who might not complete the study, construction of 90% cis was chosen based on fda guidance statistical approaches to establishing bioequivalence.12 because of the nature of normal - theory construction of 90% cis, this corresponds to performing 2 one - sided tests hypothesis at the 5% level of significance . Natural log - transformed auc12, cmax, and c12 for mvc were analyzed using a mixed effect model with treatment as fixed effect and subject as a random effect . Mvc alone was the reference treatment, and mvc plus boc and mvc plus tvr were the test treatments . Estimates of the adjusted mean differences (test reference) and corresponding 90% cis were obtained from the model . The adjusted mean differences and 90% cis for the differences were exponentiated to provide estimates of the ratio of adjusted geometric means (test / reference) and 90% cis for the ratios . Boc and tvr pk data were summarized descriptively and compared with mean historical minimum plasma concentrations (cmin) data (102 ng / ml and 1802 ng / ml, respectively).1,1316 boc and tvr data were determined to be comparable with the historical data if the mean c8 for both agents fell within the 50% range of their historical cmin values: 51204 ng / ml for boc and 9013604 ng / ml for tvr, based on simulations . Eligible volunteers were healthy adults (aged 1855 years) who had a body mass index of 17.530.5 kg / m, and a body weight of more than 50 kg . Volunteers who had used prescription or nonprescription drugs or dietary supplements within 7 days or 5 half - lives (whichever was longer) before the start of study treatment were not permitted to take part in the study . Volunteers who had used herbal supplements or hormonal methods of contraception within 28 days (6 months for depo - provera) were also not permitted to take part in the study . Volunteers with positive results for hiv-1, hiv-2, hepatitis b serology (hepatitis b surface antigen, hepatitis b core antibody), or anti - hcv serology (as determined by multi - antigen enzyme immunoassay), or who had a history of hypersensitivity to the study drugs, were excluded . This was an open - label, fixed - sequence, phase i study (nct01597895) conducted at a single site (pfizer clinical research unit, brussels, belgium). After a screening visit within 28 days before the start of treatment, volunteers received mvc 150 mg bid (every 12 hours) for 5 days (treatment period 1), followed by mvc 150 mg bid plus boc 800 mg tid (every 8 hours) for 10 days (treatment period 2), then mvc 150 mg bid plus tvr 750 mg tid (every 8 hours) for 10 days (treatment period 3), with a 10-day wash - out between periods 2 and 3 (fig . Study treatment was administered with 240 ml water at ambient temperature 30 minutes after a standard fat meal / snack (approximately 20 g fat and 500 calories). On pk assessment days (day 5 of treatment period 1 and day 10 of treatment periods 2 and 3), volunteers ate a standardized breakfast containing approximately 20 g fat and 8001000 calories and received only a single (morning) dose of mvc, as well as only the morning and afternoon doses of boc and tvr . Study personnel conducted mouth checks to ensure treatment compliance . To standardize conditions on pk sampling days, all volunteers were required to refrain from lying down [except when required for vital signs and electrocardiogram (ecg) assessments] and eating and drinking beverages other than water during the first 4 hours after dosing . Volunteers could be discontinued from the study at any time at their own request, or on the grounds of safety concerns, behavioral reasons, or inability to comply with the study activity or procedures, at the investigators' discretion . This study was conducted in compliance with the declaration of helsinki and good clinical practice guidelines established by the international conference on harmonization . The final protocol, amendments, and informed consent documentation were reviewed and approved by the study center institutional review board . Blood samples for mvc pk analysis were collected predose and at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours postdose on day 5 of treatment period 1 (mvc) and on day 10 of treatment periods 2 (mvc and boc) and 3 (mvc and tvr). Blood samples for boc and tvr pk analysis were collected predose and at 0.5, 1, 2, 3, 4, 6, and 8 hours postdose on day 10 of treatment periods 2 and 3, respectively . Samples of 4 ml were taken to provide a minimum volume of 1.5 ml plasma for pk analysis and were transferred into appropriately labeled tubes containing sodium heparin (mvc), dipotassium ethylenediaminetetra - acetic acid (k2edta) (boc), or tripotassium ethylenediaminetetra - acetic acid (k3edta) (tvr). All samples were centrifuged at approximately 1700 g for approximately 10 minutes at 4c . For mvc and boc, plasma was extracted and stored in appropriately labeled screw - capped polypropylene tubes at approximately 20c (mvc) or 70c (boc) within 1 hour of collection . Plasma extraction for tvr followed the same process, although after centrifugation approximately 1 ml plasma was transferred to an appropriately labeled screw - capped polypropylene tube containing 0.05 ml of 10% formic acid, before mixing thoroughly and being stored at approximately 70c . Formic acid solution was added to plasma to allow for accurate quantification of tvr by preventing tvr epimerization . Plasma samples were analyzed for mvc (tandem labs, west trenton, nj),10 and for boc and tvr (covance bioanalytical services, shanghai, china), using a solid - phase extraction and a validated high - performance liquid chromatography / dual mass spectrometry assay . Noncompartmental analyses were performed using standard methods with enca version 2.2 (pfizer, inc, new york, ny). Area under the plasma concentration time curve (auc) from predose (0 hours) to 12 hours (auc12; mvc) or 8 hours postdose (auc8; boc and tvr) was determined by the linear / log trapezoidal method, whereas plasma concentration at 12 hours (c12; mvc) or 8 hours postdose (c8; boc and tvr), maximum plasma concentration (cmax), and time to cmax (tmax) were determined by direct observation . All observed and volunteered adverse events (aes) were recorded and assessed by the investigator for severity and relationship to study treatment . Additional safety assessments included standard hematology, urinalysis, and chemistry laboratory assessments, physical examinations, vital signs (blood pressure and pulse) measurements, and ecgs . Orthostatic hypotension, a concentration - dependent ae observed with mvc,11 was defined as a decrease of 20 mm hg for systolic blood pressure or 10 mm hg for diastolic blood pressure 2 minutes after standing from a supine position, and may have been symptomatic or asymptomatic . A minimum sample size of 12 volunteers was required to provide 90% confidence intervals (cis) for the difference between treatments on the natural logarithmic scale of 0.1536 for mvc auc12, 0.2745 for mvc cmax, and 0.1493 for mvc c12, with 80% coverage probability . To allow for any volunteers who might not complete the study, construction of 90% cis was chosen based on fda guidance statistical approaches to establishing bioequivalence.12 because of the nature of normal - theory construction of 90% cis, this corresponds to performing 2 one - sided tests hypothesis at the 5% level of significance . Natural log - transformed auc12, cmax, and c12 for mvc were analyzed using a mixed effect model with treatment as fixed effect and subject as a random effect . Mvc alone was the reference treatment, and mvc plus boc and mvc plus tvr were the test treatments . Estimates of the adjusted mean differences (test reference) and corresponding 90% cis were obtained from the model . The adjusted mean differences and 90% cis for the differences were exponentiated to provide estimates of the ratio of adjusted geometric means (test / reference) and 90% cis for the ratios . Boc and tvr pk data were summarized descriptively and compared with mean historical minimum plasma concentrations (cmin) data (102 ng / ml and 1802 ng / ml, respectively).1,1316 boc and tvr data were determined to be comparable with the historical data if the mean c8 for both agents fell within the 50% range of their historical cmin values: 51204 ng / ml for boc and 9013604 ng / ml for tvr, based on simulations . All 14 completed treatment periods 1 and 2, but 1 volunteer withdrew during the wash - out period (between periods 2 and 3) because of an ae (severe asthmatic crisis) that was not considered to be related to treatment, and thus did not participate in treatment period 3 . The majority (n = 12/14, 85.7%) were white with the remaining 2 volunteers (14.3%) being of other races (one of hispanic ethnicity and one of asian ancestry). Volunteers had a mean (sd) weight of 79.3 (11.4) kg and body mass index of 24.4 (2.8) kg / m . Calibration standard responses were linear over the range of 0.5500 ng / ml for mvc, 252500 ng / ml for boc, and 505000 ng / ml for tvr . The between - day assay accuracy, expressed as percent relative error for quality - control concentrations in the low, medium, and high - diluted quality control samples ranged from 4.6%7.3% for mvc, 5.2%3.7% for boc, and 0.6%0.8% for tvr . Assay precision, expressed as the between - day percent coefficient of variation (% cv) of the mean estimated concentrations of quality - control samples, was 7.4% for the low (1.5 ng / ml), medium (50 and 150 ng / ml), high (375 ng / ml), and diluted (375 ng / ml) concentrations of mvc . Assay precision (% cv) for boc was 5.6% for the low (75 ng / ml), medium (250 ng / ml), high (1800 ng / ml), and diluted (12,500 ng / ml) concentrations, and for tvr was 3.8% for the low (150 ng / ml), medium (500 ng / ml), high (3600 ng / ml), and diluted (25,000 ng / ml) concentrations . Mvc plasma exposure (based on auc12 and cmax) was increased by approximately 3-fold in the presence of boc, and by approximately 8- to 9-fold in the presence of tvr (table 1; fig . 2). Mvc c12 values were approximately 3-fold higher for mvc plus boc (66.1 ng / ml), and approximately 10-fold higher for mvc plus tvr (235.5 ng / ml), when compared with mvc alone (23.8 ng / ml). Geometric means and adjusted geometric mean ratios for mvc pk parameters alone and in the presence of boc and tvr median plasma - time mvc concentrations by treatment shown by (a) linear scale, and (b) semi - logarithmic scale . Intersubject variability for mvc, as measured by the geometric% cv for auc12, cmax, and c12 was 24%36% when mvc was coadministered with either boc or tvr (table 1). Cmax was achieved within a median tmax of 2.0 (range, 1.06.0) hours when mvc was administered alone, 2.0 (range, 0.53.0) hours when mvc was administered with boc, and 3.0 (range, 2.04.0) hours when mvc was given with tvr . When coadministered with mvc, boc and tvr exposures were consistent with historical data (table 2), indicating that mvc had no notable impact on the pk profile of boc and tvr . Summary of plasma boc and tvr pharmacokinetic parameters in the presence and absence (historical studies) of mvc ae incidence was higher during treatment with mvc plus boc (100%) and mvc plus tvr (92%) compared with mvc alone (43%), and the majority of aes were considered to be treatment - related (table 3). The most common treatment - related aes occurring during treatment with mvc alone, mvc plus boc, and mvc plus tvr, respectively, were headache, dysgeusia, and fatigue, as summarized in table 3 . All aes were mild to moderate in severity, with the exception of a severe event of asthmatic crisis following the completion of treatment period 2 (mvc plus boc), which led to the discontinuation of 1 volunteer . This ae was not considered to be related to treatment but related to a pre - existing and undisclosed history of asthma . There were no deaths, serious aes, temporary discontinuations, or dose reductions because of aes in this study . No clinically significant changes in laboratory parameters, vital signs, or ecgs were reported . All 14 completed treatment periods 1 and 2, but 1 volunteer withdrew during the wash - out period (between periods 2 and 3) because of an ae (severe asthmatic crisis) that was not considered to be related to treatment, and thus did not participate in treatment period 3 . The majority (n = 12/14, 85.7%) were white with the remaining 2 volunteers (14.3%) being of other races (one of hispanic ethnicity and one of asian ancestry). Volunteers had a mean (sd) weight of 79.3 (11.4) kg and body mass index of 24.4 (2.8) kg / m . Calibration standard responses were linear over the range of 0.5500 ng / ml for mvc, 252500 ng / ml for boc, and 505000 ng / ml for tvr . The between - day assay accuracy, expressed as percent relative error for quality - control concentrations in the low, medium, and high - diluted quality control samples ranged from 4.6%7.3% for mvc, 5.2%3.7% for boc, and 0.6%0.8% for tvr . Assay precision, expressed as the between - day percent coefficient of variation (% cv) of the mean estimated concentrations of quality - control samples, was 7.4% for the low (1.5 ng / ml), medium (50 and 150 ng / ml), high (375 ng / ml), and diluted (375 ng / ml) concentrations of mvc . Assay precision (% cv) for boc was 5.6% for the low (75 ng / ml), medium (250 ng / ml), high (1800 ng / ml), and diluted (12,500 ng / ml) concentrations, and for tvr was 3.8% for the low (150 ng / ml), medium (500 ng / ml), high (3600 ng / ml), and diluted (25,000 ng / ml) concentrations . Mvc plasma exposure (based on auc12 and cmax) was increased by approximately 3-fold in the presence of boc, and by approximately 8- to 9-fold in the presence of tvr (table 1; fig . 2). Mvc c12 values were approximately 3-fold higher for mvc plus boc (66.1 ng / ml), and approximately 10-fold higher for mvc plus tvr (235.5 ng / ml), when compared with mvc alone (23.8 ng / ml). Geometric means and adjusted geometric mean ratios for mvc pk parameters alone and in the presence of boc and tvr median plasma - time mvc concentrations by treatment shown by (a) linear scale, and (b) semi - logarithmic scale . Intersubject variability for mvc, as measured by the geometric% cv for auc12, cmax, and c12 was 24%36% when mvc was coadministered with either boc or tvr (table 1). Cmax was achieved within a median tmax of 2.0 (range, 1.06.0) hours when mvc was administered alone, 2.0 (range, 0.53.0) hours when mvc was administered with boc, and 3.0 (range, 2.04.0) hours when mvc was given with tvr . When coadministered with mvc, boc and tvr exposures were consistent with historical data (table 2), indicating that mvc had no notable impact on the pk profile of boc and tvr . Summary of plasma boc and tvr pharmacokinetic parameters in the presence and absence (historical studies) of mvc ae incidence was higher during treatment with mvc plus boc (100%) and mvc plus tvr (92%) compared with mvc alone (43%), and the majority of aes were considered to be treatment - related (table 3). The most common treatment - related aes occurring during treatment with mvc alone, mvc plus boc, and mvc plus tvr, respectively, were headache, dysgeusia, and fatigue, as summarized in table 3 . All aes were mild to moderate in severity, with the exception of a severe event of asthmatic crisis following the completion of treatment period 2 (mvc plus boc), which led to the discontinuation of 1 volunteer . This ae was not considered to be related to treatment but related to a pre - existing and undisclosed history of asthma . There were no deaths, serious aes, temporary discontinuations, or dose reductions because of aes in this study . No clinically significant changes in laboratory parameters, vital signs, or ecgs were reported . A significant proportion of hiv - infected individuals are coinfected with hcv and consequently are at increased risk for severe liver disease.5 as liver fibrogenesis may be caused by stimulation of ccr5 receptors, mvc, a ccr5 antagonist, may have a beneficial effect on liver fibrosis . There is, therefore, increasing interest in using mvc as part of treatment regimens for hiv / hcv coinfected patients . Preliminary data from investigators from the university of brescia (italy) demonstrated a significant improvement in liver stiffness in 54 patients over 24 weeks when mvc 150 mg bid was added to antiretroviral regimens compared with existing regimens alone (p = 0.03).17 furthermore, an ongoing study (nct01327547) is primarily evaluating the safety of mvc in 120 hiv / hcv coinfected patients, as well as assessing the potential antifibrotic activity of mvc as a secondary objective . Boc and tvr are newly approved hcv protease inhibitors that have been shown to cause significant drug interactions . As such, many hiv protease inhibitors are not recommended to be coadministered with either boc or tvr, thus limiting treatment options in hiv / hcv coinfected patients.69 the study reported in this article was designed to investigate the effect of coadministration of boc 800 mg bid and tvr 750 mg tid on the pk of mvc 150 mg bid, and to describe the pk of boc and tvr when dosed in combination with mvc . Our results confirm that, when coadministered with either boc or tvr, overall mvc exposure is increased significantly . Tvr seemed to have a greater impact on mvc plasma exposure than boc, as indicated by an 8- to 9-fold increase in both mean mvc auc12 and cmax values after coadministration compared with a 3-fold increase with boc . The greater increase in mvc exposures observed with tvr was expected, as tvr has been shown to increase the auc of midazolam (a probe substrate for cyp3a) by 796% as compared with 430% with boc after oral coadministration of midazolam and an increase in the auc of digoxin (a probe for p - gp) by 85% as compared with 19% with boc.6,8 furthermore, a potential mechanism for the magnitude of this interaction observed with tvr may be interplay between inhibition of cyp3a / p - gp and organic ion transporter 1b1 (oatp1b1) by tvr,8 as mvc has been shown to be a substrate for oatp1b1.18,19 in vitro data suggest that tvr is a more potent inhibitor of oatp1b1 with an ic50 of 2.2 m compared with an ic50 of 18 m for boc.20,21 additionally, inhibition of oatp1b1 is more likely to occur in vivo with tvr given that the unbound cmax / oatp1b1 ic50 ratio for tvr is 0.95, whereas the ratio for boc is only 0.04.20,21 the combination of cyp3a / oatp1b1 inhibition by tvr was most likely also observed in a study where tvr was coadministered with atorvastatin, a substrate for both cyp3a and oatp1b1.8,9 in this study, tvr increased the auc of atorvastatin 7.88-fold whereas in a similar study, boc only increased the exposure of atorvastatin 2.30-fold.69 the magnitude of the mvc interaction with tvr is also consistent with that observed in a previous drug interaction study where mvc was dosed in combination with saquinavir / ritonavir (sqv / r), where mvc exposures were increased 9.77-fold.1,2 to date, tvr and sqv / r are the only 2 agents shown to increase the geometric mean mvc auc greater than 5-fold . Similarly to tvr, sqv / r is a potent inhibitor of cyp3a (increases midazolam auc 11.4-fold), an inhibitor of p - gp (increases digoxin auc by 49%) and an inhibitor of oatp1b1 (ic50 = 2.1 m).22,23 in the present study, mvc average concentrations (cavg), when dosed at 150 mg bid in the presence of tvr and boc, were 474 ng / ml and 151 ng / ml, respectively . The exposures seen in this study are within the exposure range observed in phase iii clinical studies evaluating the efficacy and safety of mvc in patients with ccr5-topic hiv-124 and are at or above the cavg exposure at which near maximal virologic efficacy is achieved with mvc (75100 ng / ml).25,26 these findings suggest that mvc should be dosed at 150 mg bid when coadministered with either boc or tvr, consistent with current dose recommendations for mvc when dosed in combination with other potent cyp3a inhibitors.1,2 however, as regulatory discussions are pending, we would suggest that prescribers should refer to your local prescribing information for mvc dosing recommendations with boc and tvr in their region . As with most drug drug interaction studies, healthy volunteers, rather than patients, were enrolled in this study . Mvc is primarily metabolized by the liver and therefore exposures have the potential to be higher in hiv / hcv coinfected patients with hepatic impairment, as hepatic damage and disease may affect cyp enzyme activity.2732 a study conducted in hiv - negative subjects with hepatic impairment demonstrated that mvc exposures in subjects with mild (child - pugh class a) and moderate (child - pugh class b) hepatic impairment had a geometric mean 25% (mild) and 46% (moderate) greater auclast and a 11% (mild) and 32% (moderate) greater cmax relative to subjects with normal hepatic function33 after a single dose of mvc 300 mg . As such, patients with moderate hepatic impairment receiving mvc with potent cyp3a inhibitors, such as boc, should be monitored closely.1,2 currently, tvr is not recommended to be dosed in patients with moderate and severe hepatic impairment.8,9 no exposure data are available for mvc in severe hepatic impairment, thus no recommendation in this population can be given at this time . In this study, mvc did not seem to cause clinically significant changes in concentrations of either boc or tvr as the mean pk exposures of boc (auc8 5404 ngh / ml; c8 80.7 ng / ml) and tvr (auc8 21980 ngh / ml; c8 1943 ng / ml) after mvc coadministration were consistent with those previously reported when boc (auc8 46017070 ngh / ml; c8 88.5111 ng / ml) and tvr (auc8 1815722300 ngh / ml; c8 15052030 ng / ml) were dosed alone.1,1316 these findings suggest that no dose adjustment for boc or tvr is warranted when coadministered with mvc . Finally, mvc coadministered with boc or tvr was generally well tolerated among the small population of healthy volunteers in this study . Although ae incidence was higher during combination treatment, the majority of aes were mild or moderate in severity, and there were no serious aes, discontinuations because of treatment - related aes, or deaths during the study . The most frequently experienced aes were dysgeusia and pruritus for the mvc plus boc combination, and headache and fatigue for the mvc plus tvr combination . Fatigue, dysgeusia, and pruritus seemed to be unique to the coadministration of mvc plus boc or mvc plus tpv and were consistent with previous findings for boc or tpv alone.69 no postural hypotension or dizziness was reported in this study despite the significant increases in mvc exposures when coadministered with boc or tvr . These findings are consistent with evidence that both boc and tvr are potent inhibitors of cyp3a, and support dosing of mvc at 150 mg bid when coadministered with either boc or tvr . When coadministered with mvc, boc and tvr exposures were consistent with historical boc and tvr monotherapy data; therefore, no dose adjustment for boc or tvr is warranted with mvc . Mvc coadministered with boc or tvr was generally well tolerated with no unexpected safety findings.
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Diabetes is a chronic disorder that affects a large segment of population and is a major public health problem . A recent who report indicates that india has the largest diabetic population (19 million in 1995) that is expected to rise to 57 million by 2025 . A commonly accepted definition of foot infection is the presence of systemic signs of infection (e.g., fever and leucocytosis) or purulent secretions or two or more local symptoms or signs (redness, warmth, indurations, pain, or tenderness). Viswanathan et al . Reported that 25% of diabetic individuals are anticipated to develop severe foot problems at some point in their lifetime that often end with amputation . Diabetic foot infections are more severe and more difficult to treat than infections in nondiabetics . Gram - negative infections are three - times more frequent in the diabetic than in non - diabetic individuals . The pathogenicity of these organisms is based on its ability to produce a variety of toxins and proteases and also on its ability to resist phagocytosis . Pseudomonas aeruginosa is commonly resistant to antibiotics, and because of this it is a dangerous and dreaded pathogen . The only antibiotic agents to which strains are regularly sensitive are cephalosporins, carbenicillin, colistin, gentamicin, polymyxin, quinolones, and streptomycin; however degrees of cross - resistance between these agents have been reported . P. aeruginosa is one of the most important microorganisms that cause clinical problems resulting from high - resistance to antimicrobial agents . Though it is rarely found in the normal flora of humans, it is frequently isolated from patients with burns, cystic fibrosis, and neutropenia . P. aeruginosa may cause severe tissue damage in diabetics and should never be ignored as insignificant in diabetic foot ulcers . Moreover, it should never be considered a contaminants or normal flora, and it should clearly be considered a pathogen, because it may result in sepsis and amputation . One of the challenges in managing p. aeruginosa infections is an inherent resistance mechanism, referred to as intrinsic resistance . Its multiplicity of resistance mechanisms may render this microbe less amenable to control by antibiotic cycling . P. aeruginosa is noted for its metabolic versatility and its exceptional ability to colonize a wide variety of environments and also for its intrinsic resistance to a wide variety of antimicrobial agents . The bacillus almost never causes infections in healthy individuals and often infects the immunocompromised . Because of its virulence and the limited choices of effective antimicrobial agents, treatments of infections by p. aeruginosa are often difficult . Though a lot of work has been carried out elsewhere pertaining to p. aeruginosa, the prevalence and the antimicrobial susceptibility patterns of p. aeruginosa from diabetes patients with foot ulcers have rarely been documented in this part of south india . Therefore, the present study has been carried out to study the prevalence of p. aeruginosa and their antimicrobial susceptibility by the kirby bauer - disk diffusion method . The study was based on 270 pus specimens received for the screening of p. aeruginosa from diabetes patients with foot ulcers attending tertiary care hospitals in and around coimbatore . Specimens included in the study were from soft tissue infection which includes foot wound and limb threatening infections specimens were obtained using aseptic techniques to avoid contamination and were promptly transported to the laboratory in a sterile swab in ice - cold conditions . The isolation and identification of test organisms was carried out by the procedures suggested by valentina and lalitha . Identification analysis like gram staining, motility, catalase, oxidase, pigment production, growth on cetrimide agar, ability to grow at 42c, gelatin hydrolysis, arginine dihydrolase, acid from hugh - leifson's glucose, and nitrate reduction tests were carried out . Antibiogram was performed using commercially available antibiotic discs (hi - media, mumbai) with a standard p. aeruginosa atcc 27853 as a positive control . Kirby - bauer, recommended by the clsi, was used for antimicrobial susceptibility testing . The identified 18 p. aeruginosa strains were tested against ampicillin (10 g), amikacin (30 g), ceftazidime (30 g), cefotaxime (30 g), ciprofloxacin (5 g), cefoperozone (75 g), co - trimoxazole (25 g), erythromycin (10 g), gentamicin (10 g), imipenem (10 g), norfloxacin (10 g), piperacillin (100 g), tobramycin (30 g), ticarcillin (75 g), and tetracycline (30 g). Of the total 270 diabetic patients suffering from foot infections, 180 were male and 90 were female . The male - to - female ratio was 2: 1 and the age of the patients ranged between 36 to 75 years . Studies conducted in chennai have shown that males were more susceptible than females in the ratio of 8: 3 . Previous studies have shown that males were more susceptible than females in the ratio of 2: 1, which is in accordance with the current study . Predominance of male over female patients as shown in the study can be explained by the fact that in our country males are exposed more to the outside environment because of their mobility as compared to females . Of the 270 pus specimens of diabetic patients with foot infections, 180 (66.6%) specimens were culture positive and the other 90 (33.3%) were negative . Among the strains, aerobic gram - negative pseudomonas species were 126 (70.0%) and other aerobic organisms comprised 54 (30.0%). From the 126 pseudomonas species, 18 (14.30%) dhanasekaran et al . Reported the prevalence of pseudomonas species to be 18.79% from a diabetic centre in chennai . In a similar study conducted in a private hospital in chennai, 29.8% strains among diabetic foot this finding shows the high prevalence of pseudomonas species and p. aeruginosa among diabetes patients with foot ulcers . The mueller hinton agar - based antibiogram - resistogram pattern study of p. aeruginosa isolated from foot ulcers of diabetes patients is shown in figure 1 . Almost all of the eighteen p. aeruginosa strains screened showed 100% resistance to ampicillin, erythromycin, and norfloxacin, similarly 83.3% resistance to piperacillin, ticarcillin, and tetracycline, 66.6% resistance to ceftazidime, imipenem, gentamicin, amikacin, tobramycin, and cotrimoxazole and 50.0% resistance to cefoperazone . Multidrug resistance for about 8 to 11 antibiotics was observed among 55.5% of the strains (table 1). Resistance was least with cefotaxime (16.6%), followed by an intermediate resistance of 66.7% observed for ciprofloxacin . India has the largest number of diabetic individuals and appreciably poor economic conditions; the study on this intrinsic resistant organism in diabetic foot infections assumes significance . The present study has shown the incidence of p. aeruginosa to be 14.3% in diabetic foot ulcers, which is significant when compared to previous studies . In accordance with earlier observations, the current study has demonstrated that p. aeruginosa strains isolated from foot ulcers are more resistant to antimicrobial agents . This may be due to the fact that the strains isolated from clinical specimens have been subjected to the selective actions of both disinfectants and antibiotics . As expected, the strains were resistant to imipenem, piperacillin, erythromycin, ticarcillin, tetracycline, gentamicin, co - trimoxazole, and amikacin indicating the emergence of multidrug - resistant strains . Antibiogram also revealed that cefotaxime and ciprofloxacin retained high levels of antipseudomonal activity and cefoperazone, gentamicin, ceftazidime, amikacin, imipenem, and tobramycin had the least activity . Ciprofloxacin and cefotaxime were found to be better choices for diabetes patients with foot ulcers in this part of the region when compared to gentamicin, imipenem, piperacillin, and other third - generation cephalosporins . The present study thus revealed the importance of p. aeruginosa from diabetes patients with foot ulcers, which is necessary for proper management of diabetes patients.
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Streptococcus pyogenes (group a streptococci) infection is diagnosed by either bacterial culture or serological testing . For the serodiagnosis, antistreptolysin o (aso) and antideoxyribonuclease b (adnase b) are the most widely accepted tests (1 - 3). The most popular and standardized serological test is still aso rather than adnase b. aso is not only useful in the diagnosis of streptococcal infections or complications, but also in the follow - up process, and in evaluating the effectiveness of treatments (4). Aso is especially helpful when throat culture technique is improper or the patient has already taken antibiotics (4). But, as aso is not always elevated after streptococcal infection or sequelae, it is necessary to add the alternative serological test . Since adnase b has a longer half - life than aso, it can be a valuable tool in the diagnosis of remote past infections (5). In cases of poststreptococcal glomerulonephritis (psgn) following skin infections or impetigo, adnase b is elevated, but aso levels do not tend to be elevated (4, 5). Previously, the adnase b test was complicated, lacked reproducibility, and the reagents were difficult to obtain (6). With the development of immunochemical techniques such as turbidimetry or nephelometry, quantitative adnase b analysis has become available (6). The upper limit of normal (uln) in normal children should be elucidated first in order to interpret data accurately obtained from the patients (7). The aim of this study was to measure aso and adnase b levels quantitatively to calculate the uln and to compare to the results of throat cultures from schoolchildren . Throat cultures and venous blood samples were taken from 266 children aged from 7 to 12 in seoul, korea . Bacterial cultures and identification were performed by bacitracin disk (0.04 u) and the latex agglutination method (a strep ad, denka seiken, tokyo, japan). Aso was measured with a chemistry autoanalyzer (hitachi 747, tokyo, japan) and adnase b was measured with a nephelometer (dade behring nephelometer 100, la jolla, ca, u.s.a . ). The reagents of aso and adnase b were purchased from daichi (tokyo, japan) and dade behring, respectively . Mean aso and adnase b were calculated by school grade, and compared with the results of the throat cultures . The uln of aso or adnase b was defined as the level of the 80th percentile of cumulative frequencies (8 - 10). The children were classified into three groups according to the results of throat culture, such as children with s. pyogenes, children with non - group a beta - hemolytic streptococci (bhs), and children with no bhs in their throats . The statistical significance of differences in aso and adnase b between each group was evaluated by student's t - test, and the correlation between aso and adnase b was determined by regression analysis . Mean aso and adnase b levels were 203 iu / ml (sd 217 iu / ml) and 251 iu / ml (sd 190 iu / ml), respectively . The ulns for aso and adnase b were determined to be 326 iu / ml and 362 iu / ml, respectively (table 1). The ranges of aso and adnase b were 1 to 1,712 iu / ml, and 77 (detection limit) to 1,616 iu / ml, respectively . Percentages of children with adnase b concentrations higher than 400 iu / ml and 500 iu / ml were 17.8 and 6.8, respectively . S. pyogenes was most frequently detected in the 3rd grade (26.3%) and the 6th grade (18.0%), but aso and adnase b levels were highest in the 5th grade . While aso levels were highest in the children with non - group a bhs, adnase b levels were significantly higher in children with s. pyogenes (table 2). The correlation equation between adnase b (y) and aso (x) was y=0.4x+173 (r=0.46). S. pyogenes produces several extracellular proteins, including streptolysin o, hyaluronidase, streptokinase, dnase, diphosphopyridine nucleotidase, and these antigens can also cause immune reactions (5). Aso tests have, lately, been most widely applied in clinical laboratory situations, using semiquantitative latex agglutination, the hemolysis inhibition method, quantitative turbidimetry, nephelometry or even the rapid chemistry autoanalyzer (9). Recently developed turbidimetric and nephelometric methods are both rapid and reproducible (6). Quantitative analyses of aso or adnase b are useful for the interpretation of a single sample if we know the uln of the population . But it is preferable to compare acute and convalescent levels, or levels both before and after treatment of streptococcal infections, rather than measuring a single level against an arbitrary standard (7). Although quantitative analysis is rapid and convenient for testing large numbers of samples, it still requires an accurate standardization, and an adequate quality of reagents . Adnase b is valuable, especially when there is little or no elevation of aso, even though the patient is infected with s. pyogenes . This is the case in which the victim develops impetigo, erysipelas, and psgn secondary to skin infection (5). Fujikawa and okuni (11) observed that aso elevation occurs only in 60% of rheumatic fever (rf). But after they added either adnase b or streptokinase test, they were able to diagnose rf with 95% accuracy . Ayoub and wannamaker (4) recommended that it is also advisable to check a few other antibodies, because aso can be normal, or throat culture can be negative, in some cases of rf or psgn . They found that they could diagnose 80% of sequelae with one antibody, but 95% with two kinds of antibodies . As there was very little correlation between aso and adnase b in this study, it appears that it would, indeed, be helpful to screen adnase b in addition to aso to diagnose s. pyogenes infections . Aso and adnase b levels were not closely related to the isolation rate of s. pyogenes in each school grade . As group c or group g streptococci can induce aso (5), the children with non - group a bhs also may exhibit higher levels of aso than children with s. pyogenes (table 2). Otherwise, these children might have a mixed infection, with non - group a bhs and s. pyogenes, or just a remote gas infection . It is very difficult to define as' healthy normal' children whose antibody levels are above the uln threshold, as that is also one of the criteria for' infection' (3). Fifty - five percent of schoolchildren in india had either aso levels or adnase b levels above 200 iu / ml (10), while 65% of children in our study measured above 200 iu / ml . It should be emphasized that children have different criteria to interpret aso or adn ase b compared to the adults . The uln of aso or adnase b varies according to age or area (1, 12, 13). The uln of aso and adnase b of children in seoul were found to be 326 iu/ ml and 362 iu / ml, respectively . (13) reported the uln thresholds of aso and adnase b by age groups, as 85 iu / ml for aso and 60 iu / ml for adnase in preschool - age children; 170 iu / ml for both aso and adnase in school - age children; and 85 iu / ml for both aso and adnase in adults . Kaplan et al . (14) reported that the uln of adnase b (400 iu / ml) was higher than that of aso (320 iu / ml) in the school - age group, a similar finding to ours . Ayoub and wannamaker (2) reported the ulns of aso and adnase b to be 166 iu / ml and 250 iu / ml, respectively . Taken together, it appears that the ulns of aso or adnase b varies according to age, study population, and detection method (9). Generally the uln of adnase b appeared to be higher than that of aso by our study and above reports . Some elementary school children without history of recent upper respiratory tract infections or skin infections manifested very high aso or adnase b levels, indicating frequent asymptomatic infections in this age group . In the recent recurrence of rf in the u.s.a ., almost half of the cases did not recall having previous pharyngitis symptoms (15), suggesting that asymptomatic infections could precede rf . Whether we should treat these asymptomatic infections or not has not been determined (1, 14, 16). In conclusion, some school children were proved, by aso and adnase b tests, to have asymptomatic s. pyogenes infections . Because the ulns of aso and adnase b were so high in schoolchildren adnase b test is an efficient method for the accurate diagnosis of streptococcal infections, because adnase b was more specifically related to throat culture results, and there was little correlation between aso and adn ase b.
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A 43-year - old caucasian german salesman was referred to our outpatient clinic from the haematologists in july 2009 . He was under their care for anaemia and had lost 10 kg of weight in 6 months . He also had increasing shortness of breath on exertion with a cough productive of yellow phlegm, increasing lethargy, dizziness on standing, and numbness in both hands . His wife had hiv infection for 6 years but had only recently disclosed her diagnosis to him . He had a minimal past medical history with previous knee cartilage repair only and no family history or children . He has travelled to mauritius, spain, and the dominican republic in the past and denies smoking, drinking very much alcohol, or indulging in recreational drugs . He denied other hiv risk factors including male partners, blood transfusions, or female partners from risk countries . Examination revealed a thin gentleman with multiple supraclavicular, axillary, and inguinal lymph nodes which were soft, nonmotile, and up to 2 cm in size . He had hepatosplenomegaly with a 1 cm liver edge and a 4 cm spleen palpated . Investigations showed a haemaglobin of 8.6 g / dl, platelets 186, mcv 76.4 his pt was prolonged at 15.7 seconds, but his lfts were normal except for a low albumin of 27 his cd4 count was 140 cells/l and the hiv-1 viral load was 710,000 copies / ml which suggested advanced hiv-1 infection and profound immunosuppression . The chest x - ray showed mediastinal and hilar lymphadenopathy with a subsequent ct chest / abdomen / pelvis revealing multiple, enlarged lymph nodes in the axilla, subcarinal, para - aortic, retroperitoneal, and mesenteric areas with a 14.5 cm spleen enlarged to the right iliac crest . He was commenced on truvada and efavirenz as standard triple antiretroviral therapy for hiv-1 infection after a full discussion on the importance of adherence, possible toxicities', and side effects . Daily 480 mg cotrimoxazole orally was started as part of pneumocystis jiroveci pneumoni (pcp) prophylaxis, fluconazole for possible disseminated fungal infection and standard intramuscular hydroxycobalamin given for the vitamin b12 deficiency . Atrophic follicles, germinal centre involution, lymphocytic depletion in paracortex: (i) + ve staining for kappa + lambda, (ii) appearances compatible with subacute / chronic stage of hiv lymphadenitis, and (iii) no evidence of lymphoma or carcinoma seen . At a 2-week review, he showed a 2 log drop in his hiv-1 viral load but complained of an itchy rash, and thus, his cotrimoxazole was switched to dapsone . Further results showed a positive latent syphilis result which was treated with 3 doses of intramuscular benzathine over 3 weeks . At a 4-week review, however, he complained of flu - like symptoms, fevers, dizzy spells, and periodic faints . His hb dropped further to 7.3 g / dl with severe renal impairment having a urea of 21.4 iu / l, creatinine 221 iu / l, and a corrected calcium of 1.94 a fanconi's syndrome secondary to the tenofovir component of his antiretroviral therapy was the likely diagnosis and was switched to abacavir instead . A penicillin allergy was also questioned, so the 3rd dose of benzathine was not given, and he was given oral 200 mg docycycline once daily for 28 days instead for the latent syphilis . Immune reconstitution inflammatory syndrome (iris) was also a possibility, but his repeat cd4 count was just 100 cells/l . He was admitted to the medical ward 3 days later as a result of worsening shortness of breath and lethargy, and although he had an improved renal function (urea 15.9 iu / l and creatinine 171 iu / l), he had pancytopenia with his hb 6.6, wcc 0.6 iu / l, platelets 97 iu / l, and 0.0 neutrophils! He was given 4 units of blood and standard neutropenic antibiotic cover . He developed violaceous lesions over his chest wall and left thigh which looked like kaposi's sarcoma (ks), and as his hb was still low at 7.0 iu / l with normal b12/folate / ferritin, ks was thought to be in his chest and gi systems . A repeat ct chest / abdomen / pelvis with contrast was done which showed there is extensive lymphadenopathy above and below the diaphragm with enlarged axillary, bilateral hilar, subcarinal, paraaortic, small bowel, mesenteric and inguinal nodes, up to 2.5 cm in the para - aortic region . There is peribronchial thickening in the right middle lobe with small bilateral pleural effusions in conjunction with right hilar lymphadenopathy? He was transferred to the john radcliffe hospital, oxford, for further management, as it was felt that complex care with possible chemotherapy and radiotherapy could be given at a tertiary centre . A splenic biopsy (figure 3) result showed widespread kaposi's sarcoma and plasma cell variant multifocal castleman's disease . A bone marrow trephine biopsy also showed leishmaniasis (figure 5) which was treated with ambisome . The initial cervical lymphnode biopsy was relooked at, and the report suggested lymph node shows changes associated with plasma cell variant of castleman's disease . Numerous human herpes virus 8 (hhv8) lymphoid cells (figure 5) and vascular proliferation which are also strongly hhv8+typically seen with cutaneous ks . An oesophagoduodenoscopy and colonoscopy confirmed gi ks, but no haematemesis or malaena seen . He responded very well to chemotherapy with 6 cycles of liposomal doxorubicin 40 mg / m + 4 weekly iv rituximab (4 courses). Apart from mild shingles which responded to high - dose acyclovir, he showed a good splenic and ct response to chemotherapy with the syphilis and leishmaniasis being successfully eradicated . Castleman's disease is a rare, benign disorder comprising of a nonclonal disease of the lymph nodes and angiofollicular hyperplasia but is more common in hiv / aids . There is usually widespread lymphadenopathy with hepatosplenomegaly, fatigue, night sweats, fever, wt - loss, anorexia, peripheral oedema, anaemia, low albumin, and peripheral neuropathy . It is associated with autoimmune hemolytic anemia, multiple myeloma, amyloidosis, and pemphigus which can lead to non - hodgkin's lymphoma . Treatment is guided by histology type and involves combination chemotherapy and antiretroviral therapy . As there is often a raised interleukin-6, novel therapies such as monoclonal anti - il-6 thus, multiple diagnoses were seen in this patient which is not atypical of someone living with hiv / aids . Multidisciplinary teams are crucial in most chronic conditions, and a low threshold for seeking expert advice and facilities should be maintained . The multiple symptoms and signs fitted most in the unifying diagnosis of multivariant castleman's disease.
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Reviewing his publications, he can be described as (i) a physician who wanted better drug dosage regimens for patients, (ii) a quantitative scientist who advocated better use of computers in medicine, (iii) a clinical pharmacologist who sought better models to better understand drug action, (iv) a statistician who looked for better methods in clinical pharmacology, (v) a visionary who was driven to seek better methods in drug development . He was a pioneer in the use of nonlinear mixed - effect models (nlmem) for better drug use, and he created the discipline of pharmacometrics . He published 234 articles, which were cited over 13,070 times (web of science, may 2013; figure 1). I first met lewis sheiner at a meeting in 1991, and we subsequently enjoyed many stimulating discussions, mainly during the advanced pharmacokinetics / pharmacodynamics (pkpd) workshops where he lectured and i was a tutor from 1999 on . After his untimely death in 2004, with several colleagues, we took over the course to continue promoting his vision of quantitative pharmacology . Lewis was for me a mentor and a friend who had a great impact on my career . He stimulated me to work in pharmacometrics, to become professor in a school of medicine, to perform research in academia, to address unmet challenges and develop new methods, to (try) to report results intelligently and correctly, to develop international scientific collaborations and friendships, to learn from others and from interdisciplinary collaboration, and to teach and promote scientific and quantitative thinking . Being a biostatistician, my main contributions in the field are the development of new statistical methods following the work of lewis sheiner . Here, i focus on three main statistical topics in nlmem: parameter estimation, model evaluation, and optimal design . For these three topics, i have had scientific discussions and various interactions with lewis sheiner . Key references to lewis's or my publications in those fields have been detailed in the slides (supplementary material 1). The first, and most significant, contribution of lewis sheiner in the field of pharmacometrics was the introduction of nlmem in pharmacokinetics for drug dosage individualization . He introduced the concept of the population approach and developed the nonmem software in 1980, implementing the fo estimation method . He later improved the methodology and expanded its scope, for instance, to discrete data . I have contributed to this area through collaboration with colleagues for development and/or dissemination of several new estimation methods, specifically nonparametric maximum likelihood, iterative two - stage and saem . My involvement in stochastic em approaches for nlmem was triggered by lewis sheiner who asked me in 2001 to investigate the potential of mcpem developed by serge guzy . In an article in the special issue of journal of pharmacokinetics and pharmacodynamics in honor of lewis sheiner mould and upton in their recent tutorial provided a list of all software developed since nonmem . These improvements in estimation methods and software tools have contributed to the development and spread of pharmacometrics . Future developments are still needed to address faster algorithms, handling of more complex data or models, and better use of computers' capacities . Very early, lewis sheiner addressed the problem of evaluation, and his most quoted paper is on measurement of predictive performance . He also introduced major concepts in modern model evaluation such as external validation, predictions errors, simulation - based diagnostic, and posterior predictive check . My main contribution to this area has been the development of new pseudo - residuals that i later termed prediction discrepancies following long discussion with lewis . The npde metric is now implemented in several software, and a specific r package was developed (http://www.npde.biostat.fr). The main topics are: external vs. cross - validation, prediction - corrected visual predictive check vs. transformed npde, extension of simulation - based diagnostics for complex data and/or complex designs, summary statistics to quantify predictability of a model, and so on . Model evaluation is a crucial step in pharmacometrics, and it is still lacking standardization and/or consensus . In 2014, at the american conference of pharmacometrics, i chaired the first meeting of the international society of pharmacometrics working group for model evaluation in pharmacometrics which is part of the standard and best practice committee . With the increased ability to apply nlmem to pkpd and clinical data, the problem of designing good studies emerged . In 1991, after work on design for dose ranging studies, lewis sheiner published the first simulation study to compare and evaluate several designs in pkpd, showing the importance in the balance between number of samples and number of patients . Evaluation of population designs by simulation is time consuming and is not suited for design optimization . That is why, with alain mallet, we developed an expression of the fisher information matrix (fim) for nlmem using fo . My main contribution in pharmacometrics is certainly all the work i perform with my team on optimal population designs . Since that first paper, we have extended the expression of the fim for more complex cases . In 2001, we developed the first r function with evaluation of the population fim (pfim; http://www.pfim.biostat.fr), and the latest version was released in april 2014 . Other software tools have been developed in the field (poped, popdes, popt, and pkstamp), and a comparison of the results for design evaluation in a pk example and in a more complex pharmacokinetic / viral kinetic model was recently performed . In 2006, basia bogacka (university of london) and i founded the multidisciplinary group population optimum design of experiments which has met every year since . The highlight was in 2011, where the meeting was held in cambridge in the isaac newton institute for mathematical sciences, uniting pharmacometricians and statisticians working in designs for mixed or generalized models . All the work and simulations performed for design in nlmem showed that design considerably affects precision of estimation . With the increased use of the results in drug development or use, the best information is needed out of each sample . Of course, prediction of standard error via the expected fim can be optimistic for design of small sizes as it will provide a lower bound of the variance, so that clinical trial simulation is important to evaluate strategic designs . Future work in that area involves fim for more complex data or design, evaluation of fim without linearization, adaptive designs, model - averaging approaches, design for individual predictions, and dosage individualization . More collaboration between statisticians and pharmacometricians is needed to help design clinical studies that provide meaningful results with feasible sample sizes . The first, and most significant, contribution of lewis sheiner in the field of pharmacometrics was the introduction of nlmem in pharmacokinetics for drug dosage individualization . He introduced the concept of the population approach and developed the nonmem software in 1980, implementing the fo estimation method . He later improved the methodology and expanded its scope, for instance, to discrete data . I have contributed to this area through collaboration with colleagues for development and/or dissemination of several new estimation methods, specifically nonparametric maximum likelihood, iterative two - stage and saem . My involvement in stochastic em approaches for nlmem was triggered by lewis sheiner who asked me in 2001 to investigate the potential of mcpem developed by serge guzy . In an article in the special issue of journal of pharmacokinetics and pharmacodynamics in honor of lewis sheiner mould and upton in their recent tutorial provided a list of all software developed since nonmem . These improvements in estimation methods and software tools have contributed to the development and spread of pharmacometrics . Future developments are still needed to address faster algorithms, handling of more complex data or models, and better use of computers' capacities . Very early, lewis sheiner addressed the problem of evaluation, and his most quoted paper is on measurement of predictive performance . He also introduced major concepts in modern model evaluation such as external validation, predictions errors, simulation - based diagnostic, and posterior predictive check . My main contribution to this area has been the development of new pseudo - residuals that i later termed prediction discrepancies following long discussion with lewis . The npde metric is now implemented in several software, and a specific r package was developed (http://www.npde.biostat.fr). The main topics are: external vs. cross - validation, prediction - corrected visual predictive check vs. transformed npde, extension of simulation - based diagnostics for complex data and/or complex designs, summary statistics to quantify predictability of a model, and so on . Model evaluation is a crucial step in pharmacometrics, and it is still lacking standardization and/or consensus . In 2014, at the american conference of pharmacometrics, i chaired the first meeting of the international society of pharmacometrics working group for model evaluation in pharmacometrics which is part of the standard and best practice committee . With the increased ability to apply nlmem to pkpd and clinical data, the problem of designing good studies emerged . In 1991, after work on design for dose ranging studies, lewis sheiner published the first simulation study to compare and evaluate several designs in pkpd, showing the importance in the balance between number of samples and number of patients . Evaluation of population designs by simulation is time consuming and is not suited for design optimization . That is why, with alain mallet, we developed an expression of the fisher information matrix (fim) for nlmem using fo . My main contribution in pharmacometrics is certainly all the work i perform with my team on optimal population designs . Since that first paper, we have extended the expression of the fim for more complex cases . In 2001, we developed the first r function with evaluation of the population fim (pfim; http://www.pfim.biostat.fr), and the latest version was released in april 2014 . Other software tools have been developed in the field (poped, popdes, popt, and pkstamp), and a comparison of the results for design evaluation in a pk example and in a more complex pharmacokinetic / viral kinetic model was recently performed . In 2006, basia bogacka (university of london) and i founded the multidisciplinary group population optimum design of experiments which has met every year since . The highlight was in 2011, where the meeting was held in cambridge in the isaac newton institute for mathematical sciences, uniting pharmacometricians and statisticians working in designs for mixed or generalized models . All the work and simulations performed for design in nlmem showed that design considerably affects precision of estimation . With the increased use of the results in drug development or use, the best information is needed out of each sample . Of course, prediction of standard error via the expected fim can be optimistic for design of small sizes as it will provide a lower bound of the variance, so that clinical trial simulation is important to evaluate strategic designs . Future work in that area involves fim for more complex data or design, evaluation of fim without linearization, adaptive designs, model - averaging approaches, design for individual predictions, and dosage individualization . More collaboration between statisticians and pharmacometricians is needed to help design clinical studies that provide meaningful results with feasible sample sizes . Although pharmacostatistical models were first developed for improvement of dosage in patients, their main area of application nowadays is in drug development . In the framework of the hype cycle (figure 2), we can view pharmacometrics in model - based drug development as having reached its plateau of productivity; however, there are still too few applications in clinical routine for patients' treatment . For instance, the first paper by sheiner on computer - aided dosage of warfarin was in 1969, and as late as 2013, papers are still published addressing that question . With new tools (smartphones and tablets) and clever implementation of bayesian forecasting, use of models for helping decision making under uncertainty another point of discussion is the relationship between pharmacometricians and statisticians, leading stephen senn to write in 2010: the battle lines were clear . It is time to bridge the gap through a better understanding of model - based approaches by statisticians and by more rigor in selecting data and models by pharmacometricians . Most deaths in the world (57 million in 2002; http://www.worldmapper.org) are preventable deaths (19 million in 2002), among which 11 million are from infectious and parasitic diseases . Pharmacometricians should redirect their expertise to focus on the disease burden affecting the developing world and work on the several important challenges in this area . To conclude, following the inspiration of our mentor, i hope that pharmacometricians and statisticians will work together and will develop (i) model - based analysis of pivotal trials, (ii) model - based treatment individualization, and (iii) model - based evaluation of treatments in the developing world . I wish and hope that the development of pharmacometrics will contribute to decrease disease burden in the word by using better treatments better targeted to each patient.
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Children have a natural tendency to put any small objects into their mouth; occasionally these objects can be ingested in the airodigestive tract; 80% of them will be stuck in the esophagus and 20% of them will lodge in the airway . In china, no statistical data on the incidence of esophageal foreign body is available, while the american association of poison control documented 182,105 incidents of foreign body ingestion by patients younger than 20 years in 1999 [1, 2]. In most cases, the majority of patients with esophageal foreign bodies are children . Unlike adults, the most common pediatric esophageal foreign bodies are blunt and round objects like coins, buttons, or button batteries . The conventional method involves removal of the foreign body under direct vision using a rigid esophagoscope under general anesthesia . In fact, the majority of blunt pediatric esophageal foreign bodies can be removed by a nonoperative foley catheter removal . In this report we describe our experience of first 17 cases removing blunt radiopaque esophageal foreign bodies by a foley's catheter without fluoroscopic guidance . We evaluated the safety and efficacy of the foley catheter technique by identifying detailed records of our first 17 cases that had undergone a foley catheter removal of blunt radiopaque foreign bodies from may 2010 to may 2013 . The materials needed for foreign body removal include a number of 14 to 18 foley catheters, a 10 ml syringe, tongue depressor, and saline water . Additionally, pediatric direct laryngoscope, rigid esophagoscope and bronchoscope, laryngeal and bronchial forceps, suction apparatus, and oxygen supply were kept ready . All procedures were performed in the examination room of the ent inpatient department . No patient is sedated . Radiographic confirmation is obtained to find out the presence, location, and shape of the foreign bodies . The balloon of the catheter is tested before insertion to make sure that it inflates symmetrically . While the catheter is inserted transorally, patients sit or are held upright position on the edge of the examination table . Children are instructed to open their mouth naturally; in uncooperative children, a tongue depressor is put intraorally and then its two sides are bristled to prevent the child from biting the catheter . The operator holds the catheter in his left hand and inserts it by his / her right hand, while advancing it inferiorly, he or she makes the action of swallowing and asks the child to follow . When the catheter passed about 2025 cm, the balloon is inflated with 5 ml air or saline . Before the catheter is withdrawn, patients are placed in a prone oblique position with head slightly out of the edge of the examination table . The balloon expands the esophageal lumen and frees the impacted foreign body; with moderate, steady traction, it pulls the foreign body out from the esophagus; with aid of gravity, it will usually fall out of the mouth . If balloon extraction fails to dislodge the foreign body because of under inflation or because the balloon is not down beyond the foreign body, correction is made with an additional 13 ml air or saline in the balloon or advancing the catheter deeper . If these corrections are unsuccessful and the foreign body will not move after 3 attempts, the technique is abandoned and the patient is referred to the rigid esophagoscopic examination under general anesthesia immediately . After successful extraction, children are monitored for 30 mins and subsequently discharged . The patents were instructed to feed the child with a soft diet and to return immediately if the child has symptoms of chest pain, fever, dysphagia, bloody saliva, respiratory difficulty, or abdominal pain . A total of 17 children (9 boys and 8 girls) with blunt esophageal foreign bodies, aged 20 months to 10 years, were included in the study . Chest roentgenograms were obtained in all the cases to identify the shape, size, and location of the foreign body; in all the cases, the impacted foreign body was a blunt and flat radiopaque object; 16 of them were metallic objects (11 coins, 2 buttons, 1 button battery, 1 key ring, and 1 heart shaped pendant) and one of them was a round and flat stone . Foreign body was lodged in thoracic esophagus in 7 cases, at the thoracic inlet in 6 cases and in the cervical esophagus in 4 cases . We were successful in 16 cases; in 12 of them the foreign body was removed in the first attempt and in 4 of them the foreign body was extracted in the second attempt by slightly increasing the volume of the balloon . We failed in one case; this child has a heart shaped pendant in the level of 2nd thoracic vertebra; after 3 unsuccessful attempts, we abandoned this method and had a rigid esophagascopic examination subsequently under general anesthesia . In the examination, nothing abnormal has been identified in the whole esophageal lumen . An immediate fluoroscopy was given in the operation room under a c arm and the foreign body was detected in the stomach . Except the child who has rigid esophagoscopy under general anesthesia, all patients were discharged well on the same day after the procedure . Rigid esophagoscopy has long been hailed as the gold standard for the removal of esophageal foreign bodies in children . If all types of esophageal foreign bodies were considered, rigid esophagoscope is the first choice of treatment; however, the subset of blunt and flat esophageal foreign bodies can be removed by a nonoperative method with a foley catheter . Foley catheter removal of blunt pediatric esophageal foreign bodies was first reported by bigler in 1966 . However, similar method existed long ago, according to the descriptions of paulus aegineta, in his book six of epitome of medicine; during the peak of the byzantine period, foreign bodies were extracted from the esophagus by having the patient swallow a small, dry sponge on a string, allowing it to expand in the stomach and then withdrawing the sponge . Bigler hypothesized in his original article that distension of the balloon of a foley catheter filled with contrast media inferior to the impacted foreign body would dilate the esophageal lumen, free the impaction, and allow safe extraction of the blunt and flat foreign bodies under fluoroscopic monitoring . Because of no need for anesthesia, removal of blunt esophageal foreign bodies by this method has become a relatively common problem shared by radiologists, pediatric surgeons, otolaryngologists, emergency department physicians, and gastroenterologists . In real practice, certain modifications were made by some scholars, like inserting the catheter transorally, replacing contrast media with saline water or air, and performing the procedure without fluoroscopy [58]. By these modifications, the whole procedure becomes simpler, the radioactive contaminations can be avoided and the patients and their parents' anxieties also can be eliminated . We used this modified technique to remove blunt esophageal foreign bodies and succeeded in 16 out of 17 cases . The main critical concern about foley catheter removal of esophageal foreign bodies was safety, because it carries certain blindness, resulting in esophageal perforation and airway compromise . However, the incidence of all complications of foley catheter removal of blunt foreign bodies has been consistently low in all published series . The largest survey of pediatric radiologists by campbell and condon included 2500 procedures with only one serious but reversible hypoxic episode . The only one case of death in the literature till now, caused by aspiration of a coin during foley catheter removal, was reported in a survey by hawkins . At the same time he reported five patients who died while undergoing esophagoscopic removal of a coin under general anesthesia . After 10 years of editing many articles on the pros and cons of foley catheter removal, berdon found that great differences in opinion existed among practitioners . He concluded that those who use the technique will continue to use it and feel comfortable: those that are not sold on the technique will remain unsold . A review of 415 cases by schunk et al . Found minor complications like epistaxis and vomiting in 8 (2%) patients and 4 (1%) patients who had the major complications of mediastinitis, transient airway compromise, respiratory distress with uneventful recovery, and esophageal laceration requiring surgery . Two of these complications occurred in patients with nonradiopaque foreign bodies, the third complication was caused by an impaction of unknown duration, and the fourth occurred after multiple attempts including simultaneous use of two foley catheters . Actually, all the 4 major complications above can totally be prevented by careful patient selection and standard performance . Foley catheter method was contraindicated to the former three cases; the fourth case was entirely the result of improper handling . All of these show that, if performed under strict inclusion criteria and standard procedure, foley catheter removal can be the first choice of treatment for blunt and flat esophageal foreign bodies . Advantages of foley catheter method including that they are (1) easy to perform and learn; (2) safe: complication rate is as low as 02% and can omit the potential complications of rigid esophagoscopy; (3) efficient: success rat is 85100%; (4) rapid: it rarely takes more than 20 minutes; (5) having no need for anesthesia: it can prevent anesthesia related complications; (6) haveing the possibility to be performed on outpatient basis . Because it can obviate the need for anesthesia and hospitalization, this method is highly cost effective . In current health expense system in our region, the mean cost for foley catheter extraction without fluoroscopy on an outpatient basis is less than 200rmb (cost for foreign body removal is 150rmb+cost for material is 35rmb); rigid esophagoscopy under general anesthesia costs at least 4000rmb, which translates into savings of 3800rmb . To ensure safety and to improve success rate, firstly, this method should be administered only for blunt and flat radioopaque foreign bodies . Some scholars suggest that blunt non radiopaque foreign bodies also should be included in the indications of this method; however, it is not easy to estimate the shape and size of this type of foreign bodies precisely, so they are preferably removed under rigid esophagoscopy . The duration of impaction should not be longer than 72 hours, because success decreased to 50% if the duration was longer than 72 hours . Button batteries are an exception; because the potential voltage burn and esophageal erosion can occur as early as 4 hours after ingestion, they should be removed endoscopically as early as possible . However, if the impaction is of less than 2-hour duration, foley catheter extraction is an acceptable alternative [13, 14]. In this series we have a 4-year - old boy who had swallowed a button battery 30 mins earlier; the battery was removed by a foley catheter in one attempt uneventfully . This method is contraindicated in the following situations: (1) if the foreign body has been impacted for more than 72 hours, (2) if the esophagus was totally obstructed by impacted foreign body, (3) if we found suspected esophageal perforation, (4) if we found multiple foreign body impaction, (5) if patient has sign of airway distress, (6) if we found sharp edged foreign bodies, (7) and if we found button batteries that impacted for more than 2 hours . Some authors noted that children younger than 1.5 years seem to be at the highest risk for esophageal edema and failure of foley catheter extraction . This may be due to the smaller caliber of the esophagus, the softer trachea that can be compressed easily, as well as the delayed diagnosis in nonverbal, and uncooperative infants . Our youngest patient was a 20-month - old child who had swallowed a flat and round stone for 4 hours; the stone was removed in one attempt with a foley catheter . Secondly, during the procedure, it is strongly recommended to maneuver the child into a prone oblique position while withdrawing the catheter to eliminate the freed foreign body to advance toward stomach or to the airway by gravity overdistention should be avoided . At first, inflate the balloon with 4 - 5 ml saline, if the balloon slips past the impacted object, try again with an additional 13 ml saline in the balloon . Stop inflation if the patient feels pain . If the impacted foreign body cannot be removed after 3 attempts, this method should be abandoned and the child should be referred to rigid esophagoscopy under general anesthesia . After 3 unsuccessful attempts in one of our cases, without obtaining another chest x - ray film, we have esophagascopic examination under general anesthesia and did not find anything abnormal in the whole esophageal lumen; subsequently we have a fluoroscopy in the operating room under a c arm and found that the impacted object was already in the stomach . So, before esophagascopic examination, it is very important to have another chest roentgenograms to see if the object moved or passed to the stomach . In up to 16% patients, the foreign body will advance into the stomach; this is not seen as a failure given that safe passage throughout the rest of the alimentary tract can usually be assumed . Thirdly, when performing this technique, it is preferable to keep pediatric laryngoscope, rigid esophagoscope and bronchoscope, suction apparatus, and oxygen supply readily available, so that we may utilize them in critical moments . We have never required any of this equipment in all of our cases; however, we think if conditions permit, this equipment will never be the fifth wheel of the coaching.
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Oxygen (o2) is essential for all aerobic organisms to produce energy via mitochondrial oxidative respiration as well as to perform other critical biological processes (koch and britton, 2008; taylor and mcelwain, 2010). Hypoxia is a condition in which tissues are inadequately oxygenated, and various physiological conditions, such as strenuous exercise or high altitudes, can result in inadequate o2 supply to support metabolic and energy requirements . Since depriving tissues of oxygen can adversely affect the function of these tissues, and consequently the cells of these tissues are significantly stressed and are unable to perform important biological processes, evolutionarily conserved adaptive processes (e.g. Hypoxia - inducible factors, hifs) are activated in order to match o2 supply with the metabolic and energy needs of the cells (semenza, 2012; weidemann and johnson, 2008). Insufficient o2 supply in cells or tissues is also a prominent feature of a number of pathological conditions including cancer, obesity and ischemic diseases . Recently, it has been shown that wounds and tissue injuries cause the injured area to become hypoxic, presumably due to the disruption of vasculature and increased o2 consumption by cells within and bordering the injured tissue (lokmic et al ., 2012). Moreover, recent preclinical studies suggest that adaptation to hypoxic stress via hif signaling plays a critical role in promoting wound healing processes (andrikopoulou et al ., 2011; hong et al ., 2014 despite that molecular and cellular mechanisms underlying hypoxic regulation of wound healing are still poorly understood, hif activation via the inhibition of oxygen sensor prolyl hydroxylase enzyme (phd) has been intensively investigated with the aim to develop novel treatment regimens for wound healing and ischemic diseases (myllyharju, 2009; 2013). Thus, it is of critical importance to improve our understanding of how hypoxic responses influence wound healing and tissue injury at the molecular level . In this review, we will focus on the mechanisms and processes regulated by hypoxia and hif signaling in wound healing and tissue injury . We will further discuss how hypoxic responses impact pathologic wound healing (e.g. Diabetic wounds) and fibrotic diseases in which wound healing processes become aberrant and fail . Finally, we will conclude with an overview of an exciting new treatment strategy that may prove effective for wound healing, tissue injury, and fibrosis . In response to the low oxygen - tension in the tissue, cells attempt to restore oxygen homeostasis within the hypoxic microenvironment . 1). Hif-1 is a heterodimeric protein complex that is composed of an and a subunit, both of which belong to the basic helix - loop - helix per / arnt / sim (bhlh - pas) family of transcription factors and are constitutively expressed in cells (wang et al ., 1995). In contrast to hif-1 proteins that are not regulated by the presence or absence of oxygen (hoffman et al ., 1991; labrecque et al ., 2013), hif-1 proteins are regulated at the posttranscriptional level by cellular oxygen tension . In non - hypoxic conditions, hif-1 proteins are rapidly degraded resulting in a half - life of less than 10 min (berra et al ., 2001). Proline residues of the oxygen - dependent degradation (odd) domain of hif-1 are hydroxylated in non - hypoxic conditions by phds (ivan et al ., 2001; jaakkola et al ., the hydroxylation of hif-1 allows the von hippel - lindau protein (pvhl), an e3 ubiquitin ligase, to bind, polyubiquitinate, and target it for proteasomal degradation (maxwell et al ., 1999; ohh et al ., 2000). The lack of available oxygen for hydroxylation by phds in hypoxic conditions stabilizes hif-1, increasing cellular levels of the protein, and facilitating its translocation into the nucleus where it heterodimerizes with hif-1. The hif-1 complex then binds to a 50-base pair cis - acting hypoxia response element (hre) in the enhancer and promoter regions of genomic dna, thereby activating hif-1 target genes (semenza and wang, 1992; wang and semenza, 1993), which are involved in hypoxic adaptation and survival processes including anaerobic glycolysis, growth factor signaling, angiogenesis, cellular mobility, and erythropoiesis . When tissue is injured the regulation of key processes by hif-1 enables the cell to efficiently adapt to the changing oxygen - tensions of the microenvironment and enables the dynamic capacity for the injured tissue to repair, remodel, and heal . The process of tissue repair and wound healing is comprised of three continuous and overlapping phases: the inflammatory phase, the proliferation phase, and the remodeling phase (hong et al ., 2014). This process requires highly orchestrated temporal and spatial regulation of various cell types and mediators in the injured tissue area . Moreover, elevated oxygen consumption by infiltrated inflammatory and stromal cells further lowers the tissue oxygen tension leading to prolonged chronic hypoxia . An early study by remensnyder and majno made a seminal observation that wounded tissue displays a steep hypoxic gradient in the cremaster muscle of rats that is strongly correlated with the level of revascularization (remensnyder and majno, 1968). Later, elson et al . Demonstrated a coordinated transient induction of hif-1 and its target genes, glut-1, pgk-1, and vegf, in epidermal wound healing (elson et al ., 2000). These initial studies highlight the temporal and spatial activation and regulation of hif-1 signaling in wound healing processes . Further, one can reason that adaption to the hypoxic microenvironment by hypoxic cells within the injured area, including inflammatory cells, keratinocytes, endothelial cells, and mesenchymal stromal cells, is essential for the cells to exert their wound healing activities . During wound healing or tissue injury repair, after platelets begin the coagulation process, monocytes infiltrate the injury or wound and start to differentiate into macrophages . At this point, macrophages become the predominant cell population in the inflammatory phase of wound healing . Biological significance of hypoxic adaptation of macrophages has been investigated utilizing a macrophage - specific hif-1 knockout animal model (cramer et al ., 2003). Macrophages displayed impaired recruitment into inflamed areas, metabolic dysfunction characterized by atp depletion, and decreased bactericidal capacity indicating that hif-1 activation is an essential determinant for macrophage survival and function in hypoxic injury microenvironments . The inflammatory phase of wound healing is also governed by macrophage - derived reactive oxygen species (ros). Although the rise in ros production is the main defense against bacterial infection by acting as intracellular signaling mediators which activate cytokine activity and angiogenesis, a balance within the injured tissue must be maintained because high levels of the ros can further damage the area due to the high reactivity of the molecules . Indeed, chronic non - healing wounds have demonstrated over production of ros resulting in oxidative stress within the tissue (schafer and werner, 2008). In this regard, hif-1-mediated switching to glycolytic metabolism from mitochondrial oxidative respiration in the hypoxic macrophages may be important for preventing the excess generation of ros and impaired wound resolution (kim et al ., 2006). In addition, in response to hypoxia, macrophages release chemotactic factors and secrete growth factors important for cell migration and proliferation leading to tissue repair . Macrophage - secreted growth factors such as vegf, platelet - derived growth factor (pdgf), fibroblast growth factor (fgf), transforming growth factor- (tgf-), and tgf- have been reported to be induced by hypoxia (murdoch et al ., 2005). In addition to being a chemoattractant for monocytes, macrophages, and neutrophils, pdgf has been shown to induce mitosis in fibroblasts and smooth muscle cells in vitro . Endothelial cell migration as well as fibroblast migration and deposition of extracellular matrix proteins (e.g. Collagen) are induced by tgf- secreted by macrophages and these events lead to the formation of granulation tissue (li et al ., 2007). These studies highlight the prominent implications of macrophage hypoxic responses in the wound healing process . The proliferative phase of wound healing consists of several processes: angiogenesis and vasculogenesis to reestablish blood supply to the injured tissue; fibroplasia, the formation of fibrous tissue, which contributes to granulation tissue development; re - epithelialization driven by keratinocyte proliferation and migration; and wound contraction mediated by myofibroblasts . Healing tissue requires new blood vessel growth, via angiogenesis and vasculogenesis, to re - establish the vasculature necessary to deliver oxygen and nutrients to the injured tissue . Hypoxia, through the accumulation of hif-1 and formation of the hif complex, activates several angiogenic growth factor genes including vegf, angiopoietin 2 (angpt2), and stromal cell - derived factor-1 (sdf-1) (ceradini et al ., 2004; forsythe et al ., 1996; multiple cell types, including fibroblasts and endothelial cells, additionally produce vegf, the key mediator of angiogenesis . Work by ahluwalia and tarnawski provides evidence that hif-1 increases in endothelial cells that line newly formed capillaries and that it co - localizes with vegf in these cells (ahluwalia and tarnawski, 2012). Angiogenesis is also stimulated by the secretion of cytokines by macrophages (murdoch et al ., 2005), the low - oxygen tension resulting from the hypoxic microenvironment, and the increased lactic acid within the tissue resulting from the high metabolic demands of the cellular influx into the damaged tissue area (remensnyder and majno, 1968; taraboletti et al ., 2006). The activation of angiogenesis allows for the re - establishment of vasculature within the injured tissue site enhancing its capacity to heal . In addition to angiogenesis, the re - establishment of vasculature within injured tissue can occur by the process called vasculogenesis where bone marrow - derived circulating endothelial progenitor cells (epcs) have the ability to form new blood vessels (ceradini and gurtner, 2005). Hypoxia, among other environmental stimuli, promotes recruiting these progenitor cells to the ischemic tissue via a hif-1 direct target, stromal cell - derived factor-1 (sdf-1) (ceradini et al . Sdf-1, also known as c - x - c - motif chemokine 12 (cxcl12), is a chemokine that attracts hematopoietic and endothelial progenitor cells expressing its receptor cxcr4 . Using a mouse model with graded soft tissue ischemia, ceradini et al . Demonstrated that reduction in the oxygen tension of tissue was directly proportionate to the expression of sdf-1 (ceradini et al ., 2004). Additionally, in vitro assay by tepper et al . Showed that epcs preferentially proliferate in hypoxic conditions (tepper et al ., 2005). Taken together, these studies suggest that hif-1 plays an important role in the localization or homing of circulating epcs to injured tissues exhibiting low oxygen tension to promote the restoration of damaged blood vessels . Another process occurring during the proliferation phase of wound healing and one that is essentially important for wound contraction is fibroplasia, the formation of fibrous tissue . The fibroblasts begin to proliferate and differentiate into myofibroblasts resulting in the generation of excess extracellular matrix (ecm) proteins aiding in granulation tissue development . Proliferation and differentiation of fibroblasts is induced by various growth factors such as fgf and tgf-, whose expression and activity can be further enhanced by hypoxic microenvironment (li et al ., 2007). Intriguingly, recent studies have shown that increased lactic acid within tissue, due to high demand of hif-1-mediated glycolytic metabolism of cells in the damaged hypoxic tissue area, is important in the activation of tgf- signaling and myofibroblastic differentiation (kottmann et al ., 2012; tuder et al ., this study revealed a previously unknown ph - dependent mechanism of tgf- activation in which hypoxia may provide fibroblasts an acidic microenvironment that enhances their differentiation to myofibroblasts . The formation of granulation tissue is followed by the reepithelialization process, which is characterized by keratinocyte proliferation and migration into the wounded area to establish a protective barrier . (2013) have recently investigated the implication of hypoxic responses in this process in keratinocyte - specific phd2 knockout mice that exhibit constitutively stable hif-1 activity . The authors demonstrated that keratinocyte phd2 ablation results in an elevated migratory capacity of keratinocytes in their skin wound healing model . This enhanced migration was mediated by hif-1-dependent integrin-3 that previously has been shown to play a critical role in wound healing (de luca et al ., 1994). Moreover, suppressing the growth inhibitory effects of tgf- signaling significantly enhanced the proliferation of phd2-null keratinocytes . In order for wounds and injured tissues to be properly repaired, each step of the wound healing cascade essentially requires a fine orchestration among various cellular components with support from a physiological microenvironmental milieu . Defects in the normal wound repair process or lack of cellular or non - cellular components leads to the development of chronic or pathologic wounds . Among many impaired wound diseases, we will describe here diabetic wounds and fibrosis focusing on how hypoxia and hif-1 signaling are implicated with the pathogenesis of these conditions . We will further discuss targeting hypoxic signaling as a potential therapeutic strategy for aberrant wound healing disorders . Impaired wound healing in diabetic patients (e.g. Diabetic foot ulcers) is one of the major medical and public health issues worldwide . More than 50% of non - traumatic lower limb amputations are caused by diabetic wound ulceration . Moreover, diabetic patients with foot ulcers are expected to have a poorer prognosis and significantly higher mortality (moulik et al ., 2003). Currently, there is no effective treatment strategy available mainly because the molecular pathogenesis that leads to wound repair failure is poorly understood . Given that hypoxia plays a significant role in physiologic wound healing processes, several groups have sought to determine how hypoxic responses and hif-1 signaling activity are affected by delayed wound healing in diabetic animal models . (2007) reported that the hyperglycemic condition was sufficient to repress hif-1 induction in hypoxic conditions . This appeared to be a proteasome - dependent mechanism as proteasomal inhibitor, mg-132, but not phd inhibitor abolished the hif-1 suppressive effect of hyperglycemia . Consistent with in vitro observations, diabetic ulcer biopsy displayed a significantly lower expression of hif-1 as compared to non - diabetic chronic ulcer tissues emphasizing the impaired hif-1 signaling in hyperglycemic diabetic wounds . In subsequent studies, pharmacological stabilization or genetic activation of hif-1 was able to improve wound healing in genetic diabetic db / db mice (botusan et al . Hif-1 activation significantly induced neovascularization that is associated with the local recruitment of bone marrow derived endothelial progenitor cells into the wound area . This highlights the clinical implication of hif-1 stabilization for the management of diabetic wounds given that impaired vasculature and blood perfusion is considered a major pathogenic mechanism . During the normal progression of tissue repair, myofibroblasts ultimately become the predominant cell population secreting collagen and other ecm proteins to restore tissue homeostasis and promote healing . Fibrosis is the aberrant wound healing process where the repair of injured tissue is deregulated causing myofibroblasts to deposit excessive amounts of ecm (e.g. Collagen) (zeisberg and kalluri, 2013). The excess ecm results in the overgrowth of tissue, hardening, and scar formation . The tissue overgrowth and hardening can lead to organ dysfunction and ultimately organ failure can occur if fibrosis advances . One of the important mechanisms underlying the pathogenesis of tissue fibrosis is epithelial - mesenchymal transition (emt) (lee and nelson, 2012). Emt is defined by the loss of epithelial cell polarity and cell - cell adhesion enabling these cells to migrate and acquire the invasive properties of mesenchymal cells . Mesenchymal cells facilitate tissue regeneration in wound and tissue healing and can promote the pathogenesis of chronic conditions, such as fibrosis . A characteristic of fibrotic tissue is reduced capillary density within the tissue or organ resulting in decreased oxygen delivery to cells . As a result, fibrotic tissue becomes hypoxic and stabilizes the transcription factor hif-1 transactivating a wide variety of genes including profibrotic genes . In support of this, recent preclinical and clinical studies show that initiation and progression of various tissue fibroses are closely correlated with hif-1 activated, or hypoxia - induced, profibrotic genes (kaminski and rosas, 2006; tzouvelekis et al ., 2007). In studies using cell culture and genetic knockout mouse models, (2007) showed that the expression of hif-1 is associated with increased renal fibrosis by positive regulation of emt . Loss of hif-1 in primary renal epithelial cells resulted in a reduced capacity to undergo emt as well as attenuated expression of profibrotic genes, including a collagen cross - linking enzyme lysyl oxidase (lox). The authors further showed that targeted hif-1 deletion in proximal tubular epithelial cells leads to reduced tubulointerstitial fibrosis in the model of unilateral urethral obstruction (uuo). Clinically, hif-1 was shown to be highly expressed in renal biopsies from chronic kidney diseases . The transdifferentiation of the tubular epithelial cells into myofibroblasts is likely a result of hif regulation of sdf-1 and its cytokine receptor, cxcr4 (barriga et al ., 2013). Additionally, hif-1 has been shown to directly induce transcription of the pro - fibrotic factors tissue - inhibitor of metalloproteinases (timp)-1, plasminogen activator inhibitor (pai)-1, and connective tissue growth factor (ctgf). Similar to vegf transcription, a cooperation between hypoxia and tgf- signaling has been suggested because the dna binding sites for hif-1 and smads are near one another in target genes (e.g. Type i collagen) of fibroblasts (lee and nelson, 2012). For example, zhang et al . Showed that the up - regulation of smad3 by hypoxia leads to the activation of tgf- signaling pathways (zhang et al ., 2003). Similarly, basu et al . Demonstrated that tgf- directed expression of type i collagen could be decreased by inhibiting hif-1 transcription (basu et al ., 2011). While the pathological processes of renal fibrosis are extensively studied, the origin of kidney myofibroblasts is still a matter of debate . Recently, renal erythropoietin producing (rep) cells were identified (pan et al ., 2011), subsequent fate mapping analysis demonstrated that myelin protein zero - cre (p0-cre) lineage - labeled cells comprised the majority of rep cells . An animal model of renal fibrosis, unilateral ureteral obstruction, revealed that most of the kidney myofibroblasts were derived from p0-cre lineage - labeled cells, but not from injured tubular epithelial cells (asada et al ., 2011). More intriguingly, together with the differentiation into myofibroblasts, p0-cre lineage - labeled cells concomitantly lose erythropoietin production, which is one of the hallmarks of chronic kidney disease . (2014) reported a strong correlation between hypoxia and the expression of fibrotic gene products including collagens and smooth muscle actin (-sma), which are representative markers of myofibroblast differentiation, in human cardiac tissues . Under hypoxic conditions, human cardiac fibroblasts exhibited pro - fibrotic features including activated tgf- signaling, which appears to be associated with global dna methylation . Intriguingly, hif-1 directly transactivated dna methyltransferase 1 (dnmt1) and dnmt3b in cardiac fibroblasts in response to hypoxia . Pharmacological or genetic inhibition of dnmt abolished the hypoxic induction of pro - fibrotic genes suggesting that hypoxic induction of hif-1 may contribute to ischemic fibrosis in cardiac tissue by epigenetic regulation of pro - fibrotic gene expression . Another major target organ for fibrotic disorder is the lung (loomis - king et al ., 2013; comparative expression profiling data of pulmonary fibrosis mouse models and idiopathic pulmonary fibrosis (ipf) patients demonstrated a significant increase of alveolar epithelial hif-1 expression in an early stage of pulmonary fibrosis (tzouvelekis et al ., 2007). Immunohistochemical detection of hif-1 in mice bearing bleomycin - induced pulmonary fibrosis and ipf patients further supports the potential implication of hif-1 in fibrotic progression . A recent study by kottman et al . (2012) showing the ph - dependent activation of tgf- signaling and myofibroblastic differentiation by increased lactic acid within pulmonary fibrotic tissue provides an insight into the mechanisms underlying hif-1-mediated fibrotic progression; aberrantly activated glycolytic metabolism may be implicated in fibrotic progression . Wound healing involves a variety of processes including inflammation, angiogenesis, vasculogenesis, fibroplasia, and reepithelialization, all of which are critically regulated by hypoxic responses (fig . Hif-1 has been repeatedly shown to accelerate physiologic wound healing processes, as well as diabetic wound healing, providing a fundamental background to hypothesize that hif-1 activation can be used to aid in the healing of normal and diabetic wounds . In light of this, inhibition of oxygen sensor proyly hydroxylase enzyme (phd), which sensitizes hif-1 for proteasomal degradation, has been intensively investigated and shown to be beneficial for diabetic wound healing (kalucka et al ., 2013; myllyharju, 2009, 2013; thangarajah et al ., 2010; zhang et al ., 2013). Thus there is a potential for the use of phd inhibitors to treat tissue injuries and wounds . For example, selective phd inhibitors, such as fibrogen s fg-4592 and fg-2216, are being developed and are currently undergoing fda approved human clinical trials for use as oral drugs (beuck et al ., 2012). Although these phd inhibitors are intended to treat anemia by acting upon hif-2-mediated erythropoietin (epo) induction, they may potentially play a role in tissue injury and wound healing . While hif-1 can accelerate normal and diabetic wound healing, it can also aggravate the pathogenic progression of fibrotic disorders such as fibrosis (higgins et al ., 2007; kimura et al ., 2008; lokmic et al ., 2012). Given that fibrotic progression is promoted by hypoxic response and its contribution to the differentiation into ecm - producing myofibroblasts, further study should be warranted to evaluate the inhibition of hif-1 signaling as a potential therapeutic strategy for fibrotic diseases.
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Historically, kidneys with congenital abnormalities were often discarded because of the perceived risk of technical complications . However, as the waiting times for kidney transplants continue to increase, transplant centers have become more aggressive at using select kidneys with congenital abnormalities . Horseshoe kidneys are now routinely used for transplantation, either as a single or split graft . Renal ectopia describes the failure of the kidney to ascend and cross the midline during development, resulting in the ipsilateral position of both kidneys . In many cases they remain fused . Although these kidneys have an increased rate of vascular and ureteral anatomical anomalies, there are select reports of use in renal transplantation . This is the first case report of the successful transplantation of a split crossed fused kidney for a combined simultaneous kidney pancreas transplantation . The transplant recipient is a 58-year - old female who developed type i diabetes mellitus at 6 months old and used an insulin pump for blood sugar control . She developed end organ damage including renal failure but was not yet dialysis dependent . She did not have hypoglemic unawareness and had good blood sugar control with her insulin pump . She was approved and listed for combined simultaneous kidney pancreas transplantation . At the time of transplantation her panel of reactive antigen (pra) was 0% and there were no identifiable hla specific antibodies . The donor was a 31-year - old male motorcyclist who collided with a concrete medium at highway speed . The donor did not have significant past medical or surgical history, except for smoking cigarettes and occasional marijuana . The donor family was not aware of his congenital renal anomaly and the patient did not have other congenital anomalies . A computed tomography (ct) scan of the abdomen done at the time of admission revealed an ectopic right kidney located in the pelvis and fused with the lower pole of the left kidney, compatible with crossed fused renal ectopia (figure 1). The serum creatinine peaked at 1.8 and was 1.4 at the time of procurement . The procurement of the kidneys, pancreas, and liver was completed in the standard fashion, flushing with custodial htk (odyssey) solution . The upper and mid polar arteries of the right kidney originated from the aorta, while the lower pole artery originated from the right common iliac artery . There were single renal veins bilaterally, which drained into the inferior vena cava . The left ureter crossed the midline and entered the bladder in the normal anatomic position . The back table dissection and parenchymal transection was completed with the assistance of the urology service . Initially we had planned to split the kidneys and use each singly for transplant (figure 2). However, the right kidney was abnormal in appearance; in particular it was very thin . The mid and lower pole arteries were very small and were not connected to the aortic patch, making them difficult to reimplant . We elected not use the renal mass en bloc, due to the complex arterial anatomy, including the number, caliber, and configuration . At that time the right kidney was split from the left, carefully transecting the isthmus using sharp dissection . A few vessels and what appeared to be a small calyx were suture ligated with vicryl suture . Next, the transection plane was closed using interrupted 0 chromic mattress sutures . Pledgets of fat were used to prevent any cut through of the sutures through the parenchyma (figure 3). The kidney was anastamosed to the external iliac artery and vein on the left side . The kidney reperfused well, with minimal bleeding after reperfusion needing only bipolar coagulation for control . The pancreas graft was anastamosed to the right common iliac artery and vein and the donor duodenum to the recipient jejunum . Immunosupression included campath induction, with prograf, cellcept, and a rapid steroid taper . A protocol pancreatic biopsy at one year demonstrated drachenberg grade iii out v acute rejection . She is now 2 years posttransplant with normal pancreatic (fasting blood sugar 86, c - peptide 2.1, hemoglobin a1c 5.4) and renal graft function (creatinine 1.0 and creatinine clearance 55). The continuing shortage of deceased donor kidneys has inspired transplant programs to search for innovative ways to increase the number of potential donor kidneys . It is now common for many centers to use en bloc pediatric kidneys, kidneys with acute renal failure, and two extended donor criteria kidneys in a single recipient [13]. Horseshoe kidneys are now routinely used as both dual and split single transplants, with results almost equivalent to deceased donor transplantation (83.3% graft survival at 5 year follow - up). The recent case reports describe the use of living donors and in situ split of the horseshoe kidney for transplantation . Congenital renal fusion anomalies are seen in 1 in 250 autopsies, with horseshoe kidney being the most common anomaly present in 1 to 600800 adults . Renal ectopia has an incidence of 1/5001200 and the ectopic kidney can be located from the pelvis (most common) to the thorax . The combination of ectopia and fusion can be found at an incidence of 1/1000 and is termed crossed fused renal ectopia (cfre). Although there are 6 described variations of cfre, the aberrant kidney is most often located inferior to the normally positioned kidney, and fusion occurs between the superior pole of the aberrant kidney to the inferior pole of the normal kidney . Most patients remain asymptomatic, and the diagnosis is made at the time of autopsy or imaging done for another indication . However, ureteral obstruction, renal calculi, and an increased risk of neoplasm have all been described . There is a single case report of transplantation with a nonfused kidneys and two case reports of crossed as a dual transplant [8, 10]. This is the first report of splitting the crossed fused renal ectopia prior to transplantation as well as in combination with a simultaneous pancreas transplant . Cfre may be diagnosed preoperatively on routine imaging or intraoperatively at the time of donation . Donors with a history of renal calculi or recurrent urinary tract infections in combination of crfe should be excluded because of the potential for postoperative obstructive complications . Familiarity with the potential anatomic anomalies associated with crfe is essential to prevent injury to the kidneys at the time of procurement . More than 70% of crfe have multiple arteries and multiple veins are also common . It is optimal for the kidneys to be procured en bloc, with consideration for transection done at the recipient transplant center . Unlike with horseshoe kidneys where the ureters are located anteriorly in a normal anatomic position, ureters from the crossed fused ectopic kidney crosses over the midline to left side to enter in the bladder in a normal anatomic position . Care must be taken to avoid ureteral transection, especially during the contra - lateral iliac dissection . Investigation of the kidneys should be on the back table to consider for possible split . Obviously, obtaining two grafts is preferable to using one, but it may not be possible secondary to anatomic anomalies . Both the aberrant and normally positioned kidney can have variant arterial supply from the upper abdominal aorta, lower aorta, and iliac arteries . If the number or size of the arteries is too complex for transplantation, splitting of the ectopic moiety (as in our case) or en bloc transplantation is recommended . In our case, if multiple renal veins are encountered during routine transplantation, most surgeons routinely ligate smaller renal veins without affecting the outflow of the kidney . However, in these cases there may not be collateral venous drainage between the two kidneys and anastamosis of even smaller veins may be necessary to prevent venous hypertension . Horseshoe kidneys and cfre are separated by a tissue band, which ranges from a thin fibrous isthmus to thick functional parenchyma . The techniques used for renal transection in partial nephrectomies are applicable to the back table splitting the fused kidneys . This includes sharp transection followed by use of the tissue link or cautery for hemostasis, as well as direct locking suture ligature of the stump . In addition, the use of hemostatic glues and materials can be used to help attain hemostasis and prevent a urine leaks . In this case, the isthmus was thin and a sharp transection followed interlocking suture closure of the defect in combination with peri - renal fat pledgets was sufficient to prevent both hemorrhage and urine leak . If the bridge of tissue is too thick to allow safe transection, the kidney can be transplanted as a single graft or en bloc . The last consideration is that the crfe kidney may be difficult to position due to the vascular reconstruction and increased renal mass . As pointed out by bailey et al ., opening of the peritoneum for positioning may be necessary if the transplant is completed through an extra - peritoneal approach . In this paper, the kidney was placed intraperitoneal and secured behind the sigmoid colon to prevent torsion . Although the use of a living donor split horseshoe kidney has been reported in the literature, the complex vascular and arterial anatomy, as well as potential compromise of the donors remaining kidney, would make living donation treacherous . However, the kidneys must be both procured and transplanted with careful attention to the anomalous vascular and ureteral anatomy, as well as graft placement to prevent torsion . If there are multiple vascular structures, or the transaction plane does not result in adequate nephron mass for both kidneys, consideration of transplanting the kidneys en bloc or resection the smaller moiety should be considered . Transplant surgeons should be familiar with these potential anatomic variations to ensure these grafts are not wasted . Long term graft survival does not appear to be limited in these otherwise healthy donor kidneys.
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Muscular ventricular septal defects account for 20% of the total ventricular septal defects in the population . Most muscular ventricular septal defects (mvsd) are single and isolated lesions that close spontaneously in their natural history . Sometimes there are many different muscular communications between the left and right ventricles, called swiss - cheese type septal defects . Muscular vsds may cause clinical signs and symptoms of pulmonary overflow with left - sided volume overload . This may result in heart failure, especially in infants with hemodynamically significant unrestricted communications . Unsuitable localization for a classic surgical closure of mvsds can be challenging in congenital heart surgery . In such situations it is not uncommon that residual shunts are still present despite several attempts to close multiple muscular defects . This results in higher mortality and morbidity and increases the risk of reoperation and ventricular dysfunction due to right or left ventriculotomy . Therefore, closure of small mvsds with no hemodynamic compromise remains controversial, facing expected spontaneous closure of 80% of the defects in the first 2 years life . Device closure of mvsds can be successfully accomplished using transcatheter technique when the child reaches a body weight that allows intervention . The complexity of ventricular septal defects in early infancy led to development of new mini - invasive techniques based on collaboration of cardiac surgeons with interventional cardiologists, called hybrid procedures . Hybrid therapies aim to combine the advantages of surgical and interventional techniques in an effort to reduce the invasiveness (e.g., cardiopulmonary bypass, cardioplegic arrest, incision trauma, groin vessel injury, and risk of tricuspid insufficiency) of a cardiac procedure . In selected babies, the trans - ventricular hybrid technique may be more feasible and advantageous, although closure of mvsds is still one of the most challenging procedures in cardiac intervention and surgery . The aim of this study was to present our approach with mvsd patients and initial results in the development of a mini - invasive hybrid procedure in the gdansk hybrid heartlink programme (ghhp) at the department of pediatric cardiac surgery, pomeranian centre of traumatology in gdansk, poland . We present different approach strategies to patients referred for surgery with the diagnosis of mvsds, individually modified with regard to morphology of muscular defects, or accompanying pathologies, requiring additional interventions . We present a group of 11 patients with the diagnosis of muscular ventricular septal defects, who consecutively entered ghhp at the department of pediatric cardiac surgery, pomeranian centre of traumatology in gdansk, poland (table 1). Six groups of patients were identified: children with isolated mvsds treated primarily with mini - invasive hybrid procedure (n=2); patients with multiple mvsds and pulmonary artery banding (n=3); children with coarctation of the aorta (coa) and muscular vsds or other complex cardiac lesions, who after initial coarctation repair and pulmonary artery banding (pa) finally underwent hybrid mvsd procedure (n=2); an infant with large perimembranous vsd and accompanying mvsd that had to be closed periventricularly after routine extracorporeal circulation (ecc) (n=1); patients with mvsd who are listed for primary hybrid mvsd closure (n=2); and 1 patient with mvsd, whose parents refused permission for treatment (n=1). This study is a part of ghhp, which was initiated in 2008 to introduce and develop mini - invasive hybrid techniques in our department . Inclusion criteria for patients with muscular septum defects were: infants with muscular vsd or multiple vsds, either isolated defects, or accompanied by other cardiac lesions necessitating operative repair; as well as patients after initial palliative treatment, who underwent pulmonary artery banding because of pulmonary overflow in early infancy . Free consent was obtained from well informed parents of all involved or potentially recruited patients in the outpatient clinic . We present 4 different strategies of hybrid mvsd closure: single - stage hybrid mvsd closure with the use of the amplatzer vsd occluder (aga med . Two - stage approach in patients with a complex multi - mvsds and neonatal critical aortic coarctation repair, when hybrid mvsd closure is the final procedure with surgical pa de - banding . One - stage hybrid approach as combination of open heart surgical closure of perimembranous vsd with a novel use of the amplatzer duct occluder ii (aga med . Routine 2-stage approach with pa banding, inducing septum hypertrophy and after reevaluation, a hybrid or percutaneous mvsd closure . The first group consisted of children with mvsds observed from birth, with significant left - to - right shunts (qp: qs>1.6:1) and signs of lv overload (lv enlargement, mitral insufficiency, left atrial enlargement, and poor contractility) on echo . In the second group were babies after critical coarctation repair supplemented with pa banding under direct pa pressure monitoring, systemic pressure, respiratory pco2 measurement, and peripheral saturation control . The definitive procedure with hybrid mvsd closure and pa de - banding was performed after meticulous evaluation of patients, including clinical status, peripheral desaturation, and echocardiographic symptoms of rv and septal hypertrophy . In the third group, perimembranous vsd was closed using ecc and muscular defects were closed periventricularly with devices during the same open heart procedure . The fourth group was babies with initial diagnosis of mvsd who underwent elective primary pa banding to prepare the patient for the final operation and pa de - banding when the defects will close after induced rv hypertrophy . Of the 11 involved patients, 2 were in generally good condition, and were regularly examined and observed as candidates for hybrid approach when the left - to - right shunt definitely appears hemodynamically significant . Criteria by which patients were excluded from periventricular device closure were the same as for standard ecc procedure (infections, contraindications for heparin administration, instable metabolic status, and renal and liver failure), as well as parental refusal to allow blood products transfusion or any cardiac procedures . The child referred for hybrid procedure is prepared for a classic cardiac procedure under general anesthesia in a routine fashion, with extracorporeal circulation ready to be commenced in case of any complications . After a classic median sternotomy, the heparin is administered in a half - dose as for cardiopulmonary bypass (1.5 mg / kg body weight) and a purse string suture is placed on the free wall of the right ventricle facing the location of the mvsd . The purse string site is precisely localized with simultaneous transesophageal echocardiography (tee) and epicardial echocardiography (ee), to find an optimal puncture point just opposite the largest muscular vsd . A gentle palpation on the right ventricle (rv) free wall is helpful in identifying the optimal location . A 6f gauge needle is inserted through the purse string suture on the rv free wall into the rv cavity, followed by a guiding wire inserted before removing the needle . The guiding wire is manoeuvred towards the mvsd to pass the septum to the lv, while the needle is removed (figure 1). It is crucial to localize the tip of the dilator to prevent any injury to the lv structures . Under tee on beating heart, the right disc is expanded in the right ventricle to commence closing the vsd (figure 2). The position of the implant is checked carefully before releasing the device with both tee and ee . The wires are removed and finally the purse string is tied (figure 3). After the operation, standard pediatric cardiac surgical perioperative monitoring is used routinely . The heparin infusion (under activated partial thromboplastin time control) is continued until an oral acetylsalicylic acid (10 mg / kg body weight / day) is administered for 36 months .. in the larger implants or an apical localization, the acetylsalicylic acid is continued longer than 6 months . The follow - up outcome after periventricular mvsd closure was analyzed in terms of success of the procedure, which is defined as adequate placement of the device, the lack of or trivial (<1 mm color jet width) residual shunt, with regard to any procedure - related late complications, patient status and psychosocial condition during the standard controls in the outpatient clinic . The child referred for hybrid procedure is prepared for a classic cardiac procedure under general anesthesia in a routine fashion, with extracorporeal circulation ready to be commenced in case of any complications . After a classic median sternotomy, the heparin is administered in a half - dose as for cardiopulmonary bypass (1.5 mg / kg body weight) and a purse string suture is placed on the free wall of the right ventricle facing the location of the mvsd . The purse string site is precisely localized with simultaneous transesophageal echocardiography (tee) and epicardial echocardiography (ee), to find an optimal puncture point just opposite the largest muscular vsd . A gentle palpation on the right ventricle (rv) free wall is helpful in identifying the optimal location . A 6f gauge needle is inserted through the purse string suture on the rv free wall into the rv cavity, followed by a guiding wire inserted before removing the needle . The guiding wire is manoeuvred towards the mvsd to pass the septum to the lv, while the needle is removed (figure 1). It is crucial to localize the tip of the dilator to prevent any injury to the lv structures . Under tee on beating heart, the right disc is expanded in the right ventricle to commence closing the vsd (figure 2). The position of the implant is checked carefully before releasing the device with both tee and ee . The wires are removed and finally the purse string is tied (figure 3). After the operation, standard pediatric cardiac surgical perioperative monitoring is used routinely . The heparin infusion (under activated partial thromboplastin time control) is continued until an oral acetylsalicylic acid (10 mg / kg body weight / day) is administered for 36 months .. in the larger implants or an apical localization, the acetylsalicylic acid is continued longer than 6 months . The follow - up outcome after periventricular mvsd closure was analyzed in terms of success of the procedure, which is defined as adequate placement of the device, the lack of or trivial (<1 mm color jet width) residual shunt, with regard to any procedure - related late complications, patient status and psychosocial condition during the standard controls in the outpatient clinic . In the total group of 11 patients with mvsd involved in ghhp, 6 children were qualified to hybrid trans - ventricular mvsd device closure . Mean age at time of hybrid procedure was 8.22 months (range: from 2.7 to 17.8 months, sd=5.1), mean body weight was 6.3 kg (range: from 3.4 to 7.5 kg, sd=1.5). An isolated mvsd appeared in only 2 children in this group, in every other case of muscular defects there were accompanying pathologies: perimembranous vsd with separate mvsd in 1 child and critical aortic coarctation (coa) in another 2 babies . In all but 1 patient, preoperative lv function was in the borderline normal range (lvef> 50%, sf> 40%), with clear evidence of pulmonary congestion in a routine chest x - ray in all infants . The size of mvsds ranged from 4 to 10 mm, with a median of 7 mm (sd=2.6). A single occluder (amplatzer vsd occluder or amplatzer duct occluder ii, aga med . Four patients, after hybrid mvsd closure, were extubated within the first 10 hours after the procedure, and 2 others required prolonged ventilation for up to 6 days due to preoperative heart failure . Six patients, after hybrid procedures, were discharged home in general good condition, no atrioventricular blocks, heart insufficiency, or pericardial effusions were observed . One patient (kk) required prolonged pleural drainage postoperatively (to postoperative day 4) due to pleural effusions . Mean follow - up is now 21 months (range: from 5 to 32 months, sd=9.6) after hybrid mvsd closure . All patients are free of any cardiac events and medication, with no neurocognition deficits or circulatory insufficiency . Residual shunts that were measured in outpatient echocardiography controls showed qp: qs<1.5:1, with the tendency to gradually decrease . In 1 girl (tt) (table 1) referred for surgical pa - debanding, who initially underwent coarctation repair and pa - banding, the left - to - right shunt appeared hemodynamically insignificant . Thus, after removing the pa banding, a hybrid procedure was abandoned . One patient (cw) (table 1) with isolated, hemodynamically significant mvsd, who was initially included into ghhp, at the age of 18 months underwent successful percutaneous mvsd closure with an amplatzer vsd occluder (aga med . Two children are still awaiting hybrid mvsd closure procedure, and 1 of them has been referred for pa banding . Parents of another girl, who received complete information about natural history and pathophysiology of mvsd, potentially adverse effects of hybrid operation, as well as of cessation of treatment, finally refused consent for any medical intervention . This report summarizes our single - centre experience in hybrid operations with periventricular muscular vsd closure . Our intention was to offer an alternative option for small, borderline babies, who are usually failing to thrive, with low body weight, and in individuals that have concomitant abnormalities that may require several staged interventions . After initial training and detailed preparation of therapeutic plans, we decided to undertake the challenge . The first successful case of intraoperative perventricular device closure on the beating heart in an infant was reported in 1998 . After that, a few authors have presented their initial experience in hybrid approach with promising results, and techniques are still evolving [813]. Following the literature on hybrid therapies, it appears that the advantages of surgical and interventional techniques, which are beneficial in borderline babies who do not meet criteria for surgery or cardiac intervention . Hybrid procedures have the advantage of avoiding cardiopulmonary bypass and complications of vascular access in small and hemodynamically unstable babies . Hybrid strategy is a reasonable alternative to transcatheter closure of mvsds, mainly because of high risk of significant complications and residual shunt when performed in small children . Also, prolonged exposure to radiation associated with this procedure carries long - term undesirable and potentially detrimental side - effects . The advantages of hybrid approach are closure of the defect under direct control, smaller risk of hemodynamic compromise and reduced device closure falls, as well as simultaneous possibility of other defects correction . In our group of patients, we performed other cardiac procedures simultaneously with the hybrid approach: surgical closure of perimembranous vsd or pa de - banding, with use of classical surgical approach via midline sternotomy . With regard to periventricular technique used for mvsd closure, the aid of an experienced echocardiographer is crucial to provide perfect imaging during procedure of implant placement, as well as for safe catheter manipulation, device delivery and deployment, and post - release position in the septum . All hybrid procedures were performed under tee guidance, but we also used an epicardial probe that was helpful in precise assessment of mvsd position and shape (figure 3). All steps of the procedure must be accomplished gently, with minimal manipulation and invasiveness, and the imaging must be perfect . There is no doubt that mvsds are frequently hidden within the rv trabeculations and therefore are difficult to localize through the standard surgical approach via the right atrium and tricuspid valve . Therefore, in 1 of the presented patients we closed perimembranous vsd using cardioplegic ecc, and after the failure to localize mvsd, we restored heart function and periventricularly closed mvsd on the beating heart with a vsd occluder . Although the device of choice in our experience is the amplatzer vsd occluder (aga med . Usa) with self - expandable double - discs made from nitinol wire mesh, the more suitable implant in this case (premature baby aged 2 months, body weight 3.4 kg) was the amplatzer duct occluder ii because its small discs, which does not interfere with intracardiac structures in a small heart . This could be an argument for the industry to provide more delicate ventricular occluding implants dedicated to very small patients . To the best of our knowledge there is still a lack of such devices available commercially . We follow the experience of gan et al, who suggest that in children with many small nearby mvsds, after the largest and most central defect device closure, the nearby smaller defect might become even smaller, making it difficult to pass the guidewire to the lv cavity . This might be due to the local compression of inserted device on the muscular bridges between smaller defects, and narrowing the diameter of unclosed defects . The body weight of hybrid patients less than 5.2 kg is thought to be associated with increased risk of complications . The smallest of our patients was 3.4 kg, a premature neonate who had successful hybrid mvsd closure without any complications . The most common complications reported in the literature on mvsd device closure are device embolization, cardiac perforation, and even intraoperative deaths . Other complications are transient loss of arterial pulse after the procedure, blood loss requiring blood transfusion, hematoma, complete heart block, ventricular tachyarrhythmia, hypotension, injury of the aortic valve, stroke, and device - related hemolysis [2,6,1618]. We believe that heart blocks can be avoided by careful patient selection and avoiding inlet type of defects . The limitations of our preliminary report are the small number of patients and short follow - up . Our promising results suggest the value of offering hybrid treatment to other patients, and encourage us to continue our ghhp . Our initial results demonstrate that hybrid procedures of periventricular muscular vsd closure appear feasible and effective for patients with unfavorable morphology, and who are unsuitable for classic surgical or interventional closure . A modern strategy combining cardiac surgery with interventional techniques provides patients with difficult clinical factors with the benefits of cooperation between cardiac surgeon and interventional cardiologist.
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It is capable of causing various clinical manifestations like pneumoniae, septicaemia, arthritis, abscess etc . And is associated with high morbidity and mortality . Cases have been reported from southeast asian countries like thailand, malaysia and vietnam etc . In india, most cases have so far been reported from the southern states like kerela and tamil nadu . Alhough not so uncommon in india but early and correct diagnosis and institution of proper antimicrobial therapy is important in order to reduce morbidity and mortality and have a favourable outcome . Here we report a case of melioidosis which probably was undiagnosed for long but was saved due to correct and timely intervention . He presented with swelling of ankles and pain, redness on right ankle for 7 days and fever, cough, breathlessness from last 1 month . Two months back he was treated for fever with ceftriaxone as his widal test was reactive . At about the same time, he was incidentally diagnosed with diabetes mellitus (dm). On treatment, though his symptoms subsided to some extent, during last 7 days he again complained of high fever, worsening breathlessness and loss of appetite . His body temperature was 102 f, blood pressure 90/70 mm hg, respiratory rate 50/min, and heart rate 128/min . Laboratory investigations revealed that multiple hematological and biochemical parameters were deranged [table 1]. His chest x - ray showed homogenous consolidation in upper left lobe and diffuse alveolar opacities in remaining part of lung [figure 1]. Chest x - ray showing left upper lobe consolidation post admission his condition further deteriorated and had clinical evidences of acute respiratory distress syndrome, deranged blood gases, and was in respiratory distress . His oxygen saturation was 78% . With this background, he was put on ventilator and started with piperacillin / tazobactum and clindamycin . On day 2 non fermenting pale colonies with metallic sheen were isolated next day on blood agar and mac conkey agar [figure 3]; the isolate was further processed on microscan walkaway 96 si . Gram negative bacilli with safety pin appearance on gram stain colonies of bukhlorderia pseudomallei on mac conkey agar on day 4, the isolate was identified as burkhlorderia pseudomallei, sensitive to imipenem, cotrimoxazole / sulfamethoxazole, tetracycline and resistant to ceftazidime . Later on, both his blood cultures and pus drained from right leg [figure 4] also grew b. pseudomallei . Based on the sensitivity report, antibiotic was changed to a combination of imipenem and doxycycline . Patient showed improvement, his fever subsided, total counts decreased, oxygen saturation was 100%, and was extubated on day 8 . Pus drained from right ankle his antibiotics were continued and after complete recovery and improvement of his liver and renal parameters he was discharged on day 20 . B. pseudomallei is an environmental inhabitant and is widely disseminated in soil, water, paddy fields, etc . It is geographically restricted to tropical and subtropical areas of australia and southeast asian countries . In india, quite a number of cases were reported though many are still underreported due to its protean manifestations . Most of these were reported from the southern part though melioidosis may be more widely prevalent . Two of the cases reported from tamil nadu actually originated from eastern part of india . Our patient was exposed to recent floods, which could be the source of infection . Review of cases in india active infection have been predisposed to occur in patients with many underlying conditions like dm, renal disease, and hiv postive . In our patient diabetes was an incidental finding during the course of investigation . Review of literature reveals successful treatment with a combination of ceftazidime and co - trimoxazole [table 2]. Our strain was resistant to ceftazidime and therefore patient was put on imipenem and doxycycline . Carbepenems have a better response against b. pseudomallei . The patient was put on maintenance therapy of doxycycline, trimethoprim sulfamethoxazole and is doing well . This case was probably missed due to lack of clinical awareness and correct microbiological diagnosis . A high index of suspicion is needed for diagnosis due to its varied clinical presentations . At the same time we were able to successfully treat the case by institution of correct antimicrobials based on microbiology feedback.
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As research in implantable biomaterial advances, the understanding and manipulation of cell - substrate interactions have increased in importance . One approach is to produce a more biomimetic construct that can recruit and control the patterning of functional cells to mimic the native tissue organization . For example, the aligned orientation of cells on extracellular matrix (ecm) plays an important role in several tissues including corneal stroma, tendons, bones, skeletal muscle, and, with significance to the present study, the vasculature . Here we demonstrate an intrinsically effective cell aligning surface fabricated from the biodegradable and cytocompatible polymers pcl, chitosan, and gelatin . The development of a small diameter vascular prosthesis (> 6 mm diameter) for arterial disease has been hampered by the mechanical compliance mismatch of the prosthesis and the native blood vessel . The mismatch is the key factor for the relatively rapid loss of patency compared to larger - diameter prostheses . In turn the mismatch is due to the fact that artificial prostheses do not mimic the layered structure of the native vessel, in which one of the layers has circumferentially aligned vascular smooth muscle cells (vsmcs) as well as extracellular matrix (ecm). The two predominant cell types within blood vessels, fibroblasts and vsmcs, could both functionally benefit cell - seeded prosthesis if recruited in an aligned orientation [1, 4]. Fibroblasts produce extracellular matrix such as collagen fibrils and elastin that confer to blood vessels most of their mechanical and structural properties . In the context of a vascular prosthesis it is beneficial to align the growth of cells to control the pattern of ecm deposition . If the scaffold of the prosthesis is biodegradable, then the construct will be eventually replaced by ecm with the desired orientation . These cells are integral to the vascular functioning through regulation of vessel tone and lumen diameter . Interestingly, these cells exist as two very distinct and changeable phenotypes: the contractile, characterized by a spindle shape and the abundant presence of alpha - smooth muscle actin (-sma), and the secretory (also referred to as synthetic), recognizable by rhomboid shape and reduced -sma . The contractile vsmc allows the changes that mediate blood pressure, by altering the vessel luminal diameter, and is not proliferative, whereas the secretory phenotype is associated with tissue remodeling, inflammation, and proliferation . The secretory phenotype is central to the pathology of neointimal hyperplasia and artery bypass failure . There is plasticity between the two states as they are not differentiation end - points [7, 8]. During the culturing of vsmc, freshly seeded vsmcs exist primarily in the contractile state, but over time the population shifts predominantly towards the secretory phenotype . For long - term patency, it is important to preserve the contractile phenotype, as only vsmcs in the contractile state are beneficial in fabricating cellularized tissue engineered blood vessel (tebv) because this phenotype minimizes the compliance mismatch . Previous studies already have demonstrated that the aligned orientation and phenotypic characteristics of cells can be guided by surface cues generated through micropatterning a surface with channels . These channels are often considerable wider and deeper than the dimensions of the cell, for example, 60 m deep and 300 m wide . Although these scaffolds encourage cell alignment the channel depth often prevents cell interaction across the interval . Cells are able to align on grooves as shallow as 150 nm; however deeper channels are more effective for cell alignment, down to depths of approximately 25 m, whereas, for groove width, as it increases cellular alignment decreases as cells lying centrally can longer sense the edges [13, 14]. Here, we construct a scaffold of parallel melt spun plc fibers . As the fiber width is 10 m, a scaffold of side - by - side fibers can be formed to orientate the cells within a groove of 10 m width with no overly large groove walls nor any extended interval between grooves, thus enabling intercellular contact . Early alignment studies employed nondegradable polymers such as channels on pdms films to produce aligned cell - orientating scaffolds . More recently, however, biodegradable polymers have been studied, since such scaffolds can be slowly replaced by native tissue and ecm which minimizes the issue of thrombosis . These polymers have been promoted as suitable for fabricating a vascular conduit and such an application is actively investigated by several groups [1620]. In this work, we have successfully produced a highly aligned melt spun pcl fiber scaffold that allows fibroblast and vsmc aligned attachment and also preserves the contractile -smooth muscle actin (-sma) expressing phenotype of the vsmc . This technique was also shown to be applicable for cell alignment on a prototype synthetic plc vascular conduit . Melt spinning of polycaprolactone was performed as described in an et al ., using a customized apparatus as shown in figure 1(a). In brief, powdered pcl (50 kda) was melted at approximately 120c and then was drawn by gravity to mandrel at 750 rpm . The fibers were adhered into place by dip coating in 5% chitosan (w / v) in acetic acid and then incubated for 5 days at room temperature to allow solvent evaporation . As a control, chitosan films were formed by coating the base of 6-well plates with 500 l of 5% (w / v) chitosan in acetic acid followed by incubation for 5 days at room temperature in a fume hood to allow solvent evaporation . A plc vascular graft was formed by dip coating a 5 mm diameter stainless steel mandrel in a 12% (w / v) plc solution dissolved on chloroform using a nima dc - mono 300 dip coating apparatus . The plc coating was incubated at room temperature for 48 hours to allow solvent evaporation . Subsequently the mandrel was placed into the melt spinning apparatus and a layer of circumferentially aligned pcl fibers was applied . The fibers were adhered using 5% chitosan solution as an adhesive as described above; following solvent evaporation the scaffold was coated in 1% gelatin to enhance cell attachment on the tubular surface . Ftir analysis was used to qualitatively characterize the functional groups introduced presented on the surface of the construct . Ftir spectra were collected with frontier ft - ir spectrometer (perkinelmer) at resolution of 4 cm and signal average of 16 scans in each interferogram over the range of 4000600 cm . Intermittent contact mode atomic force micrographs were obtained on jpk instruments nanowizard iii (aufgang c, germany) with a nanoprobe of 100 m length and a rotated monolithic silicon narrow cantilever with a force constant of 40 nm and a tapping frequency of 300 khz . The tapping mode afm conditions were as follows: scan rate was 0.4 hz, set point was 1.5 v, drive amplitude was set to 0.5 v, and the drive frequency was approximately 280 khz . Adjustments of the integral gain, proportional gain, and set point were done to minimize contact force and electronic noise as well as to maximize the features of the sample . The afm micrographs were evaluated by flattening the images (second - order) with the help of nanowizard data processing jpk software . Smooth muscle cells (smcs, lonza) or human fibroblasts were cultured up to the 8th passage in smooth muscle cell basal medium (smgm-2 media (lonza bioscience)) or fibrogro complete media (millipore), respectively . Pcl fiber films (1 1 cm) were sterilized with 70% ethanol for 1 h and then were washed away with pbs (three times). The cells were seeded on the pcl fiber films and a control surface at a density of 5 10 cells / cm . Cell - seeded fibers were cultured in a flat bottom 24-well plate for 7 days . A 5 cm section of either uncoated or aligned pcl fiber coated conduits was placed in a 6 cm diameter dish . Subsequently, 3 mls of a concentrated smc suspension (5 10 cells / ml) was slowly applied to the upper surface; after 10 min incubation at room temperature the tube was turned over and another 3 mls of the cell suspension was applied . The constructs were incubated over night at 37c in 5% co2; then the cells morphology was examined using fluorescence microscopy . The fluorescence was generated by 30-minute incubation with 2 m calcein am in pbs . Smcs in cell - seeded fibers at day 3 and day 7 were fixed in 4% paraformaldehyde for 30 min in room temperature . Following fixation, fibers were washed 3x with pbs, permeabilized with 0.1% triton x-100, and blocked using 2% bsa in pbs for 1 h at 4c . After washing 3x at room temperature in pbs and immunohistochemistry labeling on fibers and control (chitosan film) samples was performed, applying primary antibody against alpha - smooth muscle actin (monoclonal mouse anti - human), at 1: 100 dilutions in pbs / bsa / buffer at room temperature for 2 h. after washing 3 times for 10 min in pbs / bsa buffer, secondary antibodies (af 488 goat anti - mouse, invitrogen) were applied at 1: 200 dilutions in pbs / bsa buffer at room temperature for 1 h. cell nuclei were stained with dapi (4,6-diamidino-2-phenylindole) and pcl fibers were then imaged via confocal microscopy (leica, wetzlar, germany). The melt spin method produced a thin layer of highly aligned pcl fibers of 10 m width on a mandrel within approximately 15 minutes, creating aligning fibers of 10 m diameter (figures 1(b) and 1(c)). The pcl is melt spun into nonbonded, distinct fibers that readily unravels (figure 1(c)). Hence, the chitosan dip coating allows for an adhesive effect for the fibers to be removed from the mandrel as an aligned fiber mat (figure 1(d)). The fiber mat was examined by afm (figure 2(a)) and ftir (figure 2(b)). The ftir demonstrated a strong signal for the characteristic amide groups chitosan; hence the chitosan coats the fibers and becomes the principle substance cells will then interact with . The chitosan amide groups are known to help provide for a cytocompatible surface to the device . From the afm, it appears the fibers form curved grooves approximately 10 m and 10 m deep, a depth which allows cells to make contact with cells growing in neighboring grooves as seen in figures 3(a) and 3(c). In addition, the chitosan gives the scaffold a roughened surface feature, unlike the relative smooth surface of pristine pcl . Fibroblasts were seeded onto the fiber surface and readily aligned with the parallel fibers and became noticeably elongated compared to the control (figures 3(a) and 3(b)). These cells continued to proliferate until reaching 100% confluency, producing a continuous, aligned monolayer (not shown); hence the fibroblasts were able to sense each other across channels . Similarly for smcs, it was observed that after 7 days the cells adopted an elongated morphology following the orientation of the fiber, resembling the spindle - like appearance of the contractile cells (figure 3(c)). In comparison the smcs seeded on chitosan film grew without obvious orientation and present a rhomboid appearance akin to the secretory phenotype (figure 3(d)). Interestingly, unlike the fibroblasts, the fiber seeded smcs were not observed to proliferate towards confluency . The recently passaged smooth muscle cells (up to day 3) demonstrated positive staining by immunochemistry for the contractile phenotype marker protein -sma on both aligned fiber and nonaligning surface (figures 4(a) and 4(c)). However, after several days the protein was only detectable in cells on the aligned fiber surface . This observation indicates that the fiber induced both cellular orientation and prolonged -sma expression and thus promoted the retention of the contractile phenotype (figures 4(b) and 4(d)). The melt spun fibers could be readily used to pattern the surface of a prototype plc vascular conduit (figure 5(a)). It was observed 48 hours after seeding the fiber covered and fiberless plc tubes that vsmcs aligned longitudinally (figure 5(b)) on the fiberless construct whereas, on the fiber decorated tubes, the vsmc had orientated along the fibers, giving a circumferentially aligned, elongated morphology . Pcl based conduits are seen to have potential for the future fabrication of cellularized vascular prosthesis, with some successful clinical implantation . Smcs are critical for the proper functioning of a blood vessel; however the synthetic phenotype is often associated with inflammation and graft failure . Few attempts have been made to control smc behavior on a cell - seeded tube [24, 25]. Here we test techniques of surface guidance, known to influence cell behavior on 2d surfaces, for their circumferential application around a conduit tube as required on a pcl vascular prosthesis . We are able to create cell aligning grooves using a method of melt spinning pcl which successfully aligned the orientation of both fibroblasts and smcs and influenced the phenotype of smc . Furthermore, we have demonstrated that polymer tubing (fabricated from pcl) on a mandrel can be decorated with circumferentially aligned melt spun pcl . Fibers removed from the mandrel without the chitosan or gelatin bonding separated very readily . Furthermore polycaprolactone without modification has poor cell adhering properties, whereas chitosan with high deacetylation (8595% in this case) has very good cell recruiting qualities . The higher the deacetylation, the greater the amount of free cationic amino groups that encourage cell adhesion to the surface [26, 27]. The smooth pristine pcl also lacks the surface roughness that usually facilitates the establishment of cellular adhesion points, whereas the chitosan coating gives a considerably more roughened surface feature . Furthermore, the afm revealed that the chitosan covering does not fill in the space between fibers but instead leaves a distinct, curved groove that facilitates the aligned orientation of the cells . Previous studies have demonstrated that smcs are often seeded in a strongly contractile phenotype; then following a prolonged period of culture, the cells gradually become predominantly synthetic [7, 28]. Similarly in the present study we found smcs seeded on chitosan films also began with notable expression of the contractile marker -sma . This expression is considerably diminished after several days, implying a move towards the secretory . The cells seeded on the aligned fibers also showed substantial expression of -sma 24 hours after seeding, and this expression was retained after several days, indicating the preservation of the contractile state [28, 29]. The contractile phenotype in this study was assessed by the elongated cell morphology and -sma expression; the latter is an important marker due to its dramatic loss during phenotype change during in vitro culture . There are several other key characteristics and marker genes that determine the smc contractile phenotype . Chang et al . Demonstrated that smcs seeded on and elongating along similar micropatterned grooves expressed a greater number of contractile related genes and less proliferative related ones than smcs grown on flat surface, thus proving the grooves promote the retention of the contractile phenotype . The intracellular mechanism by which the contractile phenotype can be promoted in smc elongating with cell aligning channels has been examined . There is a milieu of signal transduction pathways affecting smc phenotype (as reviewed by). However studies have been carried out on the mechanism of phenotype determination by surface features . Chang et al . Detected strong activation of erk and fak, downstream signaling molecules from integrins, the surface adhesion proteins which sense the surface features . Thakar et al . They found a significant decrease in the expression of both the mrna and protein of the nuclear receptor nor-1 by elongated smcs . Fibroblasts were able to align and become confluent on the fibers whereas smcs differentiating towards contractile phenotype have a greatly reduced rate of proliferation [7, 8, 28]. To achieve confluency on the construct we expect the requirement to seed smcs at a high cell density, hence conferring immediate confluency . However, the method presented here has substantial advantages over electrospinning, such as the absence of the whipping action characteristic of electrospinning which reduces exact fiber alignment, as compared to melt spinning . To get a highly aligned structure by electrospinning a narrow rotating mandrel collector is used for fiber collection thus generating aligning surfaces of limited width and with multiple layers of piled fibers whereas our melt spinning method produces a scaffold of 10 cm length and 1-fiber thickness . However an important advantage of electrospinning over melt spinning is the delivery of cells within the fibers for immediate and simultaneous deposition onto the graft within cytocompatible polymers and solvents [34, 35]. This will become an important technique if it is found that the smcs adopt a contractile phenotype following delivery within circumferentially aligned electrospun fibers . Unlike most work on cell aligning fiber scaffolds, the current study employed micron - diameter scaffolds . It has been demonstrated that pcl can be melt spun into fibers with range of diameters from 10 to 200 m . The melt spun fibers we fabricate here have a diameter of 10 m; these generate interfiber grooves 10 m across, which matches the width of an elongated smc . In a comparable study, on microsized fibers produced by wet spinning of plga, it was found that as the diameter of fibers increases from 10 m to 242 m, the degree of orientation decreases . Hence the fibers we created by melt spinning are in the favorable width range for promoting cell alignment . In addition, the relatively low depth for the groove allows fibroblast and smcs to grow closely together as seen in figure 3, unlike other methods that produce much deeper channels or wider intervals which keep cells separated across channels . The melt spun fibers can be used to circumferentially align the smcs on a prototype plc vascular prosthesis in an elongated contractile - like phenotype thus demonstrating the effectiveness of the technique to orientate the cell both on flat 2d film and also circumferentially on the outer surface of a tube . This ability of fabricating and controlling alignment patterns on tissue engineering scaffolds will aid the production of a more physiologically relevant representation of natural tissue . Moreover, since the melt spun technique is delivered to a mandrel, it can be adapted to introduce circumferentially aligned pattern on the outer surface of a synthetic polymer tebv, when placed in the position of the rotating mandrel . This allows the alignment of cells that comprise the outer vasculature layers, that is, fibroblasts and smcs . We aim to eventually achieve the coating of fully functional, vasoresponsive smcs for the structure . However, smcs can exist at various points of differentiation between the two phenotypes . Through contact guidance features to achieve fully functional contractile smcs, we expect to involve additional steps including seeding at high density, optimized cell harvesting techniques (enzyme digestion rather than outgrowths), growth factors (such as tgf-), serum optimization, smc source, and stimulation with pulsatile force . It is unlikely that only one of these approaches in isolation can achieve the formation of a vasoresponsive smc medial layer; hence combinations may be more effective [28, 38]. In summary, the combination of aligned melt spun micron - sized fibers with a polymeric adhesive (chitosan) is a very promising substrate that enables the retention of the desired contractile phenotype of smooth muscle cells . Such a construct may be advantageously applied onto tubular constructs for forming a biomimetic blood vessel that will potentially have superior compliance matching with the native vessel.
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Parasites - a strain of a. cantonensis has been maintained in our laboratory in biomphalaria glabrata snails and sprague - dawley rats since 1980 (wang et al . Young adults and adult worms were collected on days 21 and 50 post - infection, respectively, from the cerebral tissues and pulmonary arteries of rats . The sex of these worms was determined based on their morphological characteristics: male worms are usually shorter and exhibit copulatory bursa and long spicules . Infective larvae were collected from the tissues of infected snails through digestion with 0.6% (w / v) pepsin - hcl (ph 2 - 3) for 1 h. these worms were washed with normal saline, phosphate buffered saline and distilled water and then stored at -80c for further analyses (hwang et al . This experiments performed in this study followed the recommendations of the institutional animal care and use committee of chang gung university . Small rna library construction and high - throughput sequencing - total rna was isolated from young adults of a. cantonensis (500 worms of each sex) using the tri reagent, according to the instructions of the manufacturer (molecular research center, cincinnati, oh, usa). Rna integrity was assessed via 1% (w / v) agarose gel electrophoresis, while rna purity was determined based on the absorbance recorded at 260/280 nm using an sma1000 uv spectrophotometer (merinton technology, beijing, china). Rna fragments of 18 - 30 nt in length were separated from total rna using the small rna sample prep kit (illumina, san diego, ca, usa). Following 15% novex tbe - urea polyacrylamide gel electrophoresis (page), the purified fragments were ligated to 5 and 3 rna adaptors, reverse transcribed to produce single - stranded cdnas and amplified via pcr . Rna fragments (approximately 92 bp) were isolated from the pcr products and sequenced using the illumina hiseq 2000 system (illumina, san diego, ca, usa). Bioinformatic analysis - the small rna library was analysed through a deep - sequencing small rna analysis pipeline (dsap) (dsap.cgu.edu.tw) (huang et al . 2010). Following the removal of adaptors and poly - a / t / c / g / n nt, sequences longer than 16 nt were considered clean sequence tags . Using the clustering module of dsap, the reads for each unique tag were counted as its expression abundance . To identify transfer rnas (trnas), ribosomal rnas (rrnas), small nucleolar rnas (snornas), small nuclear rnas (snrnas) and other annotated non - coding rnas (ncrnas), the unique tags were subjected to searches against the transcribed sequence library of the ncrna (rfam) database (version 10.0). The remaining sequences were compared with mature mirna sequences in mirbase (version 16) using blastn . Stem - loop rt - pcr - total rnas were isolated from infective larvae (20,000 worms), young adults (500 worms of each sex) and adult worms (50 worms of each sex). The expression levels of aca - mir-1 - 1 and aca - mir-71 - 1 were determined via modified stem - loop rt - pcr (chen et al . The stem - loop reverse transcription primer and forward primer for aca - mir-1 - 1 were 5-gtcgtatccagtgcagggtccgaggtattc - gcactggatacgactacatac-3 and 5-cgcggc - tggaatgtaaagaagt-3, respectively, and those for aca - mir-71 - 1 were 5-gtcgtatccagtgcagggt - ccgag - gtattcgcactggatacgaccgtctca-3 and 5-gcgcggtgaaagacatgg - gtagtg-3, respectively . The reverse primer employed in these assays was 5-gtgcagggtccgaggt-3. Small rnas from a. cantonensis at different developmental stages were extracted using an mirna isolation kit (geneaid, sijhih city, taipei county, taiwan) according to the instructions of the manufacturer . First - strand cdnas were synthesised using superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa). Briefly, reaction mixtures (20 l) containing the purified small rnas (160 ng), 5x first - strand buffer (4 l), individual specific stem - loop rt primers (0.5 m), dntp mix (0.5 mm), dtt (5 mm), superscript iii reverse transcriptase (10 units) and rnaseout (2 units) were incubated at 55c for 50 min, then inactivated by heating at 70c for 15 min and subsequently incubated with two units of rnase h at 37c for 20 min . Quantitative rt - pcr was performed using realq pcr master mix (ampliqon a / s, skovlunde, denmark) in the stratagene mx3000p qpcr system (agilent technologies, santa clara, ca, usa). The pcr mixtures (20 l) included the reverse transcription product for each mirna (3 l), realq - pcr 2x master mix (10 l), a specific forward primer (0.5 m) and the reverse primer (0.5 m). The rt - pcr assays were carried out with an initial step at 95c for 10 min, followed by 40 cycles of amplification, with denaturation at 95c for 30 s, primer annealing at 58c for 1 min and elongation at 72c for 30 s. the specificity of this assay was confirmed via 15% page . Finally, the expression levels of the targeted mirnas were determined through three rounds of stem - loop rt - pcr amplification and analysed according to the 2 method (livak & schmittgen 2001). Statistical analysis - the expression levels of the mirnas were expressed as the mean standard deviation . Means were compared via one - way anova and the least significance difference test was used for post - hoc multiple range comparisons . Analysis of short rnas - a total of 22,484,156 raw sequence reads were obtained through high - throughput sequencing from the small rna library generated for young adults of a. cantonensis . The removal of adapters, contaminated nt and low - quality sequences resulted in 16,880,456 (75.1%) high - quality, clean reads of 16 - 31 nt in length, averaging 22.8 nt . A majority of the sequences were 23 nt in length (46.1%), followed by lengths of 22 nt (26.3%) and 24 nt (12.1%) sequences . Among the clean reads, 10,766,590 (63.8%) they included 7,736,727 trnas (45.8%), 1,191,869 rrnas (7.1%), 228,262 snornas (1.4%), 174,633 snrnas (1%) and 1,435,099 ncrnas (8.5%) (fig . 1: classification of small rnas of angiostrongylus cantonensis young adults identified by a deep - sequencing approach . Mirna: micrornas; ncrna: non - coding rnas; rrna: ribosomal rnas; snorna: small nucleolar rnas; snrna: small nuclear rnas; trna: transfer rnas . Identification of conserved mirnas - from the remaining 7,548,965 clean reads, 252 conserved mature mirnas including 10 antisense mirnas were identified based on comparison with entities in mirbase . All of the identified mirnas were homologous to mirnas from metazoa and their lengths ranged from 20 - 24 nt, with most being 22 nt in length (117), followed by lengths of 21 nt (54), 23 nt (49), 20 nt (19) and 24 nt (13). These mirnas belonged to 90 families, in addition to which 10 antisense mirnas were discovered . The number of reads obtained was 50 or more for 53 of these mirnas, 21 - 49 for 28 mirnas, 11 - 20 for 14 mirnas, two-10 for 82 mirnas and only a single read was observed in the remaining 75 mirnas . Supplementary data lists the 53 mirnas with 50 or more reads, which belonged to 25 families . More than one mirna was identified from the following families: let-7, mir-1, mir-34, mir-87, mir-71 mir-99, mir-2, mir-9, mir-31, mir-50 and mir-103 . Aca - mir-1 - 1 exhibited the highest number of reads, at 184,946, followed by aca - mir-71 - 1 (92,433). These two highly expressed mirnas constituted 50.7% and 25.3% of the total reads (365,112), respectively, among this group of 53 mirnas . Phylogenic distribution - to analyse the evolutionary features of a. cantonensis mirnas, the identified mirnas showing 50 or more reads, from 25 families, were compared with those from other nematodes in mirbase . The families let-7, mir-1, mir-2, mir-9, mir-31, mir-34, mir-44, mir-50, mir-67, mir-71, mir-81, mir-87 and mir-124 contained members that were homologous to parasitic nematodes (ascaris suum and brugia malayi) and free - living nematodes (c. elegans, caenorhabditis briggsae, caenorhabditis remanei and pristionchus pacificus). Members of the mir-60 and mir-235 families were homologous to sequences from free - living nematodes, but not parasitic nematodes . The family mir-99 was homologous only to a. suum, while members of the mir-21, mir-29, mir-30, mir-103, mir-140, mir-146, mir-185, mir-191 and mir-320 families did not exhibit homology with sequences from other nematodes (table). Tablephylogenic distributions of conserved micrornas families of angiostrongylus cantonensis young adults with 50 or more readsfamily a. cantonensis ascaris suum brugia malayi caenorhabditis elegans caenorhabditis briggsae caenorhabditis remanei pristionchus pacificus let-7+++++++mir-1+++++mir-2+++++mir-9+++++++mir-21+mir-29+mir-30+mir-31++++mir-34++++++mir-44++++++mir-50++++++mir-60++++mir-67+++++mir-71++++++mir-81+++++mir-87+++++++mir-99++mir-103+mir-124+++++++mir-140+mir-146+mir-185+mir-191+mir-235+++mir-320+ expression of mirnas at different developmental stages - to confirm the expression of the mirnas from a. cantonensis young adults that were identified through the applied high - throughput approach and to determine the expression levels of these mirnas at different developmental stages, the levels of aca - mir-1 - 1 and aca - mir-71 - 1 transcripts were validated in a modified stem - loop quantitative rt - pcr analysis . Significant differences in the expression levels of these mirnas were observed among different developmental stages (aca - mir-1 - 1: f = 521.55, p <0.001; aca - mir-71 - 1: f = 1585.86, p <0.001). In male worms, the expression of the two mirnas was significantly higher in young adults than in infective larvae or adult worms (p <0.05). Additionally, the expression in adult worms was significantly higher than in infective larvae (p <0.05). In female worms, significantly higher expression was also observed in young adults (p <0.05). However, there was no significant difference in aca - mir-1 - 1 expression observed between infective larvae and adult worms (p> 0.05) and the infective larvae exhibited significantly higher aca - mir-71 - 1 expression than adult worms (p <0.05). Moreover, male worms displayed significantly higher expression than female worms in both the young adult and adult stages (p <0.05) (fig . 2:expression profiles of selected micrornas of angiostrongylus cantonensis young adults in different developmental stages . Fa: female adults; fya: female young adults; il: infective larvae; ma: male adults; mya: male young adults . Using a deep - sequencing approach, based on 7,548,965 reads, we identified and characterised 252 conserved mature mirnas including 10 antisense mirnas that belonging to 90 families from young adults of a. cantonensis . Previous authors identified mirnas in adult male and female worms from 592,899 and 458,447 reads, respectively (chen et al . However, no novel mirnas were discovered in either the present or previous studies . The sequences obtained from the adult male and female worms were matched to the c. elegans genome, although the percentage of perfect matches was quite low (18.94% in females and 22.58% in males). Although c. elegans and a. cantonensis are both nematodes, the former species is free - living in soil or water, whereas the latter is parasitic, being found in the pulmonary arteries of its definitive host and requiring a mollusc as an intermediate host (chen et al . We hypothesise that novel mirnas will be identified only when a reference genome for a. cantonensis becomes available . In the present study, we identified nine mirnas in young adults of a. cantonensis displaying more than a 1,000 reads: two in the let-7 family, four in the mir-1 family, one in the mir-44 family, one in the mir-71 family and one in the mir-99 family . In male adult worms, seven mirnas exhibited more than 1,000 reads: mir-1, mir-228, mir-44, mir-45, mir-71, mir-72 and mir-81 . In the female worms, 10 mirnas showed more than a 1,000 reads: mir-1, mir-2, mir-228, mir-44, mir-45, mir-60, mir-71, mir-72, mir-81 and mir-87 (chen et al . Mirr-1, mirr-71 and mir-44 show high expression in both the young adult and in adult stages . In contrast, let-7 and mir-99 are highly expressed only in young adults, whereas mir-45 and mir-81 are expressed only in adult worms . These findings indicate that a stage - specific expression of mirnas occurs in a. cantonensis . Moreover, the regulatory functions of mir-99 require further investigation . Let-7 and lin-4 are two important mirna families associated with the lifespan of c. elegans . These mirnas have been characterised as playing an essential role in the developmental timing of the worm by downregulating specific targets, such as the trim protein lin-41 and the transcription factor lin-14 (ibez - ventoso et al . Let-7 is an mirna families that was discovered in ancient animals (christodoulou et al . The let-7 family is expressed in a wide range of animals and the sequences of its members are highly conserved . However, significant variations in the size of the let-7 family occur among organisms: of the 55 organisms known to express members of the let-7 family, 13 express only one let-7 mirna, including the nematodes p. pacificus, c. remanei, c. elegans, c. briggsae and b. malayi, whereas 19 mature let-7 sequences have been identified in the zebrafish (danio rerio) (pasquinelli et al . 2003). In the present study, we identified 19 members of the let-7 family in young adults of a. cantonensis, 11 of which exhibited 50 or more reads . In adult worms, the copy number of this mirna was determined to be less than 50 in both sexes . These finding suggests the importance of let-7 in gene regulation in young adults of a. cantonensis . However, understanding the role of let-7 in the development of this parasite will require further studies . Among the mirna families found to be expressed in young adults of a. cantonensis, mir-1 displayed the highest number of total reads . Members of this family have been found in a wide range of organisms, including worms, flies, fishes, mice and humans (bentwich et al . They are evolutionarily conserved and have been characterised as playing essential roles in regulating proliferation and the differentiation of muscle development via the regulation of synaptic transmission (simon et al . 2008, the mir-71 family presented the highest number of reads in both sexes (chen et al . This family has been reported to promote longevity and stress resistance in worms (pincus et al . 2011) and is involved in the sexual maturation of female worms (gomes et al . These expression patterns suggest the different roles of mirnas at different developmental stages . Although the phylogenetic distribution has been reported for clonorchis sinensis (xu et al . 2010), there is no such information available for parasitic nematodes . The 25 conserved mirna families found in a. canto - nensis young adults showed a distribution bias . These conserved families can be divided into four groups: 13 families were homologous to sequences of other parasitic nematodes, two to sequences of free - living nematodes, but not parasitic nematodes, and one to a. suum sequences . Nine families did not exhibited any member that was homologous to either a free - living or parasitic nematode . Moreover, these mirnas observed in young adults of a. cantonensis were not found in adult worms (chen et al . It is possible that the first two groups regulate general biological or physiological functions in nematodes . The mirnas showing homology to a. suum may be specific to parasitic nematodes . As adult worms of a. cantonensis live within the central nervous system, the last group may regulate adaptive functions of worms related to this special environment, which may also cause pathological changes in the central nervous system . Analysis of the levels of aca - mir-1 - 1 and aca - mir-71 - 1 expression via the stem - loop rt - pcr revealed different expression patterns based on developmental stages and sex . These two mirnas were selected because they are highly expressed not only in young adults, but also in adult worms of both sexes . In both male and female worms, the level of expression of these mirnas increased dramatically from the levels observed in infective larvae and peaked in young adults, subsequently declining to a low level in adult worms . Overall, the expressions levels of these mirnas were found to be highest in male adult males . Similar expression patterns have been reported for 18 mirnas in adult worms (chen et al . . These mirnas may be important in regulating sex differentiation, rather than developmental stages . The lower expression levels of these mirnas observed in female worms indicate that females may require a lower degree of post - transcriptional regulation than male worms . Moreover, the higher expression levels of the mirnas detected in young adults suggest that more significant changes may occur during the young adult stage than in the adult stage . Replicate analyses were difficult in the present study because of the technical difficulties involved in obtaining sufficient sample sizes from infected animals . However, we identified 53 mirnas, belonging to 25 families, that displayed 50 or more reads . These findings provide reliable information about the global mirna expression profiles found in a. cantonensis . Although northern blotting is considered a gold - standard approach for detecting mirnas, this method is limited by its low sensitivity and difficulties in distinguishing homologous mirnas from highly similar sequences (van rooij 2011, pritchard et al . Moreover, we succeeded in confirming the expression of two mirnas initially identified via the high - throughput approach in a. cantonensis young adults through the more sensitive and specific technique of stem - loop quantitative rt - pcr . Based on the results of the present study, there are significant differences in the expression of mirnas between young adults and adult worms of a. canto - nensis . These differences are not only qualitative, but also quantitative . In the present study, we identified nine mirna families without homologous members in the available sequences of other nematodes in the adult stage . Moreover, the expression levels of mir-1 and mir-71 increase from a low expression level in infective larvae to a peak in young adults and subsequently decrease in adult worms . These results suggest that mirnas play a more important role in the regulation of biological functions in young adults than in adult worms of a. cantonensis.
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Pleural effusion (pe) is one of the most common diagnostic problems in clinical practice . Distinguishing an exudate from a transudate is the first step in the diagnostic approach of a patient with pe [2, 3]. Currently, the criteria proposed by light et al, have been generally accepted for this discrimination . However, these criteria misclassified 20% to 30% of transudates as exudates that occur mostly in patients receiving diuretic therapy . Several parameters such as pleural fluid (pf) cholesterol level, pf to serum cholesterol ratio, pf to serum bilirubin concentration ratio, alkaline phosphatase value, pleural cholinesterase, pf to serum cholinesterase ratio and serum - pleural effusion albumin gradient have been proposed in segregating the transudates from exudates more reliably than those of light's criteria . Pe can be caused by several mechanisms including increased permeability of the pleural membrane capillaries, increased pulmonary capillary pressure, decreased negative intrapleural pressure, decreased oncotic pressure and obstructed lymphatic flow . . Increased permeability of the pleural microvasculature is generally attributed to factors that are released in inflammatory pleural diseases . Angiopoietins (ang) play an important role in angiogenesis occurring under both physiologic and disease conditions . Ang-1 and ang-2 function as ligands for tie2, which is a receptor tyrosine kinase specifically expressed on endothelial cells [1315]. Ang-1 stabilizes blood vessels by promoting the interaction between endothelial cells and the surrounding extracellular matrix . Ang-2 antagonizes the stabilizing action of ang-1 by binding to tie2 competitively, which destabilizes vessels . Ang-1 has anti - inflammatory and anti - permeability properties; it blocks the expression of adhesion molecules on the endothelial cell surface, leukocyte adherence on endothelial cells and transmigration into tissues, and interleukin-8 production by endothelial cells . In addition, ang-1 inhibits vascular permeability caused by vascular endothelial growth factor (vegf) and inflammatory agents [1620]. Ang-2 destabilizes the endothelial cell monolayer integrity leading to the detachment of endothelial cells in vitro . Furthermore, ang-2 was found to induce edema formation and to exert a weak stimulatory effect on leukocyte migration when injected into a mouse paw . The aim of this study was to evaluate the application of pleural and serum angiopoietin-1 and 2 in categorizing pes into exudates and transudates in children . Our study was performed on 80 children [categorized as exudates or transudates according to the criteria of light in chest unit of pediatric department and in chest department, zagazig university hospital and outpatient clinics in the same hospital between november 2006 and april 2008 . Forty of all cases were defined as transudates and sub classified as regards etiology to heart failure (15 patients), renal hydrothorax (13 patients) and liver cirrhosis (12 patients). In exudative effusion group (40 patients) there were 11 tuberculous effusions, 17 non - tuberculous para - pneumonic effusions and 12 empyema patients . The diagnosis of heart failure was made on clinical grounds based on history, physical examination, chest radiograph, electrocardiogram with response to diuretic therapy and confirmed by echocardiographical evidence of left ventricular systolic dysfunction (left ventricular ejection fraction 40%), evidence of severe valvular disease or evidence of severe left ventricular diastolic dysfunction according to the american heart association guidelines .hepatic hydrothorax was defined as transudative effusion due to cirrhosis in presence of ascites with absence of any other causes of pe.renal hydrothorax (nephrotic patients) was defined as transudative effusion due to renal dysfunction (presence of heavy proteinuria, hyperlipidemia, hypoalbuminemia and edema) in absence of any other causes of pe.tuberculous pleuritis was diagnosed if ziehl - nelseen stains (three cases) or lowenstein jensen cultures of pf (three cases), or sputum (two cases) were positive or an exudative lymphocytic effusion with high adenosine deaminase level (> 40 u / l) (three cases) cleared in response to antituberculous therapy.parapneumonic effusions refer to patients presented with pleural exudates and associated with bacterial pneumonia which was diagnosed by clinical, radiological and laboratory findings (including positive bacterial culture in pleural fluids).empyema was considered to be present if there was a finding of a pe on the chest radiograph coupled with aspiration of frank pus . The diagnosis of heart failure was made on clinical grounds based on history, physical examination, chest radiograph, electrocardiogram with response to diuretic therapy and confirmed by echocardiographical evidence of left ventricular systolic dysfunction (left ventricular ejection fraction 40%), evidence of severe valvular disease or evidence of severe left ventricular diastolic dysfunction according to the american heart association guidelines . Hepatic hydrothorax was defined as transudative effusion due to cirrhosis in presence of ascites with absence of any other causes of pe . Renal hydrothorax (nephrotic patients) was defined as transudative effusion due to renal dysfunction (presence of heavy proteinuria, hyperlipidemia, hypoalbuminemia and edema) in absence of any other causes of pe . Tuberculous pleuritis was diagnosed if ziehl - nelseen stains (three cases) or lowenstein jensen cultures of pf (three cases), or sputum (two cases) were positive or an exudative lymphocytic effusion with high adenosine deaminase level (> 40 parapneumonic effusions refer to patients presented with pleural exudates and associated with bacterial pneumonia which was diagnosed by clinical, radiological and laboratory findings (including positive bacterial culture in pleural fluids). Empyema was considered to be present if there was a finding of a pe on the chest radiograph coupled with aspiration of frank pus . The following biochemical parameters were determined simultaneously in all samples [serum and pleural fluid] including total protein level, ldh concentration and total cholesterol concentration . Pleural fluid / serum protein ratio, pleural fluid / serum ldh ratio and pleural fluid / serum cholesterol ratio were calculated . Light's criteria were applied (the fluid was defined as exudates if it fulfilled at least one of the following criteria: pleural / serum ratio of total proteins greater than 0.5; pleural / serum ratio of total lactate dehydrogenase greater than 0.6 or pleural lactate dehydrogenase greater than two thirds of upper limit of normal for serum lactate dehydrogenase [i.e.> 400 iu / l according to the kits used). Pf cholesterol as well as the serum - pf gradients for albumin and total proteins (serum albumin or protein concentration minus pf albumin or protein concentration) were determined in all samples . A pf cholesterol 45 mg / dl and a serum - effusion albumin and protein gradient 12g / l and 31g / l respectively were consistent with a transudate . All these biochemical measurements in serum and pf specimens were carried out on hitachi autoanalizer [mode l902, japan] using standarized photometric method specifically, total protein was measured by biuret method and ldh by an optimized uv kinetic method . Pf ph was measured using arterial blood gas machine just after aspiration of pleural fluid . Blood for measurement of protein, ldh, cholesterol was drawn at the same time as pleural fluid . All pf samples underwent biochemical, cytological and microbiological analysis shortly after thoracocentesis . Both pf and serum samples were centrifuged at 4c; aliquots of supernatant were frozen at 80c until ang-1 and ang-2 levels were estimated . The levels of ang-2 in pe and serum were measured by enzyme - linked immunosorbent assay using a duoset methodology [r&d systems; minneapolis, mn]. Ang-1 protein levels were measured by a sandwich noncompetitive enzyme - linked immunosorbent assay consisting of a primary mouse antihuman ang-1 antibody and a secondary biotinylated goat antihuman ang-1 antibody [both from r&d systems]. The minimum detectable dose of the assays for ang-1 and ang-2 were 200 and 65 pg / ml respectively . In this study, all patients with effusions of undetermined origin, effusions with more than one cause, chylothorax and hemothorax were excluded . Chi - square test, student's t - test and analysis of variance (anova) test were used . Multiple comparison analysis by the least significant difference (lsd) was used to detect statistical difference between two means when anova test referred to significances . Receiver operating characteristic (roc) curve analysis was used to determine the discriminative properties of various cutoff levels of serum and pleural ang-2 levels our study was performed on 80 children [categorized as exudates or transudates according to the criteria of light in chest unit of pediatric department and in chest department, zagazig university hospital and outpatient clinics in the same hospital between november 2006 and april 2008 . Forty of all cases were defined as transudates and sub classified as regards etiology to heart failure (15 patients), renal hydrothorax (13 patients) and liver cirrhosis (12 patients). In exudative effusion group (40 patients) there were 11 tuberculous effusions, 17 non - tuberculous para - pneumonic effusions and 12 empyema patients . The diagnosis of heart failure was made on clinical grounds based on history, physical examination, chest radiograph, electrocardiogram with response to diuretic therapy and confirmed by echocardiographical evidence of left ventricular systolic dysfunction (left ventricular ejection fraction 40%), evidence of severe valvular disease or evidence of severe left ventricular diastolic dysfunction according to the american heart association guidelines .hepatic hydrothorax was defined as transudative effusion due to cirrhosis in presence of ascites with absence of any other causes of pe.renal hydrothorax (nephrotic patients) was defined as transudative effusion due to renal dysfunction (presence of heavy proteinuria, hyperlipidemia, hypoalbuminemia and edema) in absence of any other causes of pe.tuberculous pleuritis was diagnosed if ziehl - nelseen stains (three cases) or lowenstein jensen cultures of pf (three cases), or sputum (two cases) were positive or an exudative lymphocytic effusion with high adenosine deaminase level (> 40 u / l) (three cases) cleared in response to antituberculous therapy.parapneumonic effusions refer to patients presented with pleural exudates and associated with bacterial pneumonia which was diagnosed by clinical, radiological and laboratory findings (including positive bacterial culture in pleural fluids).empyema was considered to be present if there was a finding of a pe on the chest radiograph coupled with aspiration of frank pus . The diagnosis of heart failure was made on clinical grounds based on history, physical examination, chest radiograph, electrocardiogram with response to diuretic therapy and confirmed by echocardiographical evidence of left ventricular systolic dysfunction (left ventricular ejection fraction 40%), evidence of severe valvular disease or evidence of severe left ventricular diastolic dysfunction according to the american heart association guidelines . Hepatic hydrothorax was defined as transudative effusion due to cirrhosis in presence of ascites with absence of any other causes of pe . Renal hydrothorax (nephrotic patients) was defined as transudative effusion due to renal dysfunction (presence of heavy proteinuria, hyperlipidemia, hypoalbuminemia and edema) in absence of any other causes of pe . Tuberculous pleuritis was diagnosed if ziehl - nelseen stains (three cases) or lowenstein jensen cultures of pf (three cases), or sputum (two cases) were positive or an exudative lymphocytic effusion with high adenosine deaminase level (> 40 parapneumonic effusions refer to patients presented with pleural exudates and associated with bacterial pneumonia which was diagnosed by clinical, radiological and laboratory findings (including positive bacterial culture in pleural fluids). Empyema was considered to be present if there was a finding of a pe on the chest radiograph coupled with aspiration of frank pus . The following biochemical parameters were determined simultaneously in all samples [serum and pleural fluid] including total protein level, ldh concentration and total cholesterol concentration . Pleural fluid / serum protein ratio, pleural fluid / serum ldh ratio and pleural fluid / serum cholesterol ratio were calculated . Light's criteria were applied (the fluid was defined as exudates if it fulfilled at least one of the following criteria: pleural / serum ratio of total proteins greater than 0.5; pleural / serum ratio of total lactate dehydrogenase greater than 0.6 or pleural lactate dehydrogenase greater than two thirds of upper limit of normal for serum lactate dehydrogenase [i.e.> 400 iu / l according to the kits used). Pf cholesterol as well as the serum - pf gradients for albumin and total proteins (serum albumin or protein concentration minus pf albumin or protein concentration) were determined in all samples . A pf cholesterol 45 mg / dl and a serum - effusion albumin and protein gradient 12g / l and 31g / l respectively were consistent with a transudate . All these biochemical measurements in serum and pf specimens were carried out on hitachi autoanalizer [mode l902, japan] using standarized photometric method specifically, total protein was measured by biuret method and ldh by an optimized uv kinetic method . Pf ph was measured using arterial blood gas machine just after aspiration of pleural fluid . Blood for measurement of protein, ldh, cholesterol was drawn at the same time as pleural fluid . All pf samples underwent biochemical, cytological and microbiological analysis shortly after thoracocentesis . Both pf and serum samples were centrifuged at 4c; aliquots of supernatant were frozen at 80c until ang-1 and ang-2 levels were estimated . The levels of ang-2 in pe and serum were measured by enzyme - linked immunosorbent assay using a duoset methodology [r&d systems; minneapolis, mn]. Ang-1 protein levels were measured by a sandwich noncompetitive enzyme - linked immunosorbent assay consisting of a primary mouse antihuman ang-1 antibody and a secondary biotinylated goat antihuman ang-1 antibody [both from r&d systems]. The minimum detectable dose of the assays for ang-1 and ang-2 were 200 and 65 pg / ml respectively . In this study, all patients with effusions of undetermined origin, effusions with more than one cause, chylothorax and hemothorax were excluded . Chi - square test, student's t - test and analysis of variance (anova) test were used . Multiple comparison analysis by the least significant difference (lsd) was used to detect statistical difference between two means when anova test referred to significances . Receiver operating characteristic (roc) curve analysis was used to determine the discriminative properties of various cutoff levels of serum and pleural ang-2 levels . Causes of pes in 80 patients, who were included in this study, are demonstrated in table 1 . There were no significant differences as regards age, weight and gender of the studied cases (p=0.1, p=0.1 and p=0.09, respectively) (table 2). Biochemical profile of pleural fluids showed significant differences between transudates and exudates (p <0.01) (table 2). Different causes of pleural effusion in our study demographic, clinical and pleural fluid data of the studied cases ldh: lactate dehydrogenase; rbc: red blood cell pf ang-2 levels were significantly higher in pleural exudates than in transudates (p=0.01) (table 3). Pf ang-2 levels were higher than serum ang-2 levels in patients with pleural exudates and patients with transudates and that was highly significant (p<0.001)(table 3). Pf ang-1 levels were significantly lower than serum ang-1 levels both in patients with exudates and those with transudates (p<0.001), otherwise, we did not detect significant differences between our groups as regards pf and serum ang-1 levels (p=0.2 and p=0.09, respectively)(table 3). Angiopeptin 1 and 2 levels in patients with pleural exudates and pleural transudates we also compared various etiologies of pe for differences in pf ang-2 levels which were significant (p=0.01). Ang-2 levels were higher in tuberculous than in non - tuberculous pneumonic pes and empyema exudates (p=0.02). By contrast, there were no significant differences in pleural ang-2 levels between patients with pleural transudates due to different etiologies (p>0.05) (table 4). Mean level (standard deviation) of angiopeptin 2 in patients with pleural effusions of different etiologies anova and multicomparison analysis with lsd (the least significant difference) ang: angiopeptin; anova: analysis of variance aa, bb= non - significant (p>0.05); ab, ac, ad = significant (p<0.05). Although serum ang-2 levels were significantly higher in patients with exudates than those with transudates (p=0.01), serum ang-2 levels showed no differences between different etiologies of transudates or exudates (p>0.05) (table 4]. Roc curve was used to detect cutoff points for both serum and pf ang-2, differentiating between transudative and exudative pes, which were 3ng / ml and 8ng / ml respectively (fig . Predictive values of serum ang-2 cutoff point were as the following: sensitivity 90%, specificity 92.5%, positive predictive value 92.3% and negative predictive value 90.2% (table 5). Predictive values of pleural ang-2 cutoff point were as the following: sensitivity 95%, specificity 97.5%, positive predictive value 97.4% and negative predictive value 95.1% (table 5). Our results showed that there was highly significant positive correlation between serum and pleural ang-2 levels in patients presented with pe (p<0.001)(fig . Receiver operating characteristic curves for angiopeptin-2 (ang-2) levels in serum and pleural fluid to differentiate between transudative and exudative pleural effusions correlation between serum and pleural levels of angiopeptin-2 (ang-2) predictive potential of serum and pleural ang-2 cutoff points ang: angiopeptin; roc: receiver operating characteristic although pf ang-2 levels showed significant positive correlation with pf rbc count, pf nucleated cell count, pf total protein levels and pf ldh levels, it showed significant negative correlation with pf ph, pf glucose levels and pf / serum glucose ratio (table 6). Pf ang-1 levels showed highly significant negative correlations with pf protein and pf / serum protein ratio (table 6). Correlation of ang-1 and ang-2 levels with clinical and laboratory data ang: angiopeptin; pf: pleural fluid; rbc: red blood cell; ldh: lactate dehydrogenase ang-1 and ang-2 have been shown [27, 28] to be involved in the pathogenesis of a variety of human diseases . Our study revealed that pf ang-2 showed significant elevation in pleural exudates than in transudates . That coincided with kalomenidis et al study who found that pf ang-2 levels were significantly higher in pleural exudates than in transudates . Pleural inflammation exists in a mutually dependent association with hyperpermeability of the pleural vasculature and constitutes the pathogenetic basis of the vast majority of exudative pes . On the other hand, transudative pes are formed as a result of fluid extravasation that is caused by a disruption of the equilibrium of hydrostatic and/or osmotic pressures across an intact endothelial membrane . In our study pf ang-2 levels were significantly higher than serum ang-2 levels in patients with pleural exudates and patients with transudates . That agree with kalomenidis et al who suggested that ang-2 may be locally produced in the pleural cavity in patients with inflammatory pleural diseases and may play a role in the promotion of pleural inflammation and hyperpermeability and participate in the formation of exudative pes . Further, it is probable that ang-2 is mainly produced by the endothelial and perivascular cells of the pleural microvasculature, since neither mesothelial nor inflammatory cells have been reported to express ang-2 . In our study, pf ang-1 levels were significantly lower than serum ang-1 levels both in patients with exudates and those with transudates . Ang-1 is not produced in the pleural cavity in patients with either heart failure or pleural diseases characterized by inflammation and vascular hyperpermeability . Probably, the presence of ang-1 in the pleural cavity is the result of diffusion from the blood . In this study, ang-2 levels were significantly higher in tuberculous than in non - tuberculous pneumonic pes and empyema exudates . Higher levels of ang-2 in tuberculous pes than in pneumonic pes may suggest that ang-2 plays a primary role in the pathogenesis of pleural exudates of different etiologies . Inspite of significantly higher serum ang-2 in patients with pleural exudates than in those with transudates, we did not reveal significant differences between different etiologies of transudates or different etiologies of exudates as regards serum ang-2 levels . In our study, pf ang-2 levels showed significant positive correlation with pf rbc count, pf nucleated cell count, pf protein levels, pf / serum protein ratio, pf ldh levels and pf / serum ldh ratio . By contrast, there was significant negative correlation with pf ph, pf glucose levels and pf / serum glucose ratio . That coincides with kalomenidis et al who demonstrated that pf levels of ang-2 correlated with markers of vascular hyperpermeability and pleural inflammation . Both low pf ph and glucose levels are mainly due to increased metabolism in the pleural space, occurring in patients with pleural disease characterized by intense inflammation . In our study, pf ang-1 levels showed highly significant negative correlations with pf protein and pf / serum protein ratio . Both pf protein and pf / serum protein ratio are indexes of pleural vascular permeability and inversely correlated with pf ang-1, that is in line with the previously reported data showing that ang-1 is an antipermeability factor [16, 17]. In our study there was highly significant positive correlation between serum and pleural levels of ang-2 in patients presented with pe . One new and important finding in this study was the detection of two cutoff levels for both serum and pfs ang-2 with valuable discriminative properties, differentiating between transudative and exudative pes, which were 3ng / ml and 8ng / ml respectively . Predictive potentials of serum and pf levels of ang-2 cutoff levels were as the following: sensitivity 90% and 95% respectively, specificity 92.5% and 97.5% respectively, positive predictive value 92.3% and 97.4% respectively and negative predictive value 90.2% and 95.1% respectively . Mandriota and pepper detected a significant correlation between ang-2 levels and pf vegf levels which may suggest a functional association between these two growth factors in the pathogenesis of exudative pes . Kalomenidis et al speculated that ang-2 may amplify the hyperpermeability and proinflammatory signal produced by vegf . One of our limitations in this study was that we did not follow serum and pfs levels of ang-2 to detect its prognostic value . To our knowledge, this was the 1 trial to detect ang-2 cutoff points for categorization of pe to exudates or transudates . We can conclude that ang-2 levels were elevated in exudative pes and correlated with levels of markers of pleural inflammation and pleural vascular hyperpermeability . Further in vivo studies to detect the role of angiopoietin in the pathogenesis of pleural diseases and its relation to different causative agents will provide a basis for the development of novel therapeutic strategies in which ang-2 inhibitors may be used to treat patients with persistent exudative pes.
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The incidence of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (capd) has significantly decreased recently compared to rates in the past because of the development of peritoneal dialysis methods and catheter - related techniques, continuous patient education, and the establishment of antibiotic treatment guidelines; however, peritonitis remains an important cause of peritoneal dialysis failure . In peritonitis induced by a single microorganism strain, coagulase - negative staphylococcus was the most common cause of peritonitis (24.3%), followed by streptococcus, staphylococcus, escherichia coli, and pseudomonas aeruginosa . Pseudomonas stutzeri was first isolated from human spinal fluid as a gram - negative motile organism with a single polar flagella . P. stutzeri is widely distributed in the environment and rarely causes infections, but it has been isolated as an opportunistic pathogen in clinical conditions . Stutzeri - induced peritonitis in patients with capd was reported by ceri et al . Overseas, but no cases have so far been reported in korea . With regard to inflammatory diseases other than peritonitis, only one case of choroid plexitis has been reported domestically . In this report, we present a case of peritonitis by an uncommon pathogen, p. stutzeri, in a patient undergoing capd treated without catheter removal . An 82-year - old woman who had undergone capd for 1 year for the treatment of end - stage renal disease secondary to hypertension was hospitalized for a cloudy peritoneal dialysis effluent for 1 day before admission . Physical examination on admission revealed abdominal distension, decreased bowel sounds, and mild diffuse abdominal tenderness . The patient s vital signs were as follows: body temperature, 36.8 c; blood pressure, 185/88 mmhg; and heart rate, 78/min in sinus rhythm . Laboratory analysis revealed the following: white blood cell count of 10.410/mm with 89% polymorphonucleocytes; hemoglobin, 11.1 g / dl; platelet count, 30410/mm; erythrocyte sedimentation rate, 64 mm / h; c - reactive protein level, 2.92 mg / dl . The patient s blood chemistries were normal except for a creatinine level of 6.33 mg / dl, blood urea nitrogen level of 56.7 mg / dl, and albumin level of 2.8 g / dl . Chest radiography showed mild pulmonary congestion with cardiomegaly, whereas abdominal radiography revealed a mild paralytic ileus (fig . 1). Peritoneal fluid analysis revealed a leukocyte count of 9.9210/mm (polymorphonuclear leukocytes, 84%; lymphocytes, 4%). Empirical therapy was initiated with intraperitoneal cefazolin (dong - a pharmaceutical company limited, seoul, korea) and isepamicin . Because p.stutzeri grew in the culture of the peritoneal effluent performed on admission, cefazolin was discontinued from the 9 day of peritonitis, and ciprofloxacin was administered orally in addition to the initial treatment . The white blood cell count of the peritoneal fluid decreased from 9,920/mm on admission to 6/mm on the 19 day of peritonitis, and the dialysate effluent that was cloudy on admission drained clear from the 3 day of peritonitis . There were no other complications, and the patient is currently undergoing capd through the outpatient nephrology service without recurrent peritonitis . Identifying the causative microorganism is important for antibiotic selection and the determination of a treatment strategy in the management of capd peritonitis . Approximately 72% of capd peritonitis cases can be treated with antibiotics, but the catheter must be removed in the other 28% of cases because of recurrent and intractable peritonitis such as fungal infections . . Cases of p. stutzeri have been reported in the form of osteomyelitis, arthritis, endocarditis, meningitis, pneumonia, empyema, skin infections, eye infections, urinary tract infections, and diverticulitis, and susceptibility tests for several antibiotics have been performed in epidemiological and case reports . Nearly all studies involving several antibiotics and bacterial species have shown that p. stutzeri is sensitive to many more antibiotics than p. aeruginosa, its most closely related species and a well - known human pathogen . The higher sensitivity of p. stutzeri can be explained by its reduced exposure to antibiotics because of its low incidence rate in clinical environments . Despite these results, when isolated in immunocompromised hosts, p. aeruginosa and other pseudomonas spp ., including p. stutzeri, this finding implies that p. stutzeri has a range of antibiotic resistance mechanisms, such as changes in outer membrane proteins and lipopolysaccharide, and the presence of -lactamase has been described . Siva et al . Reported that pseudomonas is associated with a higher catheter removal rate and conversion to hemodialysis compared to general microorganisms, and immediate removal of the peritoneal catheter and treatment with two types of antipseudomonal antibiotics decreased mortality through a meta - analysis of 191 cases of peritonitis by pseudomonas that occurred in 20032006 in australia . P. stutzeri was identified in 15 (8%) of a total of 191 cases . Treated the patients with refractory capd peritonitis due to p. stutzeri with susceptible antibiotics and catheter removal . Tan et al . Described p. stutzeri as one of the causative pathogens of recurrent peritonitis in pediatric patients undergoing capd . However, no case has yet been reported in korea . In this case, the patient was treated with antibiotics without catheter removal because the isolated bacterium was susceptible to ciproflocaxin and isepamicin (table 1), there was no evidence of an exit site infection or tunnel infection, and her clinical symptoms improved after antibiotic administration (table 2). According to the recommendations of the international society for peritoneal dialysis, catheter removal with antibiotic treatment is recommended because peritonitis by pseudomonas species, which has the capacity to generate biofilm, is commonly associated with catheter - related infection, . In addition, like p. aeruginosa, p. stutzeri has a variety of antibiotic resistance mechanisms . Thus, the administration of two susceptible antibiotics with different mechanisms of action is required . In conclusion, management of p. stutzeri - induced peritonitis should be the same as that with p. aeruginosa . However, careful deliberation is necessary upon capd catheter removal because p. stutzeri infection progresses according to host immunity and antibiotic exposure as shown in this case.
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Nursing is experiencing a significant shortage of registered nurses around the world, particularly in developed countries such as the united states and canada [14]. This shortage is due to the increased demand for healthcare services, shortage of nursing professors, and the aging population of the nursing workforce . In ontario, canada, the shortage is intensified by the requirement for all registered nurses to be prepared at the bachelor of science of nursing (bscn) level . Most bscn programs include public health clinical experience as an intricate part of it . In order to meet this requirement, a public health department (phd) collaborated with a faculty of health sciences to develop a collaborative preceptorship model called the team preceptorship model (tpm), which was used to guide students' clinical experience in a public health setting . The purpose of this paper is to describe the strengths, limitations, and applicability of the tpm, which contributed to students' clinical experience and learning in public health nursing . Prior to reviewing the literature on collaborative preceptorship models, we examined the definitions and purposes of preceptorship to nursing education . Preceptorship is defined as a one - to - one relationship between a registered nurse (rn) and a nursing student during an intense, time - limited clinical experience to facilitate students' learning, and is supported by nursing faculty . Preceptorship is important to nursing education for several reasons: it assists nursing students to incorporate theory into practice, integrates students into the practice setting within the organization, allows the student to apply learning and internalize the role and values of the profession within a nurturing and supportive relationship, and assists in recruiting nursing students into the profession . This paper begins with a brief review of the literature, which is followed by a description of the collaborative development of the tpm, methods of evaluating the model and concludes with a description of the model's strength, limitations, and evaluation of it . In the past decade, several investigators [813] reported positive findings of collaborative preceptor models between schools of nursing and service agencies . Results of these studies showed that nursing students had a positive experience, expanded their knowledge, increased their confidence, and integrated their skills with real - life situations . These benefits have been found across a range of preceptorship models that have been developed over time . The preceptor model involving a single student being precepted by a single nurse originated in the time of florence nightingale and is commonly utilized today . According to nordgren, the preceptor model is mainly used in north america for senior nursing students during their final term of study; where one nurse - preceptor is responsible for the clinical teaching of a single student and the faculty member is responsible for supervising the general experience of the student . Similarly, in the integrated clinical preceptor model, students participate in planning their clinical experience, the preceptor acts as the clinical teacher, role model, and mentor, and the faculty member is resource for both student and preceptor . Evaluation of these models revealed positive outcomes for students: increased confidence, acquisition of skills and experiential knowledge in a specific clinical specialty, and preparation for practice following graduation; preceptors increase their scope of service (research and career development); faculty increased their productivity in research and scholarship . However, students expressed negative effects of the nordgren's model, such as lack of control over their experience and the need to be more assertive in expressing their learning needs to preceptors . Evaluation results showed positive outcomes for students and faculty such as a variety of patient care experiences for students, increased students' confidence, and faculty increased or freed - up time to address more complex issues . Alternatively, the modified clinical teaching model developed by baird et al . Involved beginning and advance students being taught in small groups by one preceptor . The faculty member was always present in the clinical area during students' clinical experience and assisted the preceptor in planning students' learning experiences . Evaluation of this model revealed several benefits for students and faculty members such as, increased contact time between students and preceptors, better usage of faculty time, and instruction of students by clinical experts . A limitation of the aforementioned models was that fewer nursing students receive individual or 1: 1 preceptor's support . In phillips and kaempfer's model, preceptors had difficulty in covering patients' assignments since preceptors were assigned according to students' needs and not unit needs . Lastly, happell developed a collaborative model between a university and health care agency . This preceptorship model was based upon the preceptor - preceptee relationship and factors which influence clinical learning from the perspective of nursing students and clinicians who taught them . The preceptor was the role model who inspired students to develop clinical skills and embrace the inherent value of nursing practice, respected the student as a member of the nursing team, and recognized them as inexperience and lacking confidence . The university and healthcare agency recognized their dual roles in the partnership and valued the preceptorship as an essential component of high - quality nursing education and provided resources to sustain it . A critical review of this model revealed several benefits for students and preceptors: students gained specialized clinical skills and received regular feedback on their performance in a positive working environment while preceptors contributed to the theoretical program and development of learning objectives for students and had designated time to preceptor students within their workload . The strength of this model was it applicability to other settings and clinical specialties . In summary, five preceptorship models were reviewed in terms of process, outcomes, and limitations . Despite their differences in design and processes, evaluation of the models indicated positive clinical outcomes for students, preceptors, and faculty . A major limitation of these models was that they were used in acute care settings and none included attention to preceptorship in community placements like public health departments . Additionally, most of the models have not taken a staff team approach to facilitate students' clinical experiences . Such a shared preceptorship approach was developed in collaboration between phd and a school of nursing that allowed students' placements in a public health setting . In order to meet this gap, a phd collaborated with the faculty of health sciences at a canadian university to design and implement a tpm, which is described in subsequent paragraphs . Team preceptorship model is an innovative model based on the premise of collaborative mentoring and is defined as nurses working together with several members of the partnership between the school and the phd, to provide public health preceptored experiences for bscn students by mutually coaching and facilitating their personal and professional growth . Each individual in the partnership is recognized for unique experiences, skills, and knowledge that he or she brings to the learning experience . Each person participates in a direct or supportive role as a member of the partnership, and each person coaches the student in interactive, interpersonal processes that involve the acquisition of appropriate skills, actions, and abilities that form the basis of professional practice gearlish . The tpm is defined by the authors as an integrated approach that fosters a reciprocal relationship amongst the student, preceptor, program staff, and faculty member to provide rich community experiences for undergraduate nursing students . In each program at the phd, a group of preceptors work as a team . Smaller programs have a team of two preceptors with two students assigned to each preceptor, whereas the larger programs have a team of four preceptors with 2 - 3 students assigned to each preceptor . Additionally, the team of preceptors and their students are supported by program staff in their respective programs . This structure allows for flexibility, creativity, and support for students and preceptors, when selecting learning experiences for students . For example, a student may be assigned to a project or activity that a program staff is responsible for if this activity fits within the students' learning objectives . Consequently, the program staff provides coaching, mentoring, and feedback to the student as well as contributes to his or her evaluation . The objectives of the tpm are to (1) significantly increase the number and proportion of bscn learners receiving public health nursing experiences, (2) create a learning environment in which the preceptor and learners feel supported (3) create an environment in which the learning responsibility is shared among all members of the team (preceptors, student placement coordinator, mentors, learners, managers, and faculty) (4) increase collaboration and communication between the university and phd, and (5) prepare nursing students with competencies for public health practice . In order to meet these objectives, students attend a two - day phd orientation program that helps to familiarize them with public health topics including policies and practices, organizational structure, services provided to the community, geographical location of communities that comprise the area of service, public health standards, nursing competencies, roles and responsibilities of the preceptors, students' responsibilities and expectations in the tpm partnership, health promotion theories, activities, and roles of the team members . During the first day of orientation, the phd coordinator gives an overview of the ph divisions (nursing and nutrition, environmental health, oral health services, emergency medical services, administration, and infant & child development). She also explains practical concerns of locations of offices, working hours, personal safety, and process for obtaining personal identification cards . The preceptor facilitates the second day of orientation, providing students with specific information regarding each program to which they are assigned . Throughout the clinical placements, students are expected to take an active role within their assigned programs (prevention of injury and substance misuse, chronic disease, infectious disease control and prevention or reproductive and child health) and are recognized and respected for their contributions as team members . Thus, the preceptors collaborate with program peers and manager to select appropriate activities and projects for each group of students . Criteria of appropriateness include ability to realistically implement projects during placement time frame and opportunity to learn the public health nursing role . An important aspect of students' learning experience is reflection which allows students to critically review and analyze their experiences and to examine alternative perspectives and solutions . The preceptor provides feedback and comments in an open, honest, and nonthreatening manner, contributing to a formative evaluation of the clinical experience . Effective feedback promotes professional and personal growth when it is provided in a respectful, supportive approach . The preceptor provides mid - term and final evaluations to each student in collaboration with faculty advisors according to school requirements . These evaluations are an integral component of the practicum experience and are based on third year evaluation criteria / competencies, to determine students' progress and achievement of the learning objectives . Role of the preceptor is comprised of providing orientation to students in their respective programs; reviewing organizational policies and procedures and code of conduct; reviewing with students the reciprocal expectations for the preceptorship such as meeting times, role of students to contacting preceptors and providing feedback; coaching and role models; communicating effectively with all members of the team; and conducting mid - term and final evaluations for students . The role of the students include attending university and agency orientation programs, reviewing expectations of placement, developing a learning plan related to the practice area in collaboration with the preceptor, maintaining open communication with preceptor and faculty advisor, and providing constructive feedback to the preceptor about the preceptorship experience and compiling with the academic expectations . Similarly, the program staff provides support to preceptors and learners, provides feedback to learners, contributes to their evaluation, and assists learners in meeting their learning needs . Additionally, the program managers support the tpm model and its implementation within the respective programs . Finally, the student placement coordinator is responsible for recruiting preceptors and organizing the preceptors' and students' orientations, facilitating preceptor and student debriefing at mid - and end of term, and liaising between the phd and university . Alternatively, the faculty's roles include communicating with student placement coordinator and preceptors, monitoring and evaluating students' progress and learning experiences, and determining the final grade . We received ethical approval from the involved organizations' ethical review boards (university and phd) prior to recruiting participants . An interview guide was created to help in facilitating the two focus groups (one for students and one for preceptors). A focus group format was useful for this project because it assisted in information recall and produced rich data . Additionally, students were comfortable in sharing with one another and phns reflected together about the preceptorship role, to generate deeper descriptions of their experiences . A faculty member moderated the discussions with the preceptor group, while a manager at the phd facilitated the discussions with the student group to ensure noncoercion in data collection and dependability of data analysis . Neither the faculty member nor the manager was associated with the clinical experience of students or recruiting of participants . At the beginning of each group, the facilitators introduced themselves and had members of the groups introduce themselves to one another . Data from the focus groups were audio taped and then transcribed by a research assistant (ra) from the university . Specific interview questions included (1) based on your experience, what do you feel are the advantages of the team preceptorship model? And (2) what do you feel are the disadvantages of the team preceptorship model? Participants were also asked to share their thoughts as they reflected on the whole clinical experience through these open - ended questions . The researchers used hsueg and shannon methods of content analysis to analyze the data to identify themes with supportive quotes . She facilitated internal consistency in the interpretation of the data by assuming the major responsibility for conducting the data analysis, making interpretative notes, and communicating with other team members as the data analysis proceeded . Then, the other researcher independently reviewed the transcripts and themes as well as made interpretative notes . Finally, the team of researchers met to discuss and verify consistency in interpretation of the data and reached consensus on the final themes . The researchers confirmed credibility of the findings by having participants review the themes and corresponding quotes to see whether they recognize the findings of the study to be true to their expectations . Thus, members' checking confirmed the accuracy of the findings [22, 23]. In the preceptor focus group, three themes emerged from the discussion of question 1 and one theme from question 2 . Each theme was described and summarized with direct quotes from participants to provide rich description of the themes . They were support for preceptors and students, good communication among team members, and collaboration among team members . The student focus group was asked the same questions regarding the advantages and disadvantages of the tpm . The tpm model was evaluated to determine its applicability and relevance to a public health setting . In the evaluation of the model, several strengths were identified: accessibility of preceptors to students, preceptors' expertise, support for preceptors and students, good communication among team members, and collaboration among team members . These positive results were found by other researchers [18, 19, 2428]. For example, myrick reported that preceptors' accessibility and expertise contributed in promoting students' critical thinking and broadened their clinical knowledge and expertise . Similarly, ferguson reported that faculty's support to preceptors facilitated their ability to evaluate students' performance, while usher et al . And yonge et al . Emphasized support from the clinical agency and fellow workers were vital to the preceptor's role . Hyrkas and shoemaker also found a positive association between preceptors' perceptions of support and commitment to their role . Additionally, latham and colleagues purported that partnership among frontline rns improved the culture of support and healthy workplace environment . Limitations of the model were that preceptors felt overworked and students felt that public health activities took too long to be implemented . Pertaining to preceptors' feelings of being overworked, coates and gormley and yonge et al . Found that the biggest barrier to effective preceptoring of students was preceptors' lack of time with students . Preceptors had acknowledged that being assigned to students meant taking extra time and effort to teach them, which consequently increased their workloads . This finding corroborated with leners et al . Who indicated that preceptors' workload was a major disadvantage and huge concern for preceptors . It is important to note that each preceptor was given one day per week of dedicated time funded by the phd to support students within the ph setting for the term . The dedicated time was used for coaching and mentoring students' activities, attending meetings with the student placement coordinator and faculty, as well as performing students' evaluations . Alternatively, students' perception of public health is accurate, in that it takes time and effort to implement social change . This perception of public health is not a limitation of the model but reveals the challenges students face when learning and implementing public health strategies to enhance the health of a population . It has clinical utility and can be adapted into other settings to increase the volume of nursing students in clinical placements . For example, the number of students' clinical placements in the phd had increased from 14 to 35 per term during the tpm implementation . Special attention should be given to students' learning during orientation to explain the length of time it takes to implement social changes in the community or society . Preceptors felt overworked and students perceived that public health activities took too long to be implemented . Firstly, administrative personnel at the phd should reduce preceptors' workload to accommodate the preceptorship role, that means, delegating some of their assigned activities to peers and increasing the dedicated time for preceptoring students . The dedicated time should be increased to one and a half days per week for the first four weeks of the clinical placement and then reduced to one day per week for the remaining eight weeks of the academic term . Increasing the time and reducing their workloads will help to relieve their stress as well as the potential for burnout . Additionally, a larger pool of preceptors should be trained and rotated every second academic year, thus preventing preceptors' burnout . On the other hand, the placement coordinator and preceptors should include theories of change such as prochaska's 5-stages of change: precontemplation, contemplation, preparation, action, and maintenance and lewin's change model: unfreeze / refreeze into students' orientation . The placement coordinator should explain and apply these models to public health practice in order to increase students' understanding of the complexity and time needed to plan and implement interventions for the population . 's article goes beyond prochaska's five stages of change by introducing motivational techniques that allow a practitioner to match his / her approach with a client or colleague to the change that the person is experiencing . Similarly, the preceptor can utilize motivational interviewing to assist students in identifying the stage of change where they are at, by applying prochaska's 5-stages of change to their present situation . They may also discuss participatory program planning, which would assist students in understanding the different types of stakeholders who collaborate with public health professionals in successfully developing and implementing health promotion interventions for the population . This paper highlights components of the tpm model, its strengths, limitations, and implications for clinical practice . The tpm is an innovative approach to teach a large group of students in a public health department . Evaluation results are consistent with principles of the tpm, which embraced collaboration among all partners and was a crucial part of the model's functioning . Based on evaluation results, it seems appropriate to move forward with permanently utilizing this model for future placements of students.
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Since the introduction of microarrays, there has been considerable interest in using whole - genome expression profiling to gain insight into cancer and to identify key genetic mediators . Although there has been some success in this regard, many efforts have been complicated by the fact that it is difficult with expression data to identify the genes affected by a condition from the hundreds to thousands of genes that exhibit changes in expression . In this work, we show that a network biology approach can be used to take on this challenge . Specifically, we show that reverse - engineered gene networks can be combined with expression profiles to identify the genetic mediators and mediating pathways associated with prostate cancer . We used an approach called mode - of - action by network identification (mni), which has previously been validated as a means to identify the targets and associated pathways of compounds (di bernardo et al, 2005). The mni algorithm operates in two phases (figure 1). In phase one, a network model of regulatory interactions is reverse engineered with a diverse training set of whole - genome expression profiles . In phase two, the network is used as a filter to determine the genes affected by a condition of interest, for example, a disease (figure 1). The highest ranked mediator genes, ranked by a z - statistic, are those whose expression is most inconsistent with the model, and this inconsistency is attributed to the external influence of the condition on those genes . Genes implicated in the advancement as well as suppression of a disease are equally likely to be identified as significant genetic mediators by the mni algorithm (see supplementary information). The mni algorithm requires that the training expression profiles influence a diversity of cell functions . As a training set, we used a total of 1144 microarray expression profiles based on 13 projects spanning seven different cancer types: adrenal, brain, breast, leukemia, lung, prostate and thyroid (materials and methods). As test conditions, we used expression profiles of 14 non - recurrent primary and nine distant metastatic prostate cancer samples (latulippe et al, 2002). Each of these samples was queried against the reconstructed network, and the resulting potential genetic mediators in each case were ranked according to the z - score statistic . A characteristic list of 100 genes for the non - recurrent primary and metastatic prostate cancer groups, respectively, was obtained by averaging the z - scores across all samples . Normal prostate and early - stage prostate cancers depend on androgens for growth and survival . As the cancer advances and metastasizes, it becomes dominated by cells that proliferate and survive independent of androgens; this effect is provoked by androgen ablation therapy (taplin et al, 1995; abate - shen and shen, 2000; feldman and feldman, 2001; navarro et al, 2002; balaji et al, 2004; shultz, 2005). Sensitivity to low levels of androgen is increased by amplification, mutations and/or elevated levels or broadened specificity of co - activators of the androgen receptors . In other cases, activation of androgen receptors occurs in the absence of androgens due to crosstalk via other signaling pathways (hobisch et al, 1995; taplin et al, 1995; abate - shen and shen, 2000; feldman and feldman, 2001; navarro et al, 2002; balaji et al, 2004; shultz, 2005). It has been shown that almost all metastatic prostate cancers shift to an androgen - independent state (abate - shen and shen, 2000; navarro et al, 2002; balaji et al, 2004). After anti - androgenic treatment, primary prostate cancers can also shift to an androgen - independent state and become recurrent (abate - shen and shen, 2000; navarro et al, 2002). In order to differentiate between the two groups optimally, we chose to analyze nine advanced - stage metastatic prostate cancer samples and 14 non - recurrent primary prostate cancer samples from latulippe et al (2002) (primary prostate cancer samples remained non - recurrent after a mean follow - up of 42 months). The above points led us to hypothesize that the ar gene would be among the top genetic mediators identified by the mni algorithm for the metastatic prostate cancer group only, indicative of its key role in androgen - independent metastatic prostate cancer . Moreover, because having amplifications, mutations and increased specificity for ar in androgen - independent prostate cancer raises the possibility that downstream genes in the ar pathway are also involved in the progression and metastasis of the disease, we further hypothesized that the ar signaling pathway would be highly enriched in the metastatic prostate cancer group . The list of the top 100 potential genetic mediators for non - recurrent primary and metastatic prostate cancer groups along with their expression change rankings is given in supplementary table 1 . The mni algorithm identified the ar gene among the top genetic mediators in the metastatic prostate cancer group but not in the non - recurrent primary prostate group . We next subjected the 100 highest ranked genes in non - recurrent primary and metastatic prostate cancer groups to enrichment analysis for the ar signaling pathway . We found that the list of the top 100 genes for the metastatic prostate cancer was enriched (p=1.5 10) for the ar signaling pathway, in contrast to that for non - recurrent primary prostate cancer, which was not enriched (figure 2). These results, which are consistent with our hypotheses, imply that the ar gene and the ar pathway are mediators of prostate cancer progression and metastasis . Figure 2 includes the 20 genes transcriptionally regulated in the ar signaling pathway, identified among the top mediators for the metastatic prostate cancer group . It is thought that these genes may play a role in the acquisition of androgen - independent growth (velasco et al, 2004). Among these 20 genes, six are well - known androgen - regulated genes (arg)ar, psma, hoxb13, nkx3 - 1, cited2, ugt2b15 (nelson et al, 2002; velasco et al, 2004)which have been shown to mediate metastatic disease progression . For example, psma (also known as folh1), prostate - specific membrane antigen 1, is used as a diagnostic and prognostic indicator for prostate cancer and is associated with prostate cancer aggressiveness and metastasis (burger et al, 2002; schmittgen et al, 2003; kinoshita et al, 2005). Hoxb13, homeobox b13, has been implicated in progression and metastasis in prostate cancer (jung et al, 2004; edwards et al, 2005; zhao et al, 2005) and found to function as an ar repressor, modulating ar signaling (jung et al, 2004). Loss of nkx3 - 1 expression, a homeodomain transcription factor, is found in 6080% of human prostate carcinomas (bhatia - gaur et al, 1999) and has been associated with advanced prostate cancer and metastasis (bowen, 2000). It has also been shown that a majority of nkx3.1;pten mice develop invasive adenocarcinoma in the lymph nodes (abate - shen et al, 2003). Cited2 is known to play a crucial role in the control of tumor hypoxia, which is associated with metastatic progression (aprelikova et al, 2006). Ugt2b15, udp glucuronosyltransferase 2 family polypeptide b15, a steroid - metabolizing protein, has been found to be differentially expressed in androgen - independent bone marrow metastases following androgen ablation therapy (guillemette et al, 1997; hum et al, 1999; stanbrough et al, 2006). We discuss the clinical significance of some of the other top mediators in supplementary information . We next focused on go - annotated pathways that were significantly overrepresented among the highly ranked genetic mediators . For our analysis, we subjected the 100 highest ranked genes identified by mni in the metastatic and non - recurrent primary prostate cancer groups, respectively, to pathway analysis based on the go biological process annotations obtained from affymetrix . The significantly enriched go - annotated pathways for metastatic and non - recurrent primary prostate cancer based on mni analysis are shown in figure 2 . These pathways fall into two categories, which are well - established processes identified as hallmarks of all cancers metabolism and immune response (weinberg and hanahan, 2000). As malignant tumors the metabolism pathway is highly enriched in both non - recurrent primary and metastatic prostate cancer groups (figure 2). Transport, indicative of high metabolism, is also significantly enriched in the metastatic prostate cancer group . The immune response is the main defense against cancer, and mni successfully identifies the inflammatory response, the immune system - related biological pathway, in the metastatic prostate cancer group (figure 2). Interestingly, some ar pathway genes are also part of the enriched go - annotated pathways . Specifically, gsta2, aldh1a3 and ugtb15 in the metabolism pathway, orm1 and ar in the transport pathway and orm1 in the inflammatory response are all androgen - responsive genes . This raises the possibility that the metabolism, transport and inflammatory response pathways in the metastatic prostate cancer group may be enriched as a result of increased activity of androgen - responsive genes in the ar signaling pathway . In order to assess the relative merits of using mni, a network - based approach, we performed pathway enrichment analysis on the top 100 ranked genes obtained by expression change alone and by using gsea, a gene set enrichment tool (http://www.broad.mit.edu/gsea/). Go biological process pathway enrichment analysis, based on expression change alone, identified proteolysis and peptidolysis as a highly enriched pathway in the non - recurrent primary prostate cancer group and muscle development, muscle contraction and cell - adhesion pathways in the metastatic prostate cancer group (figure 2). Go biological process pathway enrichment analysis using gsea did not identify any significantly enriched pathways (materials and methods). Compared to expression change alone, mni failed to identify the cell - adhesion pathway, which is important in the spread and invasion of the cancer, as a highly enriched pathway . However, mni was successful in eliminating false positives such as muscle contraction and muscle development pathways in the metastatic prostate cancer group . Mni's advantage was most apparent in predicting the ar signaling pathway as a mediator for metastatic prostate cancer . These analyses reveal the benefit of our network - based approach in that it highly ranks genes whose expression is not significantly different from normal and do not have coordinated expression, but which are relevant genetic mediators, such as the ar signaling pathway genes in the metastatic prostate cancer group . Motivated by the above findings and the ability of our approach to differentiate between non - recurrent primary and metastatic prostate cancer, we next applied the mni algorithm to nine recurrent primary prostate cancer samples (which had recurred within a mean follow - up of 42 months) from latulippe et al (2002). We hypothesized that the mni ranking of the ar gene would move up as an indication of the aggressiveness of the disease . Consistent with this hypothesis, mni ranked the ar gene 970, 155 and 9 for the non - recurrent primary, recurrent primary and metastatic prostate cancer groups, respectively (figure 3). This finding suggests that the ar gene, in the context of the reverse - engineered network, can be used as a marker for detecting the aggressiveness of primary prostate cancers . Interestingly, expression change alone ranked the ar gene 641, 668 and 207 in the respective groups (figure 3), indicating that expression change alone is incapable of capturing the differential involvement of the ar gene in recurrent and non - recurrent primary prostate cancers . In this study, we showed that a network biology approach that filters expression profiles through a reverse - engineered gene network can be used to identify the genetic mediators and mediating pathways of a disease . Specifically, we identified key genetic mediators and pathways that have been implicated in the initiation, advancement and invasion of prostate cancer . Our approach extends the utility of whole - genome expression profiling, and may be useful as a predictive tool for identifying novel genetic mediators for other cancers, such as breast cancer and leukemia . Network - based techniques (gardner et al, 2003; barabasi and oltvai, 2004; basso et al, 2005; segal et al, 2005; yeang et al, 2005) may also prove useful for providing biological insight into the etiology and progression of other diseases . Microarray data were collected from five publicly available databases: the nih gene expression omnibus (gse349, gse1431, gse1923, gse3960), oncomine (giordano_adrenal, nutt_brain, huang_breast, latulippe_prostate, huang_thyroid), ebi arrayexpress (mexp-441), broad institute cancer datasets (bhatacharjee et al lung, singh et al prostate) and the st jude research datasets (yeoh et al leukemia). The collected data were from experiments conducted on affymetrix genechip human genome 95a or 95av2; the combined microarrays have 12 600 overlapping probe sets . A total of 1144 experiments were collected based on 13 projects spanning seven different cancer types: adrenal, brain, breast, leukemia, lung, prostate and thyroid . Each experiment in the data matrix was normalized by its mean to account for experimental variation between labs, and each probe set was normalized by its average across all experiments to obtain expression changes relative to a baseline . The mni algorithm takes in as input the log - transformed expression ratios and standard errors . As there were no repeated experiments in the collected set of microarray data, the standard error was set to 1.0 for all experiments and probe sets . The mni algorithm, which is described in detail in supplementary information, takes as a training set all of the expression profiles except user - specified test profiles . We used as test profiles data from latulippe et al (2002), which includes samples from 14 non - recurrent primary prostate cancers, nine recurrent primary prostate cancers and nine metastatic prostate cancers located in the lymph node, bone, lung or soft tissue . The mni algorithm was configured to output the top 200 mediators for each sample, together with the associated z - scores for those probe sets . We set to zero the z - score for probe sets that were not within the list of the top 200 probe sets identified as mediators for a given sample . To identify a characteristic list of genes within each group (i.e., non - recurrent primary, recurrent primary and metastatic prostate cancer), the z - scores across samples and across probe sets for corresponding genes were averaged and ranked . The top 100 genes within that list were chosen to be reported as significant genetic mediators . A higher average z - score is an indication of higher number of occurrences of a gene on the lists generated by the mni algorithm in each group . We compared the mni rankings with those obtained using purely expression values . To make this comparison, we scored transcripts based on their differential expression from normal prostate tissue samples . Characteristic normal, non - recurrent primary, recurrent primary and metastatic expression profiles were created by averaging the normalized transcript expression for each of the experiments in those respective categories from latulippe et al (2002). The non - recurrent primary, recurrent primary and metastatic characteristic profiles were then divided by the normal profiles to obtain expression ratios . Pathway enrichment was performed on the top 100 genetic mediators identified for the non - recurrent primary and metastatic prostate cancer cases . The pathway annotations were based on the go biological process annotations from affymetrix for the hu95a and hu95av2 chips . Ar signaling pathway transcriptionally regulated genes were obtained from the netpath database (http://www.netpath.org/). We report significant pathway enrichments for groups with at least four members and p - values0.01 . Gsea (http://www.broad.mit.edu/gsea/) pathway enrichment was performed on the ar signaling pathway and all go - annotated pathways.
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T - wave alternans is characterized by beat - to - beat change in the morphology, amplitude and/or polarity of the t wave . Microvolt level t - wave alternans (mtwa) has been proposed to assess the abnormalities in ventricular repolarization, which favors the occurrence of reentrant arrhythmias . In 1994, a first clinical study by rosenbaum and coworkers demonstrated that mtwa is closely related to arrhythmia induction in the electrophysiology laboratory as well as to the occurrence of spontaneous ventricular tachyarrhythmias during follow - up [2, 3]. Microvolt twa analysis has been implicated as one of the strongest predictors of ventricular arrhythmias and sudden cardiac death (also superior to other non - invasive markers of ventricular repolarization, such as qtc interval prolongation) in several trials including patients with myocardial infarction, heart failure and implantable defibrillators . Two main hypotheses have been proposed for the mechanism of mtwa, the action potential restitution and the calcium cycling hypothesis . There is considerable experimental and clinical trials that do not support the restitution hypothesis [6, 7] on the other hand there is considerable evidence to support the abnormal calcium cycling hypothesi . Patients undergoing elective coronary artery bypass grafting (cabg) tend to be relatively healthy and subsequently their surgery is significantly delayed when compared to urgent cases . The cumulative pre - surgical risk of death from cardiovascular disease was found to be similar in the semiurgent and elective cabg cases due to the longer wait for surgery in the later group . The in - hospital death was also shown to be significantly higher when the surgery is delayed beyond the recommended time of 12 weeks in non - urgent cases . However there is no data that reports on the risk of sudden cardiac death in elective cabg patients with no structural heart disease . Also, none of the studies looked at the prevalence of mtwa as a predictor of sudden cardiac death (scd) in this population . Also, in patients undergoing cabg, new onset post operative atrial fibrillation (afib) is a very common complication . Patient with this complication were found to have a significant higher mortality at 10 years of follow up . Recently afib was linked to abnormal calcium handling, a mechanism proposed for abnormal mtwa tracing [12, 13]. Although mtwa and afib may share a similar underlying mechanism, there are no reports on the risk of new onset afib in patients with abnormal t wave alternans testing . We designed a prospective observational study targeting a selective population of patients undergoing elective cabg with normal systolic function . In this study we followed these patients throughout their hospital stay and looked at the incidence of new onset afib in patients with normal and abnormal mtwa tracing . All patients signed an informed consent after a detailed explanation of the study aim and purpose . The inclusion criteria included all patients referred to the cardiothoracic outpatient clinic for elective cabg, who can perform aerobic exercise, and agree to have a mtwa testing prior to the scheduled surgery . Since mtwa testing involves a submaximal treadmill exercise test, and our population involves patients with significant coronary artery disease (cad), we pre - specified a safety measure in our protocol . We only considered patients with a recent exercise stress test who reached at least 85% of the maximal predicted heart rate (220 - age) and non - limiting chest pain at maximal exercise . The exclusion criteria included any of the following criteria; age less than 18, pregnancy, left ventricular ejection fraction (lvef) less than 50%, patients on any antiarrhytmic medications, baseline electrolyte abnormalities, known history of atrial fibrillation, stage 5 chronic kidney disease (ckd), and inability to perform a submaximal treadmill test . The patients were recruited between may 2008 and february 2010 at an outpatient cardiothoracic office at staten island university hospital . In this study heartwave ii system detects the presence of mtwa by electrocardiogram (ekg) measurements during rest, exercise, and then rest again . Fourteen sensors - 7 micro - v alternans sensors and 7 standard electrodes - are placed in the frank - lead configuration . The electrodes are connected to the digital ekg amplifier that leads back to the mtwa enabled system . After checking the system for correct lead placement, the patient is directed to begin walking on a treadmill to raise the heart rate . The first aims to maintain the heart rate at 100 - 110 bpm for a total 90 seconds, while the second goal is to achieve a heart rate of 110 - 120 for the same time interval . The total time of submaximal exercise can range between 5 - 10 minutes to achieve and adequate recording . The patients were instructed to hold calcium channel blockers or beta - blockers on the day of testing to achieve adequate heart rate with minimal exercise . Based on the previously defined criteria by bloomfield et al ., mtwa tracing was interpreted as either negative or non - negative which included patient with positive or intermediate tracing . Studies have well shown that intermediate and positive mtwa tracing, have similar prognostic value [15, 16]. Each test was analyzed by the same electrophysiologist (cardiologist), and confirmed by a trained technician from the company, who was blinded to the study and patients . The hospital course of all patients included in the study was followed, and in - hospital events were recorded . This included, post surgery afib, sustained ventricular tachycardia, ventricular fibrillation, cardiac arrest, arrhythmias requiring anti - arrhythmic treatment, and hospital death . In order to assess the prevalence of atrial fibrillation in patients with a non - negative mtwa we divided the sample into two groups the first involved all patients with non - negative test mtwa (+), while the second included all with a negative test mtwa (-). We also studied variables that were shown to predict atrial fibrillation post cabg . Based on a study done by amar et . These included older age, prior history of afib, p - wave duration> 110 ms on surface ekg, and low cardiac output defined as cardiac index <2 l / min / m for> 8 h after surgery . In that study a point score was developed to predict the probability of post cabg afib . In our study we used this validated score to compare the risk of post - cabg afib in the mtwa groups (table 1). Clinical characteristic and outcome by microvolt t - wave alternans (mtwa) groups . Mtwa = microvolt level t - wave alternans; mi = myocardial infarction; dm = diabetes mellitus; gfr = glomerular filtration rate; cabg = coronary artery bypass graft; afib = atrial fibrillation . Statistical analysis . Continuous variables were expressed as a mean standard deviation (sd) and were compared using an unpaired two - tailed student s t - test . Unpaired categorical variables were compared using fisher s exact test . A probability cutoff of p <0.05 was considered significant for all statistical determinations . A multivariate logistic regression model was used to evaluate the independent contribution of baseline clinical characteristics to the development of the end point in a forward stepwise manner . At each step, a significance of 0.10 was required to enter into the model while those with probabilities less than 0.05 were considered statistically significant . All analyses were performed using spss 19.0 (spss inc ., chicago, il). Sixty three patients were initially considered for enrollment . Only 36 met the inclusion criteria that were specified in our study protocol . Of the 24 patients, 4 did not show up on the day of the mtwa testing and were dropped out of the study . Ace = angiotensin converting enzyme inhibitor; arb = angiotensin receptor blocker; ccb = calcium channel blocker . The median and mean time elapse from coronary angiography to bypass surgery was 29 and 23 days, respectively . The mean age of mtwa (+) group was 64 years slightly older than the mtwa (-) group which was 61years . Diabetes mellitus (dm) and prior history of myocardial infarction (mi) was slightly higher in the mtwa (-) group . The mean glomerular filtration rate was lower in the mtwa (+), but none of the patients had a stage 5 ckd, as this was one of the exclusion criteria . The point score that predicts the risk of post cabg afib was similar in both groups (table 1). The mean hospital stay was 4 and 7 days among mtwa (-) and mtwa (+) groups, respectively . One patient in the mtwa (+) group had a cardiac arrest post surgery and was successfully resuscitated . One (9%) patient with negative mtwa developed af in comparision to 5 (55%) paitens with non - negative mtwa . Patients with non - negative mtwa are more likely to develop af post cabg then patiens with negative mtwa (p=0.05) (table 1, figure 1). The incidence of post - coronary artery bypass graft atrial fibrillation by microvolt t - wave alternans (mtwa) groups . Logistic regresion, a non - negative mtwa was the only predictor of af (p=0.042). In madit-2 trial mtwa was shown to have a better risk stratification for scd when compared to qrs duration on ekg in patients with ischemic heart disease . In several studies, mtwa was shown to have a prognostic value after acute myocardial infarction [19, 20]. In our study we found that the prevalence of abnormal mtwa is very high when compared to previous reports on healthy adults . In this study we aimed to assess the prevalence of abnormal cardiac repolarization in patients with ischemic heart disease scheduled for elective cabg . All of our patients had a systolic function of more than 50%, and none of them had a significant ckd as we specified in our study protocol (table 1). The reported prevalence of abnormal mtwa in healthy adults ranged between 4 and 5% [21, 22]. Abnormal mtwa was also reported to be high in diabetics, in patients with uremic cardiomyopathies, and in patients on hemodialysis . Neither ischemic, nor uremic cardiomyopathy can explain the high prevalence of abnormal mtwa in our studied population (45%). Moreover, looking at other potential clinical predictors for abnormal mtwa, there was more patients with history of dm and prior mi in the mtwa (-) group . In the mtwa (+) none of those patients had a concomitant history of mi and dm . On the other hand in the mtwa (-) group, there was no statistically significant difference in any of the known clinical predictors of abnormal mtwa in both groups (table 1). For this reason, although dm and prior history of mi could have contributed to the mtwa test results in this population, it does not exclusively explain the high prevalence noted in our population . Animal studies have shown that regional myocardial ischemia in structurally normal hearts can lead to regional action potential alternans [28, 29]. Regional ischemia induced by exercise might explain the prevalence of abnormal mtwa in this population . There are no previous studies that looked at mtwa as a predictor of scd in patients with ischemia but normal systolic function . One patient with abnormal mtwa tracing had a post surgical asystole, and was successfully resuscitated . There was no recorded death in both groups at 30 months of follow up . Although our observation may suggest that patients awaiting elective cabg are at high risk of arrhythmias and scd, further studies are needed to assess the potential risk in this population before drawing further conclusion . The incidence on new onset atrial fibrillation post cabg varies between 11% and 40% depending on the study cohort and method of detection used [11, 30]. In this study patients with non - negative mtwa are more likely to develop atrial fibrillation (af) post cabg then patiens with negative mtwa with a significant p value (p=0.05) (table 1, figure 1). In multivarient logistic regresion, a non - negative mtwa was the only predictor of af (p=0.042). There is growing evidence that links atrial fibrillation to abnormal calcium handling / calcium alterans [12, 13]. Similarly, there are considerable evidence that abnormal calcium cycling / calcium alternans is the mechanism behind action potential and t wave alternans . Since mtwa tracing in patients with atrial fibrillation is not feasible, no studies have looked at the prevalence of abnormal mtwa in patient with afib . Also there no previous reports on the risk of new onset afib in patients with abnormal t wave alternans testing . Knowing that both afib and abnormal mtwa might have a common electrical mechanism we think that the risk of post - cabg atrial fibrillation might be further stratified by pre - cabg mtwa in groups with comparable other risk scores . Although this finding is very interesting we only suggest a potential association between abnormal mtwa and afib, and further studies with more patients are needed to consolidate our finding . Although this is a significant limitation we think that our results are very interesting to report especially that we targeted a very specific group of patients . We had many exclusion criteria to assure patient safety, which limited our ability to recruit a larger sample . Another limitation is that the prognosis of abnormal mtwa testing is not well established in patients with ischemic heart disease with normal systolic function . For this reason even with this high prevalence of abnormal mtwa results, the risk of scd is still unknown . Furthermore, there was no difference in mortality at any interval follow up in both groups . For this reason no assumption can be drawn on the risk of scd in this population until we have more data this pilot study provides the first clinical evidence that patients with ischemic heart disease and normal systolic function have a high prevalence of abnormal mtwa and might be at higher risk of scd . Mtwa might be studied as a potential factor in future afib risk scores . Despite our interesting findings, we suggest a multicenter study with a larger representative sample to confirm our findings.
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Gastric cancer is the one of the most prevalent reason of cancer - related death in the world . Now, gastric cancer contains 10% of cancers in the world and is one of the most common kinds of cancers (1). According to the statistics of iran cancer institute, gastric cancer is the third most common cancer between iranian women after breast cancer and the most common cancer between iranian men (26). The elementary treatment of gastric cancer in initial stages is surgery; so it is considered as the prime treatment for cancer . Chemotherapy and radiotherapy will be used as supplementary treatments, if necessary . In advanced stages of the disease, surgical procedures, radiotherapy and chemotherapy the odds of patients complete recovery depend on the surgery but the time when the disease passes through the mucous membrane, it is possible lymph nodes metastases and relapse in spite of the total surgery, which has been performed on the patient (7, 8). One of the most important prognostic indicators which is considered after surgery and for patients with gastric cancer is an increase in patients survival rate especially the 5-year survival rate . Unfortunately, because early gastric cancer causes few symptoms, the cancer is usually advanced when the diagnosis is made . So conventional treatment such as surgery, chemotherapy and radiation therapy are not impressive in increasing the patients survival rate (9, 10). For this reason, the 5-year survival rate for gastric cancer after surgery the increase in these patients survival rate after surgery involves identifying various factors, including individual, clinical, diagnostic and therapeutic . There are various statistical methods to assess the effects of various factors on survival of cancer patients including parametric and cox semi - parametric regression models . These models are divided into two basic categories: proportional hazard (ph) model and accelerated failure - time (aft) model . In the proportional hazard regression model, the effect of covariates is obtained on the hazard function . In this case, if baseline hazard is considered parametric, one of the weibull, exponential and gompertz models will be achieved . If the baseline hazard is considered non - parametric, the cox proportional hazard model will be obtained . In the accelerated failure - time regression model, the obtained models in this case include generalized gamma, log - logistic, log - normal, weibull and exponential . Weibull and exponential are the only parametric regression models which have both a proportional hazards and an accelerated failure - time representation . The proportional hazard model does not need to consider a specific probability distribution for the survival time; therefore, it is the most helpful model in analyzing survival data . But the efficiency of the model is severely dependent to proportional hazards assumption and, for this reason, the cox model is often called proportional hazards model . In occasions where the proportional hazard model is not acceptable, estimates derived from cox model will lead to an improper fitting of the model and incorrect inferences (1622). These models due to having a parametric distribution for the survival times make statistical inference more accurate and lead to an proper fitting of the model (23). Factors affecting the survival of cancer patients are often identified by cox proportional hazard model (14, 2430). Neither have these studies generally tested proportional hazards assumption nor did they try to identify a proper model as an alternative to proportional hazards model . In this study in addition to comparing various survival models as well as identifying an alternative accelerated failure - time model for the cox proportional hazards model akaike information criterion (aic) and cox - snell residuals in this historical study, 330 patients with gastric cancer with the following data were studied: 1) the patients had been hospitalized and had undergone surgery during 199599 in surgical wards of iran cancer institute 2) these patients had information in the archives of the hospital, and in their files their phone numbers and addresses were available for further follow - ups . The survival status of these patients in 2011 was determined by reopening the files as well as phone calls . The survival time of these patients after surgery was determined and those patients who were still alive at the end of study time or the ones whose information were not available after a specific time were considered right - censored . The effects of demographic variables such as age, sex, and marital status, and clinical data of the disease including lymph node metastases, liver metastases, distance metastases; disease stage (i - ii - iii - iv); and type and extent of gastrectomy (total - subtotal - distal - partial - proximal) as well as post - surgical and treatment variables including relapse and the number of supplementary treatments (surgery - radiotherapy - chemotherapy or a combination of them) on patients survival were evaluated and compared among various models . To compare different survival models, akaike information criterion (aic) and cox - snell residuals is a graphical scale for evaluating the fitness of proportional hazard and accelerated failure - time models; the short deviation of residuals from the straight line through the origin with a slope of 1, the more appropriate fitness of the survival model (18, 20, 31). Thus, akaike information criterion (aic) can be used along with cox - snell residuals . Akaike information criterion (aic) is used to measure the goodness of models fitness, and the smaller it is, the better it is (17, 18, 3234). Note that a direct comparison of the aic cannot be made between parametric models and semi - parametric model because the likelihoods differ . Akaike information criterion (aic) for the models used in this study has been calculated according to the following formula: aic=2log(l)+2(p) where p is the number of model parameters and l is the model likelihood function (17, 34, 35). The smaller the akaike information criterion (aic) is, the more efficacious the model will be in identifying the risk factors (34). Moreover, in order to facilitate the comparison of the variables variances used in the model in this study, the standardized variability, calculated as sv = se(^)^, was used to standardize the variance of estimated parameters (in this equation se() is the standard error of parameter and is the coefficient of parameter in the survival model). To determine the disease stage, stata 11 software was used for all analyses and the significance level was set at 5% . Overall, 228 patients were male (69.1%) and 315 (95.45%) were married . The mean of age was 65.41 10.56 years for women and 65.7 11.22 years for men . Two hundred thirty - nine patients (72.4%) died by the end of the study and the rest were right censored . Totally, 192 patients (58.2%) had metastases out of which 12.5% suffer from liver metastases and 66.67% suffered from lymph nodes metastases . Forty - three patients (13.03%) had a relapse and 8.48% had undergone proximal gastrectomy, 8.79% had undergone partial gastrectomy, 3.03% had undergone distal gastrectomy, 27.27% had undergone subtotal gastrectomy and 52.42% of patients had undergone total gastrectomy . The analysis of disease stage revealed that 58.79% of patients were in stage iv, 16.36% in stage iii, 18.18% in stage ii and 6.67% stage i of disease . 20.3% of patients had not received any supplementary treatments whereas, 23.03% of the patients had received one supplementary treatment, 30.61% of the patients had received two supplementary treatments and 26.06% of the patients had received three supplementary treatments . Referring to the figure analysis of cox - snell residuals for accelerated failure - time models and cox proportional hazard model represents approximately equal fitness of accelerated failure - time models compared with proportional hazard model (fig . The cox - snell residuals in the considered cox proportional hazard and accelerated failure - time models among accelerated failure - time models, the exponential, gompertz and weibull proved better fitness to the data . The comparison results of the risk factors between the cox proportional hazard and accelerated failure - time models (first category is considered as a reference group). Sv: standardized variability rr: relative risk hr: hazard ratio based on partial likelihood furthermore, table 1 shows cox proportional hazard model and accelerated failure - time models analyses of risk factors according to the standardized variations, hazard ratio (hr) and relative risk (rr) for all variables . According to akaike information criterion, the exponential (aic=969.14) and gompertz (aic=970.70) models are more efficient than other models . The results of cox proportional hazard model and accelerated failure - time models (except log - logistic and log - normal) do not show much difference in terms of variables significance . Although the hazard rate in proportional hazard model is virtually the same as the results of accelerated failure - time models, the exponential and gompertz models had better results according to akaike information criterion . Results of cox proportional hazard model and analyses of exponential, weibull, gompertz, and gamma accelerated failure - time models showed that variables of age (at diagnosis), marital status, relapse, number of supplementary treatments, disease stage, and type of surgery are among the effective factors on the survival of patients with gastric cancer (p <0.05). Unlike the similarity between proportional hazard model results and the results of accelerated failure - time models, the analysis of log - normal and log - logistic accelerated failure - time models also revealed that only variables of age (at diagnosis), marital status, relapse, and number of supplementary treatments were among the effective factors on the survival of patients with gastric cancer (p <0.05). Disease stage and type of surgery were not identified as risk factors by these models . Variables of sex, metastases, lymph node metastases, liver metastases, and distance metastases did not have any significant effect on patients survival in any of the studied models . To investigate the effect of different variables on the survival of cancer patients, most cancer researchers tend to use proportional hazard proportional hazard model rather than accelerated failure - time models . A systematic review on cancer journals indicates that only 5% of studies in which proportional hazard model was used, investigated the required assumptions for this model (37). The lack of proportional hazards assumption causes the results of model to be unreliable and biased; therefore, accelerated failure - time models such as generalized gamma, log - logistic, log - normal, gompertz, weibull and exponential can be better choices in such circumstance . As accelerated failure - time models consider a statistical distribution for survival time and they do not need proportional hazards assumption (ph), they are suitable alternatives to proportional hazard model . In this study, the results of cox proportional hazard model and accelerated failure - time models were compared to analyze the survival of patients with gastric cancer who had undergone surgery . To compare these models, cox - snell residuals and akaike information criterion (aic) were used . 1) revealed that accelerated failure - time models and cox proportional hazard model had approximately equal fitness . Among accelerated failure - time models, moreover, the analysis of models based on akaike information criterion (aic) (table 1) showed that the exponential and gompertz models were the best alternatives for cox proportional hazard model . There was not a significant difference between accelerated failure - time models and proportional hazard model in identifying factors affecting the survival of patients with gastric cancer except log - normal and log - logistic which showed higher aic than other accelerated failure - time models . The analyses of accelerated failure - time models and proportional hazard model showed that variables of age (at diagnosis), marital status, relapse, number of supplementary treatments, disease stage, and type of surgery were among the effective factors on the survival of patients with gastric cancer (p <0.05). These results are consistent with the results of many studies in this field (13, 14, 30, 38, 39). Moreover, variables of sex, metastases, lymph node metastases, liver metastases, and distance metastases did not have any significant effect on patients survival in any of the studied models . Based on the criteria presented in this study (aic & cox - snell), exponential and gompertz models are the best parametric alternatives for cox proportional hazard model . This issue is consistent with most studies conducted on patients with gastric cancer (4042). In some studies, however, weibull model has been considered as the good model but as exponential model is a specific case of weibull; again, the results of these studies are confirming the results of the present research (40). Although using cox proportional hazard model has come to the fore by most researchers in medical and cancer fields, results of accelerated failure - time models have often been more valid and have had minor bias since these models have better fitness in similar conditions due to a specific statistical distribution for the survival time and their not having need to ph assumption . Accelerated failure - time models will also be reliable alternatives to cox proportional hazard model where this assumption is not made . In addition, accelerated failure - time models may offer some benefits . Based on asymptotic results, accelerated failure - time models lead to more efficient parameters than proportional hazard model . With a reduction in sample size, relative efficiency may further change in favor of accelerated failure - time models . When empirical information is adequate, accelerated failure - time models can prepare some insights into the form of the baseline hazard . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy) have been completely observed by the authors.
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With an increase in the longevity of life and an increasingly sedentary lifestyle, there is an increased incidence of diabetes mellitus (dm2) in the elderly, who may get admitted to hospital for symptomatic or operative treatment . In the us, seven million americans are undiagnosed and 25 million are known diabetics, which might double up in the next decade . This is projected to keep increasing and is estimated to cost about 100 billion dollars per year to the tax payers . The challenge of treating diabetes in this section of population is to avoid hypoglycemia . With minimal tendency to cause hypoglycemia, canagliflozin sounds appropriate for the geriatric population . However, the drug needs to be used with caution, as it causes a drop in blood pressure and its use in the elderly needs to be closely monitored . It has a tendency to cause mycotic infections in the genital area and causes recurrent urinary infections, which may cause many unwanted side effects, especially in elderly women . For our discussion, elderly is defined as people with a chronological age of more than or equal to 65 . Articles in various international and national bibliographic indices were extensively searched, with an emphasis on canagliflozin, mycotic infections, and management of diabetes in the elderly . The various search sites included entrez (including pubmed), nih.gov, and medscape.com, webmd.com, medhelp.org, searchmedica, md consult, and google.com . Thereafter, there is a slow progressive decline of the glomerular filtration rate (gfr), even if there is no superimposed pathology . With aging there is an increased prevalence of diabetes and hypertension, which further exacerbates the picture . This also complicates the diagnosis and monitoring of diabetes in the elderly . In the elderly, hba1c should be interpreted with cynicism, keeping in view the comorbidities of the changing body physiology . In the elderly in rapid cell turnover states like hemolytic anemia, hba1c could be falsely low . During treatment with iron, b12 or erythropoietin, as in chronic kidney disease (ckd) on the other hand, in low cell turnover states, especially iron, b12 or folate deficiency or in the very elderly, when the marrow turnover is low, hba1c may be falsely high . In ckd, hba1c may be falsely high or low . When there is high carbamylated hemoglobin it may be falsely high and if erythropoietin is being used it may be falsely low [figure 1]. Pitfalls of hba1c monitoring in the elderly canagliflozin is the first oral inhibitor of sodium / glucose cotransporter 2 (sglt2) in the kidney . Sglt2 is responsible for the reabsorption of a majority of the glucose filtered by the kidneys . Canagliflozin acts by promoting loss of glucose in the urine, in a dose - dependent manner . The time taken for the drug concentration to peak is one to two hours (time to peak (tmax)). Depending on the dose used, the half - life (t) of the drug is 10.6 and 13 hours . It is metabolized by o - glucuronidation and is metabolized to inactive metabolites by uridine diphosphate glucose (udp) glucuronyltransferase (ugt). It cannot be removed by hemodialysis, owing to a large volume of distribution and high protein binding . The drug dose does not have to be adapted to hepatic dysfunction, but its use is not indicated in child - pugh c liver dysfunction . The effect of age, body mass index (bmi), gender, and race is clinically insignificant . There is no data on pregnancy, pediatric population, and nursing mothers, but it definitely causes weight loss . The most common side effects, especially in women, were recurrent urinary tract infection (uti), candida genital infections, and decreased blood pressure . These side effects are seen in about 10% of the subjects . There was a concern of carcinogenicity with canagliflozin, especially with respect to the renal tubules, adrenals, and leydig cells of the testis in rats . However, the fda concluded that these nongenotoxic effects were related to carbohydrate malabsorption and calcium imbalance . It has been used successfully in older patients with uncontrolled diabetes, in a multicenter, double - blind study by bode et al . In a trial by cefalu et al . The rate of recurrent uti and genital candida infections was higher than in the control group . Sglt2 is located in the proximal tubules of the kidney and is related to reabsorption of glucose from the proximal tubules . This leads to loss of glucose and thereby causes weight loss and improved glycemic control in an insulin - independent manner . Canagliflozin also prevents a rise in postprandial glucose due to intestinal sglt1 inhibition and an increase in the renal excretion of glucose . Vulvo vaginal candidiasis affects 75% of the women, at least once in their life time . Risk factors include employed woman, diabetes mellitus, use of an intrauterine device or spermicidal jelly for contraception and oral sex . However, there is a rise in the incidence of candida glabrata, especially in diabetic patients with recurrent vulvo vaginal candidiasis . Balanitis and belanoposthitis might be seen more so in diabetic indian men, as a majority of hindu men are not circumcised . In may 2013, fda approved canagliflozin for use in people with noninsulin - dependent diabetes mellitus (niddm) as a monotherapy or in combination with other drugs . A fixed drug combination therapy with metformin has so far not been approved by the fda . Canagliflozin is still seeking to carve a niche for itself in the management of diabetes in the elderly . Elderly diabetics often have cognitive dysfunction, and may have difficulty with self - management and may follow complicated treatment regimens . The unawareness of hypoglycemia is a major complicating factor in the management of elderly diabetics . Canagliflozin with its favorable pharmacokinetic profile and tendency for minimal hypoglycemia may aptly favor its use in the elderly . A standard approach of long - acting insulin at meal time and correctional insulin remains the standard even in the treatment of stress hyperglycemia . However, often there may be a wide fluctuation in glycemic control, which is more detrimental than sustained hyperglycemia . Canagliflozin may come in handy in these clinical scenarios when insulin cannot be used for some reason . Data regarding the use of canagliflozin for control of hyperglycemia in the elderly is minimal . Its biggest advantage for use in the elderly is its minimal tendency to cause hypoglycemia, which could lead to increased morbidity and mortality . If a patient has a risk for genital and urinary infections, these drugs must be avoided, especially in elderly females . Further clinical trials are needed to define the safety profile and refine the indications further . In general, treatment needs to be individualized in this age group, keeping in mind the comorbidities and the benefit to risk ratio . This may be worsened secondary to diuretic - like action in the renal tubules, with osmotic diuresis . It is important to continue to monitor for hyperkalemia, hypermagnesemia, hyperphosphatemia, and increase in low - density lipoprotein (ldl). The elderly are frequently on polypharmacy, which opens the door for potential drug interactions . Uridine diphosphate glucuronosyltransferase (ugt) enzyme inducers like rifampin, phenytoin, phenobarbitone, and protease inhibitors could potentially make canagliflozin subtherapeutic [table 1]. Rifampin is a non - selective inducer of ugt enzymes - ugt 1a9, ugt2b4 . The area under the curve (auc) is decreased for canagliflozin when used concomitantly with rifampin . Potential drug interactions of canagliflozin in the elderly with use of digoxin, which is a ugt enzyme inhibitor, the auc may increase by 20% for digoxin [table 1]. Also there is an epidemic of congestive heart failure (chf) and digoxin is often used in this clinical setting . Serum levels of digoxin may need to be monitored when initiating canagliflozin, especially in women, given its narrow therapeutic index . Also patients with chf would likely be on loop diuretics, which could potentially worsen the metabolic derangements associated with canagliflozin . Also it is a weak inhibitor of the p - glycoprotein (p - gp) transport system . However, it is not a substrate or inducer for the cyh or the p - gp system . Most of the oral hypoglycemics need to be adjusted as per the changing renal function . Renal insufficiency is one of the common clinical scenarios where severe hypoglycemia is seen, especially during the perioperative period and in critically ill patients . The physician should carefully monitor the renal function in the elderly, especially if there is nephropathy . The dose of canagliflozin needs to be carefully titrated with underlying renal insufficiency [table 2]. Dose reduction is recommended if the glomerular filtration rate (gfr) <60 ml / minute/1.73 m. it has been used safely in stage 3 ckd . The maximum dose for gfr <60 ml / minute/1.73 m is 100 mg daily . Use is not indicated for a gfr <45 ml / minute/1.73 m. renal insufficiency is a dynamic variable and careful monitoring of the renal function is mandated if canagliflozin is used for renal insufficiency . The dosing principles should include both evidence - based practices as well as logical empiricism, based on the experience of clinicians . A continuous need is felt to treat diabetes in a wider horizon, keeping in view the various social and behavioral aspects associated with it . However, such practices need time and more evidence - based studies, especially in critically ill and elderly diabetic surgical patients . Articles in various international and national bibliographic indices were extensively searched, with an emphasis on canagliflozin, mycotic infections, and management of diabetes in the elderly . The various search sites included entrez (including pubmed), nih.gov, and medscape.com, webmd.com, medhelp.org, searchmedica, md consult, and google.com . Thereafter, there is a slow progressive decline of the glomerular filtration rate (gfr), even if there is no superimposed pathology . With aging there is an increased prevalence of diabetes and hypertension, which further exacerbates the picture . This also complicates the diagnosis and monitoring of diabetes in the elderly . In the elderly, hba1c should be interpreted with cynicism, keeping in view the comorbidities of the changing body physiology . In the elderly in rapid cell turnover states like hemolytic anemia, hba1c could be falsely low . During treatment with iron, b12 or erythropoietin, as in chronic kidney disease (ckd) on the other hand, in low cell turnover states, especially iron, b12 or folate deficiency or in the very elderly, when the marrow turnover is low, hba1c may be falsely high . In ckd, hba1c may be falsely high or low . When there is high carbamylated hemoglobin it may be falsely high and if erythropoietin is being used it may be falsely low [figure 1]. Canagliflozin is the first oral inhibitor of sodium / glucose cotransporter 2 (sglt2) in the kidney . Sglt2 is responsible for the reabsorption of a majority of the glucose filtered by the kidneys . Canagliflozin acts by promoting loss of glucose in the urine, in a dose - dependent manner . The time taken for the drug concentration to peak is one to two hours (time to peak (tmax)). Depending on the dose used, the half - life (t) of the drug is 10.6 and 13 hours . It is metabolized by o - glucuronidation and is metabolized to inactive metabolites by uridine diphosphate glucose (udp) glucuronyltransferase (ugt). It cannot be removed by hemodialysis, owing to a large volume of distribution and high protein binding . The drug dose does not have to be adapted to hepatic dysfunction, but its use is not indicated in child - pugh c liver dysfunction . The effect of age, body mass index (bmi), gender, and race is clinically insignificant . There is no data on pregnancy, pediatric population, and nursing mothers, but it definitely causes weight loss . The most common side effects, especially in women, were recurrent urinary tract infection (uti), candida genital infections, and decreased blood pressure . These side effects are seen in about 10% of the subjects . There was a concern of carcinogenicity with canagliflozin, especially with respect to the renal tubules, adrenals, and leydig cells of the testis in rats . However, the fda concluded that these nongenotoxic effects were related to carbohydrate malabsorption and calcium imbalance . It has been used successfully in older patients with uncontrolled diabetes, in a multicenter, double - blind study by bode et al . In a trial by cefalu et al ., canagliflozin bettered glimepiride in both the doses . The rate of recurrent uti and genital candida infections was higher than in the control group . Sglt2 is located in the proximal tubules of the kidney and is related to reabsorption of glucose from the proximal tubules . This leads to loss of glucose and thereby causes weight loss and improved glycemic control in an insulin - independent manner . Canagliflozin also prevents a rise in postprandial glucose due to intestinal sglt1 inhibition and an increase in the renal excretion of glucose . Vulvo vaginal candidiasis affects 75% of the women, at least once in their life time . Risk factors include employed woman, diabetes mellitus, use of an intrauterine device or spermicidal jelly for contraception and oral sex . However, there is a rise in the incidence of candida glabrata, especially in diabetic patients with recurrent vulvo vaginal candidiasis . Balanitis and belanoposthitis might be seen more so in diabetic indian men, as a majority of hindu men are not circumcised . In may 2013, fda approved canagliflozin for use in people with noninsulin - dependent diabetes mellitus (niddm) as a monotherapy or in combination with other drugs . A fixed drug combination therapy with metformin has so far not been approved by the fda . Canagliflozin is still seeking to carve a niche for itself in the management of diabetes in the elderly . Elderly diabetics often have cognitive dysfunction, and may have difficulty with self - management and may follow complicated treatment regimens . The unawareness of hypoglycemia is a major complicating factor in the management of elderly diabetics . Canagliflozin with its favorable pharmacokinetic profile and tendency for minimal hypoglycemia may aptly favor its use in the elderly . A standard approach of long - acting insulin at meal time and correctional insulin remains the standard even in the treatment of stress hyperglycemia . However, often there may be a wide fluctuation in glycemic control, which is more detrimental than sustained hyperglycemia . Canagliflozin may come in handy in these clinical scenarios when insulin cannot be used for some reason . Data regarding the use of canagliflozin for control of hyperglycemia in the elderly is minimal . Its biggest advantage for use in the elderly is its minimal tendency to cause hypoglycemia, which could lead to increased morbidity and mortality . If a patient has a risk for genital and urinary infections, these drugs must be avoided, especially in elderly females . Further clinical trials are needed to define the safety profile and refine the indications further . In general, treatment needs to be individualized in this age group, keeping in mind the comorbidities and the benefit to risk ratio . This may be worsened secondary to diuretic - like action in the renal tubules, with osmotic diuresis . It is important to continue to monitor for hyperkalemia, hypermagnesemia, hyperphosphatemia, and increase in low - density lipoprotein (ldl). The elderly are frequently on polypharmacy, which opens the door for potential drug interactions . Uridine diphosphate glucuronosyltransferase (ugt) enzyme inducers like rifampin, phenytoin, phenobarbitone, and protease inhibitors could potentially make canagliflozin subtherapeutic [table 1]. Rifampin is a non - selective inducer of ugt enzymes - ugt 1a9, ugt2b4 . The area under the curve (auc) is decreased for canagliflozin when used concomitantly with rifampin . Potential drug interactions of canagliflozin in the elderly with use of digoxin, which is a ugt enzyme inhibitor, the auc may increase by 20% for digoxin also there is an epidemic of congestive heart failure (chf) and digoxin is often used in this clinical setting . Serum levels of digoxin may need to be monitored when initiating canagliflozin, especially in women, given its narrow therapeutic index . Also patients with chf would likely be on loop diuretics, which could potentially worsen the metabolic derangements associated with canagliflozin . Also it is a weak inhibitor of the p - glycoprotein (p - gp) transport system . However, it is not a substrate or inducer for the cyh or the p - gp system . Most of the oral hypoglycemics need to be adjusted as per the changing renal function . Renal insufficiency is one of the common clinical scenarios where severe hypoglycemia is seen, especially during the perioperative period and in critically ill patients . The physician should carefully monitor the renal function in the elderly, especially if there is nephropathy . The dose of canagliflozin needs to be carefully titrated with underlying renal insufficiency [table 2]. Dose reduction is recommended if the glomerular filtration rate (gfr) <60 ml / minute/1.73 m. it has been used safely in stage 3 ckd . The maximum dose for gfr <60 ml / minute/1.73 m is 100 mg daily . Use is not indicated for a gfr <45 ml / minute/1.73 m. renal insufficiency is a dynamic variable and careful monitoring of the renal function is mandated if canagliflozin is used for renal insufficiency . The dosing principles should include both evidence - based practices as well as logical empiricism, based on the experience of clinicians . A continuous need is felt to treat diabetes in a wider horizon, keeping in view the various social and behavioral aspects associated with it . However, such practices need time and more evidence - based studies, especially in critically ill and elderly diabetic surgical patients . Canagliflozin could accentuate side effect profile, if its dose is not adjusted to renal function . Also given the tendency of canagliflozin to decrease blood pressure, it might be prudent to avoid its use in patients with chf, until further clinical trials are available to refine the indications further . Given the high incidence of genital mycotic infection it might be prudent to avoid the use of canagliflozin in the elderly, who have a tendency for recurrent uti or genital mycotic infections.
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Several authors recently reported a substantial number of bare - metal stent (bms)-related restenosis cases presenting as acute coronary syndromes (acs)1)2) and several case reports providing intravascular ultrasound (ivus) evidence of pla - que rupture at in - stent neointima.3)4) although ivus is widely used to evaluate plaque composition, it has inherent image qu - ality limitations when compared with optical coherence tomography (oct) for the assessment of soft tissue components and subtle surface changes in atherosclerotic lesions.5)6) here, we present a patient with acs in whom a combination of ivus and oct imaging showed atheromatous changes and plaque rupture of neointima 10 years after bms implantation . A 58-year - old man was admitted with unstable angina and complaining of new onset substernal chest pain at rest . The patient had a history of prior percutaneous coronary intervention (pci) at this hospital due to angina pectoris ten years ago . At that time, the coronary angiogram (cag) showed 90% stenosis at the proximal portion of the left anterior descending coronary (lad). A bms (nir, 3.7516 mm, medinol, boston scientific, tel aviv, israel) was implanted at the proximal - portion of the lad and there was no evidence of in stent restenosis (isr) upon follow - up cag nine months after index pci . During ten years of follow - up at the local clinic, he had not complained of chest pain while on his medical treatment; however, periods of substernal chest pain at rest had recurred from one month before admission . Cag on this admission revealed evidence of focal stenosis with luminal haziness on the previously stented proximal lad and a de novo lesion at the distal left circumflex (lcx) artery (fig . 1, upper panel). Upon quantitative coronary analysis, the previously stented lesion had a diameter stenosis of 65% . Ivus images demonstrated a well expanded previously deployed bms and remarkable neointimal proliferation causing luminal narrowing (minimal luminal area=3.32 mm, plaque burden=75.9%). Of interest was a suspicious cavitary - shaped ruptured plaque in the neointima (fig . 1, middle panel). For more detailed evaluation of the suggested ruptured plaque in ivus, we conducted oct at the isr site, which showed evident proximally located thin fibrous cap atheroma (tfca) with lipid core and a ruptured plaque with tiny multiple ulcerations of neointima that were proximally located (fig . A new - generation drug eluting stent (xience v, 4.028 mm, abbott, il, usa) was successfully placed to cover the previously stented lesion, and post - stenting ivus revealed a well expanded previous isr lesion (minimal stent area=7.31 mm). A 2.7523 mm stent was also successfully deployed in the de novo lcx lesion . At the nine months follow up, cag showed patent stented lesions without evidence of isr, and the patient has been clinically stable for 18 months after pci . A 58-year - old man was admitted with unstable angina and complaining of new onset substernal chest pain at rest . The patient had a history of prior percutaneous coronary intervention (pci) at this hospital due to angina pectoris ten years ago . At that time, the coronary angiogram (cag) showed 90% stenosis at the proximal portion of the left anterior descending coronary (lad). A bms (nir, 3.7516 mm, medinol, boston scientific, tel aviv, israel) was implanted at the proximal - portion of the lad and there was no evidence of in stent restenosis (isr) upon follow - up cag nine months after index pci . During ten years of follow - up at the local clinic, he had not complained of chest pain while on his medical treatment; however, periods of substernal chest pain at rest had recurred from one month before admission . Cag on this admission revealed evidence of focal stenosis with luminal haziness on the previously stented proximal lad and a de novo lesion at the distal left circumflex (lcx) artery (fig . 1, upper panel). Upon quantitative coronary analysis, the previously stented lesion had a diameter stenosis of 65% . Ivus images demonstrated a well expanded previously deployed bms and remarkable neointimal proliferation causing luminal narrowing (minimal luminal area=3.32 mm, plaque burden=75.9%). Of interest was a suspicious cavitary - shaped ruptured plaque in the neointima (fig . 1, middle panel). For more detailed evaluation of the suggested ruptured plaque in ivus, we conducted oct at the isr site, which showed evident proximally located thin fibrous cap atheroma (tfca) with lipid core and a ruptured plaque with tiny multiple ulcerations of neointima that were proximally located (fig . A new - generation drug eluting stent (xience v, 4.028 mm, abbott, il, usa) was successfully placed to cover the previously stented lesion, and post - stenting ivus revealed a well expanded previous isr lesion (minimal stent area=7.31 mm). A 2.7523 mm stent was also successfully deployed in the de novo lcx lesion . At the nine months follow up, cag showed patent stented lesions without evidence of isr, and the patient has been clinically stable for 18 months after pci . Although many cardiologists are under the impression that neointimal growth after bms implantation has benign consequences, this case shows unstable clinical features and consistent neointimal atheromatous changes and plaque rupture findings on ivus and oct . In the past, post - mortem and endarterectomy specimens had shown extensive extracellular matrix accumulation with low rates of cellular proliferation in bms - related isr lesions,7 - 9) which were believed to have benign clinical presentation as stable angina . Although awareness of very late stent thrombosis is usually associated with the drug - eluting stent (des) era, it is also pertinent to the bms era . A large retrospective study reported that the cumulative incidence of stent thrombosis after bms implantation was 0.5% at 30 days, 0.8% at 1 year, 1.3% at 5 years, and 2.0% at 10 years.2) another retrospective study of 1,186 cases of bms - related isr reported that more than one - third presented as acute myocardial infarction or unstable angina.1) these studies suggested that stent thrombosis and acs long after bms implantation might be attributable to neointimal plaque rupture . Recently, an interesting report on assessment of very late stent thrombosis after both des and bms using ivus demonstrated that neointimal atherosclerotic changes and plaque ruptures were the main cause of bms - related very late stent thrombosis.10) another postmortem pathologic study revealed that atherosclerotic changes of neointima that occur significantly earlier and more frequently with des, were also found with bms.11) despite several previous reports suggesting atheromatous changes in neointima after stenting and case reports of ivus - documented ruptured plaque in neointima, there have been few reports of atherosclerotic progression with ruptured plaques of in - stent intima confirmed by both ivus and oct . Because of its high resolution of approximately 10 - 20 m, which is approximately 10-fold greater than that of ivus, oct can be a valuable tool for evaluation of intravascular plaque characterization that cannot be detected by cag and ivus.5) in this case, we found a suspicious plaque rupture in the neointima using ivus . However, oct imaging allowed detection of more detailed atheromatous changes of neointima such as multiple tiny ulcerations and a tcfa with a large lipid core, which could not be precisely discriminated in ivus images . Oct has a higher sensitivity than ivus for characterizing lipid - rich plaques, and the higher resolution of oct allows improved visualization of soft tissue components and detection of the cause of acs such as plaque rupture or erosion when compared with ivus images.5)6) we anticipate that neointimal atherosclerotic changes might be more easily detected before plaque rupture using a combination of oct and ivus than ivus alone . In summary, this case provides in vivo evidence of atheromatous neointimal changes and ruptured plaque using combination of ivus and oct imaging modalities.
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Aberrant cytokine expression accompanies both lymphoid and myeloid malignancies and is believed to contribute to disease phenotype and outcome . Recent studies in diffuse large b - cell lymphoma and primary myelofibrosis (pmf) have provided proof - of - concept in this regard by demonstrating phenotypic correlates and a powerful prognostic value for plasma levels of certain cytokines; circulating levels of interleukin (il)-2r, interleukin (il)-8, il-12, il-15 and c x c motif chemokine 10 (cxcl10) in pmf and il-2r, il-8, il-10, il-12, cxcl9 and cxcl10 in diffuse large b - cell lymphoma were independently associated with worse survival . In addition, in pmf, jak2v617f - positive patients, compared to their mutation - negative counterparts, were more likely to display higher plasma levels of il-1ra, il-2r, il-6, il-12, hepatocyte growth factor (hgf), cxcl10 and monokine induced by interferon (ifn)- other phenotypic correlates included increased levels of (i) il-6 and il-8 with constitutional symptoms, (ii) hgf with marked splenomegaly, (iii) il-2r, il-8, il-10, macrophage inflammatory protein (mip)-1 and monocyte chemotactic protein-1 with red cell transfusion dependency, (iv) il-2r, hgf and cxcl10 with leukocytosis and (v) cxcl10 with thrombocytopenia . Furthermore, a recent report suggested that anemia response to pomalidomide treatment in pmf was predicted by lower levels of circulating monocyte chemotactic protein-1, il-2r, il-15 and il-8 . The above - mentioned observations in pmf strongly suggest that specific cytokines contribute to specific disease symptoms and poor survival, and are also predictive of therapeutic response . Whether or not the particular nosogenic cytokines represent the host's immune response to clonal myeloproliferation or a tumor - specific autocrine process is yet to be clarified . Similarly, the detrimental effect on survival might be mediated by either exacerbation of co - morbid conditions, such as cardiovascular disease, or by promoting clonal evolution and/or leukemic transformation . The latter are consistent with the general concept of a direct link between inflammation and oncogenesis . Myelodysplastic syndrome (mds) and pmf are both clonal stem cell diseases and share a number of clinical features, including ineffective erythropoiesis, bone marrow neoangiogenesis and an increased risk of leukemic transformation . These similarities, as well as the known role of immune dysregulation in mds development and/or progression, led us to hypothesize that an altered plasma cytokine profile in mds is both phenotypically and prognostically relevant and may also provide additional pathogenetic and therapeutic insights . To that end, in the current study, we performed a comprehensive plasma cytokine analysis in 78 patients with primary mds . The main objectives of the study were: (i) describe the spectrum of abnormalities in cytokine levels and their comparison with normal controls; (ii) identify phenotypic and prognostic correlates of abnormally expressed cytokines; and (iii) compare plasma cytokine signatures of mds and pmf . The current study was approved by the mayo clinic institutional review board . All patients provided informed written consent for study sample collection as well as permission for use in research . Inclusion to the current study required availability of archived plasma, bone marrow aspirate and biopsy and cytogenetic information at the time of first referral to the mayo clinic . The diagnoses of mds and its subclassification were according to the world health organization criteria . To assure mature survival data, follow - up information was updated in june 2011 through review of patient histories and correspondence, social security death index or a telephone call to the patient or their local physician; date of last follow - up' reflected this time point and not the last time a patient was seen at the mayo clinic . Peripheral blood was collected under a mayo clinic protocol for patients with myeloid malignancies and standard procedures were followed to centrifuge samples at 4 c and store aliquots at 80 c . Concentrations of 30 plasma cytokines were analyzed in duplicates using multiplex bead - based luminex technology (invitrogen, carlsbad, ca, usa): il-1, il-1ra, il-2, il-2r, il-4, il-5, il-6, il-7, il-8, il-10, il-12, il-13, il-15, il-16, il-17, epidermal growth factor, eotaxin, fibroblast growth factor - basic, granulocyte macrophage colony - stimulating factor (gm - csf), granulocyte colony - stimulating factor, hgf, ifn-, ifn-, ifn--inducible protein 10/cxcl10, monocyte chemotactic protein-1, monokine induced by ifn-, mip-1, mip-1, regulated on activation normally t - cell expressed and secreted (rantes), tumor necrosis factor- and vascular endothelial growth factor . Measurements were performed on a luminex 200 analyzer (luminex corporation, austin, tx, usa) and resulting data were evaluated using the xponent software (luminex corporation). The observed intensities of duplicate samples were averaged and mapped to a fitted curve derived from a serial dilution series of known cytokine standards; observed intensities below and above the standard range were marked as 0% and 100%, respectively . All statistical analyses were considered clinical and laboratory parameters obtained at the time of first referral to the mayo clinic, which usually coincided with the time of plasma collection for cytokine analysis . Differences in the distribution of continuous variables between categories were analyzed by either mann whitney (for comparison of two groups) or kruskal wallis (comparison of three or more groups) test . Overall survival analysis was considered from the date of first referral to the mayo clinic (that is, date of plasma collection) to date of death (uncensored) or last contact (censored). Date of leukemic transformation replaced date of death, as the uncensored variable, for estimating leukemia - free survival . Overall and leukemia - free survival curves were prepared by the kaplan meier method and compared by the log - rank test . The jmp statistical package (sas institute, cary, nc, usa) was used for all calculations . The study population comprised of 78 consecutive patients with primary mds (median age, 72 years; range 4489 years; 67% male) for whom an archived plasma sample collected at the time of referral to the mayo clinic was available for cytokine analysis (table 1). In all, 64 (82%) patients were not receiving mds - directed therapy at the time of sample collection, and the remaining were receiving erythropoiesis - stimulating agents (that is, recombinant erythropoietin) alone . In total, 57 (73%) patients were treatment - nave; nine (11%) and five (6%) patients had previously received hematopoietic growth factors and hypomethylating agents, respectively . The international prognostic scoring system (ipss) distribution was as follows: low (28%), intermediate-1 (47%), intermediate-2 (18%) and high (5%); the corresponding distribution per the revised ipss (ipss - r) was as follows: very good 15 (19%), good 33 (43%), intermediate 13 (17%), poor 3 (4%) and very poor 13 (17%). The world health organization subgroups were as follows: refractory cytopenia with multilineage dysplasia / refractory cytopenia with multilineage dysplasia and ringed sideroblasts: 29 (37%); refractory anemia with excess blast-1: 13 (17%); refractory anemia with excess blast-2: 16 (21%); refractory anemia / refractory anemia with ringed sideroblasts: 8 (10%); mds with isolated del(5q): 7 (9%); mds - unclassified: 3 (4%); and refractory cytopenia with unilineage dysplasia (refractory thrombocytopenia): 2 (3%). Other risk - relevant information, such as the proportion of patients with red cell transfusion dependency, unfavorable karyotype and thrombocytopenia, is outlined in table 1 . Plasma cytokine levels were measured in 78 patients with primary mds (table 2) and 35 normal controls . Compared to normal controls, the levels of 19 cytokines were significantly different for mds patients: all were increased except for rantes (table 2). Phenotypic correlates in mds included increased levels of il-8 with higher ipss risk category, red cell transfusion dependency, higher bone marrow blast count and female gender; increased levels of cxcl10 with the presence of circulating peripheral blood blasts and thrombocytopenia; decreased levels of eotaxin with ipss - defined unfavorable karyotype; increased levels of rantes with thrombocytopenia; and decreased levels of il-17 with red cell transfusion dependency; with other associations as outlined in table 2 . Median follow - up from the time of plasma sample collection was 24 months (range, 0114 months); for the 32 living patients, the median follow - up was 31 months (range, 0114 months). During this period, 46 deaths (59%) a number of clinical and laboratory parameters were identified as being associated with shortened survival by using cox proportional hazards regression models (table 3). Of the 18 cytokines that were abnormally elevated in mds patients, only three cytokines were associated with shortened survival on univariate analysis: cxcl10 (p<0.01), il-6 (p=0.02) and il-7 (p=0.02) (table 3). All three cytokines maintained their individual significant association with shortened survival when the cox model was adjusted for the ipss or ipss - r (table 3). Consistent with this finding, there was no significant difference in cxcl-10, il-7 or il-6 levels between ipss low- and high - risk subgroups, or refractory anemia / refractory anemia with ringed sideroblasts /isolated on multivariable analysis that included the three cytokines, increased levels of cxcl10 (p<0.01) and il-7 (p=0.02) were identified as predictors of shortened survival, whereas il-6 maintained borderline significance (p=0.07). The survival association for cxcl10 (p=0.02) and il-7 (p=0.04) remained significant when the cox model was adjusted for ipss or ipss - r, age, red cell transfusion dependence and thrombocytopenia (il-6 levels remained borderline significant in this analysis). Based on the strength of their association with survival, a model for risk categorization of mds patients based on increased plasma levels of il-6, il-7 and cxcl10 was developed . For consistency with a previous similar analysis in pmf patients, a cytokine level that exceeded 3 standard deviations from the normal mean (> 3 s.d .) Mds patients with normal plasma levels of il-6, il-7 and cxcl10 lived significantly longer (n=20; median survival 76 months) as compared to those with increased levels of one or more of the three cytokines (n=57; median survival 25 months) (p<0.01) (figure 1). The risk - categorization model retained its predictive significance when adjusted for ipss (or ipss - r), red cell transfusion dependence and thrombocytopenia using a cox proportional hazards regression model (p=0.03; risk ratio=2.7, 95% confidence interval=1.18.2). In the present cohort, complete follow - up information (up to june 2011) including cause of death (where applicable) was available for 50 mds patients (64%); of these, six patients (12%) were confirmed to have undergone leukemic transformation . Leukemia - free survival was significantly associated with increased il-6 level (p=0.01) and borderline associated with increased cxcl10 level (p=0.09), but not with increased il-7 level (p=0.8). The association between inferior leukemia - free survival and increased il-6 was not accounted for by age, transfusion dependence, ipss, ipss - r, world health organization mds category, thrombocytopenia, ipss - defined karyotype or bm blast category (p<0.05). The present findings were not affected when the analysis was extended to the entire cohort; that is, the remaining 28 mds patients were included after censoring for leukemic transformation at the date of last follow - up (data not shown). Given recent data regarding a similar significant association of cytokines with specific phenotypic features as well as clinical outcome (overall and leukemia - free survival) in pmf, we sought to compare plasma cytokine levels between the two diseases (that is, mds and pmf). Of the 31 cytokines interrogated, plasma levels of all except il-2, il-5, il-8, granulocyte macrophage colony - stimulating factor, monocyte chemotactic protein-1 and vascular endothelial growth factor were significantly different between the two disease groups (table 4). The mds are a clinically and molecularly heterogeneous group of clonal hematopoietic stem cell disorders that are singularly characterized by peripheral blood cytopenias from ineffective hematopoiesis and an increased but variable risk of leukemic transformation . Dysregulation of the immune system is thought to play an important role in mds pathogenesis by promoting the development and/or progression of disease . The best evidence in this regard comes from the proven efficacy of immunosuppressive therapies such as anti - thymocyte globulin, cyclosporin a and alemtuzumab in at least a subset of mds patients where autoimmunity is the central pathophysiological mechanism, and also the frequent concurrence of autoimmune conditions with mds . Further evidence comes from a large, population - based, case control study using registry data, which demonstrated that chronic immune stimulation, represented by a wide range of infectious or autoimmune conditions, was significantly associated with an increased risk of developing mds or acute myeloid leukemia . Various aspects of immune dysregulation have been studied in mds, including the antigen - driven expansion of autoreactive cd8 + t cells of restricted v repertoire that are capable of suppressing hematopoietic colony growth in vitro . Evidence for impaired immune surveillance comes from the presence of dysfunctional natural killer (nk) cells with decreased cytolytic activity possibly reflecting expansion of clonal nk cells and/or decreased expression of nk activating receptors, and also from the presence of increased numbers of peripheral blood t regulatory cells (defined as cd4 +, cd25 foxp3 + t cells) of polyclonal origin, particularly in advanced mds patients . Another possible mechanism is the inflammatory cytokine - driven expansion of myeloid - derived suppressor cells that may further contribute to the development of immune tolerance in mds . Mds patients exhibit an abnormal cytokine milieu that likely stems from the interaction of the mds clone with bone marrow stromal cells and infiltrating immune effector cells that underpins the persistent low - level inflammatory state . Although much of the attention has been focused on the study of individual myelosuppressive / pro - apoptotic or pro - inflammatory cytokines such as tumor necrosis factor-, tumor growth factor-, ifn- or il-6, only two previous studies to our knowledge have attempted to comprehensively profile circulating cytokine chemokine levels in mds patients . In one study of 162 acute myeloid leukemia and 100 mds patients who profiled 27 cytokines, a combined analysis of both patient groups showed that serum (not plasma) levels of csf3, il-1ra, il-8, il-12, il-15, cxcl10, tumor necrosis factor - and il-17 were significantly elevated relative to normal controls . Unexpectedly, the expression profiles of 24 of the 27 cytokines were statistically indistinguishable between acute myeloid leukemia and mds patients . Although individual cytokine levels were not correlated with clinical or laboratory features, elevated levels of il-4 and mip-1 were associated with longer survival in mds; the latter observation was however not confirmed within the context of other known prognostic factors in mds in a multivariate analysis . In the other study of 33 aplastic anemia and 57 mds patients, plasma levels of tumor necrosis factor -, il-6, mip-1, mip-1 and hgf although distinct cytokine signature profiles were identified for aa and mds patients, there were no data presented regarding clinical or laboratory correlates of individual cytokines or any information regarding association of cytokine levels with clinical outcome . The current study adds significant novel information relative to the two aforementioned studies of cytokine profiling in mds . First, we measured cytokine levels in plasma, which may be an advantage given the significant differences in levels of soluble protein factors between plasma relative to serum due to activation of the coagulation cascade in the latter . Second, the vast majority of mds patients in our study were not receiving active therapy at the time of sample collection and none were receiving disease - modifying therapy that could potentially alter the disease - inherent baseline cytokine profile . Third, the follow - up of patients in our cohort was mature, thereby allowing for full analysis of the effect of cytokine levels on overall survival and leukemia - free survival . Fourth, availability of a fully annotated clinical database allowed a detailed assessment of association of cytokine levels with clinical or laboratory features . Fifth, updated prognostic information per the ipss - r was incorporated and the added prognostic impact of cytokine levels on clinical outcome was confirmed within this context . Sixth, a full comparison of cytokine profiles between mds and pmf was conducted given the disease similarities in terms of anemia prevalence, presence of constitutional symptoms and increased risk of bone marrow failure and leukemic transformation . In the current study of mds patients, il-8 and cxcl10 stand out as the most relevant cytokines in terms of phenotypic correlations and cxcl10, il-7 and il-6 in terms of their prognostic impact . Shortened overall survival was predicted by increased levels of the latter three cytokines independent of conventional prognostic factors in mds that included ipss or ipss - r, age, red cell transfusion dependence and thrombocytopenia . This suggests a significant pathogenetic contribution of the aforementioned cytokines in disease development and/or progression that is independent of the mds clone and confirms the importance of dysregulated immunity in mds . Accordingly, the cytokine data could be effectively combined into a model for risk categorization, wherein normal levels of all three cytokines identified mds patients with significantly longer survival relative to those with elevated levels of one or more cytokines . Although the predictive value of this model was confirmed to be independent of known prognostic factors in mds for the overall cohort, the analysis was not extended to ipss subsets given the limited number of patients in the current study . Death in mds patients results from the competing phenomena of worsening co - morbidities, bone marrow failure and leukemic transformation . In an analysis of the association of specific cytokines with the risk of leukemic transformation, we found that two of the three cytokines that were associated with inferior overall survival (il-6, and to a lesser extent cxcl10) were also predictive for inferior leukemia - free survival in mds, independent of previously established prognostic factors in this regard . This observation suggests an additional permissive role of specific cytokines in the process of clonal evolution in mds and identifies at least one reason for the inferior survival of patients with increased cytokine levels . Additional studies are needed to ascertain whether elevated cytokine levels are also implicated in excess deaths due to increased progression of co - morbidities such as cardiovascular disease, and so on . Observations from the current study reinforce the concept of distinct and prognostically relevant plasma cytokine signatures in hematological malignancies . Although the plasma cytokine profile between mds and pmf is substantially dissimilar, it is interesting to note that cxcl10 was identified as an independent prognostic indicator in both diseases and also large - cell lymphoma . The particular observation suggests a general host response to a disease state with an aggressive biology rather than a tumor - specific cytokine release . Also of note is the observation that the other prognostically relevant cytokines were different between mds (il-6, il-7) and pmf (il-2r, il-8, il-12, il-15) or large - cell lymphoma (il-2r, il-8, il-10, il-12, cxcl9). In this instance, some of the nosogenic cytokines might be under the control of a tumor - specific autocrine process and therefore also relevant to disease pathogenesis . With regards to mds - relevant cytokines, il-7 was initially identified as a growth factor for b - lymphocyte progenitors and is now known to be an important regulator for many aspects of b - cell lymphopoiesis . Il-6 is a pro - inflammatory cytokine, which has an important role in activating the immune system, particularly in the transition from an innate to an acquired immune response . Cxcl10 is a chemokine induced by ifn-, and is also classified as an inflammatory cytokine; this chemokine binds to cxcr3 and regulates the immune response through the activation and recruitment of various immune - competent cells . The observations from the current study may also have therapeutic implications, an aspect that is exemplified by the known activity of drugs such as thalidomide and lenalidomide that have broad immunomodulatory activity in mds . Patients with mds face not only shortened survival, but also a major compromise in quality of life as a result of frequent red blood cell transfusion requirement and other complications such as infections and bleeding . It is possible that the specific aforementioned cytokines or their downstream signaling intermediates can be therapeutically targeted to alleviate clinically relevant aspects of mds.
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Although cardiac surgery procedures fall into the category of so - called clean procedures, they remain burdened with the risk of infectious complications . Postoperative sternal wound infection (swi) occurs in 0.5 - 8% of patients and is associated with high mortality (14 - 47%), especially in the event of deep sternal wound infection (dswi), which is the most serious form of sternal wound infection . The reasons behind the complications are multifactorial and may be endogenous or exogenous in nature . The occurrence of postoperative infection may be related to patient characteristics (advanced age, diabetes, generalized atherosclerosis, circulatory insufficiency) or surgery parameters (duration lasting several hours, use of extracorporeal circulation, operative wound extent, necessity of employing electrocoagulation during hemostasis, peri- and postoperative blood loss requiring blood product transfusion). Applying antibiotic locally in the form of collagen - gentamicin sponge appears to be the perfect solution . Notwithstanding, although the sponge is used in cardiac surgery in more than 50 countries, its efficacy in preventing swi is still being disputed . Gram - positive and gram - negative bacteria, such as staphylococcus aureus, staphylococcus epidermidis, pseudomonas aeruginosa, and escherichia coli, are the most commonly encountered microorganisms that infect bone and soft tissue . In cardiac surgery, being able to adhere to foreign material, such as catheters or stainless steel wires used for sternal closure, they synthesize an extracellular polysaccharide, producing a biofilm that is difficult to eliminate . In such cases, the foreign body has to be removed in order to allow proper treatment of the infection . Other pathogens isolated from sternal wounds include escherichia coli, klebsiella, enterobacter cloacae, and acinetobacter . Propionibacterium acnes has also been identified as a microbal agent of swi, which can influence sternal instability after fixation [2, 6]. This natural antibiotic produced by micromonospora purpurea is characterized by a wide spectrum of bactericidal action with regard to oxidative gram - negative bacteria and some gram - positive microorganisms . As has been demonstrated, staphylococci are sensitive to high concentrations of gentamicin [59]. Its bactericidal action is dependent on the concentration of the agent at the infection site . The mechanism of action is based on the blocking of protein biosynthesis, as aminoglycosides bind permanently to subunit 30s of the bacterial ribosome . Biological distribution is dependent on factors including age, obesity, renal function, or the presence of ascites in patients . Gentamicin, like other aminoglycosides, is not well absorbed when administered orally due to its cationic nature; therefore, its administration needs to be parenteral intravenous or intramuscular [7, 10]. In the case of intravenous administration, the agent's bioavailability is 100%, and its maximum serum concentration is reached as early as 30 minutes after injection . Its biological half - life time is 2 hours for adults, and therapeutic concentration is achieved at the range of 4 - 10 mg / l; however, intravenous administration of> 2 mg / l is considered to be toxic for the patient . The bioavailability of gentamicin administered intramuscularly is the same as in the case of intravenous administration, but a longer period of time (30 - 90 minutes) is required to reach maximum serum concentration . Within 24 hours, gentamicin is expelled by the kidneys in unchanged form, accumulating in the lysosomes of kidney proximal tubular cells and causing their apoptosis in clinically relevant doses aminoglycosides have good chemical stability and do not commonly cause allergic reactions . In comparison to -lactam antibiotics, which are widely used in perioperative prophylaxis, aminoglycosides have a significantly narrower operating range, but they rarely induce bacterial resistance . If bacterial resistance to gentamicin does occur, it mostly consists in the breaking down of the antibiotic by various enzymes . An important characteristic of aminoglycosides is their ability to act in synergy with other antibacterial agents . The synergy effect and the ability to rapidly kill microorganisms can be observed in combination therapy, substantiating their clinical efficacy . Combining them with -lactam antibiotics can be employed in the treatment of serious infections by gram - negative bacteria or in the treatment of infections caused by gram - positive bacteria, such as staphylococcus aureus, enterococci, or streptococcus viridans endocarditis . Combining gentamicin with ultrasound is being successfully used to kill biofilm - producing bacteria, such as escherichia coli and pseudomonas aeruginosa, which are commonly found on implants, dialysis catheters, cardiac pacemakers, as well as on artificial vascular prostheses and heart valves . The therapeutic capabilities of systemic antibiotic administration are limited, while the local application of antibiotics is associated with numerous advantages . Most importantly, high agent concentration at the implantation site is ensured and minimum inhibitory concentration (mic) is retained during the treatment, while adverse effects are minimized without reaching a toxic blood concentration . At present, various biodegradable and nondegradable antibacterial agent carriers are being used . One of the first biomaterials available on the market was poly(methyl methacrylate) (pmma) used in the form of bone cement or granules; the currently employed materials include hydroxyapatite (granules or porous scaffold), the natural polymer chitosan (membranes, microgranules, thermosensitive gel, sponges), and bovine tendon collagen (sponges) [7, 1215]. Collagen sponges resorbed after the release of the drug are widely used as carriers for active substances . Some of the conducted in vitro studies demonstrate that the kinetics of releasing the antibiotic from this matrix may reach as much as 95% after as little as 1.5 hours, in comparison to 8% achieved by pmma carriers . Local application results in the achievement of a high concentration of the therapeutic agent within several hours, which is then retained for up to 4 days . Owing to these kinetic properties and to the activity of the antibiotic within a set time period, the development of microorganism resistance progresses very slowly . Moreover, collagen may produce scaffolds for fibrin deposition, which results in the healing of tissue defects and acceleration of wound healing . Gentamicin - collagen sponge (gmcs) is frequently used in surgery in order to prevent wound infections and to treat local infections of bone and soft tissue [1, 4]. The site of surgical access, such as sternotomy, is rarely affected by infection; however, if it does take place, it is associated with the risk of further serious complications . For this reason, additional prophylaxis is crucial, particularly in the case of high - risk patients . The local application of gentamicin in the form of an absorbable collagen sponge ensures gradual release of the agent within a short time period, which improves wound healing without the nephrotoxic or ototoxic effect of aminoglycosides [1, 14]. One of the studies by friberg et al . Has demonstrated that administering 260 mg of gentamicin in between the parts of the closed sternum results in a high antibiotic concentration within 8 - 12 hours after the surgery . It may be applied substernally, presternally, or in between the parts of the cut sternum . Substernal implantation allows a high gentamicin concentration to be achieved in this region, with concurrent lower antibiotic concentration in the presternal area . This results in a significant reduction in mediastinal deep sternal wound infections (dswi) and a slight reduction in superficial sternal wound infections (sswi). In turn, in presternal application, the wires are partially covered, which prevents the adhesion of coagulase - negative staphylococci present in the physiological flora of the skin . It appears, therefore, that the best option is to place the matrix in between the halves of the cut sternum, which allows for a balanced release of the antibiotic in the vicinity of the sternal closure . There remains, however, a risk of reoperation due to sternal dehiscence and bleeding . Literature data indicate that additional cardiac surgery interventions due to bleeding were required by approximately 4% of patients with sponges applied in this manner and by 2.3% of controls . Certainly, a too thick layer of the sponge applied during sternal closure may lead to such complications; therefore, in order to reduce the high risk of postoperative bone marrow bleeding and sternal instability, a single antibiotic matrix, properly cut to match the margins of the wound, should be used (figs . 1 and 2) [1, 5, 9]. Scheme a gentamicin - collagen sponge, properly cut to size and placed in between the halves of the sternum and substernally (longitudinal and cross section) [5, 17] a gentamicin - collagen sponge cut with sterile scissors to fit the shape of the wound, inserted under and in between the halves of the sternum after a cabg procedure the literature presents the advantages of the local application of antibacterial agents in surgery . Efficacious use of sponges in prophylaxis provided to cardiac surgery patients has been presented by friberg et al ., who examined 2000 patients undergoing coronary artery bypass grafting (cabg). In the group implanted with the sponges, postoperative complications in wound healing occurred in 4.3% of patients, as compared to 9% of patients in whom only standard perioperative antibiotic prophylaxis was used . Reports from a hospital in germany also corroborate the advantageous effect of gmcs (2.54% vs. 6.5%), especially with regard to dswi reduction (0.56% vs. 3.52%). A medical center in the czech republic also reported a positive effect of this type of prophylaxis on the incidence of swi (8.3% vs 19.8%). By applying a gentamicin - collagen sponge locally in between the parts of the surgically cut sternum and modifying the sternal closure procedure, a significant reduction has been observed with regard to swi cases caused primarily by cons bacterial strains, with the exception of propionibacterium acnes bacteria refractory to aminoglycoside treatment . Furthermore, redness and secretion at the surgical access site were reduced during the wound healing process in comparison with the control group . Reported 12.3% of surgical site infections within 5 years in a clinical hospital in france . Reoperation due to dswi was required by 13.8% of patients, in comparison to 12.6% of patients implanted with gmcs . Therefore, the use of the sponges did not significantly limit the development of deep infection . Similar observations were made by raja et al . ; in their study, gentamicin did not limit the occurrence of dswi (2.1% vs. 3.1%), but it did influence the reduction of sswi (2.1% vs. 6.2%). Data collected from as many as 48 hospitals in the usa indicate the lack of a significant difference in terms of reducing infection between the study group and controls (8.4% vs. 8.7%) with regard to both superficial and deep sternal wound infection . As underscored by the authors, multicenter studies often do not reflect results obtained in individual cases . The differences in the results may also be explained by ethnic and regional differences, as well as by the associated distribution of pathogens with varying mechanisms of resistance . For example, the american studies included a small percentage of bacterial strains that turned out to be resistant to gentamicin, including staphylococcus epidermidis . Therefore, the differences in bacterial strains distribution condition the efficacy of agents used in prophylaxis . Despite advanced surgical techniques, a body of science concerning the pathogenesis of surgical site infections, and the use of perioperative antibiotic prophylaxis, the incidence of these infections remains unchanged . Thus, more effective methods of prevention are required, particularly because of the rising population age and obesity, the number of type 2 diabetes patients, and the rapid emergence of resistant microorganisms . While evidence indicating that local prophylaxis administered in the form of gentamicin - collagen sponge implanted at the end of cardiac surgery procedures may reduce the incidence of swi does exist, it appears crucial for clinical practice to conduct further observation and assessment of the capabilities of gmcs due to differing opinions . Although their use is recommended for all adult patients undergoing heart surgery, it can be of greatest benefit to high - risk patients (e.g. With diabetes and/or bmi> 25).
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Vaccination using antigens expressed by endothelial cells lining the tumor vasculature represents the most attractive vaccination strategy because immunization using this approach may prevent the growth of any solid tumor . Therefore, endothelial cells can be used as a source of antigens used in the development of a universal cancer vaccine (ucv). However, utoimmune - mediated damage to microvessels, the primary targets of anticancer endothelial cell - based vaccination strategies, may lead to side effects, namely, autoimmune - mediated damage of microvessels in healthy tissues [2, 3]. Therefore, antigen compositions constituting a ucv that are distinct from antigens expressed by endothelial cells in normal tissues need to be designed to prevent the elicitation of undesired autoimmunity . Recently we described the interactions between tumor - induced endothelial cell surface heterogeneity and endothelial cell escape from cell - mediated immune responses [4, 5]. These data sets suggested that an efficient autologous vaccine could be designed utilizing surface antigens expressed by cultured human microvascular endothelial cells (hmec) if their tumor - induced cell surface profile and the profile of target hmec were similar . In this scenario, the efficacy of the autologous vaccine would exceed 18 (i.e., 18 tumor endothelial cells will be destroyed before 1 endothelial cell in normal tissue is destroyed). Antigen compositions intended for vaccination were based on a specifically derived set of hmec natural cell surface antigens distinguished by the abbreviation santavac (set of all natural target antigens for vaccination against cancer). Although the design of these studies was mainly intended to describe an autologic type of santavac, it was found that alloantigen compositions also may be efficiently used for anticancer vaccination . In one case, the killing rate of target hmec using allogeneic surface antigens related directly to the in vitro design of the allogeneic universal vaccine with efficacy of targeting equal to 4 . This efficacy provides a therapeutic window where tumor hmec could be killed before hmec of normal tissues are adversely affected . Unfortunately, the efficacy of the allogeneic santavac in these studies was characterized by a limited number of experiments . To fill this gap, additional cytotoxicity assays (cta) were performed in the present study to complement the ucv design based on the santavac . The possibility of excluding a patient's own biomaterial from the vaccine preparation simplifies the development activities and increases the value of the allogeneic santavac vaccines . Two abdominal subcutaneous adipose tissue biopsies were obtained from female patients (4050 years old) undergoing open abdominal surgical procedures at the national medico - surgical center (moscow, russia). The protocol was approved by the research ethics committee and the patients provided written informed consent . The biopsy specimens were transported to the laboratory, and endothelial primary cultures were established using magnetic beads coated with anti - cd31 monoclonal antibody (dynabeads cd31 endothelial cells, invitrogen, life technologies, carlsbad, ca, usa) as described previously . Culture media (medium mcdb 131 and microvascular growth supplement, gibco, thermo fisher scientific, waltham, ma, usa) supplemented with 10% fbs (fetal bovine serum) (paa laboratories, dartmouth, ma, usa), 50 g / ml streptomycin, 50 u / ml penicillin, 2 mm glutamine (gibco), and 12 u / ml heparin (sigma - aldrich, st . Louis, mo, usa) were changed every 2 - 3 days and after the first passage . Cells were grown to 65% confluence and used in future experiments . To obtain hmec with tumor - induced phenotypes, cell cultures were incubated for 4 days in culture medium (mcdb 131, fbs, streptomycin - penicillin, glutamine, and heparin) supplemented with 5%, 15%, and 25% of tumor - conditioned medium . Cells were visualized using a lei dm5000b microscope (leica microsystems, buffalo grove, il, usa). Primary fibroblast cultures were established from an adult skin biopsy (45-year - old woman; donor provided written informed consent) as described by ritti and fisher . Primary cultures were cultured in dmem (gibco) supplemented with 10% fbs, 5 g / ml streptomycin, 5 u / ml penicillin, and 2 mm glutamine at 5% co2 at 37c and third passage cells were used to obtain fibroblast - associated antigens (faa). Tumor - conditioned medium was collected from hepg2 (human hepatocellular carcinoma cells, atcc, manassas, va, usa; cell lines have been authenticated by cell proteomic footprinting) as described by folkman et al . . Media were conditioned for 48 h, collected, centrifuged for 10 min at 600 g, and filter - sterilized (0.2 m). Tumor - conditioned medium was then concentrated 10x using centriplus centrifugal filter devices ym-3 (millipore, merck kgaa, darmstadt, germany) and used in experiments . In order to determine the optimal concentration of tumor - conditioned medium required to provide different tumor - induced stimuli to hmec, the 10x tumor - conditioned medium was added to hmec seeded in the wells of a 96-well plate at different concentrations (0, 10, 20, 30, 40, and 50% in mcdb 131 medium supplemented with streptomycin - penicillin, glutamine, and heparin). After 3 days in culture, cells were counted in wells using trypan blue staining to determine the concentration of tumor - conditioned medium that induced weak (stimuli just a little higher than in the control), moderate (half of the maximum), and strong (a little more than related to maximum) stimulation . Endothelial cells were stained with phycoerythrin- (pe-) conjugated mouse anti - hvegfr-2 igg1 (clone 89106, r&d systems, minneapolis, mn, usa) or pe - conjugated mouse anti - human cd62e igg1 (clone 68 - 5h11, bd pharmingen, becton dickinson, san jose, ca, usa). For isotype control, cells were stained with pe - conjugated mouse igg1 (r&d systems, clone 11711, or bd pharmingen, clone mopc-21, resp . ). Flow cytometry was performed on a bd facscalibur flow cytometry system (becton dickinson) and the data analyzed using cell quest software (becton dickinson). Hmec or fibroblasts grown to 65% confluence were washed 5x with hbss before being treated with 0.2 g / ml trypsin (15,000 u / mg, promega, madison, wi, usa) in hbss . A 0.5 ml trypsin solution was added to each well of a 6-well plate, incubated for 20 min at 37c in saturated humidity, then collected again, and centrifuged (600 g for 5 min). The resulting supernatant contained cell surface targets and was considered a solution of santavac or fibroblast - associated antigens (faa), respectively . Briefly, fresh peripheral blood mononuclear cells (pbmcs) from healthy donors were isolated using ficoll - hypaque (paneco, moscow, russia) gradient centrifugation and were then allowed to adhere to 12-well culture plates for 1 h. nonadherent cells were collected and centrifuged, and cell pellets were mixed with autologous serum containing 10% dmso and stored in liquid nitrogen . Cryopreserved, nonadherent pbmcs, which also are considered as peripheral blood lymphocytes, were later used as a source of effector cells (cytotoxic t lymphocytes, ctl) for cytotoxicity assays . The adherent cell fraction was cultured in rpmi-1640 (gibco) supplemented with 10% fbs, streptomycin - penicillin, and glutamine in the presence of 0.075 g / ml granulocyte macrophage colony - stimulating factor (neostim, 1.67 10 me, fds farma, uk) and 1000 u / ml interleukin-4 (sigma - aldrich). After 6 d in culture, santavac (0.5 ml) or faa (0.5 ml) were added to each well of a 12-well culture plate with immature dc (3 10 cells / well in 1 ml of culture medium) and dc were matured with 1000 u / ml tumor necrosis factor- (sigma - aldrich) for 48 h. matured, santavac - loaded or faa - loaded dc were then used to stimulate ctl . Santavac - loaded dc (3 10 cells / well) in 12-well culture plates were combined with 6 10 autologous nonadherent pbmcs (1: 20) in 1 ml of rpmi-1640 medium (containing 10% fbs, streptomycin - penicillin, and glutamine) supplemented with 30 u / ml of clinical grade human interleukin-2 (ronkoleukin, biotech, st . After incubation for nine days, nonadherent pbmcs containing stimulated ctl were washed by centrifugation and used as effector ctl in cytotoxicity assays . Hmec (5 10 cells / well) were seeded into 48-well plates, which yielded 3 10 cells / well after 72 h. effector ctl were then added to hmec at an effector: target ratio of 20: 1 . On the third day, target hmec were washed to remove ctl and attached hmec were trypsinized and viability detected using trypan blue exclusion . The number of nonstimulated and tumor - stimulated hmec in the presence or absence of effector ctl stimulated with faa - loaded dc was used as controls . Cta data were used to calculate the in vitro efficacy of the allogeneic santavac formulation, namely, efficacy type i, denoted as efficacy i and calculated as a ratio of the number of nonstimulated cells in control wells (i.e., hmec) to the number of tumor - stimulated cells in experimental wells, and efficacy type ii, denoted as efficacy ii and calculated as a ratio of the number of tumor - stimulated cells in control wells (i.e., hmec, hmec, or hmec) to tumor - stimulated cells in experimental wells . Figures 1(a) and 1(b) show primary hmec cultures isolated from fat biopsies obtained from donors 1 and 2, respectively . Facs analysis (figure 1(c) for hmec donor 1; data for donor 2 are not shown) revealed that the vegfr-2 endothelial cell marker is associated with almost 90% of the cells during the first passage following isolation . No growth of contaminating fibroblasts or mesothelial cells was detected, demonstrating that primary hmec cultures were successfully established . Additionally, facs analysis of cd62 confirmed the endothelial nature of the primary cultures after activation using tumor - conditioned medium (figures 1(d) and 1(e)). The optimal concentration of tumor - conditioned medium required to provide hmec stimulation was determined next . Hmec were cultured for 3 days in the presence of different concentrations of tumor - conditioned medium . Culture medium containing tumor - conditioned volumes of 5%, 15%, or 25% elicited weak (stimuli just a little higher than in the control), moderate (half of the maximum), or strong (a little more than related to maximum) levels of hmec stimulation, respectively (figure 2). The immunologic properties of respective santavac compositions were evaluated by loading dc with corresponding santavac as a means of activating and stimulating human cytotoxic t lymphocytes (ctl) against target hmec . Ctl stimulated with fibroblast - associated antigen- (faa-) loaded dc incubated in the presence of target hmec were used as controls . On day 3, surviving target hmec were identified using trypan blue exclusion . A subtle improvement in cytotoxicity was observed when ctl were stimulated with faa - loaded dc . Dc loaded with hmec and stimulated with 15% or 25% tumor - conditioned medium elicited effective immune responses measured by high death rates of target hmec (figure 3). Notably, ctl stimulated with dc loaded with antigens from hmec (superscript represents the percentage of tumor - conditioned medium used to stimulate hmec) was also most effective against hmec target cells (in this case almost all target cells were dead). The target hmec were most efficiently killed by ctl stimulated with dc loaded with antigens from hmec . Santavac efficacy i indicates that in vitro modeled vaccine safety was 17.3, achieved using antigens derived from hmec and hmec used as targets . Santavac efficacy ii indicates that the in vitro modeled capacity to arrest tumor growth was ~60, also achieved by santavachmec (hereinafter [santavac is santavac generated from hmec for cta][hmec used as target cells in same cta]). Development of cell - based vaccines focuses on the elicitation of immune responses against target cells expressing native antigens [12, 13]. Cell surface targets are prioritized for vaccine design [14, 15] and are accessible to proteases whose byproducts could be isolated following in vitro proteolytic cleavage . Previously, it was shown that the antigenic essence of cells, which may be used in cell - based vaccines in contrast to whole cells, could be prepared by proteolytic cleavage of cell surface targets [16, 17]. The composition of this antigenic essence, which was established by the proteomic footprinting [8, 18], directly defined target cell killing rates in cta that represent an in vitro anticancer vaccination model . Santavac formulations can be mixed with different adjuvants and their immunogenicity and safety tested in vivo as ucv . The present study expanded on the selection of alloantigens used in the preparation of santavac vaccines . The primary benefit of utilizing alloantigens as vaccine components is the possibility of excluding the patient's biomaterial from the vaccine preparation, thereby simplifying vaccine development, lowering the cost, and facilitating translation into clinical practice . Although autogenic santavac induced much higher target cell killing rates in cta than alloantigens, alloantigens may also be highly efficient . Alloantigen compositions that induced low tumor killing rates also exhibited low killing rates of healthy tissues providing the required therapeutic window for their application . From the perspective of estimating vaccine efficacy (defined by target cell destruction in the absence of damage to healthy tissues), the immune response elicited by alloantigens was safer and the following previously discovered observations relating to endothelial cell heterogeneity were considered: (i) the tumor influence on hmec was not specific to the tumor type and hmec heterogeneity was a result of differences in strength of this influence; (ii) there was a linear dependence between target cell killing rates and the similarity of cell surface profiles of target cells and cells used to generate surface antigens for targeting the immune response; and (iii) the increase in tumor - induced changes at the hmec surface led to decreased immunogenicity of hmec surface antigens . In addition, one particular observation from previous experiments suggested that the strongest changes to the hmec surface were induced by hepg2 cells . This research was therefore designed to measure target cell killing rates in cta where alloantigens were derived from hmec stimulated to grow following stimulation by hepg2 that possessed a different signal strength (from weak to strong stimuli). It was therefore expected that the cta experiments would reveal the maximum efficacy of allogeneic santavac in vitro . Figure 2 showed how tumor stimuli strength was selected to provide hmec with the diversity of tumor - induced surface profiles . Weak stimuli corresponded to the tumor - conditioned medium that induced an hmec proliferation rate slightly higher compared to proliferation of control hmec . Moderate stimuli corresponded to the percentage of tumor - conditioned medium which provided hmec proliferation at half the maximum rate . Strong stimuli corresponded to the percentage of tumor - conditioned medium used which provided hmec the stimuli to proliferate at a high rate . Cta revealed that hmec with tumor - induced surface changes may be efficiently targeted by allogeneic santavac . This phenomenon was consistent with previously published data describing hmec heterogeneity that established the foundation for the development of the santavac . Tumor cells induced unidirectional changes to the hmec surface profiles resulting in a more similar antigen profile between target cell surface antigens and the surface antigen profile of cells used to generate antigens needed to target the immune response . As a consequence, the observed efficacy of santavac generated from hmec and hmec (i.e., santavac and santavac, resp .) Was sufficiently higher than the efficacy observed for control cells (i.e., hmec) and santavac . The high similarity between antigens present in santavac and the cell surface antigens expressed by target cells explains this observation . The fact that target cell killing of santavachmec was sufficiently higher than that of santavachmec can be explained by one above - mentioned statement: that immunogenicity decreases with increasing tumor - induced changes to the hmec antigen surface profile . Therefore, moderate tumor - induced changes to hmec surface antigens would be preferable in the context of vaccine design resulting in efficacy i equal to 17.3 (safety) and efficacy ii equal to 60 (capacity to arrest tumor growth). In this report efficacy i allowed for an in vitro estimation of the number of tumor vasculature endothelial cells that would be destroyed before one normal tissue endothelial cell would be destroyed . Efficacy ii allowed for an in vitro estimation of the vaccine efficacy in the context of suppression of hmec proliferation of the tumor vasculature and primarily is a reflection of the potential for the vaccine to arrest tumor growth; that is, it describes the vaccine's therapeutic effect . It should be noted that stimuli of different strengths would be expected in vivo due to gradual diminishing growth stimuli in relation to increasing distance from the tumor cells . Therefore, it can be expected that hmec with different target surface profiles, including profiles related to hmec, will also be present in the tumor - associated vasculature . Future studies in the field of vaccine development using allogeneic santavac are required; however, in vitro data presented in this report demonstrated that the allogeneic santavac was a perfect candidate for the development of a ucv with outstanding efficacy and safety . The santavac formulation described achieved efficacy equal to 17 and 60 in relation to in vitro prediction of vaccine safety and capacity to arrest tumor growth, respectively . Criteria critical to the development of such efficient allogeneic santavac are defined in this paper and may be used for preparing ucv for clinical trials.
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Atmospheric aerosol particles influence the global climate through their two - fold impact on earth s radiative balance . They scatter incoming solar radiation directly, but they also affect the climate indirectly by acting as condensation nuclei for cloud droplets (ccn), therefore affecting the radiative properties and lifetime of clouds . The ccn grow to become cloud droplets by condensation of water vapor, and this growth is controlled by the ability of the droplets to uptake the condensing water vapor molecules . Therefore an understanding of the underlying condensational growth process is necessary to achieve correct concentrations of cloud droplets in climate models . The key quantity controlling the growth of submicrometer aerosol particles is the mass accommodation coefficient m, which is defined as the fraction of vapor molecules hitting the particle surface that will be accommodated by the condensed phase . The mass accommodation of water molecules on water surfaces has been extensively studied both experimentally and with molecular dynamics (md) simulations . However, the value of m reported by different experimental studies has varied in the range 0.011 . A recent sensitivity analysis of results measured by a number of ensemble and single particle techniques has suggested that the values of the mass accommodation coefficients retrieved from these studies are consistent with a value larger than 0.5 . The wide range of experimental values is contrasted by md simulations which have consistently resulted in a unity mass accommodation coefficient for water . Part of the difficulty in determining the mass accommodation coefficient is linked to the fact that the coefficient can be defined in different ways . While the definition given above seems simple, the mass accommodation coefficient can be understood in at least two ways: either as surface accommodation where all molecules that are not directly scattered are considered to be accommodated or as bulk accommodation where mass accommodation is defined as the fraction of the molecules arriving at the surface that are absorbed to the bulk . The experimental results on mass accommodation and evaporation are usually interpreted with the aid of various condensation models . These models are generally based on a combination of the kinetic gas theory and macroscopic mass and heat transfer theories . In this work we compare md simulations to these kinetic condensation models to shed light on how compatible the two approaches actually are for the accommodation of water molecules onto water surfaces . One of the key complications in the interpretation of the laboratory experiments is that they always probe net condensation or evaporation processes, thus requiring quantification of simultaneous evaporation and condensation processes . Md simulations, on the other hand, provide a means to study these two processes separately, and at constant temperature . Basic kinetic condensation models estimate the condensational flux from the kinetic theory of gases, correcting for diffusional effects in the gas phase, where needed . The description considers the flux as occurring directly between the gas and condensed phases (see figure 1) without any specific consideration of the processes occurring in the surface region . The condensational flux consists of molecules arriving at the surface from a distance that is of the order of the molecular mean free path . In these kinetic models the mass accommodation coefficient is present as the condensation coefficient, multiplying the maximum kinetic collision rate to the liquid surface . Based on the equality of the net condensation and evaporation fluxes in equilibrium, the condensation coefficient is typically considered to be equal to the evaporation coefficient . As a next step from these simple theoretical considerations, more sophisticated models which also explicitly include the surface processes have been presented . Developed a new kinetic multilayer model for gas particle interactions in aerosols and clouds (km - gap). In km - gap the aerosol gas system is divided into several layers (figure 1), with corresponding fluxes between each layer . The mass accommodation coefficient is present as separately defined surface and bulk accommodation coefficients that appear as parameters of the model . Md simulations provide a means to investigate these descriptions of the condensation / evaporation process on a molecular basis (figure 1). Schematic figure illustrating the different levels of theory from kinetic condensation models through km - gap to md . The adsorption flux in km - gap (jads) is equivalent with the condensation flux of the general kinetic condensation model, and the km - gap desorption flux (jdes) is equivalent with the evaporation flux . Md simulations provide a straightforward way to determine m, as the trajectories of individual molecules can be followed throughout a simulation and the fraction of accommodated molecules can be simply evaluated . The md mass accommodation simulations consist of shooting individual gas phase molecules toward a surface and determining the subsequent fate of those molecules (see section 2.4). To our knowledge, so far in pure water mass accommodation md the surface in question has always been a planar surface . At typical atmospheric conditions, however, only the growth of the smallest nanodroplets (of submicrometer size) are in fact sensitive to mass accommodation processes . Droplets in the few - nanometers range are accessible for present - day md, and the surface curvature could have an effect on the mass accommodation process, being especially important for small atmospheric droplets . We study the potential effect of surface curvature by conducting md simulations on the accommodation of water on nanodroplets and comparing the results to similar simulations for a planar surface . A further complication related to a md mass accommodation coefficient, and in fact to a molecular level m in general, is the fact that a fraction of the evaporating molecules observed during the mass accommodation simulations can be due to the incoming molecules inducing an exchange evaporation of a surface molecule . If this phenomenon has a nonnegligible effect, the assumed equality of the condensation and evaporation coefficients may not hold . While the exchange evaporation cases should also be considered as nonaccommodation, lacking any clear temporal and spatial definition on when the evaporation is induced by the incoming molecules, it is impossible to assign an individual evaporation event to either exchange or thermal evaporation . However, the thermal evaporation rate is independent of the properties of the surrounding vapor phase, and it can be determined by simulating evaporation from the liquid into a vacuum . We compare the evaporative flux into a full vacuum with that of a mass accommodation simulation to yield estimates on what fraction of the evaporated molecules should be counted as exchange evaporation and thus inducing a feedback between the condensation and evaporation processes . The md simulations were performed using the gromacs molecular dynamics software . The tip4p - ew water potential was used . The simulation time step was 1 fs, the temperature was controlled through the bussi thermostat, and particle - mesh ewald summation was used for the long - range part of the coulombic interactions . The initial configurations consisted of either a liquid slab with the surfaces in the xy - plane and the simulation box elongated in the z - direction, or a liquid droplet located at the center of the simulation box . The various simulation configurations are collected in table 1 . For each initial condition a total of 1000 incident molecules were generated at 10 ps intervals at a distance of about 1.5 nm from the target surface and were assigned velocities from the maxwell boltzmann velocity distribution corresponding to the temperature in question . For the planar cases, the incident molecules were introduced at alternating sides of the slab at random x, y - coordinates and the center of mass velocity of the molecule was set toward the center of the surface . Consequently, the procedure results in a variety of incident angles and orientations . For the droplet cases, the incident molecules were placed at randomly selected locations around the droplet and the initial velocity was set toward the center of mass of the droplet . After the generation of the tenth incident molecule the simulation was continued for a further 20 ps before the simulation was terminated and a new mass accommodation simulation was started with a fresh starting configuration . The total simulation time for a single condition was thus 11 ns, the setup following the steps of morita et al . The necessity of restarts becomes especially clear in the case of the droplet simulations, as the droplet radius needs to be constant . The number of molecules denotes the molecules in the target bulk liquid at the start of the simulation . We have also performed simulations without the impacting molecules present, that is, simulations where the system only consists of the slab or droplet, in order to study the evaporation from the surface without incoming gas phase molecules affecting the situation . This is not a true vacuum case as the evaporated molecules are allowed to travel across the periodic boundary of the simulation box and, thus, eventually return to the liquid . However, the resulting evaporation flux should represent a reasonable approximation of the evaporation flux to a true vacuum when considering the small amount of evaporating molecules in the studied temperature range . These runs were performed for 20 ns of simulation time for all conditions used in the mass accommodation simulation except for the larger droplet, which is omitted because of the relatively high computational cost of a 10 000 water molecule system . The kinetic gas theory can be used for modeling the net condensation / evaporation to / from an aerosol particle if the knudsen number kn, which is the ratio between the mean free path of the vapor molecules and the particle radius rp, is considerably larger than 1 . In this free molecular regime, the net condensational mass flux (in molecules per second) to a droplet with surface area a (m) is thus given by1where v is the average thermal velocity of a molecule (m / s), c is the gas phase density (1/m) far from the droplet (about one mean free path away and further), and ca is the gas density at the droplet surface . The here is the condensation coefficient, which is equal to the evaporation coefficient in an equilibrium case . This equality is usually assumed to hold in general, thus yielding the form of eq 1, where the first term refers to the forward condensation flux to the particle and the second term refers to the evaporation from it . The condensation coefficient can also be called the mass accommodation coefficient, as it is by definition the ratio of the actual condensational mass flux and the collision flux . At this level of theory (see figure 1), it is not possible to address whether the condensation coefficient requires that the colliding molecule should end up in the bulk or merely stick to the surface as the surface is not treated explicitly . Based on the collision flux and the requirement for the net flux between the gas and liquid phases to be zero at equilibrium, the evaporative flux from a surface with an area a is given by2where pe is the equilibrium vapor pressure (pa), k is the boltzmann constant (j / k), and t is the temperature (k). Equation 2 is derived from considering an equilibrium situation between the condensed and vapor phases, but as the thermal evaporation rate is a property of only the condensed phase, the equation is usually assumed to be valid regardless of the vapor conditions above the surface . As mentioned above, the free molecular regime equations hold if kn 1, while as the knudsen number approaches unity and diminishes to values considerably less than 1, the flux expressions need to be corrected for gas phase diffusional effects . This is done in practice by multiplying the free molecular regime flux by a correction factor, yielding a general flux expression of the form3one of the most widely used transition regime correction factors was derived by fuchs and sutugin, and is given by (see, e.g., ref (7))4this factor follows from a fit to a numerical solution to the boltzmann equation for a condition where the diffusing species is lighter than the background gas . Figure 2a illustrates the kinetic collision flux density, that is jcond, kin / a with = 1, as a function of water vapor pressure . For the four different temperatures used in this study (268, 273.15, 290, and 300 k) the lines lie practically on top of each other, so for clarity only the line corresponding to the lowest temperature is included . The dashed lines show the flux densities corresponding to the conditions in our mass accommodation simulations, with the squares highlighting the corresponding pressures . As can be seen, the generation of incident molecules every 10 ps effectively corresponds to a range of very high supersaturations of 10200 . Figure 2b shows the mean free path of water molecules as a function of pressure . Again, the lines corresponding to the three highest temperatures are omitted for clarity . The circles denote the equilibrium vapor pressures at our model temperatures, while the squares indicate the mean free paths corresponding to the effective pressure created by the incident molecules . As can be seen, even with the relatively high supersaturations represented by these simulations the mean free paths are orders of magnitude longer than the 1.5 nm distance from where the incident molecules begin their trajectory to the surface . The corresponding knudsen numbers for our simulated conditions are thus on the order of about 30 to a few hundred, thus justifying the use of the free molecular regime condensation equations for our comparisons . In general, there are two possible approaches to set up the mass accommodation md simulations: the one employed here (and in, e.g., ref (18)) where several molecules are generated before restarting, or restarting for every incident molecule (e.g., ref (21)). Because of computational limitations, both necessarily represent a high ratio of collisions / simulation time and thus correspond to high supersaturations . In the latter case, however, the surface is refreshed and is thus not subject to continuous bombardment . (a, top) molecular flux density as a function of vapor pressure at t = 268 k. other temperatures used in this work are omitted as they result in overlapping lines . Circles correspond again to the equilibrium vapor pressures at the various temperatures, while the squares correspond to the md simulation conditions . While the kinetic condensation models assume that the molecules arriving at the surface originate on average from the distance of the mean free path, creating the incident molecules in md closer to the surface does not hamper the determination of the mass accommodation coefficient in any way; for this purpose only the fate of the molecules at the surface is needed, not the long journey beforehand . The km - gap model is based on the kinetic model framework of pschl, rudich, and ammann and treats explicitly the steps of mass transfer from gas to condensed phase including gas diffusion, surface bulk exchange, and bulk diffusion of water molecules (see figure 1). The model divides the gas phase and the bulk condensed phase into a number of layers, and the surface is described by two layers: the quasistatic surface layer and the sorption layer . The flux between the aerosol and the gas phase occur only between the sorption layer and the near - surface gas phase . Compared to the simplified picture of section 2.2 (figure 1, left), where mass transfer is only described by the condensation and evaporation fluxes between the condensed and gas phases, km - gap adds a comprehensive set of additional fluxes to the picture with an additional pair of fluxes describing the transport between each layer (figure 1). The transport from the near - surface gas phase to the first (near - surface) bulk layer is not only controlled by the condensational flux to the sorption layer, but also by the transport between the sorption and quasi - static surface layers and between the quasi - static surface layer and the near - surface bulk . Determining the condensational flux to the surface in km - gap follows from the collision flux in the same way as for the simple kinetic models, and for gas molecules of a given species the collision flux is given by5where cgs is the near - surface gas phase concentration of the species in question . Uptake of the gas molecules will cause a depletion in the near - surface gas phase, establishing a concentration gradient in the gas phase, and thus uptake will be influenced by gas phase diffusion . This is addressed by adjusting the concentration with a diffusion correction factor bg = cgs / cg . Following the fuchs and sutugin transition regime correction, the correction factor is given by6where kn is the knudsen number and is the uptake coefficient, defined as the ratio of the net flux between the gas and the condensed phases (as defined by eq 1) and the collision flux . Finally, the adsorption flux (corresponding to the condensation flux in eq 1) in km - gap can be written as7where s is the surface accommodation coefficient . The surface accommodation coefficient in eq 7 is formally identical to the definition of the condensation coefficient given in section 2.2 . Km - gap also provides a bulk accommodation coefficient b as an output parameter which describes the probability of a gas phase molecule entering the bulk . However, as outlined above, b does not enter the equations arising from gas phase kinetics in the km - gap treatment . Unlike the kinetic models where the mass accommodation coefficient appears in equations concerning mass fluxes, in md the coefficient the possible fates of gas phase molecules arriving at the surface can be roughly divided into four outcomes, which are scattering, desorption, adsorption, and absorption . Both scattered and desorbed molecules return to the gas phase, the difference being that desorbed molecules spend some time on the surface before doing so . Adsorbed and absorbed molecules remain in the liquid, with absorbed molecules ending up in the bulk liquid and adsorbed molecules on the surface . There are a few ways in which the mass accommodation coefficient can be defined with this classification . It is clear that the scattered molecules cannot be considered accommodated in any definition . Lacking any definite criteria on the time an incoming molecule has to stay on the surface, the definition for a surface accommodation coefficient is8this definition is in agreement with the surface accommodation coefficient as it appears in km - gap . However, if the mass accommodation coefficient is understood as the fraction of incoming molecules that are absorbed into the bulk liquid, we might use the formula9where the correction factor pk is10 this correction factor is introduced because the limited simulation time prevents following the trajectories of the adsorbed molecules until they are either absorbed or desorbed . In the case for water, both in our present work and in for example ref (19), no occurrences of desorption of the incoming molecules is observed . This makes the two definitions presented above identical over the time scales of our md simulations . From the classification above it is clear that these definitions consider only the fate of incoming molecules, and the possible exchange evaporation, that is, the evaporation of a surface molecule induced by an incoming molecule, is not taken into account . However, from the point of view of the mass fluxes to and from the surface, it is irrelevant if the outgoing molecule is the same as the incoming one . In principle the exchange evaporation could be taken into account in the above definitions by assigning to this new category what would otherwise be classified as absorption or adsorption, but the problem remains that these events would need to be distinguished from thermal evaporation governed by eq 2 . Indeed, even performing a separate set of evaporation simulations to observe differences in the evaporation rate provides only an idea of the magnitude of this effect, not a way to classify individual evaporation occurrences . The md simulated evaporation rates for the planar surfaces are plotted as a function of temperature in figure 3 for both the evaporation and mass accommodation simulations . For comparison, figure 3 shows the theoretical prediction given by eq 2, with the equilibrium vapor pressures based on the simulated values for tip4p - ew water reported by vega et al . While vega et al . Report values for pe down to 245.5 k, their reported fit for pe with the form ln(p) = a + b/(t + c) is for temperatures above 300 k. we have therefore for our purposes fitted the lower temperature end (<400 k) of their simulated pressures using the same functional form . The value for the mass accommodation, or evaporation, coefficient in eq 2 is set to 1 . Figure 4 shows the evaporation rates for different droplet sizes at t = 273.15 k. the droplet radii given, 1.92 and 4.14 nm for the 1000 and 10 000 molecule droplets respectively, are the equimolar radii of the droplets . For equilibrium vapor pressures above a curved surface, the kelvin effect must be accounted for, which requires knowledge of the liquid density and surface tension . We have used the recent values given by sakamaki et al . At 273 k for the tip4p - ew model . For convenience these values are also listed in table 2 . Evaporation rate as a function of droplet size at t = 273.15 k. equilibrium vapor pressures pe from our fit to data from ref (34) (see text), and surface tension and liquid density l from ref (35). For comparison, figure 3 shows also the prediction of eq 2 when the equilibrium vapor pressure of real water is used . It should be stressed that for a meaningful comparison between simulated rates and the prediction of eq 2 one must look at the points obtained using the simulated pe, not the experimental one . In order to get an error estimate from the md simulations, we note that the evaporation process is a poisson process, and we calculate the 1 error for the observed number of evaporation events during the total simulation time . If there is exchange evaporation taking place within the mass accommodation simulations, the observed evaporation is a combination of two processes and this error treatment is too simplified . The error bars shown are nonetheless found by treating both simulation sets in the same manner, since this will help clarify whether the differences seen could just be a product of the shorter simulation time in the mass accommodation simulations . Figures 3 and 4 show that the theoretical evaporation rate from eq 2 results in values that are reasonably close to the evaporation rates seen in simulations . Moreover, the same qualitative behavior as a function of temperature and droplet size is seen in both simulation and theory . A notable exception would appear to be the dip seen when going from 268 to 273.15 k in the mass accommodation simulations . However, considering both the theoretically expected evaporation rates that are quite close to each other for the two temperatures and the relatively large and overlapping error bars which follow from the small number of evaporation events (see table 3), it is strongly suggested that the dip follows from poor statistics rather than an actual feature of the temperature dependence of the evaporation rate . The differences between the evaporation rates from mass accommodation and evaporation simulations appear to mostly be within statistical uncertainty for the lower temperatures, implying that the exchange evaporation does not have a significant role in these simulation conditions . For the higher temperatures, however, the evaporation simulations start to exhibit notably lower evaporation rates, remaining closer to the theoretical prediction which describes only thermal evaporation . It is therefore conceivable that a fraction of the evaporating molecules in the mass accommodation md simulations at higher temperatures could be attributed to exchange evaporation . As was seen from figure 2, the mass accommodation simulations correspond to very high supersaturations as a consequence of the relatively frequent generation of incident molecules . Thus, the assumption that the evaporation flux is independent of the condensation flux can be considered to be a good one for most, if not all, realistic natural situations for water . However, the exchange evaporation apparent in figure 3 is notable only at the higher temperatures, which actually correspond to lower supersaturations than the lower temperature cases (see figure 2). We conclude that the supersaturation where the effect becomes nonnegligible is temperature dependent, and the importance of exchange evaporation for other types of molecules should be further investigated . Although the agreement between theory and simulation is fairly good (within a factor of 2), the theoretical prediction consistently underestimates the simulated evaporation rate . The error bars in the simulated values are of course quite large, but the sensitivity of eq 2 to the input values should also be considered . As is already set to 1, changing the value of the evaporation coefficient will only make the agreement worse (i.e., the value cannot exceed unity). The temperature and surface area are fixed in the simulations, so we are left with a possible underestimation of pe as a source for the discrepancy . In figure 5 the equilibrium vapor pressures of vega et al . Are compared to values obtained by solving pe from eq 2 using the md simulated evaporation rate . We stress that the validity of the equilibrium vapor pressures of vega et al . Is not questioned, figure 5 merely illustrates the change in pe that would be required to have the simulated and theoretical evaporation rates in figures 3 and 4 agree . The equilibrium vapor pressure obtained through eq 2 is off by a factor of 2.7 for t = 268 k and by less than a factor of 2 for the other temperatures, equivalent to a difference of a few hundred pascals . The error bars are also in the range of a few hundred pascals, but as the pressures from eq 2 result consistently in an overestimation, it is unlikely that a longer simulation time and improved evaporation statistics would result in the vapor pressures coming into better agreement . Comparison of simulated equilibrium vapor pressure of tip4p - ew water with approximated value obtained through the evaporation rate . For the droplet cases, errors in the surface tension and bulk liquid density might also explain some of the disagreement between the theoretical and simulated evaporation rates . For the value of surface tension an error estimate of 0.4 mn / m has been given, which translates to about a 0.51 pa uncertainty in the droplet pe . No error estimates were given for the liquid density, but even when considering a very generous error estimate of 10 kg / m, a change in pe of only about 0.51.5 pa would result . Thus, the errors in surface tension and bulk liquid density do not improve the consistency between theoretical and simulated evaporation rates for the droplets . The surface region can be identified from a density profile in a md simulation as the part where the density changes from the bulk liquid value to the gas phase value; see figure 6 . As a more rigorous definition for the surface, the region where the density is between 90 and 10% of the liquid value is commonly used . The km - gap model on the other hand describes the surface with two layers, the quasistatic surface layer and the sorption layer (see figure 1). The rate of evaporation is described by the desorption lifetime d, which is the mean time an evaporating molecule spends in the sorption layer before evaporating into the gas phase . As a test case in ref (23), the km - gap model was used to simulate the experimental water vapor condensation and droplet growth in the expansion chamber work of winkler et al . In their experiments, monodisperse ag particles with a diameter of 9 nm and particle number concentration of 4381 cm were used as condensation nuclei and humidified under an initial supersaturation of 37.5% at 268 k and 737 torr . A value of d = 35 ps was used in ref (23) for the desorption lifetime, based on earlier md simulations . The value is, however, based on only a handful of desorption events of incoming molecules, of which not all were water molecules, and which were obtained using the 9010 surface definition . Example density profile (t = 300 k) where the area between black vertical lines is the km - gap sorption layer . The red lines denote where the density falls to 90% and 10% of the bulk liquid value . To identify where the location of the sorption layer is in terms of the md density profile, we take advantage of the quantity denoted by s in the km - gap model . S is the ratio between the actual surface concentration of the sorption layer and the maximum surface concentration of water molecules . We take the outer limit of the sorption layer to be located at the distance where the density has fallen to the fraction of the corresponding bulk liquid value that is indicated by s, which for the water condensation example in ref (23) is s 7 10 . The thickness of the sorption layer is set to 0.3 nm, since in km - gap the sorption layer has a fixed width of one molecular diameter . The black lines in figure 6 show the region of the md density profile that corresponds to the km - gap sorption layer . As can be seen, the outer limit of the sorption layer is quite far from the bulk liquid phase and the sorption layer lies entirely outside the 9010 surface region . This was to be expected, as the km - gap surface region includes also the quasi - static surface layer located between the sorption layer and bulk condensed phase . Due to the dense vapor that follows from the frequently generated incoming molecules, the density does not fall in the mass accommodation simulations of the planar interface to the small value indicated by s . This is however not a problem in the evaporation simulations, and the location identified from the evaporation simulations is used also for the mass accommodation simulations when calculating the d related to the sorption layer . Table 4 collects the values for the mean lifetime before evaporation when the 9010 surface definition is used . The times the evaporating molecules spend in the surface are calculated starting from the last time the molecules enter the surface before evaporation without entering the bulk in between . Evaporation is here taken to occur when the molecule enters the constant density vapor phase . We find that, while individual evaporating molecules occasionally spend 35 ps or even longer in the 9010 surface region, the mean lifetime is around 10 ps or less . To be consistent with km - gap this results in a d that is on average below 1 ps for all of the various simulated conditions . The longest time observed is only about 2 ps, but these are a minority among the evaporation events with only six such occurrences among the total number of 134 evaporation events observed throughout all of the simulations . The number of events listed here is not exactly the same as the total number of evaporations since on three occasions the molecule was in the surface from the start of the simulation until evaporating . To examine the effect of the value of d on the predictions of km - gap, we have simulated water vapor condensation using km - gap with various d values, again in comparison to experimental data from winkler et al . We investigate the impact of d on droplet growth with d values of 1, 10, and 100 ps . As shown in figure 7a, km - gap reproduces the observed growth very well and the exact value of d has practically no effect on particle growth . This is because particle growth is limited by gas phase diffusion and subsequent surface accommodation . However, d does have a critical effect on the surface coverage of water molecule s as shown in figure 7b . Increase of d by an order of magnitude leads to roughly an order of magnitude increase in s: s is 2 10 with d = 1 ps, increasing to 2 10 with d = 100 ps . (a) water droplet growth curve with different desorption lifetimes of h2o (d) (1, 10, and 100 ps) in comparison with the experimental data by ref (10). (b) surface coverage of h2o . (c) surface and bulk mass accommodation coefficients (s and b; left axis) and uptake coefficient of h2o (; right axis) with d = 1 ps . Figure 7c shows the temporal evolution of the surface and bulk mass accommodation coefficients (s and b) and uptake coefficient (). S stays at 1 whereas b 0.998, which means that about 99.8% of water molecules that collide with the surface eventually enter the bulk . The almost exact agreement between the values of s and b indicates that the transport of water molecules from surface to bulk is a very fast process due to the high bulk diffusivity of water molecules of 10 cm s. drops from 1 to 0.1 very quickly and afterward continues decreasing to 0.01, corresponding to a slowing down of particle growth . Water molecules are always desorbing from the surface, and the balance between adsorption and desorption of water molecules determines the value . As time progresses, the net flux decreases and the uptake coefficient decreases . Thus, for water, the surface and the bulk accommodation coefficient values as predicted by km - gap are nearly identical, and the problem reduces to the simple kinetic condensation problem . When identifying the equivalent of the km - gap sorption layer within the md simulations, we used the value of the sorption layer surface coverage s taken from the km - gap runs of ref (23). Figure 7b shows, however, that the value of s changes when d is changed, and the location of the sorption layer should be moved accordingly . This makes it impossible to determine a definite location for the sorption layer in md, and therefore also d: a new value of d will then again change the value of s calculated from km - gap . In practice, however, we can be quite confident that d should be around 1 ps . While they are orders of magnitude different from each other, all of the s values seen in figure 7b are small . Thus, when used to determine the md equivalent of the sorption layer, each results in a sorption layer located close to the bulk gas and completely outside the 9010 surface region . Then, for any of these values of s, the sorption layer is located so far from the bulk liquid that an evaporating molecule in a md simulation will not spend more time in the sorption layer than is required to cross it, of the order of 1 ps . The introduction of the md equivalent of the km - gap sorption layer was done here for the purpose of connecting the md picture to km - gap terminology, and therefore the md sorption layer is utilized only in this section . Using eq 9 to determine the mass accommodation coefficient requires one to choose a definition for the surface . For this purpose we adopt the 9010 definition (figure 6), which is consistent with previous mass accommodation md work . We find that the water mass accommodation coefficient, as defined by eq 9, is close to unity not only for the planar surfaces but also for the droplet surfaces, as is seen in figure 8 . The number of scattered molecules is particularly small (see table 3), to the extent that we see no scattered molecules among the 1000 incoming molecules for the planar surface in simulations at the two lowest temperatures . The triangles in figure 8 are calculated when taking the exchange evaporation into account where the number of evaporated molecules that are assigned to the exchange evaporation are determined from the ratio of evaporation rates from the evaporation and mass accommodation simulations . For completeness the results using the exchange evaporation are plotted also for the two lower temperatures, even though the difference in evaporation rates could clearly be accounted for by statistical error in these two cases (see figure 3). There seems to be a slight decrease in as the temperature grows and a slight increase as the droplet size grows . The decreasing trend as temperature rises is in line with earlier experimental results and with simulation results . This behavior is intuitively expected as higher temperature corresponds to on average more energetic incoming molecules . Note that the region where the accommodation coefficient drops to values considerably below unity in refs (26) and (38) is at temperatures close to the critical temperature, that is, at temperatures much higher than those in the present study or in experiments . Mass accommodation coefficient as a function of temperature (top) and droplet size (bottom). The circles in the top follow from only counting scattered molecules as nonaccommodated; the triangles take also into account the difference in evaporation rate between mass accommodation and evaporation simulations . The mass accommodation coefficient remains practically unity even when the exchange evaporation is taken into account . However, it is interesting to note that for t = 300 k the number of nonaccommodated molecules due to exchange evaporation is about 4 times the number of scattered molecules, making the exchange evaporation the dominant type of nonaccommodation under these conditions . However, translating these findings to other substances with mass accommodation coefficients smaller than 1 is speculative . If the magnitude of the evaporation events caused by the incoming molecules is similar to what it is for water, the number of scattered molecules could outnumber the evaporated by a clear margin . On the other hand, it might be that the exchange evaporation is also enhanced in these situations . For accommodation of a molecule on a surface that consists of a different molecular species, it seems natural to only take into account the fate of the incoming molecular species when calculating the mass accommodation coefficient, even if the adsorption leads to an increase in the evaporation of the surface species . While it might seem worthwhile to use the sorption layer also when determining m, the fact that the md sorption layer is here so removed from the condensed phase makes it ill - suited for the task . According to the definitions in section 2.4, an incoming molecule that has passed through the surface is classified as absorbed into the bulk liquid, but from figure 6 it is clear that any molecule that has just traveled past the md sorption layer can hardly be considered to reside in the bulk liquid phase . We have performed molecular dynamics simulations of water mass accommodation on both planar and droplet surfaces as well as evaporation simulations with the same set of simulation conditions . These included a planar surface at four different temperatures and droplets at t = 273.15 k with radii of 1.92 and 4.14 nm, with the latter size considered in the mass accommodation simulations only . Our simulated water evaporation rates are in qualitative agreement with the evaporation rate given by simple kinetic condensation equations, but this theoretical expression consistently underestimates the observed rates . While a longer simulation time might result in a better quantitative agreement, the systematic difference between the two makes this doubtful . On the other hand, a change of a few hundred pascals in the equilibrium vapor pressure would be needed to bring the theoretical prediction to the same level as simulations . In the two highest of the studied temperatures, t = 290 k and t = 300 k, the evaporation rate in the md mass accommodation simulations is elevated compared to the evaporation simulations . This effect can be attributed to exchange evaporation; that is, an incoming molecule will remain in the condensed phase but causes a surface molecule to evaporate . Such behavior is at odds with the commonly used assumption that the evaporation mass flux is independent of the condensational mass flux, but it should be noted that the relatively frequently generated incident molecules cause the simulation conditions to correspond to very high supersaturations of the order 10200 . The comparison of md evaporation rates and theoretical free molecular regime evaporation rates suggests that the evaporation coefficient for water is unity, which is in agreement with earlier md work where the evaporation coefficient has been calculated from the evaporation fluxes in liquid vapor equilibrium and vacuum evaporation simulations . This is at odds, however, with experimental raman thermometry results, which point to a value below unity . The md simulations also provided an opportunity to refine the average desorption lifetime that is required as an input parameter for the detailed kinetic flux model km - gap . Using the surface coverage of the km - gap sorption layer, the region in the md simulations that corresponds to this km - gap sorption layer is identified . From the time that evaporating molecules spend in this region, we find the average desorption lifetime to be around 1 ps . Although this new d differs by an order of magnitude from the earlier value used in km - gap, the model succeeds in describing the condensational growth of water droplets from the experiments of ref (10). In fact, the droplet growth is described equally well with desorption lifetimes ranging from 1 to 100 ps, indicating that the growth is limited by gas phase diffusion and surface accommodation rather than desorption . Thus the simple kinetic treatment (see figure 1, left panel) is sufficient for describing pure water . This is likely not the case for a situation where the transport between surface and bulk is less efficient, for example, for a water droplet coated with organic molecules . A mass accommodation coefficient between 0.99 and 1 is found for all studied planar interfaces, which is in accordance with previous md studies . Using a droplet as the target surface also produces a coefficient that is practically unity, even though the coefficient appears to get slightly (<0.5%) lower as the droplet radius decreases . The mass accommodation coefficient gets slightly (0.2%) smaller as temperature increases, which is in agreement with experiments . When the exchange evaporation is taken into account, the decrease with increasing temperature is somewhat more pronounced, resulting in a value of 0.99 at the highest temperature . The effect of exhange evaporation is included by comparing the evaporation fluxes in mass accommodation and evaporation simulations and attributing the difference to molecules evaporated by the exchange method and, consequently, as nonaccommodated . The molecular level definitions of the mass accommodation coefficient conventionally used in md consider only the fate of the incoming molecules . However, when kinetic condensation models for aerosol growth are used, the picture is not a molecular level one but instead is one of mass fluxes, and then the mass accommodation coefficient acts as a factor that effectively decides the magnitude of the condensation and evaporation fluxes . In this case, the information that the coefficient is expected to contain includes the possible exchange evaporation.
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Chronic obstructive pulmonary disease (copd) is a disease state characterized by airflow limitation that is not fully reversible according to the global initiative for chronic obstructive lung disease guidelines.1 irreversible airflow limitation is defined as the proportion of forced vital capacity (fvc) in 1 second, ie, forced expiratory volume in 1 second (fev1)/fvc ratio, being <70% after inhalation of 2-agonist . However, copd is not a simple homogenous disease clinically defined only as irreversible airflow limitation . For example, copd has some morphological phenotypes on chest high - resolution computed tomography (hrct), and each phenotype is associated with clinical characteristics and different responsiveness to bronchodilators.2,3 moreover, copd can coexist with asthma which may also cause irreversible airflow limitation,4 although copd and asthma are two quite different and independent diseases . Asthma may be a risk factor for the development of copd.5 in particular, copd and asthma may overlap and converge in older people.6,7 however, the potential for overlap of the individual obstructive airway disease syndromes, including copd and asthma, has received less attention.8 for this study, it was hypothesized that copd patients with asthmatic symptoms may have different clinical features than copd patients without asthmatic symptoms . The aim of this study was to clarify the features of copd patients with asthmatic symptoms, and these findings may therefore play a role in developing the optimal strategy for the management of copd with asthmatic symptoms . A total of 63 patients with stable copd, who showed fev1/fvc <70% and fev1 <80% of the predicted value after inhalation of a 2-agonist (moderate to very severe copd) and were seen at the outpatient clinic of shinshu university hospital (matsumoto, japan) from february 2007 to july 2009, were recruited for this study . The diagnosis of copd was based on the clinical history and symptoms, including dyspnea on exertion and pulmonary function characterized by irreversible airflow limitation (fev1/fvc <70% after inhalation of a 2-agonist) in accordance with the global initiative for chronic obstructive lung disease guidelines.1 all subjects had smoking - related copd without 1-antitrypsin deficiency, and had a smoking history of more than 30 pack years . All patients with copd without asthmatic symptoms had no history of asthma or asthmatic symptoms (copd without asthma group). All patients with copd with asthmatic symptoms had experienced asthmatic symptoms such as episodic breathlessness, wheezing, cough, and chest tightness worsening at night or in the early morning (copd with asthma group). The exclusion criteria included the presence of a respiratory tract infection or exacerbation of copd and/or asthma during the preceding 3 months . In total, 46 patients were in the copd without asthma group and 17 patients were in the copd with asthma group . Most of the patients used short - acting 2-agonists as needed to relieve dyspnea two to three times per week, but did not use them on the day that the pulmonary function tests were performed . The study was approved by the institutional research ethics committee of shinshu university school of medicine (matsumoto, japan), and all patients gave written informed consent to participate . Information was obtained from each patient on the history of the current illness including complications and history of smoking, physical and laboratory examinations, pulmonary function tests including reversibility of airflow limitation by 2-agonist (20 g of inhaled procaterol hydrochloride), arterial blood gas analysis, analysis of inflammatory cells in induced sputum, and the findings on chest hrct . The patients were treated with inhaled corticosteroid (ics; 400 g / day of inhaled fluticasone propionate) for 23 months . Spirometry and measurements of the diffusing capacity for carbon monoxide (dlco) were performed using a pulmonary function testing system (chestac-55v; chest co, ltd, tokyo, japan). Fev1 was measured before and 20 minutes after inhalation of 2-agonist (20 g of procaterol hydrochloride) by aerosol (metered - dose inhaler) with a spacer (meptin; otsuka pharmaceutical, tokushima, japan) to evaluate the reversibility of airflow limitation . The functional residual capacity was measured using a body box (medgraphics model 1085; medical graphics corp, minneapolis, mn), after which the subjects immediately inspired to total lung capacity and expired maximally to residual volume, thus allowing calculation of lung volume and residual volume / total lung capacity . The pulmonary function tests were performed by two special technicians according to the american thoracic society criteria . Sputum induced by the inhalation of hypertonic saline was collected as described previously.9 briefly, all subjects inhaled a 2-agonist and 3.5% hypertonic saline nebulized with an ultrasonic nebulizer (ne - v10b; omron corporation, tokyo, japan). The nebulization was continued for at least 10 minutes and stopped after 15 minutes or earlier if a 2 ml sputum sample of good quality was obtained . The collected sputum was then separated from contaminating saliva by macroscopic examination, and any mucus plugs were removed from the dish to a sterile plastic container, after which the volume of the sample was determined . The sample was incubated with an equal volume of hank s balanced salt solution containing 1 mm dithiothreitol (sigma - aldrich company ltd, poole, united kingdom) at 37c for 15 minutes . The residual mucous was removed and the eluent was used to obtain total and differential cell counts . The total cell count, except that of squamous cells, was determined with a standard hemocytometer (fuchs rosenthal; erma, tokyo, japan), normalized for weight and expressed as cells 10/g wet weight of sputum . Cell smears were prepared with a centrifuge (autosmear; sakura keishi kabushiki kaisha, tokyo, japan) and stained with may - grnwald - giemsa (merck, darmstadt, germany) for differential cell counting . The slides were coated and then 500 cells were counted to determine the differential leukocyte count . A helical ct scanner (lightspeed vct; ge healthcare, waukesha, wi) was used for conventional contiguous scanning with a slice thickness of 10 mm to screen for chest abnormalities followed by hrct scanning at full inspiration (at total lung capacity level) with 1 mm collimation (120 kvp, 200 ma, pitch 1.0). Four 1 mm thick slices were obtained at three anatomical levels at full inspiration: near the superior margin of the aortic arch, at the level of the carina, and at the level of the orifice of the inferior pulmonary veins . The low attenuation area (laa) was visually scored in each bilateral lung field according to the method of goddard et al.10 total scores were calculated and the severity of emphysema was graded as: score 0, laa <5%; score 1, 5% laa <25%; score 2, 25% laa <50%; score 3, 50% laa <75%; and score 4, the severity of emphysema was graded in accordance with the sum of the scores for six dimensions: grade 0, total score = 0; grade 1, total score = 16; grade 2, total score = 712; grade 3, total score = 1318; and grade 4, total score = 1924 . The patients were classified according to the visual hrct findings as: absence of emphysema phenotype, which showed little emphysema and at least laa grade 1, and emphysema phenotype, which showed apparent emphysema (at least grade 2). Bwt in all lung fields was graded visually as reported previously:11,12 grade 0, none; grade 1, <50% adjacent pulmonary artery diameter; and grade 2, 50% adjacent pulmonary artery diameter . The patients without bwt showed bwt of grade 0, and patients with bwt showed bwt of more than grade 1 . Hrct images were analyzed independently by two pulmonologists with no knowledge of the patients clinical status . The values shown in the text and tables are the mean standard error of the mean . The data distribution of the variables in the groups was first assessed with bartlett s test . Data for the variables that showed a normal distribution were compared using the parametric student s t - test . Data for the variables that did not show a normal distribution were compared using the nonparametric mann cutoff values of the sputum eosinophil count for detecting copd with asthma were calculated from receiver operator characteristic curve analysis, with sensitivity and specificity determined in each case . All statistical analyses were performed with the use of a windows - compatible software program (statflex version 5.0; artech ltd, osaka, japan). A p value of less than 0.05 was considered to be significant in all statistical analyses . A total of 63 patients with stable copd, who showed fev1/fvc <70% and fev1 <80% of the predicted value after inhalation of a 2-agonist (moderate to very severe copd) and were seen at the outpatient clinic of shinshu university hospital (matsumoto, japan) from february 2007 to july 2009, were recruited for this study . The diagnosis of copd was based on the clinical history and symptoms, including dyspnea on exertion and pulmonary function characterized by irreversible airflow limitation (fev1/fvc <70% after inhalation of a 2-agonist) in accordance with the global initiative for chronic obstructive lung disease guidelines.1 all subjects had smoking - related copd without 1-antitrypsin deficiency, and had a smoking history of more than 30 pack years . All patients with copd without asthmatic symptoms had no history of asthma or asthmatic symptoms (copd without asthma group). All patients with copd with asthmatic symptoms had experienced asthmatic symptoms such as episodic breathlessness, wheezing, cough, and chest tightness worsening at night or in the early morning (copd with asthma group). The exclusion criteria included the presence of a respiratory tract infection or exacerbation of copd and/or asthma during the preceding 3 months . In total, 46 patients were in the copd without asthma group and 17 patients were in the copd with asthma group . Most of the patients used short - acting 2-agonists as needed to relieve dyspnea two to three times per week, but did not use them on the day that the pulmonary function tests were performed . The study was approved by the institutional research ethics committee of shinshu university school of medicine (matsumoto, japan), and all patients gave written informed consent to participate . Information was obtained from each patient on the history of the current illness including complications and history of smoking, physical and laboratory examinations, pulmonary function tests including reversibility of airflow limitation by 2-agonist (20 g of inhaled procaterol hydrochloride), arterial blood gas analysis, analysis of inflammatory cells in induced sputum, and the findings on chest hrct . The patients were treated with inhaled corticosteroid (ics; 400 g / day of inhaled fluticasone propionate) for 23 months . Spirometry and measurements of the diffusing capacity for carbon monoxide (dlco) were performed using a pulmonary function testing system (chestac-55v; chest co, ltd, tokyo, japan). Fev1 was measured before and 20 minutes after inhalation of 2-agonist (20 g of procaterol hydrochloride) by aerosol (metered - dose inhaler) with a spacer (meptin; otsuka pharmaceutical, tokushima, japan) to evaluate the reversibility of airflow limitation . The functional residual capacity was measured using a body box (medgraphics model 1085; medical graphics corp, minneapolis, mn), after which the subjects immediately inspired to total lung capacity and expired maximally to residual volume, thus allowing calculation of lung volume and residual volume / total lung capacity . The pulmonary function tests were performed by two special technicians according to the american thoracic society criteria . Sputum induced by the inhalation of hypertonic saline was collected as described previously.9 briefly, all subjects inhaled a 2-agonist and 3.5% hypertonic saline nebulized with an ultrasonic nebulizer (ne - v10b; omron corporation, tokyo, japan). The nebulization was continued for at least 10 minutes and stopped after 15 minutes or earlier if a 2 ml sputum sample of good quality was obtained . The collected sputum was then separated from contaminating saliva by macroscopic examination, and any mucus plugs were removed from the dish to a sterile plastic container, after which the volume of the sample was determined . The sample was incubated with an equal volume of hank s balanced salt solution containing 1 mm dithiothreitol (sigma - aldrich company ltd, poole, united kingdom) at 37c for 15 minutes . The residual mucous was removed and the eluent was used to obtain total and differential cell counts . The total cell count, except that of squamous cells, was determined with a standard hemocytometer (fuchs rosenthal; erma, tokyo, japan), normalized for weight and expressed as cells 10/g wet weight of sputum . Cell smears were prepared with a centrifuge (autosmear; sakura keishi kabushiki kaisha, tokyo, japan) and stained with may - grnwald - giemsa (merck, darmstadt, germany) for differential cell counting . The slides were coated and then 500 cells were counted to determine the differential leukocyte count . A helical ct scanner (lightspeed vct; ge healthcare, waukesha, wi) was used for conventional contiguous scanning with a slice thickness of 10 mm to screen for chest abnormalities followed by hrct scanning at full inspiration (at total lung capacity level) with 1 mm collimation (120 kvp, 200 ma, pitch 1.0). Four 1 mm thick slices were obtained at three anatomical levels at full inspiration: near the superior margin of the aortic arch, at the level of the carina, and at the level of the orifice of the inferior pulmonary veins . The low attenuation area (laa) was visually scored in each bilateral lung field according to the method of goddard et al.10 total scores were calculated and the severity of emphysema was graded as: score 0, laa <5%; score 1, 5% laa <25%; score 2, 25% laa <50%; score 3, 50% laa <75%; and score 4, laa 75% . The severity of emphysema was graded in accordance with the sum of the scores for six dimensions: grade 0, total score = 0; grade 1, total score = 16; grade 2, total score = 712; grade 3, total score = 1318; and grade 4, total score = 1924 . The patients were classified according to the visual hrct findings as: absence of emphysema phenotype, which showed little emphysema and at least laa grade 1, and emphysema phenotype, which showed apparent emphysema (at least grade 2). Bwt in all lung fields was graded visually as reported previously:11,12 grade 0, none; grade 1, <50% adjacent pulmonary artery diameter; and grade 2, 50% adjacent pulmonary artery diameter . The patients without bwt showed bwt of grade 0, and patients with bwt showed bwt of more than grade 1 . Hrct images were analyzed independently by two pulmonologists with no knowledge of the patients clinical status . The values shown in the text and tables are the mean standard error of the mean . The data distribution of the variables in the groups was first assessed with bartlett s test . Data for the variables that showed a normal distribution were compared using the parametric student s t - test . Data for the variables that did not show a normal distribution were compared using the nonparametric mann cutoff values of the sputum eosinophil count for detecting copd with asthma were calculated from receiver operator characteristic curve analysis, with sensitivity and specificity determined in each case . All statistical analyses were performed with the use of a windows - compatible software program (statflex version 5.0; artech ltd, osaka, japan). A p value of less than 0.05 was considered to be significant in all statistical analyses . There were no significant differences in age, gender, body mass index, brinkman index, history of sinusitis, or history of noxious particles or gases other than tobacco between the two groups (table 1). The prevalence of history of allergic rhinitis was significantly higher in the copd with asthma group . A laboratory analysis showed no cases of 1-antitrypsin deficiency, and no significant difference was observed in the serum 1-antitrypsin and serum total immunoglobulin e levels between the two groups . Seven of the copd patients with asthmatic symptoms (41.2%) had a history of exposure to noxious particles or gases other than tobacco; three patients (17.6%) to asbestos and four patients (23.5%) to agrochemical compounds . Eighteen of the copd patients without asthmatic symptoms (39.1%) had a history of exposure to noxious particles or gases other than tobacco; eight patients (17.4%) to asbestos and ten patients (21.7%) to agrochemical compounds . There were no significant differences in the vital capacity, fev1, or fev1/fvc (table 2). There were no significant differences in lung hyperinflation expressed by increased residual volume and total lung capacity between the two groups . There was no significant difference in the increases in fev1 in response to 2-agonist between the two groups (table 3). The increases in fev1 in response to treatment with ics were significantly higher in the copd with asthma group . Twelve patients in the copd without asthma group (26.1%) and eleven patients in the copd with asthma group (64.7%) showed a reversibility of airflow limitation, which was defined as an increase in fev1 of> 12% and 200 ml from baseline values, in response to treatment with ics for 23 months, and none of the patients showed fev1/fvc 70% following the treatment with ics . There was no significant difference in the total cell counts in induced sputum . However, the eosinophil counts in induced sputum were significantly higher in the copd with asthma group . Figure 1 shows the relationship between the increases in fev1 in response to treatment with ics and sputum eosinophil counts . A significant correlation was found (r = 0.42, p = 0.0006). Receiver operator characteristic curve analysis was performed to determine the sputum eosinophil count for detecting copd with asthma . The sensitivity and specificity of the sputum eosinophil count for detecting copd with asthma were 82.4% and 84.8%, respectively, when the cutoff value for the sputum eosinophil count was 2.5% . No significant difference was found in the score of laa between the two groups (table 4). Thirty - eight of 46 patients in the copd without asthma group and 13 of 17 patients in the copd with asthma group were classified into the emphysema phenotype . The remaining eight patients in the copd without asthma group and four patients in the copd with asthma group were classified into the absence of emphysema phenotype . The prevalence of patients with bwt was significantly higher in the copd with asthma group . Furthermore, the prevalence of patients with bwt was significantly higher in the copd with asthma group than in the copd without asthma group only in the patients with the emphysema phenotype . A significant correlation was observed between the increases in fev1 in response to treatment with ics and the grade of bwt (r = 0.41, p = 0.0007). There were no significant differences in age, gender, body mass index, brinkman index, history of sinusitis, or history of noxious particles or gases other than tobacco between the two groups (table 1). The prevalence of history of allergic rhinitis was significantly higher in the copd with asthma group . A laboratory analysis showed no cases of 1-antitrypsin deficiency, and no significant difference was observed in the serum 1-antitrypsin and serum total immunoglobulin e levels between the two groups . Seven of the copd patients with asthmatic symptoms (41.2%) had a history of exposure to noxious particles or gases other than tobacco; three patients (17.6%) to asbestos and four patients (23.5%) to agrochemical compounds . Eighteen of the copd patients without asthmatic symptoms (39.1%) had a history of exposure to noxious particles or gases other than tobacco; eight patients (17.4%) to asbestos and ten patients (21.7%) to agrochemical compounds . There were no significant differences in the vital capacity, fev1, or fev1/fvc (table 2). There were no significant differences in lung hyperinflation expressed by increased residual volume and total lung capacity between the two groups . There was no significant difference in the increases in fev1 in response to 2-agonist between the two groups (table 3). The increases in fev1 in response to treatment with ics were significantly higher in the copd with asthma group . Twelve patients in the copd without asthma group (26.1%) and eleven patients in the copd with asthma group (64.7%) showed a reversibility of airflow limitation, which was defined as an increase in fev1 of> 12% and 200 ml from baseline values, in response to treatment with ics for 23 months, and none of the patients showed fev1/fvc 70% following the treatment with ics . However, the eosinophil counts in induced sputum were significantly higher in the copd with asthma group . Figure 1 shows the relationship between the increases in fev1 in response to treatment with ics and sputum eosinophil counts . A significant correlation was found (r = 0.42, p = 0.0006). Receiver operator characteristic curve analysis was performed to determine the sputum eosinophil count for detecting copd with asthma . The sensitivity and specificity of the sputum eosinophil count for detecting copd with asthma were 82.4% and 84.8%, respectively, when the cutoff value for the sputum eosinophil count was 2.5% . No significant difference was found in the score of laa between the two groups (table 4). Thirty - eight of 46 patients in the copd without asthma group and 13 of 17 patients in the copd with asthma group were classified into the emphysema phenotype . The remaining eight patients in the copd without asthma group and four patients in the copd with asthma group were classified into the absence of emphysema phenotype . The prevalence of patients with bwt was significantly higher in the copd with asthma group . Furthermore, the prevalence of patients with bwt was significantly higher in the copd with asthma group than in the copd without asthma group only in the patients with the emphysema phenotype . A significant correlation was observed between the increases in fev1 in response to treatment with ics and the grade of bwt (r = 0.41, p = 0.0007). The current study found the increases in fev1 in response to treatment with ics were significantly higher in the copd with asthma group, although no significant difference was found in the increases in fev1 in response to 2-agonist between the two groups . Both the peripheral eosinophil counts and sputum eosinophil counts were significantly higher in the copd with asthma group . Chest hrct showed that the prevalence of patients with bwt was significantly higher in the copd with asthma group . A significant correlation was observed between the increases in fev1 in response to treatment with ics and sputum eosinophil counts, and between the increases in fev1 in response to treatment with ics and the grade of bwt . Receiver operator characteristic curve analysis revealed 82.4% sensitivity and 84.8% specificity of sputum eosinophil count for detecting copd with asthma, using 2.5% as the cutoff value . Asthma is traditionally characterized by an eosinophilic inflammation that affects all the airways but not lung parenchyma, and it is linked to airway hyperresponsiveness.13 however, asthmatic patients that smoke develop pathological features similar to copd.14 some patients with copd may demonstrate features of asthma, such as a mixed inflammatory pattern with increased eosinophils,15 and an increased sputum eosinophil count has been reported to be related to an improvement in fev1 following treatment with ics in copd.16 the peripheral eosinophil counts and sputum eosinophil counts were significantly higher, and the reversibility due to a response to the treatment with ics was better in the copd with asthma group in the current series . These results suggest that copd patients with asthmatic symptoms also had features of asthma such as a mixed inflammatory pattern with increased eosinophils . A significant correlation was observed between the increases in fev1 in response to treatment with ics and sputum eosinophil counts, thus suggesting that high sputum eosinophil counts might be a good predictor of response to ics . Pulmonary function testing with bronchodilators has been used to differentiate asthma from copd, with copd demonstrating a smaller response.17 however, some patients with copd showed reversibility, which is defined as an increase in fev1 of> 12% and 200 ml from baseline values, in response to bronchodilators.18 the degree of reversibility of airflow limitation is no longer recommended for diagnosis, differential diagnosis with asthma, or predicting the response to longer treatment with bronchodilators or glucocorticosteroids according to the global initiative for chronic obstructive lung disease guidelines.1 the current study found no significant difference in the increases in fev1 in response to 2-agonist between the two groups, and this result was consistent with the findings of previous reports . The major differences in airway remodeling between asthma and copd are the thickening of the reticular layer under the basement membrane with submucosal infiltration of a large number of eosinophils and the proliferation of mucosal blood vessels found in asthma . On the contrary, it seems that fixed airway obstruction is the final result of structural changes of peripheral airways and lung parenchyma in copd.13,19,20 therefore, these diseases have different pathologies of airway remodeling . However, it is difficult to clinically differentiate between copd and asthma in some patients with irreversible airflow limitation.13 airway remodeling is thought to be the main cause of irreversible airflow limitation in older asthmatic patients . The presence of a normal diffusing capacity for dlco may be useful to differentiate asthma with airway remodeling from copd . However, copd has morphological phenotypes on chest hrct and some patients with copd that show normal capacity for dlco are classified into the absence of emphysema phenotype.2,3 there is a possibility that the copd with asthma group included asthmatic patients with airway remodeling in this study, because it is sometimes difficult to clinically differentiate such patients from copd patients who are classified into the absence of emphysema phenotype . Nonetheless, ics should be considered earlier as a potential treatment in patients who are clinically diagnosed to have copd with asthmatic symptoms . While dlco was significantly lower in the copd with asthma group, this may be because the mean value of laa score was lower in the copd with asthma group . Alternatively, this may be because asthmatic patients show relatively higher values of dlco because allergic inflammation may affect pulmonary circulation.21 hrct used to quantify abnormalities of the airways due to airway remodeling in asthma, and the hrct scan score is correlated with the severity of asthma and airflow obstruction.2224 the absence of emphysema phenotype and emphysema with bwt phenotype are associated with a better responsiveness to treatment with ics in copd patients.2,3 the prevalence of patients with bwt was significantly higher in the copd with asthma group on chest hrct in the current series . A significant correlation was observed between the increases in fev1 in response to treatment with ics and the grade of bwt . The current results were consistent with those of previous reports and suggest that bwt on chest hrct might be a good predictor of response to ics . One limitation of this retrospective study is that the assessment of emphysema was done by a visual scoring method, rather than using a software - based quantification of emphysema . However, the reproducibility of such visual scoring has been demonstrated in a previous report.3 another limitation is the lack of statistical power because the sample size was small for the absence of emphysema phenotype (n = 4 in the copd with asthma group and n = 8 in the copd without asthma group) which is generally lower, accounting for only about 22% of copd.2,3 another limitation of these findings is associated with the fact that while many statistical differences and correlations were observed, these results by themselves do not imply cause and effect . Copd patients with asthmatic symptoms had some clinical features such as high peripheral eosinophil counts, high sputum eosinophil counts, preserved diffusing capacity, high prevalence of bwt on chest hrct, and better reversibility responsive to treatment with ics . Ics should thus be considered earlier as a potential treatment in copd patients with asthmatic symptoms . High sputum eosinophil counts and bwt on chest hrct might therefore be a good predictor of response to ics.
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Hiv cohorts were drawn from eurocoord (www.eurocoord.net), a european union (eu)funded network of excellence that includes most european hiv cohorts [7, 8]. Only cohorts considered national that is, multicenter and not restricted by risk group were included . Hiv cohorts and surveillance agencies in austria, belgium, denmark, france, germany, greece, italy, the netherlands, spain, sweden, and the united kingdom took part (supplementary data 1). Continuums of hiv care were constructed for each country using national - level hiv case surveillance data and hiv clinical cohort data . Four stages of the continuum of hiv care were estimated for 2013, the most recent year of data available (table 1). Standardized definitions used to estimate the human immunodeficiency virus continuum of care abbreviations: art, antiretroviral therapy; ecdc, european centre for disease prevention and control; hiv, human immunodeficiency virus . Stage 1 was defined as the estimated total number of plhiv in each country by the end of 2013 . Several countries had no out - migration data or could only make assumptions about the proportion who out - migrated (supplementary data 2). Where feasible, back - calculation models that estimate hiv incidence and the undiagnosed fraction from routinely collected hiv case surveillance data were used . For consistency, plhiv estimates generated using a back - calculation modeling tool developed by the ecdc were prioritized . If this was not appropriate (eg, due to incomplete case surveillance data), similar back - calculation methods tailored to countries own data were used (4 countries), either to estimate the total number of plhiv directly, or to estimate the undiagnosed population, combined with surveillance or survey - based estimates of the diagnosed population [1113]. Otherwise, alternative approaches included multiparameter evidence synthesis incorporating case surveillance and prevalence survey data (1 country), or surveillance / survey - based estimates (1 country) (supplementary data 2). Adult prevalence was calculated using eurostat population denominators for 2013, excluding children <15 years . Stage 2 was defined as the proportion of all plhiv, estimated as above, ever diagnosed, excluding deaths and out - migrations (supplementary data 2). Ideally, the diagnosed population was derived from cumulative hiv case surveillance data to the end of 2013 (3 countries). Where this was not feasible (eg, surveillance systems that started recently or changed over time in geographic coverage), alternative approaches were used . These included estimating the diagnosed fraction from the ecdc hiv modelling tool (2 countries); combining estimates of the diagnosed population in care and not in care by triangulating data sources (1 country); use of national cohort data that is, the number of patients diagnosed and in care, where linkage to care is expected to be extremely high (3 countries); statistical modeling using recent hiv case surveillance data to estimate new hiv diagnoses for all years (1 country); or infectious disease clinic survey - based estimates (1 country). A range of uncertainty was calculated by dividing the number diagnosed by the lower / upper confidence limits for the number of plhiv, to reflect the uncertainty in estimating stage 1 . Stage 3 was defined as the proportion of those diagnosed, as above, who have ever initiated art, regardless of prevailing treatment guidelines, antiretroviral regimens or number of drugs, or treatment interruptions or discontinuations . Patients known to have died or out - migrated by the end of 2013 were excluded, as were patients with unknown year of diagnosis if it was unclear they were diagnosed before the end of 2013 . Those with unknown art status or unknown year of art initiation were assumed to be untreated by the end of 2013 . Minimum and maximum estimates were calculated based on assumptions about patients lost to follow - up (ltfu) to the cohort and whether they were likely to be receiving care in noncohort centers, or lost to care entirely and, therefore, likely not on art and unsuppressed . For the maximum estimate, patients ltfu were excluded, and for the minimum estimate they were included and assumed to be untreated, unless their records indicated art initiation . Ltfu was defined as no clinic interaction 1 july 201231 december 2013 and, therefore, no art or viral load (vl) data . Clinic interaction was based on any laboratory measurement, drug start date, or other evidence of an hiv clinic visit . Stage 4 was defined as the proportion of those ever on art, as above, with a vl measurement 200 hiv rna copies / ml, or below the assay detection limit, at their last visit 1 july 201231 december 2013 . This vl threshold was chosen to allow for improvements over time in the lower limit of detection of the assay . Cohort data were used to calculate minimum and maximum estimates, and the midpoint between the 2 . Patients ltfu (ie, no recent vl measurements) were excluded for the maximum estimate and included for the minimum estimate (assumed to be unsuppressed). Patients with no vl measurements 1 july 201231 december 2013, but classified as engaged in care based on other laboratory measurements, drug start dates, or clinic visits were assumed to be adherent to art and suppressed . Country - level results were compiled and combined, and weighted averages calculated for each stage to construct a summary continuum for the region based on all 11 countries (supplementary data 3). Percentages were calculated using the previous stage as the denominator, as well as using a single denominator of plhiv . All participating clinical cohorts obtained ethics approvals from local ethics committees, national data agencies, or institutional review boards . Surveillance data are collected under the authority of the public health agencies that abide with strict confidentiality and privacy data protection laws . Hiv cohorts were drawn from eurocoord (www.eurocoord.net), a european union (eu)funded network of excellence that includes most european hiv cohorts [7, 8]. Only cohorts considered national that is, multicenter and not restricted by risk group were included . Hiv cohorts and surveillance agencies in austria, belgium, denmark, france, germany, greece, italy, the netherlands, spain, sweden, and the united kingdom took part (supplementary data 1). Continuums of hiv care were constructed for each country using national - level hiv case surveillance data and hiv clinical cohort data . Four stages of the continuum of hiv care were estimated for 2013, the most recent year of data available (table 1). Standardized definitions used to estimate the human immunodeficiency virus continuum of care abbreviations: art, antiretroviral therapy; ecdc, european centre for disease prevention and control; hiv, human immunodeficiency virus . Stage 1 was defined as the estimated total number of plhiv in each country by the end of 2013 . Several countries had no out - migration data or could only make assumptions about the proportion who out - migrated (supplementary data 2). Where feasible, back - calculation models that estimate hiv incidence and the undiagnosed fraction from routinely collected hiv case surveillance data were used . For consistency, plhiv estimates generated using a back - calculation modeling tool developed by the ecdc were prioritized . If this was not appropriate (eg, due to incomplete case surveillance data), similar back - calculation methods tailored to countries own data were used (4 countries), either to estimate the total number of plhiv directly, or to estimate the undiagnosed population, combined with surveillance or survey - based estimates of the diagnosed population [1113]. Otherwise, alternative approaches included multiparameter evidence synthesis incorporating case surveillance and prevalence survey data (1 country), or surveillance / survey - based estimates (1 country) (supplementary data 2). Adult prevalence was calculated using eurostat population denominators for 2013, excluding children <15 years . Stage 2 was defined as the proportion of all plhiv, estimated as above, ever diagnosed, excluding deaths and out - migrations (supplementary data 2). Ideally, the diagnosed population was derived from cumulative hiv case surveillance data to the end of 2013 (3 countries). Where this was not feasible (eg, surveillance systems that started recently or changed over time in geographic coverage), alternative approaches were used . These included estimating the diagnosed fraction from the ecdc hiv modelling tool (2 countries); combining estimates of the diagnosed population in care and not in care by triangulating data sources (1 country); use of national cohort data that is, the number of patients diagnosed and in care, where linkage to care is expected to be extremely high (3 countries); statistical modeling using recent hiv case surveillance data to estimate new hiv diagnoses for all years (1 country); or infectious disease clinic survey - based estimates (1 country). A range of uncertainty was calculated by dividing the number diagnosed by the lower / upper confidence limits for the number of plhiv, to reflect the uncertainty in estimating stage 1 . Stage 3 was defined as the proportion of those diagnosed, as above, who have ever initiated art, regardless of prevailing treatment guidelines, antiretroviral regimens or number of drugs, or treatment interruptions or discontinuations . Patients known to have died or out - migrated by the end of 2013 were excluded, as were patients with unknown year of diagnosis if it was unclear they were diagnosed before the end of 2013 . Those with unknown art status or unknown year of art initiation were assumed to be untreated by the end of 2013 . Minimum and maximum estimates were calculated based on assumptions about patients lost to follow - up (ltfu) to the cohort and whether they were likely to be receiving care in noncohort centers, or lost to care entirely and, therefore, likely not on art and unsuppressed . For the maximum estimate, patients ltfu were excluded, and for the minimum estimate they were included and assumed to be untreated, unless their records indicated art initiation . Ltfu was defined as no clinic interaction 1 july 201231 december 2013 and, therefore, no art or viral load (vl) data . Clinic interaction was based on any laboratory measurement, drug start date, or other evidence of an hiv clinic visit . Stage 4 was defined as the proportion of those ever on art, as above, with a vl measurement 200 hiv rna copies / ml, or below the assay detection limit, at their last visit 1 july 201231 december 2013 . This vl threshold was chosen to allow for improvements over time in the lower limit of detection of the assay . Cohort data were used to calculate minimum and maximum estimates, and the midpoint between the 2 . Patients ltfu (ie, no recent vl measurements) were excluded for the maximum estimate and included for the minimum estimate (assumed to be unsuppressed). Patients with no vl measurements 1 july 201231 december 2013, but classified as engaged in care based on other laboratory measurements, drug start dates, or clinic visits were assumed to be adherent to art and suppressed . Stage 1 was defined as the estimated total number of plhiv in each country by the end of 2013 . Several countries had no out - migration data or could only make assumptions about the proportion who out - migrated (supplementary data 2). Where feasible, back - calculation models that estimate hiv incidence and the undiagnosed fraction from routinely collected hiv case surveillance data were used . For consistency, plhiv estimates generated using a back - calculation modeling tool developed by the ecdc were prioritized . If this was not appropriate (eg, due to incomplete case surveillance data), similar back - calculation methods tailored to countries own data were used (4 countries), either to estimate the total number of plhiv directly, or to estimate the undiagnosed population, combined with surveillance or survey - based estimates of the diagnosed population [1113]. Otherwise, alternative approaches included multiparameter evidence synthesis incorporating case surveillance and prevalence survey data (1 country), or surveillance / survey - based estimates (1 country) (supplementary data 2). Adult prevalence was calculated using eurostat population denominators for 2013, excluding children <15 years . Stage 2 was defined as the proportion of all plhiv, estimated as above, ever diagnosed, excluding deaths and out - migrations (supplementary data 2). Ideally, the diagnosed population was derived from cumulative hiv case surveillance data to the end of 2013 (3 countries). Where this was not feasible (eg, surveillance systems that started recently or changed over time in geographic coverage), alternative approaches were used . These included estimating the diagnosed fraction from the ecdc hiv modelling tool (2 countries); combining estimates of the diagnosed population in care and not in care by triangulating data sources (1 country); use of national cohort data that is, the number of patients diagnosed and in care, where linkage to care is expected to be extremely high (3 countries); statistical modeling using recent hiv case surveillance data to estimate new hiv diagnoses for all years (1 country); or infectious disease clinic survey - based estimates (1 country). A range of uncertainty was calculated by dividing the number diagnosed by the lower / upper confidence limits for the number of plhiv, to reflect the uncertainty in estimating stage 1 . Stage 3 was defined as the proportion of those diagnosed, as above, who have ever initiated art, regardless of prevailing treatment guidelines, antiretroviral regimens or number of drugs, or treatment interruptions or discontinuations . Patients known to have died or out - migrated by the end of 2013 were excluded, as were patients with unknown year of diagnosis if it was unclear they were diagnosed before the end of 2013 . Those with unknown art status or unknown year of art initiation were assumed to be untreated by the end of 2013 . Minimum and maximum estimates were calculated based on assumptions about patients lost to follow - up (ltfu) to the cohort and whether they were likely to be receiving care in noncohort centers, or lost to care entirely and, therefore, likely not on art and unsuppressed . For the maximum estimate, patients ltfu were excluded, and for the minimum estimate they were included and assumed to be untreated, unless their records indicated art initiation . Ltfu was defined as no clinic interaction 1 july 201231 december 2013 and, therefore, no art or viral load (vl) data . Clinic interaction was based on any laboratory measurement, drug start date, or other evidence of an hiv clinic visit . Stage 4 was defined as the proportion of those ever on art, as above, with a vl measurement 200 hiv rna copies / ml, or below the assay detection limit, at their last visit 1 july 201231 december 2013 . This vl threshold was chosen to allow for improvements over time in the lower limit of detection of the assay . Cohort data were used to calculate minimum and maximum estimates, and the midpoint between the 2 . Patients ltfu (ie, no recent vl measurements) were excluded for the maximum estimate and included for the minimum estimate (assumed to be unsuppressed). Patients with no vl measurements 1 july 201231 december 2013, but classified as engaged in care based on other laboratory measurements, drug start dates, or clinic visits were assumed to be adherent to art and suppressed . Country - level results were compiled and combined, and weighted averages calculated for each stage to construct a summary continuum for the region based on all 11 countries (supplementary data 3). Percentages were calculated using the previous stage as the denominator, as well as using a single denominator of plhiv . All participating clinical cohorts obtained ethics approvals from local ethics committees, national data agencies, or institutional review boards . Surveillance data are collected under the authority of the public health agencies that abide with strict confidentiality and privacy data protection laws . National estimates for the total number of plhiv by the end of 2013 ranged from 5500 in denmark to 153400 in france, corresponding to a prevalence of 0.12% and 0.29%, respectively (table 2). Prevalence was lowest in austria and sweden (both 0.09%), and highest in spain (0.36%). Estimates for 4 stages of the human immunodeficiency virus continuum of care for 2013, by country percentages shown for stages 2, 3, and 4 are out of the previous stage . Percentages in the final column are calculated out of the total plhiv (1). Estimates were constructed using standardized methods and may differ from previously published results and official national statistics due to differences in data sources, definitions, and time periods [2024]. Abbreviations: art, antiretroviral therapy; ci, confidence interval; hiv, human immunodeficiency virus; plhiv, people living with human immunodeficiency virus . Adult hiv prevalence was estimated by dividing the number of plhiv by eurostat population denominators for adults aged 15 years in 2013 . Estimated ranges for the percentage diagnosed were calculated by dividing the number diagnosed by the upper and lower confidence limits for stage 1 (plhiv), to reflect the uncertainty in the estimate for stage 1, unless otherwise indicated . Estimate for plhiv generated using austrian cohort data, which cover approximately 76% of people living with hiv in austria . Estimated range (ci not available), informed by the ecdc modelling tool and triangulation with other estimates . Minimum estimates are not applicable due to the methodology and data sources used to derive the population in care in france . Upper estimates were used to substitute the (missing) minimum estimates when calculating the combined estimates for the proportion on art and proportion virally suppressed in the 11 european union countries . Range for plhiv in italy calculated using the 95% ci for the undiagnosed estimate and, separately, a range of uncertainty for the number diagnosed and lost from care . The 95% ci, reflecting the uncertainty in estimating the diagnosed population nationally in spain, using a statistical model . Surveillance and survey - based estimate for plhiv; cis were therefore not available for the estimate of plhiv, nor was a range available for the diagnosed estimate . However, in sweden, the number diagnosed is reliably estimated from the national cohort and surveillance data, for which there is no under- or delayed reporting . Point estimate of 7000 plhiv used to substitute the (missing) upper and lower limit when calculating the overall range for the percentage diagnosed in the 11 european union countries combined . There was variation across the countries in the proportions estimated for each stage . In 2013, of all plhiv, the proportions diagnosed ranged from 78% in greece to 91% in denmark, with 2 other countries (italy and sweden) also reaching 90%, and austria just below this threshold at 88% . Of those diagnosed, the proportions on art range from 76% in spain to 96% in belgium . Five other countries (austria, denmark, france, the netherlands, and sweden) achieved 90% on art . Of those on art, the proportions virally suppressed were 81% in all countries, with the highest proportion estimated at 93% in both denmark and sweden . France and the netherlands also achieved 90% virally suppressed . Only 2 countries, denmark and sweden, achieved 90% for each of the 3 continuum stages using our standardized definitions . Of the total plhiv, denmark and sweden reached 73% virally suppressed, with france and the netherlands nearing this target, at 72% and 70%, respectively . Overall, 674500 people were estimated to be living with hiv in the 11 eu countries by the end of 2013 (prevalence = 0.22%). Overall, the proportions at each stage were 84% of plhiv diagnosed (79%90%); 84% of those diagnosed on art (81%87%); and 85% of those on art with viral suppression (76%91%) (figure 1). Of the total plhiv, 60% were estimated to be virally suppressed . The greatest drop between successive stages of the continuum was observed between the number of plhiv and the number diagnosed, with 16% of undiagnosed individuals falling out of the continuum . Continuum of human immunodeficiency virus (hiv) care in 11 european union countries (austria, belgium, denmark, france, germany, greece, italy, the netherlands, spain, sweden, and united kingdom) for 2013 . Weighted averages, accounting for the number of hiv - infected individuals at each stage in each country, were taken across all countries for each stage. *percentages out of the previous stage . * * percentages among all people living with hiv by the end of 2013 . Abbreviations: art, antiretroviral therapy; plhiv, people living with human immunodeficiency virus; vl, viral load . National estimates for the total number of plhiv by the end of 2013 ranged from 5500 in denmark to 153400 in france, corresponding to a prevalence of 0.12% and 0.29%, respectively (table 2). Prevalence was lowest in austria and sweden (both 0.09%), and highest in spain (0.36%). Estimates for 4 stages of the human immunodeficiency virus continuum of care for 2013, by country percentages shown for stages 2, 3, and 4 are out of the previous stage . Percentages in the final column are calculated out of the total plhiv (1). Estimates were constructed using standardized methods and may differ from previously published results and official national statistics due to differences in data sources, definitions, and time periods [2024]. Abbreviations: art, antiretroviral therapy; ci, confidence interval; hiv, human immunodeficiency virus; plhiv, people living with human immunodeficiency virus . Adult hiv prevalence was estimated by dividing the number of plhiv by eurostat population denominators for adults aged 15 years in 2013 . Estimated ranges for the percentage diagnosed were calculated by dividing the number diagnosed by the upper and lower confidence limits for stage 1 (plhiv), to reflect the uncertainty in the estimate for stage 1, unless otherwise indicated . Estimate for plhiv generated using austrian cohort data, which cover approximately 76% of people living with hiv in austria . Estimated range (ci not available), informed by the ecdc modelling tool and triangulation with other estimates . Minimum estimates are not applicable due to the methodology and data sources used to derive the population in care in france . Upper estimates were used to substitute the (missing) minimum estimates when calculating the combined estimates for the proportion on art and proportion virally suppressed in the 11 european union countries . Range for plhiv in italy calculated using the 95% ci for the undiagnosed estimate and, separately, a range of uncertainty for the number diagnosed and lost from care . The 95% ci, reflecting the uncertainty in estimating the diagnosed population nationally in spain, using a statistical model . Surveillance and survey - based estimate for plhiv; cis were therefore not available for the estimate of plhiv, nor was a range available for the diagnosed estimate . However, in sweden, the number diagnosed is reliably estimated from the national cohort and surveillance data, for which there is no under- or delayed reporting . Point estimate of 7000 plhiv used to substitute the (missing) upper and lower limit when calculating the overall range for the percentage diagnosed in the 11 european union countries combined . There was variation across the countries in the proportions estimated for each stage . In 2013, of all plhiv, the proportions diagnosed ranged from 78% in greece to 91% in denmark, with 2 other countries (italy and sweden) also reaching 90%, and austria just below this threshold at 88% . Of those diagnosed, the proportions on art range from 76% in spain to 96% in belgium . Five other countries (austria, denmark, france, the netherlands, and sweden) achieved 90% on art . Of those on art, the proportions virally suppressed were 81% in all countries, with the highest proportion estimated at 93% in both denmark and sweden . France and the netherlands also achieved 90% virally suppressed . Only 2 countries, denmark and sweden, achieved 90% for each of the 3 continuum stages using our standardized definitions . Of the total plhiv, denmark and sweden reached 73% virally suppressed, with france and the netherlands nearing this target, at 72% and 70%, respectively . Overall, 674500 people were estimated to be living with hiv in the 11 eu countries by the end of 2013 (prevalence = 0.22%). Overall, the proportions at each stage were 84% of plhiv diagnosed (79%90%); 84% of those diagnosed on art (81%87%); and 85% of those on art with viral suppression (76%91%) (figure 1). Of the total plhiv, the greatest drop between successive stages of the continuum was observed between the number of plhiv and the number diagnosed, with 16% of undiagnosed individuals falling out of the continuum . Continuum of human immunodeficiency virus (hiv) care in 11 european union countries (austria, belgium, denmark, france, germany, greece, italy, the netherlands, spain, sweden, and united kingdom) for 2013 . Weighted averages, accounting for the number of hiv - infected individuals at each stage in each country, were taken across all countries for each stage. *percentages out of the previous stage . * * percentages among all people living with hiv by the end of 2013 . Abbreviations: art, antiretroviral therapy; plhiv, people living with human immunodeficiency virus; vl, viral load . The 11 eu countries included in this study, constituting roughly three - quarters of the eu population and three - quarters of hiv diagnoses in the eu in 20052014, are nearing the unaids 90 - 90 - 90 target, well ahead of 2020 . Although few countries achieved 90% for each stage, based on our standardized definitions, more than half had reached, or were close to, the target for at least 1 stage . Further improvements are also expected to have occurred since 2013, following recent changes in treatment guidelines . However, reducing the undiagnosed proportion remains the biggest barrier to achieving this goal, with the largest drop between successive stages of the continuum observed at this first stage . To our knowledge, this is the first attempt to standardize definitions and derive continuum of care estimates for the eu . Our estimates may differ from previously published results and official national statistics due to differences in data sources, definitions, and time periods, although these differences are relatively minor [2025]. Unaids estimates for the number of plhiv in 2013, derived using spectrum / epp software with hiv prevalence data and most suitable for countries with generalized epidemics, were only reported for 4 of the countries in our study . Our estimates, based primarily on back - calculation modeling and routinely collected hiv case surveillance data, strengthen data availability for this stage and provide valuable information for hiv program monitoring and planning . We observed the highest hiv burden in france, spain, italy, and the united kingdom, accounting for the majority of plhiv in this region, concurring with earlier reports . Losses from the continuum occurred between all stages, but were greatest between stages 1 and 2 . Overall, 16% of plhiv were undiagnosed, indicating that further efforts are required to improve hiv testing rates, particularly among most at - risk populations . Late presentation remains a major concern in europe, with around half of new diagnoses presenting with a cd4 count <350 cells/l [18, 28]. A systematic review published in 2011 suggested that rapid testing and counseling in community settings, community - based peer counseling campaigns, and expansion of opt - out testing policies may be effective interventions to improve hiv testing rates in men who have sex with men in high - income countries . Provision of rapid hiv tests in pharmacies, and provider - initiated hiv testing in general practice or individuals presenting with indicator conditions [31, 32], may offer further opportunities to increase testing uptake . Widening legislation for and increasing access to self - testing and self - sampling are likely to increase testing, but must be coupled with channels for linkage to care . The lowest proportions of diagnosed individuals on art were estimated in spain, italy, greece, and the united kingdom . National treatment guidelines are likely to play a key role here . For example, in 2013, treatment guidelines in greece, spain, and the united kingdom recommended art initiation in patients with cd4 counts of 350 cells/l . The proportion on art is expected to improve once the recent changes in guidelines are implemented . Lack of, or delayed, linkage to care following hiv diagnosis is a possible explanation . Although patients in high - income countries are usually linked to care within 3 months of diagnosis, delays among specific subgroups have been reported [16, 33]. Failure to achieve viral suppression after starting art may reflect poor adherence, treatment interruptions or discontinuations, or insufficient time to achieve suppression for those recently initiating art . Increasing awareness of the continuum of care for example, through national treatment and/or service delivery guidelines and providing evidence - based recommendations to improve the testing and care environment, may also improve the care continuum . Use of the hiv modelling tool facilitated the standardization of estimates for plhiv, but applying the same approach to countries with different hiv surveillance systems was not always possible due to insufficient historical case surveillance data availability in some countries . Triangulation of data sources provides one possible solution, for example, summing estimates of the undiagnosed population with cohort or survey - based estimates of the diagnosed population in care / not in care . Difficulties capturing out - migration or linking surveillance or cohort datasets to population migration and death registries were additional challenges . Misclassification of vital status or out - migration will potentially overestimate the number still alive and living in a country . Few countries in our study had access to reliable out - migration data (supplementary data 2), with linkage to population registries usually precluded by the lack of unique identifiers . Where possible, adjustments were made using estimated levels of out - migration . In the long term, efforts to improve the recording of vital status and out - migration in surveillance databases, as well as linkage to registries via unique identifiers, are needed . In some cases, lack of reliable in - migration data also complicated modeling of hiv incidence and the separating of earlier infections from new infections occurring after arrival within the country . Estimating proportions using cohorts that are not representative of the diagnosed population nationally may introduce bias, so efforts are required to understand and correct for this . The cohorts in our study were large, including national cohorts with near complete coverage of the diagnosed population, and were fairly representative (supplementary data 1). Ideally, estimates derived using cohort data would be adjusted by calculating and applying weights based on the distribution of demographic variables in cohort and surveillance datasets . Namely, the assumptions that are made about whether they are still in care, taking art and virally suppressed, or truly lost from care and unsuppressed . Assuming all have been lost from care entirely would underestimate retention in care and the proportion suppressed, as suggested by a clinical audit in the united kingdom . Ideally cohorts would collect and update data on patients who transfer to other clinics, although this is challenging in practice . In the absence of reliable patient transfer data, plausible limits should be calculated based on varying assumptions, as we have done, with the true value likely to lie between these limits . Collaborations formed between cohort investigators and surveillance agencies facilitated the construction of hiv continuums from plhiv to viral suppression . We attempted to standardize methods to enhance comparability between countries, and to generate summary estimates for the region . However, complete standardization was not possible, given the different limitations in data availability and quality in each country, as well as inherent differences in cohort inclusion criteria . For example, the italian and spanish cohorts require participants to be art - naive at baseline (supplementary data 1). Although the use of cohort data improved the internal consistency of the estimates, we were unable to link surveillance and cohort datasets in most countries to maximize internal consistency . For some countries we were unable to distinguish between those diagnosed and those linked to care (ie, enrolled in a cohort), although linkage to care is expected to be very high . Additionally, our cross - sectional definitions do not address the timeliness of reaching each stage, or time spent at each stage, for example, time since starting art . However, our definitions provide a snapshot of the continuum in 2013 that is simple to interpret and communicate to policy makers . Treatment discontinuations or interruptions were not accounted for, which may result in overestimating the proportion on art . However, a sensitivity analysis conducted for a few countries, restricting the definition of on art to a record of art between 1 july 2012 and 31 december 2013, made little difference to the overall proportions of plhiv who were virally suppressed . Finally, our study omitted 17 eu countries, mainly from eastern and central europe as national cohort data were lacking, and, as such, estimates for the whole eu region may be lower than those presented here . The 11 eu countries in our study are nearing the unaids 90 - 90 - 90 target, with more than half having achieved 90% for 1 or more stages of the continuum . The main barrier to achieving this goal appears to be reducing the proportion undiagnosed . These data provide useful comparisons to governments and healthcare planners, but must be interpreted in context of the limitations and key challenges above, as well as cohort and country differences . Enhancements to data sources and methods are required to derive accurate estimates for national - level continuums of care, to facilitate comparisons between countries, and to generate regional and global estimates . Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
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Longevity and outcome of total knee arthroplasty (tka) are dependent on the restoration of mechanical axis and ligamentous balance in both flexion and extension1,2). To improve the accuracy of alignment and clinical outcomes, technologies such as computer - assisted surgery, patient matched instrumentation and robotic surgery are increasingly being used3). Although many studies have found superiority of these new technologies in achieving better alignment compared to the conventional methods, it still remains questionable whether these improvements result in better clinical outcomes3). These technologies increase the cost3,4), and cost effectiveness studies have not demonstrated their superiority over the conventional instrumentation4). Limitation of conventional instrumentation in restoration of correct femoral component rotation5) has been considered as the commonest culprit for abnormal patellofemroal kinematics and patient dissatisfaction6,7,8). This limitation of conventional instrumentation is probably attributable to flexion resection in a fixed degree of external rotation to the posterior condylar line (pcl). Studies have identified that the posterior condylar angle (pca) is not constant in all patients and it varies according to individuals and the pathology affecting the knee9). Thus, if we know the patient specific pca, then the use of an appropriate flexion cutting jig could reduce the number of outliers . Preoperative or intraoperative computed tomography (ct) scan was advocated to identify patient specific axial distal femoral geometry10). However, use of ct scan adds to the cost and there are availability issues . In addition, ct scan - based methods are not considered real time methods as they do not take cartilage thickness into account while measuring the pca11). In this article, we propose simple, easily reproducible, real - time and radiation - free methods to identify patient specific pca during tka . We tried to compare the accuracy of our methods with the currently available gold standard tool, i.e., ct scan1). Ct scans of 26 patients were randomly selected from our departmental database irrespective of their primary complaints . All the necessary measurements were done on the axial ct images using k - pacs software (www.k-pacs.net, dr.med.andreas knopke, germany) by a single investigator . We measured the angle between the clinical transepicondylar axis (tea, the line joining the most prominent points of the medial and lateral epicondyles) and the pcl by using the software's algorithms as shown in fig . 1 . In order to implement our method, we measured the distance between both epicondyles, i.e., the length of the tea and also the lengths of the perpendicular lines connecting lateral and medial epicondyles to pcl; these perpendicular lines were respectively named the lateral line (ll) and medial line (ml) (fig . 1). The measurements (length of tea, ll, and ml) obtained by the k - pacs were used to construct a right angled triangle with the angle between tea and pcl (the angle) representing the pca . This angle can be calculated by trigonometric formula, =tan (ab / bc) as described in fig . 3) based on the previously obtained measurements on a paper and then subtracted a rectangle out of it, and hence left with a right angled triangle . The angle at the base of this right angled triangle was measured with a protractor . We used paired samples t - test for comparison of the mean measurement values of the different methods . Comparisons between k - pacs measured pca and trigonometrically measured pca and between the k - pacs measured pca and protractor measured pca were done . An intraclass correlation coefficient (icc) analysis was also performed . A p - value of <0.05 was considered statistically significant . The measurements (length of tea, ll, and ml) obtained by the k - pacs were used to construct a right angled triangle with the angle between tea and pcl (the angle) representing the pca . This angle can be calculated by trigonometric formula, =tan (ab / bc) as described in fig . 3) based on the previously obtained measurements on a paper and then subtracted a rectangle out of it, and hence left with a right angled triangle . The angle at the base of this right angled triangle was measured with a protractor . We used paired samples t - test for comparison of the mean measurement values of the different methods . Comparisons between k - pacs measured pca and trigonometrically measured pca and between the k - pacs measured pca and protractor measured pca were done . An intraclass correlation coefficient (icc) analysis was also performed . A p - value of <0.05 was considered statistically significant . The data collected were the angle measured by k - pacs, base length tea, ll, and ml . These measurements were applied to obtain the trigonometrically measured pca (method 1) and protractor measured pca (method 2), which are summarized in table 1 . The mean value of the angle between the pcl and the clinical tea, i.e., pca, measured by the k - pacs was 6.27 (range, 0 to 12) with a standard deviation of 2.97. the mean value of trigonometrically measured pca was 6.23 (range, 0 to 11.11) with a standard deviation of 2.90 and of protractor measured pca was 6.31 (range, 0 to 12) with a standard deviation of 2.85. we analysed the mean pcas obtained by the three methods using the paired samples t - test . The null hypothesis for statistical analysis was that there was no statistically significant difference between the k - pacs measured pca and trigonometrically measured pca (pair 1) and between the k - pacs measured pca and protractor measured pca (pair 2). The p - values were found to be 0.726 and 0.746 for pair 1 and 2, respectively . The p - value was> 0.05 and the null hypothesis was accepted as true in each case, and hence there was no statistically significant difference between the measurements by the proposed methods and by the k - pacs . The results of icc analysis of the k - pacs measured pca and the pcas measured by our methods are summarized in tables 2 and 3; there were statistically significant correlations (p=0.000) between pcas measured by the proposed methods and by k - pacs . Thus, one can infer that the angles measured by our methods correlate well with that measured by the ct - based k - pacs . Restoration of axial rotation of the femoral component in relation to tea is a critical prerequisite for normal patellofemoral and tibiofemoral kinematics and balanced flexion gap in tka9,12,13,14,15,16,17,18,19). However, restoration of accurate rotation of the femoral component has been a challenge and failure to do so may to lead to patient dissatisfaction7,9,13,20,21). Despite the recognition of the importance of rotational alignment restoration, there is a paucity of methods which could be used during surgery for this concern . The available methods for estimating the rotational position of the femoral component during surgery rely on secondary reference axes14,16,22). Geiger and parsch10) used intraoperative ct scan for intraoperative determination of the correct rotational alignment before the implantation . They reported satisfactory results by use of intraoperative ct scan, but the cost, extra surgical time and radiation exposure need to be taken into consideration before routine employment of such a method . Preoperative ct scan has been considered as gold standard to identify the tea1), but the same may not be replicated intraoperatively . In addition, it may not be possible and economically viable to get a preoperative ct scan for every case for pca evaluation . Moreover, the preoperatively determined axis based on the ct scan may not accurately be reproduced intraoperatively as was shown by van der linden - van der zwaag et al.23) in their ct - based study on rotational alignment accuracy of the femoral component in tka using computer - assisted orthopaedic surgery . One reason for this discrepancy might be the fact that preoperative ct scan does not take into account the thickness of the cartilage on the condyles11). We think that a method that allows for intraoperative measurement of the axis is better suited to take this factor into account . For intraoperative application of the methods proposed by us, one would need a trained assistant capable of drawing a quadrilateral geometric figure based on the measurements provided by the surgeon and measuring the angle between lines using goniometer / protractor or trigonometric method with a scientific calculator which is available in all computers as well as smart phones . As per our proposed method, during surgery, the knee is flexed to 90 after the distal femoral cut and the operating surgeon then identifies the medial and lateral epicondyles and marks both points with a sterile marker pen . A ruler is placed tangential to the posterior aspects of the two condyles; this represents the pcl . The surgeon then places another ruler on the medial epicondyle mark and drops it perpendicular to the first ruler . The two marks on the first ruler (pcl) thus act as a limb of the quadrilateral with the tea forming one of the other limbs of the quadrilateral . One thus gets the measurements of all the four sides of the quadrilateral as in fig . 4 . Now the assistant has to draw the quadrilateral based on these measurements on a paper; most often it is trapezoidal in shape unless the pcl and tea are parallel . After drawing the quadrilateral, we have proposed methods to assess the pca intraoperatively such that it could be measured in real time without need for many instruments . In the present study, we applied the methods on ct scan image and evaluated the accuracy of each method in reference to the gold standard . We made an effort to statistically validate the accuracy of manual measurements with the software based measurements . All of the three methods resulted in similar angles and the mean angle measured by one method was not found to be statistically significantly different from that measured by the other method . Based on the statistical analysis, one can infer that trigonometrically and protractor measured pca are equally accurate as the k - pacs measured pca using ct scan . Importantly, the angles measured by our methods correlate well with that measured by the k - pacs . As regards the actual utility of the study, it needs to be highlighted that we have put forward a method to measure pca accurately and easily so that needful modification can be made while using posterior condylar axis as reference for flexion resection . The major limitation of the study is that we did not conduct this study on patients and just proposed methods . As the angles measured by k - pacs and our methods correlate well, we consider that these methods would actually be useful in real life . However, prior to application of these methods, they need to be validated by actual intraoperative studies or cadaveric studies . One more point of contention may be the abnormality in the shape of the condyles secondary to pathologies like osteoarthritis and rheumatoid arthritis which may give different readings on the ct scan and on the table during tka . The very fact that cartilage thickness that ct scan does not take into consideration is reflected by our methods gives some theoretical advantage to our methods . The other limitation is the wide interobserver variability in the identification of tea as was stated by berger et al.1) in 1993; however, in contradiction, a recent report by oussedik et al.24) in 2013 found more familiarity and accuracy in tea identification . In conclusion, we introduced simple, easily reproducible, real - time and radiation - free methods to measure the pca during tka . These methods can obviate the need for obtaining a preoperative ct scan for identification of patient specific pca.
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Contemporary restorative dentistry relays on the durable adhesive joint for long survival of composite restorations . Although etch and rinse adhesives are considered the gold standards, they are technique sensitive . Thus, isolation of the working field via rubber dam application is a prime requisite . Unfortunately, contamination of the adherent with saliva or blood represents a problem in adhesive dentistry . This occurs when rubber dam isolation is encroached in deep subgingival areas and while managing children with copious salivation . When contamination of the bonding site occurs, several consequences take place as postoperative sensitivity, caries recurrence, discoloration, and restoration dislodgement . With two - step etch and rinse adhesives, most studies were directed toward contaminant - removing options of etched substrate while scanty focused on cleansing treatments if contamination occurred at subsequent stages . Ari et al . Reported that contamination should be avoided regardless the affected step to avoid a reduction in bond strength t. park and lee suggested that blot drying of saliva could retain passable bond strength to etched dentin . Contrary researchers suggested that rinsing off the contaminant, particularly blood, although improved the bond strength, could not regain uncontaminated values . This study conducted to assess saliva and blood effect on etch and rinse adhesive and to evaluate cleansing treatments via micro - shear bond strength (sbs) and scanning electron microscope (sem). The null hypothesis tested that no effect of postcontamination cleansing treatments on bonding to dentin at different stages . Adper single bond (sb) plus (hema, bis - gma, vitrebond copolymer, ethanol / water, photo - initiator) and nanofilled filtek z250 composite (3 m espe, st . The crowns mounted horizontally in molds of 15 mm diameter and 18 mm height, using self - curing resin with labial surfaces upward . Surfaces were ground flat using diamond disc (komet, rock hill, usa) in low speed under water to expose superficial dentin then polished using carbide paper 600-grit to obtain uniform smear layer . Specimens were randomly grouped (n = 12/group .) According to contaminated step and sub - grouped according to cleansing treatments [table 1 and figure 1]. All specimens, except group a, were troughed inciso - cervically into two halves . Each mesial half received fresh human saliva (s) collected from the same donor 2 h after breakfast, while the distal half received fresh venous blood (b) collected with a disposable needle from the same donor . Plastic tubes 5-fr (feeding tube, integral medical products, china) with 0.7 mm diameter and 2 mm height, were mounted on the dentin surface . The composite was packed into each tube under gentle pressure over cellophane strip then light - cured according to manufacturers guidelines using elipar ii unit (3 m espe, st . Each half received two tubes away by at least 3 mm whereas group a received two microcylinders only . All specimens were stored for 24 h in an incubator at 37c and 100% humidity . Treatments at different bonding steps diagram depicting experimental protocol among the test groups the plastic tubes were removed using sharp blade then each specimen was screwed to the lower fixed compartment of testing machine (lrx - plus; lloyd instruments ltd ., uk) with 5 kn load . A loop wire, 0.014 in, wrapped around each microcylinder flushing with the resin - dentin interface and aligned with the loading axis of the machine upper movable compartment . A shearing load applied to each assembly at 0.5 mm / min crosshead speed until failure . Failures classified as adhesive if occurred at the interface, cohesive as observed within dentin substrate or composite resin, and mixed when adhesive and cohesive fractures detected simultaneously . Two representative specimens per group were examined . Specimens were gold sputtered under vacuum (ladd sputter coater, bal - tec, scd005, germany) then examined under sem (philips, holland). The data were analyzed using spss (version 16.0) software package (spss inc ., chicago, il, usa) with significance level set at p 0.05 . One - way anova evaluated cleansing treatments effects and tukey's post hoc test for multiple comparisons . The impact of saliva or blood contaminants assessed using independent t - test . Specimens were randomly grouped (n = 12/group .) According to contaminated step and sub - grouped according to cleansing treatments [table 1 and figure 1]. All specimens, except group a, were troughed inciso - cervically into two halves . Each mesial half received fresh human saliva (s) collected from the same donor 2 h after breakfast, while the distal half received fresh venous blood (b) collected with a disposable needle from the same donor . Plastic tubes 5-fr (feeding tube, integral medical products, china) with 0.7 mm diameter and 2 mm height, were mounted on the dentin surface . The composite was packed into each tube under gentle pressure over cellophane strip then light - cured according to manufacturers guidelines using elipar ii unit (3 m espe, st . Each half received two tubes away by at least 3 mm whereas group a received two microcylinders only . All specimens were stored for 24 h in an incubator at 37c and 100% humidity . The plastic tubes were removed using sharp blade then each specimen was screwed to the lower fixed compartment of testing machine (lrx - plus; lloyd instruments ltd ., uk) with 5 kn load . A loop wire, 0.014 in, wrapped around each microcylinder flushing with the resin - dentin interface and aligned with the loading axis of the machine upper movable compartment . A shearing load applied to each assembly at 0.5 mm / min crosshead speed until failure . Failures classified as adhesive if occurred at the interface, cohesive as observed within dentin substrate or composite resin, and mixed when adhesive and cohesive fractures detected simultaneously . Specimens were gold sputtered under vacuum (ladd sputter coater, bal - tec, scd005, germany) then examined under sem (philips, holland). The data were analyzed using spss (version 16.0) software package (spss inc ., chicago, il, usa) with significance level set at p 0.05 . One - way anova evaluated cleansing treatments effects and tukey's post hoc test for multiple comparisons . The impact of saliva or blood contaminants assessed using independent t - test . Table 3 presents group b cleansing treatments where re - etching showed the highest sbs . Meansstandard deviation of microshear bond strength values (mpa) of contaminated bonding stages meansstandard deviation of microshear bond strength values (mpa) of contaminated etching cleansing treatments meansstandard deviation of microshear bond strength values (mpa) of contaminated unpolymerized adhesive cleansing treatments meansstandard deviation of microshear bond strength values (mpa) of contaminated polymerized adhesive cleansing treatments figure 2 illustrates that predominant failure of group b was adhesive mode while cohesive failure in composite prevailed groups c and d. sem used to understand cleansing treatments effect on substrate topography [figures 3 and 4]. Saliva deposits observed in sb, sb1, sb3, c2 and sd (saliva contamination after adhesive polymerization) while blood remnants are notable in bb (blood contamination after dentin etching), bb1, bb3 and bd (blood contamination after adhesive polymerization). Failure mode distribution among the test groups scanning electron microscope photomicrograph of etched dentin substrate (single bond) contaminated with saliva, (bb) contaminated with blood, (sb1/bb1) rinsing of contamination, (sb2/bb2) re - etching, (sb3/bb3) sodium hypochlorite application . Black arrow points red blood cell (1000) scanning electron microscope photomicrograph of (c2) alcohol and (c3) acetone effect on contaminated adhesive before its polymerization, (sd / bd) saliva or blood contamination of adhesive after polymerization, (sd3/bd3) re - etching, (sd4/bd4) re - etching then rebonding . Black arrows point red blood cells (1000) scanning electron microscope photomicrograph depicting red blood cells (3500) micro - shear strength test considered effective for measuring variation in bonding under different conditions . Small specimens allowed several readings from the single tooth and provided harmonious stresses yielding lesser data dispersion . Bonding steps start with etching which selectively decalcify intertubular and peritubular dentin leaving collagen mesh for adhesive impregnation then polymerization . However, others reported negligible moisture effect (particularly saliva) over bonding . The difference attributed to different adhesives composition and experimental design using micro - leakage or diverse loading tests . Chang et al ., and de carvalho mendona et al ., reported that rinsing failed to remove blood due to greater proteins macromolecules contents which resist rinsing and prevent adhesive permeation . Naocl application, for <60 s, showed fractional reversing of contamination due to its nonspecific proteolytic action which eradicates organic remnants without negatively effecting bonding . Whereby, re - etching regained adequate bonding due to acid denaturation of organic remnants rendering weak affinity to underlying substrate becoming easily washed . When unpolymerized adhesive contaminated, its conversion becomes affected as a result of hydrophilic hema molecules which retain water within the adhesive limiting chain growth during polymerization, producing a plasticizing effect in polymer and oxidation of pendant c = c bonds . In addition, higher blood viscosity diminish light permeation and adhesive polymerization . According to the present result, acetone application successfully restored bonding strength when contamination affected unpolymerized adhesive . However, acetone possess additional ability to remove monomer and denature plastics (polymers), accordingly was able to remove contaminated unpolymerized adhesive leaving perspicuous bonding surface . Furthermore, contamination of polymerized adhesive permits glycoproteins adherence to air - inhibited adhesive surfaces forming a physical barrier preventing co - polymerization between adhesive and composite resin . In agreement with furuse et al ., it was observed that etching of contaminated cured adhesive, created areas devoid from adhesive coverage since etching removed contaminant residue and peeled off adhesive coating [sd3/bd3, figure 4]. Thus, rebonding after re - etching aided in the refurbishing of patent adhesive for bonding . Therapy, adhesive failures predominated etched contaminated substrate while cohesive mode prevailed affected adhesive stages . Contamination reduced bonding strength to dentin where blood yielded more negative effect than salivato enhance bonding; re - etching then rebonding are recommended with contaminated etching or polymerized adhesive while acetone and rebonding with affected uncured adhesives . Contamination reduced bonding strength to dentin where blood yielded more negative effect than saliva to enhance bonding; re - etching then rebonding are recommended with contaminated etching or polymerized adhesive while acetone and rebonding with affected uncured adhesives.
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Iga nephropathy (igan) is the most common primary glomerulonephritis in the western world [1, 2]. Gross haematuria bouts preceded by an upper respiratory infection, hypertension and proteinuria and microhaematuria of variable degrees are the most characteristic clinical findings . Dominant or co - dominant mesangial deposits of iga, with igg and c3, are the typical findings in renal biopsy immunofluorescence . It is believed that repeated exposure to environmental factors (viruses, bacteria, etc .) Causes overstimulation of b - cell subsets in the tonsils, bone marrow and intestinal lymphoid tissue, favouring the production of iga1 with deficient galactose residues . This circulating abnormally glycosylated iga1 induces the synthesis of autoantibodies and the formation of circulating immune complexes, which are deposited in renal mesangium causing local inflammation and the appearance of proteinuria and haematuria . However, many of the mechanisms involved in the abnormal glycosylation of iga1 or in the different spectrum of clinical manifestations remain unknown . Current guidelines recommend treatment with renin - angiotensin - aldosterone system (raas) blockers to reduce proteinuria, and in cases with persistent proteinuria> 1 g / day for> 46 months in spite of optimized raas blockade, a cycle of oral corticosteroids is advised . Aggressive immunosuppressive therapy, similar to that used in anti - neutrophil cytoplasmic antibody (anca) vasculitis, with corticosteroids and oral or intravenous cyclophosphamide followed by azathioprine has been tried in rapidly progressive, crescentic forms of igan . The rate of igan progression is very variable, but after 25 years of follow - up, 3050% of patients develop end - stage renal disease (esrd). The presence of minimal (<500 mg / day) or no proteinuria generally indicates a good long - term prognosis, whereas hypertension is a contributing factor for the progression of renal impairment . Acute kidney injury can accompany gross haematuria episodes; renal function recovery is usually observed after the disappearance of macrohaematuria, but it can be incomplete in elderly patients and in those with prolonged macrohaematuria bouts (> 10 days) [9, 10]. A particularly rapid progression to esrd has been reported in a minority of igan patients (<10%) [1113]. Histopathological findings in these patients are in some instances reminiscent of vasculitis, due to the presence of cellular / fibrous crescents, fibrinoid necrosis and arteriolar damage [13, 14]. Anca, mostly anca - myeloperoxidase (mpo), have been found in a minority of igan patients, even in patients showing no crescents in renal biopsy . Given the relationship of igan with henoch schnlein purpura, it has been suggested that crescents in a patient with biopsy - proven igan could be interpreted as a limited form of renal vasculitis, but the true relevance and significance of anca in the pathogenesis and course of anca - positive igan patients are not yet fully defined . Recent findings strongly indicate that the complement system has a deep pathogenic influence in igan . Studies of genetic susceptibility have described loci that predispose to igan, as well as mutations and polymorphisms in genes encoding factor h and factor h related proteins . Some of these polymorphisms appear to be protective for the development of igan [1820]. Serum levels of c3 are usually normal or slightly decreased in most patients, but deposits of c3 and c4d are found in a substantial proportion of cases, suggesting that the alternative and the mannose deposits of c4d in mesangium and c3d in peritubular cells have been associated with a more aggressive form of the disease . Studies in the japanese population have found that a serum iga: c3 ratio> 3:1 or 4:1 or high serum levels of c4 binding protein could have an impact on prognosis [24, 25]. Toxic and pro - inflammatory substances released by red blood cells in the tubular lumen during gross haematuria episodes activate the complement system, further aggravating tubulointerstitial damage [26, 27]. Taken together, these findings strongly support a role for complement activation in the pathogenesis and progression of igan . Iga, immunoglobulin a; ic, immune complex; pr3, proteinase-3; mpo, myeloperoxidase . Iga, immunoglobulin a; ic, immune complex; pr3, proteinase-3; mpo, myeloperoxidase . In this issue of ckj, two interesting papers regarding aggressive and rare forms of igan are presented . In the first, yang et al . Describe the clinical and histological features, the response to treatment and the renal outcomes of 20 igan patients with positive serology for anca (anca+). Of 1729 igan patients studied between 1997 and 2013 in whom anca serology was available, 20 (1.2%) were anca+ . This low percentage is in accordance with previously published studies [15, 16]. One of the strengths of the study is the comparison of igan / anca+, igan / anca and anca - associated vasculitis (aav) patients . Igan / anca+ patients showed several similarities to aav patients . Compared with igan / anca patients, igan / anca+ were older and had worse baseline renal function, more lung and systemic involvement and more fibrinoid necrosis in renal histology . Igan / anca+ patients also had a higher frequency of gross haematuria than igan / anca and aav patients . Despite their more aggressive presentation, igan / anca+ patients responded better to immunosuppression than igan / anca patients, with a higher dialysis withdrawal post - immunosuppression (75 versus 0%, p = 0.01) and with a non - significant trend for less esrd at 6 months (0.77 versus 0.26 events / person / year, p = 0.09). At the end of follow - up the study also compared igan / anca+ and igan / anca patients showing crescents in renal biopsy, with a group of crescentic non - igan / anca+ patients . Again, the number of events (esrd) at 6 months was lower in patients with crescentic igan / anca+ when compared with crescentic igan / anca patients (0.31 versus 2.94 events / person / year, p = 0.015). This poorer response to immunosuppressive therapy in patients with crescentic igan / anca had been shown by the same group in a multicentre cohort study, the baseline serum creatinine being the main predictor of renal prognosis . The results of the study by yang et al . Suggesting that patients with igan / anca+ are more responsive to immunosuppression than igan / anca patients had already been described in other studies . However, it should be considered that the percentage of glomerular sclerosis was clearly higher in igan / anca patients than in the other groups (9.1 versus 3.6% for igan / anca+ and 0% for aav, p = 0.002), and the finding of extensive glomerulosclerosis could have induced a more restrictive use of immunosuppression in these patients . In fact, igan / anca patients received cyclophosphamide and corticoids less frequently (64.9 versus 95% in patients iga / anca+ and 95% for patients with aav). Proper adjustment by the degree of glomerular sclerosis would have allowed a more proper assessment of the benefits of immunosuppression . Other limitations of the study are the absence of classification according to the type of anca (pr3 or mpo) and the lack of information about changes in the levels of anca, given that both of these have important prognostic implications [30, 31]. Since gross haematuria was more frequent in igan / anca+ patients, the recovery of renal function that usually follows the disappearance of gross haematuria may have influenced the apparent better response to immunosuppression in this group . Adjustments according to the degree and duration of haematuria have been desirable . In spite of the retrospective nature of the study and the relatively small number of patients, the adjusted comparison by the presence of crescents, the two control groups (igan / anca and aav) and the comparison between subgroups of patients with crescents strengthen the interest of the study in comparison with the anecdotal case reports and small series of patients previously published . If the coexistence of anca in igan patients represents overactivation of certain types of autoantibody - producing b - cells, or a second independent disease, cannot be elucidated in this type of study . In another study, ring et al . Described the case of a young man with a schnlein henoch purpura with crescents and deposits of iga, c3 and c4 in the renal biopsy, who presented as a rapidly progressive glomerulonephritis (rpgn) resistant to therapy with cyclophosphamide, steroids and plasmapheresis . As rescue therapy, despite the lack of evidence of serological or genetic abnormalities in the complement system, treatment with eculizumab was initiated (4 weekly doses of 900 mg and a final dose of 1200 mg). A control biopsy at 11 months showed initial signs of chronicity and some cellular crescents . Due to the role of complement in the pathogenesis of igan, anti - complement therapies could have a place in the treatment of igan, particularly in aggressive cases resistant to currently recommended therapies . Positive responses to eculizumab have been reported in complement - mediated glomerulonephritis [33, 34], and the effectiveness of blocking c5a receptors in anca vasculitis is now being evaluated by clinical trials . In all these diseases, abnormal activation of complement plays an important role as initiator and amplifier of kidney damage [3638]. Serum levels of c5b-9 complex, a promising biomarker to monitor complement activation and the response to eculizumab in atypical haemolytic uremic syndrome cases, were not measured in this case . Whether the improvement in renal function may have been more complete and sustained with longer eculizumab treatment and if measurement of serum c5b-9 levels could contribute to monitor both disease activity and response to therapy are interesting questions that need further studies . However, the high cost of eculizumab therapy is an important factor to be considered . Larger series of patients with longer follow - up and, ideally, controlled clinical trials comparing eculizumab with other therapies in aggressive types of igan are needed . Only such studies may offer solid conclusions about the possible role of eculizumab in rapidly progressive igan . Anca, anti - neutrophil cytoplasmic antibody; aza, azathioprine; cs, corticosteroids; iga, immunoglobulin a nephropathy; mmf, mycophenolate mofetil; pe, plasma exchange; rtx, rituximab . Anca, anti - neutrophil cytoplasmic antibody; aza, azathioprine; cs, corticosteroids; iga, immunoglobulin a nephropathy; mmf, mycophenolate mofetil; pe, plasma exchange; rtx, rituximab . Clinical features of iga nephropathy with serum anca positivity: a retrospective case control study.
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Malperfusion of coronary arteries due to acute stanford type a aortic dissection is particularly problematic because it may be obscured by myocardial ischemia or infarction.1) acute myocardial infarction due to extension of an acute stanford type a aortic dissection is an infrequent, but devastating event.2)3) several case reports of stanford type a aortic dissections in combination with myocardial infarctions present prior to surgery have been published.2 - 6) we report here an interesting case involving an acute aortic dissection in which coronary malperfusion progressed postoperatively . A 59-year - old woman was admitted to the emergency room of a local hospital with the sudden onset of severe chest pain radiating to her back . She had a history of hypertension and a cerebrovascular accident . At the time of presentation 1). The initial electrocardiogram (ecg) showed a normal sinus rhythm without significant st - t changes (fig . An emergent chest ct scan was performed under the suspicion of an acute aortic dissection, which showed an acute stanford type a aortic dissection (fig . Emergent ascending aortic replacement surgery using a graft was performed with the patient under general anesthesia using a median sternotomy . Upon opening the pericardium, a hemopericardium was noted . The aortic root was incised, and an intimal tear was identified just above the sinus of valsalva . Sedation and mechanical ventilation were maintained, and she was transferred from the operating room to the intensive care unit (icu). In the icu her blood pressure and cardiac output remained low in spite of sufficient inotropic support . An ecg showed st segment elevation in leads ii, iii, and avf, and in the precordial leads, suggesting broad myocardial ischemia (fig . 4). In addition, transthoracic echocardiography showed regional wall motion abnormalities in the territories of the left anterior descending artery (lad) and left circumflex artery (lcx), and moderately decreased lv systolic function . These results suggest coronary artery malperfusion, probably caused by progression of the aortic dissection into the left coronary artery . Accordingly coronary angiography revealed no significant luminal narrowing in the right coronary artery, but almost complete collapse of the lumen of the lad (fig . The collapsed lumen was reopened, but it subsequently recollapsed without forceful contrast injection . A similar finding we reasoned that this life - threatening coronary artery occlusion was caused by pulsatile compression of the false lumen of the left coronary artery dissection . A zotarolimus - eluting stent was deployed in the mid - portion of the lad and a bare metal stent (tsunami, 3.530 mm) was deployed in the proximal portion of the lad . Three bare metal stents (driver, 3.030 mm; tsunami, 3.030 mm; and driver, 3.018 mm) were successfully deployed from the distal portion to the proximal portion of the lcx . However, coronary blood flow was not improved . Thus, direct stenting at the left main stem, and a bare metal stent (driver, 3.518 mm) was successfully deployed . The final angiogram of the left coronary artery showed thrombolysis in myocardial infarction (timi) flow grade 3 in the lad and lcx . Follow - up coronary angiography was performed 45 days after percutaneous coronary intervention (pci) because the patient remained unstable . Dyspnea was new york heart association (nyha) class iii / iv and follow - up two - dimensional echocardiogram (2d - ucg) revealed a severe decrease in lv systolic function . Follow - up coronary angiogram showed significant in - stent restenosis at the proximal portion of lad (fig . Study showed the remaining coronary artery dissection in the diagonal branch of the lad (fig . Repeat balloon angioplasty using a 4.020 mm balloon (apollo) was performed at the site of in - stent restenosis . The final angiogram of the lca showed timi flow grade 3 in the lad and lcx . A follow - up coronary angiogram was performed 8 months after the pci and showed no significant in - stent restenosis (fig . 8). Coronary malperfusion associated with aortic dissection is relatively rare, but when it occurs, it is generally fatal.2)7) retrograde dissection of the aortic root reaching the coronary ostium was noted in 7% of necropsies according to a previous study.8) in acute stanford type a aortic dissection, acute myocardial ischemia and infarction due to this mechanism are severe complications, which potentially lead to irreversible myocardial damage . Therefore, coronary artery ischemia and concomitant coronary artery bypass grafting (cabg) have been reported to be significant risk factors of postoperative mortality.9) to salvage such moribund patients, aggressive coronary revascularization concomitant with aortic repair is essential.2) in the present case, myocardial infarction occurred immediately postoperatively, but the patient showed no previous signs of myocardial ischemia prior to the operation . Generally, myocardial ischemia caused by acute aortic dissection is apparent soon after its onset, and signs of myocardial ischemia are present prior to surgery.2 - 6) when a stanford type a aortic dissection extends to the left coronary artery after aortic replacement surgery, catastrophic hemodynamic changes occur, which frequently result in sudden death.10) treatment of a coronary artery dissection depends on its severity . Three main types of coronary lesion can be caused by ascending aortic dissection, depending on the extent: type a, ostial dissection; type b, dissection with a coronary false channel; and type c, circumferential detachment with an inner intussuscepted cylinder.3) however, these types are not easily differentiated . In the present case, the coronary artery ostium appeared intact, as in a type a coronary dissection . A fragile intima may have been damaged by a needle, arterial blood flowed into the false lumen, and coronary artery dehiscence resulted if management of the false lumen was inadequate . Accordingly, type a coronary artery dissection progresses to type b or c dissection, and blood from the aortic false channel progresses into the coronary ostium and creates an extension of the false lumen into the coronary artery . The mechanism of coronary flow impairment was attributed to compression in diastole of the true lumen by obstructing the false channel . Finally, the dissection may have encircled the affected coronary ostium and caused malperfusion by directly obstructing coronary blood flow . In types b and c dissections, it is not difficult to decide whether to undertake concomitant cabg . However, the role of concomitant cabg in type a dissection remains controversial.3) in the present case, myocardial ischemia should have been suspected when circulatory collapse was observed after terminating cardiopulmonary bypass, and at this stage immediate revascularization by concomitant cabg or pci was necessary . Although no definite method for treating an infarcted myocardium has been established, the restoration of coronary circulation as quickly as possible appears to be important . In the present case, we decided to perform immediate revascularization by emergent pci rather than cabg as an optional approach because pulsatile compression of the left coronary artery by coronary arterial dissection resulted in a life - threatening situation at the time of coronary angiography . It has been reported that an ivus study can promptly diagnose direct extension of a dissecting flap into the coronary arteries and subsequent coronary malperfusion in potentially fatal conditions.11) once coronary malperfusion has occurred, the success of myocardial salvage depends on how quickly the malperfusion is recognized . Transesophageal echocardiography is one of the most sensitive and specific modalities for diagnosing acute aortic dissection and coronary malperfusion, even during surgery.12) on the other hand, the role of coronary angiography before emergency repair of an acute aortic dissection remains controversial.13) although coronary artery malperfusion associated with acute aortic dissection after surgery is relatively rare,14) it is possible for acute stanford type a aortic dissections . Furthermore, an immediate decision is required to perform additional cabg if required . In the present case, emergent pci may have been an optional approach given the critical, unstable patient postoperatively . An ivus study could also provide additional information about the cause of coronary artery malperfusion.
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Marine dissolved organic matter (dom) is one of the largest active carbon pools on earth and, thus, an important component of the global carbon cycle . The earth s continents are a major source of terrigenous dom to the oceans, which is transported with other constituents such as iron (fe) via riverine and submarine groundwater inputs . Groundwater - based fluxes, however, are not well constrained because before entering the coastal ocean, groundwater passes through a biogeochemically active environment, the subterranean estuary (ste). While dom has been found to coagulate with fe(iii) (oxy)hydroxides at redox boundaries in other environments, dom coagulation processes in stes have not yet been investigated, despite much already being known about fe cycling in stes . If fe dom coagulation is prominent in stes, which are characteristic of sandy shores that cover up to two - thirds of the world s ice - free coastlines, then such processes would be of global importance in terms of modulating the transport of terrigenous constituents from land to the ocean, with consequential implications on the global carbon cycle . Stes are coastal aquifers where meteoric groundwater mixes with recirculating seawater . To date, no coherent database of the global coverage of stes exists, but the occurrence of submarine groundwater discharge (sgd), the advective flux of terrestrial and marine groundwater into the ocean that originates from the ste, has been reported worldwide . It has been estimated that the annual discharge of freshwater via sgd amounts to 2400 km / year, with the majority of it passing through unconfined aquifers such as permeable beach sediments prior to entering the coastal ocean . Because sgd can be highly enriched in nutrients, its constituent fluxes are estimated to be on the same order of magnitude as that of global surface runoffs, even though the freshwater volume flux amounts to only 610% as compared to rivers . Sgd is also a significant source of dom to the coastal environment, as well as of fe, a redox active trace metal and an important micronutrient for primary productivity in marine sediments . In stes, changes in redox potential are expected to serve as a main control for the removal or mobilization of fe and other nutrients . Chambers and odum first used the term iron curtain for an iron hydroxide precipitation zone on tidal freshwater marsh creekbanks, where reduced iron, fe(ii), from advectively discharged porewater was oxidized to fe(iii) upon coming into contact with the oxygenated surface waters of the tidal creeks and consequently scavenging dissolved phosphate . Later, charette applied the expression to describe ferric hydroxide precipitates at redox interfaces within the ste . This iron curtain may vary spatially and temporally, depending on tidal amplitudes and long - term sea level changes, and acts as a barrier for chemical species such as phosphate and barium . The iron curtain has mostly been investigated with regard to the retention of inorganic constituents from submarine groundwater; however, various studies have described oxidative fe dom coagulation in peat bog samples and found that highly aromatic, typically terrestrial dom molecules are more likely to coagulate with iron than are aliphatic compounds typically found in aged marine dom . Recently showed that iron from iron - rich hydrothermal vent fluids, as it coagulates with dom, leads to a decrease in dissolved organic carbon (doc) and changes in dom molecular composition in the surrounding seawater . In addition to the molecular properties described by riedel et al . And gomez - saez et al ., lv et al . Also reported an affinity of higher - molecular weight compounds (> 500 da) for coagulation with a variety of iron oxyhydroxides . They revealed that the extent of dom coagulation was related to the type of iron substrate, with amorphous ferrihydrites scavenging more dom than other iron oxyhydroxide minerals . In their studies of the organic geochemistry of advective porewater systems of the german wadden sea, seidel et al . Recently postulated that fe dom interactions had affected the observed molecular properties of dom . However, to the best of our knowledge, no experimental data about the reactivity of dom with iron within the ste exist, although stes probably occur ubiquitously along coastal shorelines and may affect land dissolved fe concentrations in the ste are often in the micromolar range, which are equal to or larger than normal doc concentrations in ste porewaters, and dom coagulation potentials of 5080% can be achieved for fe / dom ratios between 0.8 and 1.2 . The iron curtain as a major biogeochemical barrier within the ste thus has the potential to modulate the global transport of terrigenous constituents from land to the ocean . Here, we hypothesize that the iron curtain affects the quality and quantity of advective porewater dom fluxes from stes into the coastal ocean by retaining specific molecular fractions of terrigenous dom . Fractionation of dom can be investigated on a molecular level with new nontargeted analytical technologies, in particular ultra - high - resolution mass spectrometry using soft ionization [electrospray ionization fourier transform ion cyclotron resonance mass spectrometry (esi - ft - icr - ms)]. Whereas traditional analytical methods target only small groups of compounds, esi - ft - icr - ms allows the identification of specific molecules on a broader scale . This method allowed researchers to determine molecular dom fingerprints to track water masses, follow the molecular succession of microbial remineralization, and identify the molecular formulas of dom subject to change under experimental conditions . We studied coagulation of iron with dom from a set of porewater samples from the northern beach of spiekeroog island, where an ste is formed by the terrestrial freshwater lens and the tidally recirculating seawater . We characterized both noncoagulating and coagulating fractions of dom by molecular analysis with esi - ft - icr - ms . We collected porewater samples from three different sites along a transect perpendicular to the shoreline and one seawater sample as a reference . The stations were chosen to yield representative samples from the ste with varying amounts of fresh groundwater and recirculating seawater, and oxic to anoxic conditions . We performed precipitation experiments with samples that contained fe at micromolar concentrations, either naturally or through addition of an fe spike . We characterized the dom molecular composition of all samples before and after their exposure to oxygen and subsequent precipitation of fe(iii) (oxy)hydroxides, identifying both noncoagulating and coagulating dom fractions . All samples were desalted via solid - phase extraction prior to molecular analysis via esi - ft - icr - ms . Spiekeroog is one of the east frisian islands in the german north sea and part of the unesco world heritage area wadden sea . Peat and clay layers within the sand dunes confine its precipitation - fed fresh water lens at a depth of 40 m. spiekeroog is exposed to a mesotidal regime (24 m tidal range). With its complex redox zonation, spiekeroog has formerly proved to be a suitable sampling site for studies on the biogeochemistry of stes . Previously, numerical models from spiekeroog south beach, a sheltered site facing the wadden sea, revealed that tidal pumping produced an upper saline plume (usp) with an 100 m lateral extension and an 20 m depth . Groundwater from the fresh water lens discharged near the low tide water line, forced to a narrow tube . The model data were backed up by in situ geochemical transects, which in addition indicated a complex redox zonation, for example, two fe reduction zones associated with organic matter decomposition at the drift line as well as at the low tide water line (lwl). Transect data from a shallow subterranean estuary at spiekeroog north beach recently indicated the occurrence of a similar usp at this exposed beach site . Both studies from the north and south shores of spiekeroog have described the ste as a biogeochemical reactor: they showed a succession of denitrification and iron and manganese reduction zones along the groundwater flow path from land to sea . Analogous to the south beach, a transition zone in the north beach ste with high fe concentrations was described near the lwl . The zone spanned a width of approximately 150200 m and was bordered by sharply decreasing porewater fe concentrations, probably because of oxidation processes forming fe(iii) (oxy)hydroxide precipitates . A more detailed study of the north beach circulation system, including a numerical model, is currently in revision . On the basis of these previous findings, samples were taken at the north beach site of the island, at 5346n, 0743e, in the vicinity of umweltzentrum wittblten . Our sampling beach very likely represents the redox and iron gradients that are typical of many stes worldwide . Sites were selected on the basis of ref (22) and a preliminary study in november 2013 (see the supporting information), in which concentrations of nutrients and fe had been measured in porewaters over the range of the study site to define the fe(ii)/fe(iii) transition zone of the local ste . Porewater (pw) samples were taken at three different sites along a transect perpendicular to the shoreline, with increasing distances from the lwl toward the sand dunes (figure 1): pw1pw3, with distances from the lwl of 100, 250, and 450 m, respectively . Pw1 and pw2 were located within the intertidal zone; pw1 was closest to the lwl and pw2 at the edge of the intertidal zone . Pw3 was the most landward site and located outside the tidal zone, 50 m from the beginning of the foredune belt colonized by marram grass . Two seawater samples (sw) were taken from the surf zone during high tide . (a) sampling scheme at 5346n, 0743e, where lwl is the low tide water line on the day of sampling . (b) spiekeroog island, with the sampling area as shown in panel a marked with a red box . Pw denotes sites of porewater sampling and sw sites of seawater sampling . At each of the three beach sites, viton tubes were attached to stainless steel push point samplers placed into the sediments at a depth of 0.52 m. at each spot prior to dom sampling, small volumes of porewater were first sampled with a polyethylene syringe to determine salinity and fe concentrations . Salinity was measured with a standard tetra - con 925 multi 3430 (wtw) conductivity measuring cell, and fe was detected by adding 1 ml of porewater to 1.5 ml clear safe - lock tubes (eppendorf) containing 100 l of a ferrozine solution . Iron concentrations were then estimated in the field by visually comparing the emerging color from the ferrozine reaction with a color table . More precise fe determination was performed later in the laboratory (see below). Afterward, 2 l of porewater was taken from the selected spots by applying a gentle vacuum with a hand pump . The samples passed through an inline filter (a 3.0 m millipore isopore polycarbonate membrane filter on top of a 0.2 m pall polyethersulfon membrane filter, both 47 mm in diameter) into acid - washed 2 l polycarbonate (pc) bottles (nalgene). The bottles were fitted with vacuum caps and pc two - way valves and had been flushed and filled with ar gas to a slight overpressure before sampling to prevent oxygen contamination and subsequent uncontrolled fe(iii) precipitation . The sw samples were also collected in acid - washed 2 l pc bottles but filtered approximately 2 h later back in the laboratory by vacuum as described above for pw1pw3 . After the first round of filtration, subsamples for doc and trace metals were extracted from the pc bottles with acid - washed syringes attached to the vacuum valves . All subsamples were acidified with hcl (merck, suprapur) to ph 2 and stored in acid - washed hdpe bottles at 4 c in the dark ., carl roth, 1% in ultrapure water) and 0.01 m hydrochloric acid (suprapur) for 1 week each and rinsed repeatedly with ultrapure water prior to use . All other nonglass material (filter membranes, filter holders, tubing, sampling bottle caps, etc .) Were soaked in 0.14 m hno3 (p.a .) For several weeks and rinsed repeatedly with ultrapure water before being used . Samples for analyses of trace metals, doc, and solid - phase extraction of dom were acidified to a final concentration of 0.01 m hcl (suprapur). To prevent any fe in solution from precipitating, the filtered control samples in the pc bottles were acidified in situ to ph 2 with hcl . This is also the ph necessary for solid - phase extraction as described below . The sw samples were filtered in the laboratory and thereafter split into two sets, with one of them being acidified to serve as a control and the other being prepared as a treatment sample as follows . After all samples had been brought back to the laboratory, fe was roughly quantified in the ferrozine subsamples by visual comparison with an fe ferrozine calibration series (5100 m) prepared prior to sampling . In the porewater closest to the lwl (pw1), natural fe was present as indicated by the in situ ferrozine test; in all other samples, no visible fe(ii)ferrozine coloration was found . Fe from an fecl2 (emsure, merck) spike solution enriched with a suprapur hydroxylamine hydrochloride solution to prevent any fe from oxidizing (honh2hcl, merck) was then added to all treatment samples of pw2, pw3, and sw in amounts approximating the concentrations of those estimated in pw1 (80 m). The hydroxylamine hydrochloride concentration was just high enough to keep the spike in solution . After a short exposure to atmospheric oxygen, the bottles were capped, shaken, and left standing for 24 h. the samples were then shaken again, and each of the treatment samples was filtered again (through an inline filter such as in the field), split into four acid - washed 2 l pc bottles, and acidified to ph 2 with suprapur hcl . After filtration, all filters were rinsed and desalted with ultrapure water and stored at 4 c . The precipitates were later gently dissolved in 0.01 m hcl (ph 2) and are termed precipitate samples . Doc concentrations were measured on a shimadzu (toc - vcph and tnm-1) analyzer, using high - temperature catalytic oxidation, against a calibration series and a deep sea reference standard provided by d. hansell (university of miami, miami, fl). Concentrations of nutrients [sum of nitrate and nitrite (nox), ammonium, and phosphate] and fe from ferrozine treatments of the porewater samples were measured by uv / vis microplate spectroscopy (multiscan spectrum, thermo scientific) at the icbm microbiogeochemistry laboratory at the university of oldenburg (oldenburg, germany). Nutrients were measured with a method modified from refs (37) and (38), and fe was measured according to ref (35). Total fe and mn were measured by inductively coupled plasma optical emission spectrometry (icp - oes) (icap 6000, thermo scientific) coupled with an argon humidifier (t 2100 br); resultant data were analyzed with iteva software . Calibration was performed with nist traceable standard solutions and verified using slew-3 as reference material . For esi - ft - icr - ms analysis, samples must be salt - free and concentrated to a carbon concentration of> 10 mg l. all samples were thus extracted via solid - phase extraction (spe) according to the method described in ref (29); 1 g varian bond elut ppl cartridges were used for extraction . Their solid phase is a modified styrene - divinylbenzene (sdbv) polymer that is capable of also retaining some polar analytes, including phenols . Prior to use, the cartridges were soaked with methanol (uplc / ms grade, biosolve bv) overnight and rinsed twice with ultrapure water, twice with methanol, and twice with 0.01 m hcl (suprapur). All samples were acidified to ph 2 with hcl prior to extraction to prevent fe(iii) precipitation outside of the control of the experimental setup and to increase the extraction efficiency for the organic acids and phenols that are present . After the samples had been passed over the spe resin, the cartridges were rinsed with 100 ml of 0.01 m hcl and dried with ar gas . The absorbed dom samples on the cartridges were then immediately eluted with 6 ml of methanol into precombusted amber glass vials . Prior to analysis by esi - ft - icr - ms, all extracts were diluted with ultrapure water and methanol to yield a 15 ppm solution in a 1/1 (v / v) methanol / water mixture . Extracts with lower doc concentrations, such as the precipitate samples, were concentrated by evaporation and redissolved with a 1/1 methanol / water mixture . Extraction efficiencies were calculated after determining doc concentrations in both the original water samples and the extracts (after complete evaporation of the methanol and redissolution in ultrapure water) and were 43 7% for all filtrate and control samples and samples were measured on a bruker solarix esi - ft - icr - ms instrument (bruker daltonik gmbh, bremen, germany), equipped with a 15 t magnet system . All samples were analyzed in negative electrospray ionization (esi, bruker apollo ii) mode . All instrument settings and data processing approaches, including molecular formula assignments, are described in detail in ref (21). All filtrate and control samples were measured over the course of 2 days in randomly mixed order . To test the reproducibility and stability of the esi - ft - icr - ms analysis, a dom extract of north equatorial pacific intermediate water (neqpiw) this sample is an in - house reference material for aged, marine dom . On the basis of the suggestions made by stenson et al . And the double bond equivalence (dbe) was determined using the method described by stenson et al ., and the aromaticity index was determined in its modified version using the method described by koch and dittmar (aimod). After molecular formulas were assigned, data were further statistically analyzed by using the software package r (version 3.1.1, 2014 - 04 - 10). To test for differences in molecular composition between filtrate and control samples, a mann whitney - u test (= 0.05) was applied, the nonparametric version of the t test for the determination of significant differences between two data sets with non - normally distributed residuals . The precipitate samples were not included in the statistical analyses described above but were finally used to compare the presence and absence of molecular formulas between filtrate and precipitate fractions . Nutrient and doc concentrations varied substantially within only 1 m sampling distance for control and treatment samples from the same station . In particular, doc displayed no specific trend, as it varied more between close locations at a given station than between stations . In some of the experiments (pw1 and pw2), bulk doc concentrations were higher after than before the precipitation treatments . We excluded these doc data from further consideration because of the risk of contamination with volatile organics . For example, the more landward pw2 and pw3 stations were enriched with nox, whereas station pw1 was clearly located in an iron reduction zone, with nh4 being the dominant form of dissolved inorganic nitrogen . Total dissolved fe was completely removed from the filtrate samples after the second filtration step, while we found precipitated fe(iii) (oxy)hydroxides in the precipitate samples . Thus, we investigated the molecular composition of solid - phase - extracted dom in the fe(iii) (oxy)hydroxide precipitates, as well as in the filtered permeate, to track experimentally induced changes not evident in bulk doc data . Control and pretreatment: after the first filtration, mean values of three injections . Post - treatment: after coagulation and the second filtration, mean values of duplicate samples, three injections each . Natural reduced iron concentration according to the ferrozine test, as measured at the sampling spot prior to taking the esi - ft - icr - ms samples . Total dissolved iron or manganese, as measured via icp - ms directly from the esi - ft - icr - ms samples, after coagulation and filtration of the treatment samples . Pw1 treatment samples were not measured via icp - ms because of their high fe and mn concentrations; thus, icp - oes results are given . Not detected (below the detection limit). At the threshold of the detection limit . We identified 2066 molecular formulas, representing 34% of all 6015 molecular masses detected by esi - ft - icr - ms . Unassigned masses are isotopologues of assigned molecular formulas or contain combinations of elements not considered in this study . By mann whitney - u testing on all 6015 masses, we found that 423 compounds were responsible for significant differences between filtrate and control samples (p <0.05); 267 of these 423 compounds were missing or had significantly lower intensities in the mass spectra of the filtrate samples, suggesting that they coagulated with iron and were filtered out from the filtrate samples . To 217 of these 267 detected masses (4% of all 6015 compounds and 11% of all 2066 molecularly described compounds) were we able to assign molecular formulas . In the precipitate, 114 of these 217 missing compounds in the filtrate could be detected (2% of all 6015 compounds and 6% of all 2066 molecularly described compounds). It should be noted that we were not able to fully redissolve the precipitate from the filter holders by the acidification processing step . We identified c18.5h21.1o8.2n0.1s0.3 as the average formula for all molecules present in the control samples and c21.8h20.8o11.8n0.0s0.0 for all molecules that were missing in the filtrate residual solution of the treatment, indicating that they had been removed during precipitation . C18.2h21.2o7.8n0.1s0.3 is the average formula for all compounds that remained in the residual filtrate after coagulation . The reference deep sea standard, neqpiw, in comparison, had a molecular formula of c19.9h24.0o8.8n0.0s0.3 (table 2). Molecular classifications are from the work of koch and dittmar, antl temkiv et al ., and seidel et al . Dom extract of north equatorial pacific intermediate water, an in - house reference material for aged, marine dom; here given as present in at least five of eight neqpiw standard measurements . We found that iron coagulation specifically affected large molecules with a relatively high oxygen content . The mean mass and oxygen content of the molecules that were removed from the filtrate samples (470 da, 12 o) were substantially higher than the mean mass of all described molecules as found in the control samples (380 da, 8 o) and the residual filtrate (395 da, 8 o). Dbe was much higher for the coagulated molecules (12) than for molecules in the control samples and the residual filtrate (both 9), suggesting a preferential coagulation of molecules with a higher content of unsaturated fractions . As another indicator for rather unsaturated, oxygenated molecules undergoing coagulation, coagulated molecules tended to have a relatively high o / c and low h / c ratio (0.5 and 0.9, respectively) as compared to all identified molecules in the controls (0.4 and 1.1, respectively) and residual filtrates (0.4 and 1.2, respectively) (figure 2). Abundance frequency plots of molecular formulas in categories of o / c and h / c element ratios (van krevelen diagrams), showing the distribution of all detected molecular formulas in the respective sample: (a) present in all controls, (b) retained in the filtrates, (c) with relative intensities in the filtrate samples statistically significantly lower than those in the control samples, i.e., thought to have coagulated with iron, and (d) the coagulating molecules from panel c that were also detected in the precipitate samples . The color code in the background shows the different molecular categories according to antl temkiv et al . : (i) combustion - derived polycyclic aromates (pcas), (ii) soil - derived polyphenols and pcas with aliphatic chains, (iii) soil - derived humics (i.e., phenolic and highly unsaturated compounds), and (iv) unsaturated aliphatic compounds . Our findings of preferential coagulation of large, oxidized, aromatic molecules with a high content of unsaturated fractions are in general agreement with previous reports from a variety of different environmental and experimental setups ., who, in a similar experiment in a hydrothermal vent system, found a selective enrichment of sulfuric groups in their iron - dom coagulate precipitate samples, we did not find preferential coagulation of nitric or sulfuric dom; they were also not found missing in the filtrate or present in the precipitate samples . It should be noted though that gomez - saez et al . Worked with samples naturally much higher in organic sulfur, and many terrigenous dom fractions may have already been removed as they worked with seawater samples, which potentially leaves less competition for fe oxide surfaces from remaining other (marine) organic matter fractions . We found no general preferential coagulation of molecules classified as combustion - derived (black carbon) or highly saturated compounds, but it was the large, oxidized fraction within these compound groups that coagulated . In general, compounds that are characteristic of terrigenous dom (categories ii and iii in figure 2) preferentially coagulated . No evidence of the precipitation of compounds that share molecular formulas with sugars, peptides, or aliphatic molecules was found . Like any analytical technique, spe combined with ft - icr - ms even though the analytical window of this combination of techniques is unsurpassed for molecular dom analysis, not all compounds are detectable . In a study by hawkes et al ., experimental modifications of dom moved a molecularly defined fraction out of the analytical window, which was clear evidence of substantial molecular modifications . Several past studies (e.g., seidel et al .) Have, however, shown that our approach is not selective toward marine or terrigenous (polyphenolic) compounds . Our extraction efficiencies are at the lower reported end for ppl - spe but are still within the range of similar samples . Our findings for dom fractions prone to coagulation with iron are consistent with previous descriptions from peat - bog samples by riedel et al . We additionally show that these processes of preferential coagulation of terrigenous dom work in terrestrial and marine environments . On the basis of our experimental setup, we were able to estimate the coagulation potential of all studied porewater samples at each sampling site . We achieved this by calculating the cumulative difference of relative molecular abundances between control and filtrate samples for each site . To present our data, we describe three molecular categories that coagulated most with iron: group ii and iii according to antl temkiv et al . And all molecules with an o / c ratio of> 0.35 and a h / c ratio of <1.0 (figures 2 and 3). Overall, the extent of coagulation decreased along the studied transect from land to sea (figure 3). We suggest that, as dom from terrestrial sources is transported through the ste, fractions more prone to iron coagulation (such as polyphenols, pcas with aliphatic chains, and phenolic and highly unsaturated compounds) precipitate with iron, so that the extent of coagulation overall decreases with increasing distance from land to sea . Relative coagulation potential for the four different sampling sites, from the dunes (pw3) to the sea (sw). Dark brown bars show data for group ii, soil - derived polyphenols and pcas with aliphatic chains . Light brown bars show data for group iii, soil - derived humics, i.e., polyphenolic and highly unsaturated compounds, both as according to antl temkiv et al . Light gray bars show data for samples with an o / c of> 0.35 and a h / c of <1.0, i.e., large unsaturated molecules with a high oxygen content . The coagulation potential is calculated as the cumulative difference in relative molecular abundances (esi - ft - icr - ms signal intensities) between control and filtrate samples per sampling site, each shown relative to the highest potential found (i.e., pw2 group iii). Ocean trends in the extent of coagulation contribute in a systematic way to the apparent heterogeneity of the ste . Compositional differences in dom from the various groundwater end members may have contributed to the observed trend . Further, the retention of metal - sensitive dom fractions in the ste is intrinsically linked to sediment redox chemistry, which is not directly dependent on salinity . Found highly aromatic, terrigenous dom in an intertidal porewater seepage zone on the southern shore of spiekeroog, downstream of several iron oxidation reduction zones in the ste . They suggested on the basis of their findings that fe - dom coagulation in the ste is a reversible process . Similarly, concomitant dissolution of fe and doc under anoxic conditions and fast subsequent flocculation after the re - establishment of oxic conditions were reported from incubation studies with boreal lake sediments . Therefore, movements of the freshwater saltwater boundary over tidal, seasonal, or even geological time scales, which influence advective flow rates and the supply of organic matter for microbial processes in the ste, will also impact the dynamics of the iron curtain . For example, microbial reduction of iron, triggered by the supply of fresh, labile marine dom from a spring bloom, could trigger the release of older, more aromatic dom of meteoric origin into the coastal water column . We therefore hypothesize that the ste has the potential to act as intermediate storage, rather than only as a net sink, for terrigenous aromatic dom compounds . In this study dom coagulation, a process previously studied on the molecular level only in terrestrial and hydrothermal vent systems, occurs at coastal beach sites, potentially on a global scale . We found preferential coagulation of fe with dom of a molecular composition typical for soil - derived dom, such as highly oxidized polyphenols . Dom coagulation in stes potentially has major implications as stes occur globally, and dom is an essential component of the global carbon cycle . Our study supports the hypothesis of the iron curtain selectively modulating the advective groundwater fluxes of organic carbon from land to sea . Whereas esi - ft - icr - ms allows for detailed qualitative molecular analyses, the quantitative relevance of iron dom coagulation in stes should be further investigated by additional techniques; for example, terrestrial marker compounds can be used for quantification with liquid chromatography, or the sediments can be leached to quantify the attached dom . As we do not know the time scale on which coagulated dom is buried in coastal and shelf sediments, we cannot determine if stes act as a temporal storage or an effective long - term sink for terrigenous dom . Deep stes might have different dynamics such as longer residence times, which may influence the forms of dom that can potentially coagulate and the ultimate fate of sedimentary bound dom . The role of iron in the preservation of organic matter in sediments is well - known . Our study indicates that terrigenous dom may also be trapped and preserved in the rusty carbon sink on its subterranean way from land to ocean . Many open questions about the global relevance of this process and the relevant time scales involved for burial and potential remobilization remain and should be the focus of future studies.
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The human immunodeficiency virus (hiv) infects cd4 t lymphocytes and macrophages, eventually inducing the depletion of cd4 t cells, which is the defining feature of the acquired immune deficiency syndrome (aids). It is not clear precisely how the virus exploits the host cell to maximize viral particle production, but evidence is accumulating that hiv activates the cellular transcription machinery to achieve this aim . While biochemical approaches have been extensively employed to study the intracellular response to hiv infection, the advent of lymphocyte microarrays has provided a powerful new tool to help illuminate the extensive effects of hiv on host - cell transcriptional responses . Biochemical studies have demonstrated that hiv is capable of modulating a variety of signal transduction pathways in the host cell at multiple stages in the infection process, beginning at entry when it engages two transmembrane receptors, cd4 plus either of the chemokine receptors ccr5 or cxcr4, thereby activating intracellular protein tyrosine kinases . Indication that hiv gene products influence signaling processes in host cells also comes from analyses of transgenic mice that express portions of the hiv genome and display a variety of abnormalities, ranging from altered t - cell maturation to the development of a systemic disease similar to aids . Because the long terminal repeats (ltrs) of hiv contain consensus recognition motifs for the nf-b and nfat families of transcriptional transactivators, it has been speculated that hiv may have evolved mechanisms to potentiate cellular activation pathways, thereby augmenting expression of its own genome . Until now, however, there has been limited understanding of how hiv exerts control over specific transactivation responses of the host cell . Because hiv employs host factors that are vital for its replication cycle, the virus may have evolved means of modulating their expression levels during infection, so as to favor its own replication . One crucial host factor is the well - characterized transcription - elongation factor complex ptefb, which is recruited to the nascent hiv transcript by the rna - binding tat protein encoded by the virus . This complex contains the cyclin - dependent kinase cdk.9 and cyclin t1, and it phosphorylates the carboxy - terminal - domain repeats of rna polymerase ii, activating the polymerase and thus allowing processive transcription of the hiv genome . Other host proteins are required to facilitate transport of unspliced viral rna from the nucleus to the cytoplasm and to enhance viral assembly and release . The identity of most of the host proteins involved in facilitating hiv replication has yet to be determined, however . Recent studies suggest that hiv infection can influence the expression of many host genes, and some of these may indeed have critical roles in the hiv replication cycle . Among the various hiv gene products implicated in modulation of cell signaling, nef appears to be the most potent . The nef gene, expressed rapidly and abundantly following infection, is a major virulence factor both in vitro and in vivo . Rhesus macaques infected with simian immunodeficiency virus (siv), a close relative of hiv, rarely progress to disease if the viral nef gene is deleted . It has also been shown in infected macaques that there is very strong selective pressure for sivs with nef open reading frames . In humans, members of a cohort of individuals infected with a nef - deleted form of hiv have remained disease - free for many years . In primary t - cell cultures, hivs with intact nef genes replicate much better than nef - defective viruses . While the precise function of the nef protein has remained elusive, the presence of an amino - terminal myristoyl linkage and a proline - rich sh3-binding domain suggest that it may interact with host proteins at the plasma membrane . Recent work has confirmed that these two regions of nef are required for its association with lipid rafts, cholesterol - rich membrane microdomains that concentrate potent signaling mediators . One functional consequence of nef expression in t cells, as elucidated from in vitro studies, may be to enhance the levels of secreted interleukin-2 (il-2, a growth factor) during activation . Nef has also been shown to associate with the -chain of the t - cell - receptor complex (tcr-) and concomitantly to induce expression of fas ligand, one of the mediators of apoptosis in differentiated cells, an outcome that may account for the high levels of apoptosis associated with hiv infection . In this and other studies, nef was also found to complex with a serine / threonine protein kinase, which in some cases has been identified as belonging to the pak (p21-activated kinase) family . In their recent immunity paper, simmons et al . Describe results of an extensive microarray survey of expression levels of nef - responsive loci . As a model system, they have developed a clone of the t - cell - leukemia cell line jurkat that expresses nef in a tetracycline - inducible manner, thus minimizing the cytopathic effects of nef in long - term cultures . Using arrays containing 3,528 genes, simmons et al . Observed that a panoply of loci were up - regulated at multiple time points after nef induction (figure 1a). Most remarkably, they observed significant induction of mrnas encoding many transcription factors that positively regulate the viral ltr, including nfatc, nf-b p52 and p100, irf-1 and irf-2, c - fos, and jun - d . The cytokines transforming growth factor (tgf-) and il-4, which may function in an autocrine and/or paracrine manner to promote ltr transactivation, were also elicited, along with mip-1 and mip-1, chemokines previously shown to be up - regulated by nef expression in macrophages . Because these soluble factors are likely to influence host gene responses, it will be interesting in future studies to distinguish transactivation cascades that are autonomous from those requiring new protein synthesis . Host cofactors implicated in potentiating other stages of the hiv life cycle, from the processing of viral transcripts to virion budding, were also elicited in response to nef expression . It was also confirmed that the tat - cofactor cdk9 was elevated in nef - expressing cells . That these findings point to a role for nef in enhancing some of the earliest stages of the viral life cycle is particularly intriguing in the context of a recent study showing that nef transcripts are produced prior to integration of the virus into the genome of resting t cells, resulting in accumulation of nef protein that primes viral synthesis upon t - cell activation . In view of previous work implicating nef in signaling at the plasma membrane, the ability of nef to influence expression of many loci suggested that it might function as an upstream regulator of multiple divergent cascades . To explore this issue, simmons et al . Compared nef - responsive genes with those modulated when the t - cell line was activated with antibodies against the t - cell antigen - receptor (tcr) complex . Surprisingly, the spectrum of genes exhibited 97% overlap, indicating that a major function of nef may be to trigger the conventional t - cell activation program . When nef was induced during antibody stimulation, the same loci were activated even more potently, with the exception of targets unique to either inducing factor . Among the targets triggered only by nef are several genes that may aid viral progression, including those encoding the transcription - elongation factor tat - sf1, the transcription factor irf-2, and the small nuclear riboprotein u1 snrnp a. in contrast, stimulation with anti - tcr antibody but not with nef induced two factors, the cytokine il-16 and the transcription factor yy1, that are thought to negatively regulate viral transcription . How a single viral gene can achieve such remarkable specificity will need to be addressed in future studies . Paper, nef expression also results in down - modulation of numerous positive effectors, including the kinases pkc- and zap-70, the phospholipase plc-2, and 40s ribosomal protein . One of the advantages of the in vitro system of simmons et al . For example, numerous jurkat mutants that lack the expression of proteins involved in t - cell activation have been generated . Inducibly expressed nef in two such lines, one that lacked tcr- and another deficient in zap-70, a tyrosine kinase recruited to the tcr- chain upon activation . Expression profiling revealed that both proteins are required for the full - spectrum nef response; in each of the mutant lines approximately half of the gene targets were not induced . A similar magnitude of inhibition was achieved in wild - type jurkat cells in which nef was expressed in the presence of the drug cyclosporin a, which blocks the more downstream nfat effector calcineurin . Intriguingly, the genes inhibited by cyclosporin a only partially overlapped with those inhibited in the mutant jurkat lines . Caveats that must be considered when interpreting these results, however, are that the mutant lines may have undergone adaptive changes and the levels of nef protein in different jurkat lines may not be identical . Different levels of nef expression, and different nef alleles, may elicit dramatically different outcomes, as suggested by studies using both in vitro and in vivo models . Whereas the work of simmons et al . Paints a picture of nef as a' master switch' of cellular activation, a parallel study by corbeil et al . This group infected the cem t - cell line for various lengths of time (eight time points, from 0 to 72 hours) with high levels of replication - competent hiv . After analyzing microarrays containing 6,800 loci, they found that productive infection was associated with complex patterns of up- and down - regulated genes (figure 1b). On average, more genes were induced early in infection (up to 24 hours) than later, when the cytotoxic effects of the virus resulted in dramatic repression of approximately one third of expressed host genes (33% of cells were apoptotic 72 hours after infection). Among the genes augmented at consecutive early time points were interferon- (ifn-) and its target mxb, which serve anti - viral functions . Nfib-2, which encodes a factor involved in the transcription of both viral and cellular genes, was up - regulated, as confirmed by real - time pcr . Perusal of the supplementary data reveals that a multitude of host genes are strongly up - regulated at individual time points, although the relevance and reproducibility of these findings remain uncertain until confirmed . Other activated loci appeared to reflect a state of genotoxic stress, including the gene gadd45, which is induced by dna damage . Both the mrna encoding the proapoptotic mediator bax and the protein itself were up - regulated in infected cells, as were numerous caspases . It is worth noting that an earlier survey of hiv-1-induced genes by differential display revealed a variety of up- and down - regulated host genes, including some responses consistent with a cytopathic outcome ., it is apparent that nef down - regulated the anti - apoptotic bcl-2 gene while up - regulating the proapoptotic mediator bad . Utilization of replication - competent hiv to study effects on host genes has advantages as well as disadvantages . The most obvious merit is that the system is likely to reflect in vivo outcomes better (although certainly the in vivo cellular microenvironment will exert a profound influence on gene responses). The data obtained by this method are more difficult to interpret and dissociate from stress responses associated with apoptosis, however . Additional studies are now needed to dissect out the contributions of individual viral proteins by employing a variety of mutated hiv strains . Given that the envelope glycoprotein of hiv is cytotoxic, it will also be interesting to compare strains lacking expression of this product . Replication - defective hiv coated with envelopes specific for ccr5 or cxcr4 can be compared to replication - defective hiv enveloped with vesicular stomatitis virus (vsv) glycoprotein to address the provocative question of whether viral entry by means of different receptors may itself influence expression of various genes . Whereas viral products such as nef may have adapted to activate loci that favor viral progression, the acute phase of infection induces a cellular stress program associated with a generalized dampening of host - cell transcription and a shift towards the induction of the proapoptotic machinery . The effects on host gene responses of defined nef mutations, changes in nef expression, and substitution of nef alleles can be powerfully addressed in future microarray experiments . Moreover, it will be crucial to examine the effects of clinical hiv isolates in primary t cells, as well as macrophages, in which the virus also perturbs activation cascades . As enlarged panels of gene arrays become available, more comprehensive genome scanning will be possible . These studies might ultimately be extended to examine the effects of human genetic polymorphisms on hiv gene responses . Together, these approaches will prove crucial in developing new therapies that seek to suppress and eliminate hiv . A) expression of nef in t cells results in the induction of numerous viral permissivity factors as well as genes associated with t - cell activation . The induction of secreted factors, such as il-4 and tgf-, may generate secondary signals that favor viral replication, while the up - regulation of chemokines such as mip-1 and mip-1 may attract cells to the site of infection, perhaps aiding transmission of the virus . Although the precise mechanisms through which nef modulates signaling have not been elucidated, it has been implicated in exerting controls on a number of membrane - proximal pathways . (b) infection of t cells with intact hiv induces gene responses that are more complex and include the up - regulation and suppression of functionally diverse host genes . As infection proceeds, host - cell genes are increasingly suppressed, and there is a shift toward the induction of factors associated with a cytopathic response, including those regulating apoptosis and responses to genotoxic stress . The anti - viral gene ifn- is induced relatively early in infection, and may modulate a variety of response cascades . Hiv may elicit these transcriptional changes through a wide variety of mechanisms, either directly (though viral factors such as nef, env, and tat), or indirectly, by inducing cellular stress during the acute phase of infection.
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Hepatitis c virus infection has been reported to be the most common blood born pathogen all over the world . In egypt, infection with hcv has become the most important public health problem nowadays with the overall prevalence of anti - hcv in egypt in 1993 was 13.6% haemodialysis patients are at a high risk of infection by many blood borne pathogens . Some studies on haemodialysis patients in the united states reported an anti - hcv seroprevalence of 20% in adults and 18.5% among children . However a higher prevalence was reported from egypt 7080% . Hcv infections among patients on haemodialysis were attributed to several risk factors including blood transfusion . A number of studies had revealed a significant correlation between the patients who received blood transfusion and the risk of acquiring hcv infection . The nosocomial risk factors play an important role in hcv infections among patients on haemodialysis, these factors are related to dialysis machines and dialyzers which include dialyzers membranes and haemodialysis ultrafiltrate, reprocessing of dialyzers, and dialysis machines . Haemodialysis staff was found also to be an important factor in transmission of hcv infections among patients on haemodialysis . In order to control the diffusion of hcv in haemodialys units some authors recommended using a separate section to dialyze hcv+ patients . A total of 83 hcv seronegative end stage renal disease (esrd) patients; who were all the seronegative patients receiving regular haemodialysis during 36 months of the study (20082010) in four different haemodialysis units from three different governorates in egypt were included . Group 1 (n = 27, 18 males, 9 females, mean age 47.14 14.8 yeras, and mean duration on dialysis 70.1 28.9 months) included patients under regular haemodialysis in unit a and unit b, both units are following strict isolation program for hcv seropositive patients by using dedicated areas, machines, and dedicated health - care workers; 12 patients from (unit a), suez hospital (health insurance organization), suez governorate, 15 patients from (unit b),vacsera haemodialysis unit, giza governorate . Group 2 (n = 56, 29 males, 27 females, mean age 50.7 14.0 years, and mean duration on dialysis 60.4 22.01 months) included patients under regular haemodialysis in unit c and unit d, both units are not following strict isolation program for hcv seropositive patients, 21 patients from (unit c), new qalioub hospital, qalioubiah governorate, and 35 patients from (unit d), el - hamdiah el - shazliah haemodialysis unit, giza governorate . During the survey period, all subjects received treatment three times a week . The exclusion criteria included those who did not complete the period of study in their units; either due to death, leaving to other hemodialysis units, or after kidney transplantation . In addition, anti - hcv positive individuals in units a and b have been dialyzed on separate dedicated machines . A standardized form was used to collect data on age, sex, history of hepatitis / jaundice, length of time on hemodialysis treatment, number of previous transfusions, tattooing, intravenous drug use, and household contact with hepatitis / jaundice . Blood samples were collected from all patients and sera were separated and tested for hcv antibodies using elisa technique third generation . In this test, diluted patient specimens and controls were incubated in microwells coated with recombinant polypeptides of the structural and nonstructural regions of hcv . If hcv antibodies are present in a specimen or control, they bind to the antigen coated microwell . Excess sample was removed by a wash step and the enzyme tracer then added to the microwells and was allowed to incubate . Excess enzyme tracer then removed by a wash step, and a chromogen / substrate solution was added to the microwells and was allowed to incubate . If a sample contains hcv antibodies, the sample turns to a blue colour (650 nm). The blue colour turns to yellow (450 nm) after addition of the stop solution . Testing for levels of transaminases (alt, ast) was done for all the patients including the seroconverted patients . Prevalence, incidence, odds ratios, pvalues, and 95% confidence intervals (ci) were calculated to assess differences between studied groups and detect possible risk factors among studied populations . As shown in table 1 and figure 1 there was a significantly higher incidence of hcv seroconversion in patients receiving haemodialysis in units not following strict isolation program for hcv seropositive patients (24 out of 56 patients; 42.9%) than those receiving haemodialysis in units following strict isolation program for hcv seropositive patients (4 out of 27 patients; 14.8%) (p - value <0.05). Table 2 shows that there was no significant results between seroconverted and not seroconverted patients as regards their age, sex, occupation, marital status, and their education level . Analysis of risk factors in table 3 showed that isolation of hcv seropositive patients was associated with a significantly low relative risk for hcv antibody seroconversion (odds ratio 0.23, p value <0.05), while blood transfusion and duration on regular hemodialysis more than 60 months both were associated with a significant high relative risk for hcv antibody seroconversion (odds ratio 4.05 and p value <0.05) and (odds ratio 2.39 and p value <0.05), respectively . As shown in table 4 the multivariate regression analysis of predictors for seroconversion revealed that the most effective predictors in hcvab seroconversion were the duration on regular hemodialysis and the isolation of hcv seropositive . Hcv infection still remains a major problem among patients on maintenance hd . The immune suppression seen in this patient population, resulting in an absence of clinical and biochemical evidence of liver disease, the importance of prevention of hcv infection and control is due to its well - documented progression to hepatic cirrhosis, liver malignancies, and liver failure . The prevalence of hcv infection varies greatly among patients on hd from different geographic regions . In a review of data published in 1999, wreghitt described a range from 4% in the uk to 71% in kuwait for hcv prevalence among the hd population . In egypt, the prevalence of hcv antibodies in hemodialysis patients was found to be ranging from 52.3 to 82.3% . Several studies have reported nosocomial patient - to patient transmission of hcv infection among hd patients [13, 14]. As a result, in 2001 the cdc recommends that special precautions should be observed in dialysis units, including wearing and changing of gloves and waterproof gowns between patients, systematic decontamination of the equipment circuit and surfaces after each patient treatment, and no sharing of instruments (e.g., tourniquets, stethoscope, blood pressure cuff) or medications (e.g., multiuse vials of heparin) among patients . Although some studies found that nosocomial spread of hcv declined when hcv - infected patients were treated in dedicated hd units [15, 16], other investigators could control nosocomial spread of hcv by strict application of hygienic precautions without isolation of hcv - infected subjects or machine segregation [17, 18]. In our study we found that the incidence of hcv seroconversion is significantly lower in the group of patients within units implementing isolation programs of the hcv - infected patients than those who had no isolation of the hcv - infected patients . The duration on regular hemodialysis was found to be a significant predictor for hcv seroconversion in hd patients; a result that is consistent with that in a study in brazil demonstrated that patients on hd for more than three years had a 13.6-fold greater risk of hcv - positivity compared to subjects with less than one year hd treatment . Albeit the use of erythropoietin from the late 1980s reduced the need for blood transfusions among hd patients . Furthermore, the current risk of transfusion - associated hcv is approximately one in every two million people as reported by o'brien et al ., in 2007; mainly after the introduction of nucleic acid amplification testing for the screening of blood donors has markedly reduced the risk of hcv transmission through blood product transfusion, however blood transfusion was found in our study to have still a significant relative risk for hcv seroconversion in hd patients (odd's ratio 4.05). In hd units with a high prevalence of hcv infection, strict isolation of hcv+ patients in combination with implementation of universal prevention measures are recommended to avoid burden of virus transmission and morbidity.
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Smartphones provide various conveniences, such as sending and receiving e - mail, accessing the internet, and engaging in entertainment1 . The number of smartphone users has increased dramatically in recent years because of these conveniences . However, the use of smartphones is reportedly associated with physical symptoms in some users2 . Some researchers have suggested that frequent smartphone use can lead to the use of a non - neutral neck posture or the development of musculoskeletal disorders3, 4 . Berolo, wells, and amick4 surveyed a canadian university population and reported that the duration and frequency of use of mobile handheld devices were related to the prevalence of neck pain . A cause of neck disorders among smartphone users may be the ability to reposition the display of the mobile device, such as on a desk or below the shoulder5 . Many people may use smartphones with the head shifted forward and the smartphone placed near the waist or lap while in a sitting position6 . This flexed neck posture can increase the moment of the cervical spine and induce muscle strain in adjacent portions of the cervical spine7 . Although smartphone use is associated with causal factors of neck pain or musculoskeletal disease, fewer experimental studies have been performed on the effects of smartphone use than on the effects of visual display terminal (vdt) work . Maintenance of a non - neutral neck posture, such as a flexed posture, is a well - known cause of neck pain8 . In a previous study, small forward shifts of the head in the sagittal plane increased the load on the supporting structures and activated the neck muscles7 . Harrison et al.9 found that the compressive load on the cervical discs in the neck - forward flexed position was 10 kg greater than that in the upright neck position . These biomechanical variations or the presence of neck pain can induce proprioceptive deficits in the cervical region . Szeto, straker, and osullivan10 reported that more symptomatic than asymptomatic individuals used a flexed neck position while using a vdt . Therefore, we postulated that smartphone users with neck pain might more frequently utilize a non - neutral neck posture than asymptomatic users . Many previous studies have investigated the alterations in cervical movement patterns during computer use11, 12 . However, few experimental studies have addressed how smartphone use changes cervical movement patterns . Additionally, no study has reported the effect of mild neck pain (mnp) on smartphone use . Thus, this study compared alterations in the cervical spine posture of young adults with and without mnp during smartphone use . We hypothesized that the cervical spine of young adults with mnp would be more flexed than that of asymptomatic young adults during smartphone use . For this study, 27 young adults (12 male, 15 female) who had used a smartphone for at least 1 year were recruited from the university of gimhae, south korea, by print media advertisement . All subjects who were included in the study had experienced cervical symptoms during smartphone use within the last year . Individuals with a history of neck trauma or surgery or with a medical diagnosis of fibromyalgia, cervical radiculopathy, a systemic illness, or a connective tissue disorder were excluded from this study . All subjects were grouped into either the mnp group or the control group based on their neck disability index (ndi) scores (> 8 and 8). The ndi involves a 10-item questionnaire regarding the effects of neck pain and symptoms during a range of functional activities . Each subject was instructed to select one of six options concerning the severity of each item (05). The ndi score was calculated as the total score multiplied by two (score range = 0100). The intraclass correlation coefficient of the ndi scoring method in the korean language version is reported to be excellent, at 0.906 . The inje university faculty of health science human ethics committee granted approval for this study, and all subjects provided their written informed consent prior to participation . Kinematic data of the upper cervical (uc) and lower cervical (lc) angles in the sagittal plane during smartphone use were collected using an ultrasound - based motion analysis system (cms20; zebris medical gmbh, isny, germany). For measurement of the uc and lc flexion angles, four active single markers were attached, one each over the zygomatic bone, tragus, first thoracic spinous process, and sternum13 . Each subject was seated on an adjustable - height chair without a backrest, with the knee and hip joints at 90 angles and the feet on the floor . The subject initially adopted a neutral cervical posture and held the smartphone in both hands while sitting . The neutral position was defined as a cervical posture without rotation, lateral bending, or excessive cervical lordosis in the sitting posture, but with slight lumbar lordosis and a relaxed thorax . Straker, jones, and miller7 reported that the discomfort score was higher among workers performing vdt work in a flexed neck posture . In the present study, all subjects were instructed to maintain their preferred shoulder and arm postures with the exception of placing the arm and hand on the thigh during the experiment . The subjects freely used the messenger application or the internet on a galaxy sii phone (shw - m250s; samsung electronics co. ltd ., each subject performed three test trials with a 3-min rest period between trials . To analyze the cervical angle, the raw kinematic data was collected at a 10-hz sampling rate and converted to ascii files . Then, the flexion angles were analyzed using microsoft excel 2010 (microsoft corp ., a negative cervical angle was defined as cervical flexion . In each of the three trials, the flexion angle data points of 100, 200, and 300 s of smartphone use the mean of the three trial values was analyzed to determine the uc and lc flexion angles during smartphone use . The cervical flexion angles were analyzed using 2 (groups) 3 (time) analysis of variance with repeated measures of both factors . A greenhouse - geisser correction was used to adjust the degrees of freedom whenever an inequality of sphericity was rejected by mauchly s test . Differences in the ndi score and anthropometric characteristics between the two groups were analyzed using the independent t - test . Statistical analyses were performed using spss version 20.0 for windows (ibm corp ., armonk twenty - seven subjects were recruited from among young adults who used a smartphone in korea . The ndi score of the mnp group was significantly higher than that of the control group (table 1table 1.summary of the anthropometric characteristics and neck disability indices of the study participantscharacteristiccontrol group(n = 13)mnp group(n = 14)sex (n, male)66age (years), mean sd20.6 1.620.6 1.5height (cm), mean sd167.3 6.9168.0 8.0weight (kg), mean sd60.7 10.761.0 12.4neck disability index (%), mean sd3.3 2.616.9 7.1*mnp, mild neck pain; sd, standard deviation . No significant differences in age, height, or weight were observed between the mnp and control groups . Table 1 shows the mean uc and lc flexion angles of subjects with and without mnp at each measured time point during smartphone use . The cervical spine flexion angles were significantly higher in the mnp group than in the control group (p <0.05). The uc of subjects with mnp exhibited greater flexion than that of subjects without mnd (p <0.05). The variations in the degrees of lc flexion in the mnd group were significantly greater than those in the control group (p <0.05). There were no significant differences in the uc and lc segments over time between the two groups (table 2table 2.cervical spine angle of subjects with and without mnp according to timesegmentcontrol group (n = 13)mnp group (n = 14)100 s200 s300 s100 s200 s300 supper cervical spine2.68 1.462.82 2.213.20 2.474.95 3.015.85 3.025.87 3.63lower cervical spine3.99 2.575.14 2.555.84 2.938.19 5.059.20 3.869.95 4.22mnp, mild neck pain, mean sd . In the present study, the mnp group exhibited greater uc and lc flexion angles than the control group during smartphone use . We found differences in the position of the cervical spine during smartphone use between individuals with and without mnp . Young adults with mnp exhibited greater flexion of their cervical spines when using a smartphone than users without mnp . These findings indicate that young adults with mnp experience difficulty in maintaining a neutral neck posture during smartphone use . Possible explanations for the increased neck flexion angles in subjects with mnp include neck pain altering the motor control of the neck muscles10 . Based on these findings, it appears that induction factors of neck pain occurred in control subjects during consistent use of the smartphone . Thus, we believe that cognition of posture and maintenance of a neutral neck posture are important for subjects both with and without neck pain . Our findings demonstrated differences in the cervical flexion angles with time during smartphone use . The cervical flexion angles were influenced in the uc and lc regions by the passage of time during smartphone use . Possible explanations for the effect of time on the cervical angle include the subjects maintaining a static sitting posture during smartphone use . They reported that cervical angles showed gradual increase of flexion with time in asymptomatic subjects during vdt work . However, some studies have suggested that subtle but significant differences in these angles might correct postural malalignment associated with musculoskeletal disorders15, 16 . Considering the findings of these previous studies, we suggest that the significant changes in the cervical flexion angles observed in the present study might reduce mechanical stress on the cervical spine, even though the differences in the angles were subtle . A strength of this study was that the characteristics of the cervical posture of subjects with mnp during smartphone use were considered, and that this study demonstrated that subjects with mnp cannot maintain a neutral cervical posture as well as control subjects can . First, we did not measure the flexion angle of the lumbar spine during smartphone use . We speculate that lumbar flexion might induce cervical flexion because the lumbar spine is connected in a chain - like manner to the cervical spine17 . However, this study focused on the posture of the cervical spine in subjects with mnp during smartphone use . Changes in lumbar flexion combined with cervical flexion when using a smartphone in a seated position should be assessed in future studies . Electromyography should be performed to examine the muscle activity of the cervical erector spinae in relation to the duration of smartphone use . Further research is needed to identify alterations in cervical flexion angles depending on the grade of neck pain during smartphone use . In conclusion, our findings indicate that individuals with mnp adopt a posture of greater neck flexion than individuals without mnp when using a smartphone . Our findings suggest that young adults with mnp must be aware of their posture and modify their non - neutral cervical alignment when using a smartphone . To reduce the risk of developing severe neck pain, clinicians should instruct smartphone users to maintain a correct neck posture.
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In total, 126 participants were recruited for the study by advertising in the area of copenhagen and at www.forsgsperson.dk . Both men and women with a body mass index (bmi) of 1840 all participants were apparently healthy adults with no known chronic illnesses . After having received verbal and written information, all participants gave written consent about the study in accordance with the declaration of helsinki . The study was carried out at the department of nutrition, exercise and sport, faculty of science, university of copenhagen, denmark, and was approved by the local ethics committee (h - b-2009 - 071). At entry into the study, anthropometric measurements were performed in the morning after an overnight fast (> 10 h) and abstention from alcohol and physical exercise for 24 h. body weight was measured on an electronic scale while the participants were wearing light clothing and no shoes (tanita bwb-600, japan). At the first visit, height was measured to the nearest 0.5 cm by using a wall - mounted stadiometer (seca, hultafors, sweden) without shoes . The quantitative ffq was designed to measure the habitual intake of dietary fiber during the previous month . It included 60 questions in total concerning the intake of foods from eight main food groups which contain dietary fibers, that is, foods containing cereals, fruits, vegetables, legumes, lentils, and nuts . A list of food items included in the questionnaire is given in table 1 together with the estimated dietary fiber content of each food group as well as sub - groups . The dietary fiber content of the foods was based on the national food composition database (foodcomp.dk, national food institute, danish technical university) for fruits and vegetables, legumes, lentils, dried fruits, and nuts . For breads and other cereals, a comprehensive list of brand - specific foods was made based on availability in supermarkets . All foods were then sub - categorized based on dietary fiber content into the groups listed in table 1 . The questionnaire's options for frequency of consumption was given as times per day (either 1, 2, 3, or more than 3), times per week (either 12, 34, or 56), less than once per week, or never . When consumption was more than three per day, the subject was asked to write down the frequency . When consumption was given as an interval, for example, 12 times per week, a mean value of 1.5 was used for calculating the intake . When consumption was either less than once per week or never, contribution from this food item was disregarded . After each question of frequency, the responder was asked about the average portion size of each time of consumption . Portion size was presented in household units, for example, slices, pieces, spoonful, or the portion size was estimated using pictures of four different portion sizes . Mean daily intake of total fiber as well as dietary fibers from cereals, fruits and vegetables, and other sources was calculated . The ffq, which is in danish, may be obtained from the authors for use in other studies . Specific food items and their dietary fiber content used in the calculation of daily dietary fiber intake . Frequency of consumption was given as times per day, 12, 34, 56 times per week, daily, less than once per week or never, depending on the food item . Six months after completing the first ffq (ffq1), a small proportion of the participants were asked to fill in a second ffq (ffq2). All recorded foods and beverages were entered into the dankost 3000 dietary assessment software which is based on the same food composition database as used in the ffq (foodcomp.dk, national food institute, danish technical university) (dankost 3000, version 2.5, danish catering center, herlev, denmark), and mean daily total intake of energy, fat, carbohydrates, protein, alcohol, and total dietary fiber was calculated for each 7-day registration period . All statistical analyses and calculations were performed using the statistical analysis system software package, version 9.3 (sas institute inc ., data from the ffq and dr were compared using several different methods . Dietary fiber intake as assessed by dr and ffq was compared using an ancova model, where sex and method were modeled as fixed variables, subject as a random variable, and age and bmi were included as covariates . Pearson correlation coefficients were computed to measure the strength of the relationship between the two measurement methods . Altman plot was performed with difference between ffq and dr plotted against mean dietary intake of the two methods . The plot was used to assess homogeneity of the individual data and to evaluate if under-/overreporting was relative to average dietary fiber intake . Also, participants were classified into tertiles of dietary fiber intake according to both methods (ffq and dr) to assess the ability of the method to correctly group individual participants . Difference between the first and second ffq estimates (ffq1 and ffq2) of dietary fiber intake was investigated using an ancova model, where ffq (1 or 2) were modeled as a fixed variable and subject as a random variable . Additionally, pearson's correlation coefficient was measured as a measure of the relationship . In total, 126 participants were recruited for the study by advertising in the area of copenhagen and at www.forsgsperson.dk . Both men and women with a body mass index (bmi) of 1840 all participants were apparently healthy adults with no known chronic illnesses . After having received verbal and written information, all participants gave written consent about the study in accordance with the declaration of helsinki . The study was carried out at the department of nutrition, exercise and sport, faculty of science, university of copenhagen, denmark, and was approved by the local ethics committee (h - b-2009 - 071). At entry into the study, anthropometric measurements were performed in the morning after an overnight fast (> 10 h) and abstention from alcohol and physical exercise for 24 h. body weight was measured on an electronic scale while the participants were wearing light clothing and no shoes (tanita bwb-600, japan). At the first visit, height was measured to the nearest 0.5 cm by using a wall - mounted stadiometer (seca, hultafors, sweden) without shoes . The quantitative ffq was designed to measure the habitual intake of dietary fiber during the previous month . It included 60 questions in total concerning the intake of foods from eight main food groups which contain dietary fibers, that is, foods containing cereals, fruits, vegetables, legumes, lentils, and nuts . A list of food items included in the questionnaire is given in table 1 together with the estimated dietary fiber content of each food group as well as sub - groups . The dietary fiber content of the foods was based on the national food composition database (foodcomp.dk, national food institute, danish technical university) for fruits and vegetables, legumes, lentils, dried fruits, and nuts . For breads and other cereals, a comprehensive list of brand - specific foods was made based on availability in supermarkets . All foods were then sub - categorized based on dietary fiber content into the groups listed in table 1 . The questionnaire's options for frequency of consumption was given as times per day (either 1, 2, 3, or more than 3), times per week (either 12, 34, or 56), less than once per week, or never . When consumption was more than three per day, the subject was asked to write down the frequency . When consumption was given as an interval, for example, 12 times per week, a mean value of 1.5 was used for calculating the intake . When consumption was either less than once per week or never, contribution from this food item was disregarded . After each question of frequency, portion size was presented in household units, for example, slices, pieces, spoonful, or the portion size was estimated using pictures of four different portion sizes . Mean daily intake of total fiber as well as dietary fibers from cereals, fruits and vegetables, and other sources was calculated . The ffq, which is in danish, may be obtained from the authors for use in other studies . Specific food items and their dietary fiber content used in the calculation of daily dietary fiber intake . Frequency of consumption was given as times per day, 12, 34, 56 times per week, daily, less than once per week or never, depending on the food item . Six months after completing the first ffq (ffq1), a small proportion of the participants were asked to fill in a second ffq (ffq2). All recorded foods and beverages were entered into the dankost 3000 dietary assessment software which is based on the same food composition database as used in the ffq (foodcomp.dk, national food institute, danish technical university) (dankost 3000, version 2.5, danish catering center, herlev, denmark), and mean daily total intake of energy, fat, carbohydrates, protein, alcohol, and total dietary fiber was calculated for each 7-day registration period . All statistical analyses and calculations were performed using the statistical analysis system software package, version 9.3 (sas institute inc ., data from the ffq and dr were compared using several different methods . Dietary fiber intake as assessed by dr and ffq was compared using an ancova model, where sex and method were modeled as fixed variables, subject as a random variable, and age and bmi were included as covariates . Pearson correlation coefficients were computed to measure the strength of the relationship between the two measurement methods . Altman plot was performed with difference between ffq and dr plotted against mean dietary intake of the two methods . The plot was used to assess homogeneity of the individual data and to evaluate if under-/overreporting was relative to average dietary fiber intake . Also, participants were classified into tertiles of dietary fiber intake according to both methods (ffq and dr) to assess the ability of the method to correctly group individual participants . Difference between the first and second ffq estimates (ffq1 and ffq2) of dietary fiber intake was investigated using an ancova model, where ffq (1 or 2) were modeled as a fixed variable and subject as a random variable . Additionally, pearson's correlation coefficient was measured as a measure of the relationship . Normal weight, overweight, and obese individuals were included with a mean bmi of 25.75.4 kg / m . Also, all age groups were represented ranging from 18 to 60 years with a mean age of 32.311.0 years . The mean intakes of dietary fiber estimated by the dr and ffq1 were 28.19.4 and 24.89.9 g / day, respectively (table 2), thus the estimated dietary fiber intake was ~12% lower when using the ffq compared to the dr (p<0.001) after adjusting for sex, age, and bmi . Excluding sex, age, and bmi from the model only bmi was associated with dietary fiber intake so that increased bmi was associated with decreased dietary fiber intake (p=0.01). As expected, cereals were the main contributor of dietary fiber followed by fruit and vegetables . The estimated dietary fiber intake ranged from 7 to 50 g / day, thus providing sufficient range in fiber intake to assess the relative validity . Characteristics of participants (n=125) bmi, body mass index; dr, dietary record; ffq, food frequency questionnaire . Pearson's correlation coefficient between the estimated dietary fiber intake between the two methods was 0.63, p<0.001 (y=0.5975 x+13.243), where dietary fiber intake estimated by ffq (y) is a function of dietary fiber intake estimated by dr (x), (see fig . A relationship between the two methods is established and the strength hereof is considered moderate . Scatterplot of estimated dietary fiber intake (g / day) for ffq versus dr (n=125). Altman plot showed that the mean difference between the two methods (ffq from dr) was 3.28.3 g / day, and that for any new subject a difference inside the range of 13.419.8 g / day can be expected with 95% certainty (fig . 2). A regression line of the plot resulted in a negative slope (=0.05), but as the estimate was not significant (p=0.56), it can be assumed that the underestimation related to the ffq is independent of the average dietary fiber intake . Altman plot showing the relationship between the difference (diff) in estimated dietary fiber intake of the two methods and the mean estimated dietary fiber intake (mean) (n=125) with 95% confidence limits of the estimate as well as 95% prediction limits (2 sd). The individuals were classified into tertiles of dietary fiber intake according to either dr or ffq method . A total of 62% (78 subjects) was classified into the same tertiles of intake according to both methods (data not shown). However, only 5% (6 subjects) was misclassified into the opposite extreme of the tertiles . In total, 12 participants (7 women and 5 men) completed a second ffq approximately 6 months after completing the first one . The dietary fiber intake in this sub - group ranged from 8 to 48 g / day . The total dietary fiber intake from ffq1 and ffq2 was highly correlated (r=0.95, 95% ci 0.83; 0.99, p<0.001) and was very similar for the first and second ffq (22.24.0 and 23.34.1 g / day, respectively) (p=0.42). Also, the contribution of dietary fibers from different sources did not vary between the two ffqs (data not shown). The difference from ffq1 in total dietary fiber intake was less than 25% for 9 out of 12 of the participants . The mean intakes of dietary fiber estimated by the dr and ffq1 were 28.19.4 and 24.89.9 g / day, respectively (table 2), thus the estimated dietary fiber intake was ~12% lower when using the ffq compared to the dr (p<0.001) after adjusting for sex, age, and bmi . Excluding sex, age, and bmi from the model only bmi was associated with dietary fiber intake so that increased bmi was associated with decreased dietary fiber intake (p=0.01). As expected, cereals were the main contributor of dietary fiber followed by fruit and vegetables . The estimated dietary fiber intake ranged from 7 to 50 g / day, thus providing sufficient range in fiber intake to assess the relative validity . Characteristics of participants (n=125) bmi, body mass index; dr, dietary record; ffq, food frequency questionnaire . Pearson's correlation coefficient between the estimated dietary fiber intake between the two methods was 0.63, p<0.001 (y=0.5975 x+13.243), where dietary fiber intake estimated by ffq (y) is a function of dietary fiber intake estimated by dr (x), (see fig . A relationship between the two methods is established and the strength hereof is considered moderate . Scatterplot of estimated dietary fiber intake (g / day) for ffq versus dr (n=125). Altman plot showed that the mean difference between the two methods (ffq from dr) was 3.28.3 g / day, and that for any new subject a difference inside the range of 13.419.8 g / day can be expected with 95% certainty (fig . 2). A regression line of the plot resulted in a negative slope (=0.05), but as the estimate was not significant (p=0.56), it can be assumed that the underestimation related to the ffq is independent of the average dietary fiber intake . Altman plot showing the relationship between the difference (diff) in estimated dietary fiber intake of the two methods and the mean estimated dietary fiber intake (mean) (n=125) with 95% confidence limits of the estimate as well as 95% prediction limits (2 sd). The individuals were classified into tertiles of dietary fiber intake according to either dr or ffq method . A total of 62% (78 subjects) was classified into the same tertiles of intake according to both methods (data not shown). However, only 5% (6 subjects) was misclassified into the opposite extreme of the tertiles . In total, 12 participants (7 women and 5 men) completed a second ffq approximately 6 months after completing the first one . The dietary fiber intake in this sub - group ranged from 8 to 48 g / day . The total dietary fiber intake from ffq1 and ffq2 was highly correlated (r=0.95, 95% ci 0.83; 0.99, p<0.001) and was very similar for the first and second ffq (22.24.0 and 23.34.1 g / day, respectively) (p=0.42). Also, the contribution of dietary fibers from different sources did not vary between the two ffqs (data not shown). The difference from ffq1 in total dietary fiber intake was less than 25% for 9 out of 12 of the participants . Based on pearson's correlation coefficient and the bland altman plot, a relationship is established between dietary fiber intake measured by ffq and dr, and the relative validity of the ffq is considered moderate . In the present study, pearson's correlation coefficient was 0.63 and classification into tertiles showed 62% of the subjects to be in the same category in both methods . Previous studies evaluating ffqs specific for assessing dietary fiber intake (11, 12) found lower percentages of the participants to be classified into the same category when comparing ffq and dr . However, this might be explained by their division into more groups as they classified subjects into quartiles and quintiles . Only sasaki et al . (11) reported the correlation coefficient (spearmen's) which compared to the present study was quite low; 0.50 for men and 0.44 for women (11). A validation methodology, similar to the one used in the present study, was applied in a validation study by ross et al . (13), where a whole - grain ffq was evaluated by comparison to a 3-day weighed food record . They reported a pearson's correlation coefficient of 0.75, and when classifying into tertiles, 72% of the subjects were in the same category in both methods (13). Compared to few other studies that had validated ffq specific for assessing dietary fibers, the relative validity of the present ffq developed for assessing dietary fiber intake in danish adults appears comparable . When further comparing our results with a validation of a previous ffq that was also developed for danish adults but was assessing the general diet (14), we believe that the present ffq provides more accurate information about dietary fiber intake . In the study by tjnneland et al . (14), the ffq was validated against two times 7 days of weighed diet records . For dietary fiber intake, the study reported a pearson's correlation coefficient of 0.39 for men and 0.53 for women when adjusted for total energy intake (14), which are estimates lower than the correlation coefficient of 0.63 obtained in this study . Intake of dietary fiber - rich foods is usually associated with a healthy life style . Therefore, an ffq build to assess dietary fiber intake is prone to overestimation, as participants are more likely to over - report consumption of healthy food . However, in the current study, the estimated dietary fiber intake was lower using the ffq compared to the dr and the underreporting for ffq was found to be independent of the average dietary fiber intake . Two recent validation studies of self - administered quantitative ffq showed significant overestimation of dietary fiber intake; 32% when compared to 7 days dr (15) and 57% for women and however, these studies evaluated several micro- and macronutrients along with dietary fibers . Among other ffqs developed specifically for determining dietary fiber intake in adults, one study found no difference in mean intake when compared to dr (11), while another found that crude and energy - adjusted dietary fiber intake was significantly underestimated in the ffq compared to a 4-day dr (12). Together with previous studies, the present study indicates that overestimation of dietary fiber intake in general diet ffqs can be overcome by using ffqs developed specifically to assess dietary fiber intake . A possible reason for the observed underestimation of dietary fiber intake in the ffq might be that the questionnaire does not cover the full diversity of the dietary fiber containing products consumed by the participants . This is, however, not considered to be the case of the present ffq, as it includes eight different food groups covering the main sources, recognized by surveys of danish adults normally contributing to the dietary fiber intake (9). But the grouping of foods and the use of standard portion sizes in the ffq might cause a lower average estimate of dietary fiber intake to be used for the calculations . Also, it is considered that especially vegetables used in mixed dishes are neglected in the reporting, when intake is measured by recall; thus contributing to the underestimation . Additionally, the type of reference method used when evaluating the relative validity might explain parts of the differences in measurements . The ffq was designed to reflect the food pattern of the last month, whereas the 7-day food recording resembles only this particular week, thus a deviation from the habitual pattern during these 7 days may influence differences between the two methods on an individual level . However, as the dr method covers both week days and the weekend, the method takes into consideration one of the major sources of between - days variations, and the influence on the estimates on a group level is considered to be minimal . This is based on a good correlation between total dietary fiber intake measured by the two ffqs, a narrow 95% ci of the correlation coefficient and that no difference between ffq1 and ffq2 was present . It can thereby be presumed that the within - subject variation is low compared to the between - subject variation captured by this ffq . The reproducibility has not been evaluated for previous dietary fiber ffqs (11, 12), but an ffq designed for measuring whole - grain intake evaluated the reproducibility to be good based on a correlation coefficient of r=0.75 (13). Generally, correlations noted in literature between subsequent measurements of ffq in adults range from r=0.50.8 (17), and compared to this, the reproducibility correlation of the present study (r=0.95) must be considered very good . The higher correlation estimate in the present study might be explained by the nutrient of interest in the ffq . Since dietary fibers are found in many foods consumed frequently, the within - subject variability will be lower than for nutrients found in only few foods that are consumed occasionally . Additionally, the time interval between ffq1 and ffq2 in this study was about 6 months . As several months separate ffq1 and ffq2, there might be a minor seasonal variation in the responses but on the other hand this time frame prevented introduction of sequence or training effects . It can be argued that fruit and vegetable intake varies with season; however, the enrolment into the study was not limited to a short period of time, thus reflecting more than one season . Also, whole - grain rye bread, which was the single food contributing most to total fiber intake (data not shown), is consumed throughout the year although with different spreads; thus, effect of different seasons is limited . A major strength of the study was the large group of participants, who represented the general population with a wide range in age and bmi as well as varied dietary fiber intake . This increases the external validity of the results, although a wide range in dietary fiber intake likely has improved the correlation . Furthermore, the present ffq also provides a tool with possibility to associate specific fiber sources with outcomes . A limitation of the study is however the small number of participants with data available for assessment of reproducibility of the ffq (n=12); thus, our ability to assess this accurately may be limited . The ffq, evaluated in the present study, is applicable for the danish adult population but might also be relevant for adult populations in other countries with similar food patterns, especially in populations where rye is a major source of dietary fiber intake . When evaluating relative validity, the use of dr as reference method does not optimally determine the actual habitual intake, as previous studies have shown both undereating and underreporting when using dietary records for measuring energy intake (18, 19). Both ffq and dr are self - reporting methods and are affected by some of the same errors caused by observation and reporting effects . Therefore, a good agreement between the two methods does not certainly reflect good validity, but may to some degree also indicate similar errors in the two methods . However, as the dr method excludes recall bias and do not rely on conceptualization of portion size, which are major errors present in ffqs, it is a suitable reference method when evaluating the relative validity of the ffq (7). However, improvement in the research of biomarkers could in the future reveal validated quantitative biomarkers of dietary fiber intake, whereby the dependence of self - reported reference methods for validation could be minimized . Based on pearson's correlation coefficient and the bland altman plot, a relationship is established between dietary fiber intake measured by ffq and dr, and the relative validity of the ffq is considered moderate . In the present study, pearson's correlation coefficient was 0.63 and classification into tertiles showed 62% of the subjects to be in the same category in both methods . Previous studies evaluating ffqs specific for assessing dietary fiber intake (11, 12) found lower percentages of the participants to be classified into the same category when comparing ffq and dr . However, this might be explained by their division into more groups as they classified subjects into quartiles and quintiles . Only sasaki et al . (11) reported the correlation coefficient (spearmen's) which compared to the present study was quite low; 0.50 for men and 0.44 for women (11). A validation methodology, similar to the one used in the present study, was applied in a validation study by ross et al . (13), where a whole - grain ffq was evaluated by comparison to a 3-day weighed food record . They reported a pearson's correlation coefficient of 0.75, and when classifying into tertiles, 72% of the subjects were in the same category in both methods (13). Compared to few other studies that had validated ffq specific for assessing dietary fibers, the relative validity of the present ffq developed for assessing dietary fiber intake in danish adults appears comparable . When further comparing our results with a validation of a previous ffq that was also developed for danish adults but was assessing the general diet (14), we believe that the present ffq provides more accurate information about dietary fiber intake . In the study by tjnneland et al . (14), the ffq was validated against two times 7 days of weighed diet records . For dietary fiber intake, the study reported a pearson's correlation coefficient of 0.39 for men and 0.53 for women when adjusted for total energy intake (14), which are estimates lower than the correlation coefficient of 0.63 obtained in this study . Intake of dietary fiber - rich foods is usually associated with a healthy life style . Therefore, an ffq build to assess dietary fiber intake is prone to overestimation, as participants are more likely to over - report consumption of healthy food . However, in the current study, the estimated dietary fiber intake was lower using the ffq compared to the dr and the underreporting for ffq was found to be independent of the average dietary fiber intake . Two recent validation studies of self - administered quantitative ffq showed significant overestimation of dietary fiber intake; 32% when compared to 7 days dr (15) and 57% for women and however, these studies evaluated several micro- and macronutrients along with dietary fibers . Among other ffqs developed specifically for determining dietary fiber intake in adults, one study found no difference in mean intake when compared to dr (11), while another found that crude and energy - adjusted dietary fiber intake was significantly underestimated in the ffq compared to a 4-day dr (12). Together with previous studies, the present study indicates that overestimation of dietary fiber intake in general diet ffqs can be overcome by using ffqs developed specifically to assess dietary fiber intake . A possible reason for the observed underestimation of dietary fiber intake in the ffq might be that the questionnaire does not cover the full diversity of the dietary fiber containing products consumed by the participants . This is, however, not considered to be the case of the present ffq, as it includes eight different food groups covering the main sources, recognized by surveys of danish adults normally contributing to the dietary fiber intake (9). But the grouping of foods and the use of standard portion sizes in the ffq might cause a lower average estimate of dietary fiber intake to be used for the calculations . Also, it is considered that especially vegetables used in mixed dishes are neglected in the reporting, when intake is measured by recall; thus contributing to the underestimation . Additionally, the type of reference method used when evaluating the relative validity might explain parts of the differences in measurements . The ffq was designed to reflect the food pattern of the last month, whereas the 7-day food recording resembles only this particular week, thus a deviation from the habitual pattern during these 7 days may influence differences between the two methods on an individual level . However, as the dr method covers both week days and the weekend, the method takes into consideration one of the major sources of between - days variations, and the influence on the estimates on a group level is considered to be minimal . This is based on a good correlation between total dietary fiber intake measured by the two ffqs, a narrow 95% ci of the correlation coefficient and that no difference between ffq1 and ffq2 was present . It can thereby be presumed that the within - subject variation is low compared to the between - subject variation captured by this ffq . The reproducibility has not been evaluated for previous dietary fiber ffqs (11, 12), but an ffq designed for measuring whole - grain intake evaluated the reproducibility to be good based on a correlation coefficient of r=0.75 (13). Generally, correlations noted in literature between subsequent measurements of ffq in adults range from r=0.50.8 (17), and compared to this, the reproducibility correlation of the present study (r=0.95) must be considered very good . The higher correlation estimate in the present study dietary fibers are found in many foods consumed frequently, the within - subject variability will be lower than for nutrients found in only few foods that are consumed occasionally . Additionally, the time interval between ffq1 and ffq2 in this study was about 6 months . As several months separate ffq1 and ffq2, there might be a minor seasonal variation in the responses but on the other hand this time frame prevented introduction of sequence or training effects . It can be argued that fruit and vegetable intake varies with season; however, the enrolment into the study was not limited to a short period of time, thus reflecting more than one season . Also, whole - grain rye bread, which was the single food contributing most to total fiber intake (data not shown), is consumed throughout the year although with different spreads; thus, effect of different seasons is limited . A major strength of the study was the large group of participants, who represented the general population with a wide range in age and bmi as well as varied dietary fiber intake . This increases the external validity of the results, although a wide range in dietary fiber intake likely has improved the correlation . Furthermore, the present ffq also provides a tool with possibility to associate specific fiber sources with outcomes . A limitation of the study is however the small number of participants with data available for assessment of reproducibility of the ffq (n=12); thus, our ability to assess this accurately may be limited . The ffq, evaluated in the present study, is applicable for the danish adult population but might also be relevant for adult populations in other countries with similar food patterns, especially in populations where rye is a major source of dietary fiber intake . When evaluating relative validity, the use of dr as reference method does not optimally determine the actual habitual intake, as previous studies have shown both undereating and underreporting when using dietary records for measuring energy intake (18, 19). Both ffq and dr are self - reporting methods and are affected by some of the same errors caused by observation and reporting effects . Therefore, a good agreement between the two methods does not certainly reflect good validity, but may to some degree also indicate similar errors in the two methods . However, as the dr method excludes recall bias and do not rely on conceptualization of portion size, which are major errors present in ffqs, it is a suitable reference method when evaluating the relative validity of the ffq (7). However, improvement in the research of biomarkers could in the future reveal validated quantitative biomarkers of dietary fiber intake, whereby the dependence of self - reported reference methods for validation could be minimized . To sum up, we have shown that the used ffq was able to rank danish adults adequately according to their intake of dietary fiber, however with moderate underestimation of dietary fiber intake when evaluating the relative validity . Thus, we believe that the developed ffq is a valuable method to be used in epidemiology as well as a screening tool when performing human intervention studies on dietary fiber . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
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The thoracic angle is the primary curve of the vertebral column which is comprised of 12 vertebrae . The thoracic kyphosis angle increases with age and the increase is greater in females than in males [2, 3]. Hyperkyphosis or increase in thoracic curve greater than normal range is one of prevalent spinal disorders . Biomechanical data suggest that an increase in the thoracic kyphosis may be associated with significantly higher spinal loads and trunk muscle force in upright stance and this might accelerate degenerative process which in turn leads to further spinal dysfunction and pain . An increase in thoracic kyphosis has also been associated with diminished physical function, impairment of respiratory function [6, 7], an increase in cervical pain [8, 9], headaches, and shoulder discomforts such as subacromial pain syndrome [11, 12]. In spite of frequent studies on normal range of thoracic angle for example in willner and johnson study the least pronounced kyphosis was seen at the age of 1012 years and mean kyphosis angle in 8 and 16 years was 35 and 44, respectively . Propst proctor and bleck in a study on children aged from 2 to 20 years old reported an angle of 27 (range of 2133) as normal . The magnitude of thoracic kyphosis in the study of voutsinas and macewen averaged 38.l in white boys and 34.3 in black boys and 38.5 in white girls and 36.9 in black girls . In adults, kyphosis curve values varied according to different investigators, with the range of ~3537 [16, 17], but such studies were conducted in heterogeneous populations . Using two standard deviations reported that normal kyphosis is ranged from 20 to 50. such wide variations may be partly related to factors that affect spinal curve measurement such as difference in the instrument used and sample characteristic (age, sex,), and limitations researchers have been faced with and partly related to factors that have been claimed to have influence on the curve such as age, sex, geographical region, and race . Although there are quite different studies around the world on ranges of kyphosis, it seems that each region needs its specific values . The aim of this study was to define normal kyphosis range in 1318-year - old male students living in kermanshah, a city in the west of iran . The ethical board of kermanshah university of medical sciences approved this study . For this purpose, 582 subjects were chosen across male students aged 13 to 18 years living in kermanshah . Using random cluster sampling 6 guidance schools, 6 high schools were chosen . In each school, for every age group (1318), at least 16 students were defined randomly . Exclusion criteria were having neuromuscular disorders, scoliosis, history of surgery, or any diseases on spinal column . Using adams test, participants were asked about history and sign of spinal surgery . For measuring the kyphosis, students were asked to stand comfortable and look forward in a way that body weight would be divided equally on both feet (arms beside the body). In this position, using surface anatomy and by palpation, spinous processes of 2nd and 12th thoracic vertebrae (t2 and t12) were determined and marked by a marker (figure 1). A flexible ruler was placed on thoracic curve covering the two marked points and pushed gently on back so there was no gap between the skin and the ruler (figure 2). Keeping this position, a second coworker marked the adjacent points to t2 and t12 spinous processes on the ruler . Without making any change in the curve induced in the flexible ruler, it was put on a piece of paper . Using a pen, the curve was drawn and those two points were reflected on the paper . After removing the ruler a straight line was drawn connecting t2 and t12 points . Magnitude of the angle of thoracic curve was measured: (1)=4 arctang(2hl). Before starting the study, number of subjects in each age group and their height and weight characteristics are shown in table 1 . Using anova statistic test (p <0.001) it was found that mean height and weight of subjects in age groups were significantly different . By performing the measurement 2 times with 5 minute interval, intraclass correlation coefficients (icc) were found high (86%); therefore a good reliability of this test was confirmed . Table 2 shows mean kyphosis angle with 95% confidence interval in each age group (1318). Mean thoracic kyphosis for each age group was 13 (34.41), 14 (34.86), 15 (35.79), 16 (36.49), 17 (35.84), and 18 (35.55). When the whole population is considered, the average kyphosis was 35.49 sd 7.83, ranging from 12.05 to 68.84. this study would indicate that 95% confidence interval of normal kyphosis was between 19.83 to 51.15. using anova statistic test (p <0.001) it was found that mean kyphosis of subjects in 1318-year groups was significantly different too . With increase in age from 13 to 16 years old, the magnitude of mean of kyphosis increased from 34.41 to 36.49 and then there was a mild decrease reaching to 35.5 by the age of 18 years old (figure 3). The least thoracic angle was 12.05 observed in students who were 13 years old and the biggest was 68.84 in those who were 16 years old . The goal of this study was determination of thoracic kyphosis normalcy in male children and adolescents ranging from 13 to 18 years of age for regional population . Most researchers believe that there is an increase in thoracic kyphosis in the age of 10 to 20 in both sexes . Fon et al . Stated that the increase in the kyphotic curve with age is not unexpected, because of the associated changes in the soft tissues and mineral content of the bones with the progression of years . An association of progressive increase in spinal curvatures with gradual compression wedging of the vertebrae and its narrowing of the intervertebral discs has been described . Age - related increases in thoracic kyphosis can be attributed to overloading spinal soft tissue, particularly to the intervertebral disk with aging, during the growth period ., mac - thiong et al ., poussa et al ., and cil et al . In their articles have reported the least pronounced kyphosis at the age of 1012 years, but, because we did not choose subjects in this range, the least kyphosis in our study was seen in the 13-year - old group . The biggest magnitude of mean curve was seen in 16-year - old age group that may be due to pubertal growth . A positive correlation was seen between the velocity of growth and the range of the kyphosis in willner and johnson, poussa et al ., and cil et al for example, willner and johnson, poussa et al ., and giglio and volpon reported some decreases at 15, 14, and 15 - 16 years of age, respectively . The age for this decrease in each research was different which can be due to natural differences in subjects (race, lifestyle, age of puberty related to geographic region, etc . ), or margin of errors demonstrated in using different measurement methods . Radiography (cobb method) is the most commonly used method to assess sagittal spinal curves . Some studies using radiography including takemitsu and harada in a study on 519 students reported 37 for mean thoracic kyphosis . (measuring superior border of the upper end vertebra as well as along the inferior border of the lower end vertebra) found 25.11 for 1019-year - old boys . Measured the thoracic kyphosis from the upper end plate of t3 to the lower end plate of t12 in 88 boys and girls of 1018 years of age . . In a study on 121 subjects of 519 years of age stated the mean angle 33.00 with normal range (mean 2sd) of 2050. in mac - thiong et al . Study, values of 38.3 and 44.2 were reported for children younger than 10 years old and subjects of 10 years of age or older (both sexes), respectively . And cil et al . In a study of both male and female showed the average to be 45.8 for 1012 and 53.3 for 1315 years of age . Although radiography is the most commonly used, this method is not ideal for systematic population studies because of its high cost and exposure of subjects to ionizing radiation . Some radiograph images are difficult to analyze, as it may be hard to precisely identify the beginning of the kyphotic curve because of the shoulder girdle and rib overlap . To overcome these limitations, thoracic curve was clinically evaluated by other instruments . Willner and johnson studied the thoracic kyphosis in 1101 healthy children in consecutive age groups between 8 and 16 years of age using spinal pantograph . Their mean values for different ages were 13 (31.9), 14 (37.1), 15(35.6), and 16 (37.4). Results for boys in 11, 12, 13, and 14 years of age were 27.8, 28.0, 30.9, and 30.0, respectively . Widhe measured the thoracic curve using kyphometer on 46 males of 15 - 16 years and the mean value was 33.7. and giglio and volpon using spinal pantograph showed 33, 38, 35, 32, 36, and 37 for thorasic kyphosis in 13, 14, 15, 16, 17, and 18 year old boys, respectively . To investigate the comparative validity and the intra- and interrater reliability of thoracic kyphosis measurements using the flexicurve method, teixeira and carvalho conducted a study in which the thoracic kyphosis was evaluated from sagittal radiography of the thoracic column using cobb's method and by means of the flexicurve method . Estimates of intertrial and interrate reliability of spine curvature measures acquired using the digital inclinometer and flexicurve ruler also were similar . As different instruments with different methods are used in each study and because of subject's variation in different regions and races, there is a wide range of results for mean thoracic kyphosis . In nine mentioned studies on 10 to 20 years of age boys, it ranged from 25.11 to 53.3. our results in the present study (from 34.41 to 36.49) are in agreement with them . Generally, the values driven by radiographic method are obviously more than those taken by noninvasive methods using flexicurve ruler in measuring thoracic kyphosis which is ideal for systematic population studies, a norm was established for male students aged 13 to 18 years old which can be used as normal values for local and regional purposes.
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The adult sorghum chafer, pachnoda interrupta, is a polyphagous herbivore that feeds on fruits and flowers of several plant species, e.g., banana (musa spp . ), mango (mangifera spp . ), acacia (acacia spp . ), orange (citrus sinensis), and papaya (carica papaya) (clark and crowe 1978; hiwot 2000). Fermentation products, e.g., residue (dregs) from tella, an ethiopian beer - like beverage, are highly attractive to the beetles (ministry of agriculture and ethiopian agricultural research organization 1999). Tella is a spontaneously fermented beer (no yeast is added) that is brewed using water, flour of sorghum or other cereals, malt of barley or wheat, with crushed leaves of shiny - leaf buckthorn, rhamnus prinoides eschsch . Pachnoda interrupta adults also feed on the herbaceous weed abutilon, abutilon figarianum, and food crops such as pearl millet, pennisetum glaucum and sorghum, sorghum bicolor (schmutterer 1969; grunshaw 1992; jago 1995; sastawa and lale 2000). During the early 1990s, the sorghum chafer emerged as a key pest on sorghum in ethiopia (hiwot 2000). Mean percent loss of sorghum yield due to p. interrupta infestation can be as high as 70% (yitbarek and hiwot 2000). Efficient control methods for this pest insect are lacking, but trapping by using fruit (mainly musa spp .) As bait shows promise for decreasing the pest population (ministry of agriculture and ethiopian agricultural research organization 1999). The efficiency of trapping could be improved by using better traps and synthetic, standardized attractants (wolde - hawariat et al . Previous field experiments indicate that compounds commonly found in the odor profile of flowers and fruits have potential as attractants for p. interrupta, with high levels of attraction especially to methyl salicylate and eugenol (wolde - hawariat et al . Single compounds are efficient lures for many scarab species that feed on fruits and flowers (donaldson et al . 1990), and studies have shown a high degree of overlap between active compounds identified from different food sources (stensmyr et al . 2001). Continued efforts in field testing of novel synthetic attractants for the japanese beetle, popillia japonica, have led to the development of lures far outperforming initial versions (potter and held 2002). In search of attractants for pest insects, trial - and - error field screening of compounds has led to the identification of powerful attractants, e.g., for p. japonica (potter and held 2002). A commonly employed method in search of field attractants 1975), which allows the identification of antennally active compounds in the volatile blends emitted by hosts . Gc - ead has led to the identification of several powerful kairomonal field attractants (see e.g., linn et al . Few studies, however, have employed single sensillum recordings (ssr) to identify behaviorally active compounds (stensmyr et al . 2001; larsson et al . 2008), even though ssr may be a highly sensitive tool in detecting physiologically active components in the volatile profiles of host plants (wibe 2004), including compounds that do not elicit detectable gc - ead responses (blight et al . 1995 ssr usually has been employed as a means of describing and understanding the sense of olfaction, specifically the olfactory receptor populations of antennae and palps (see e.g., larsson et al . We employed gc - ead on volatile collections from sorghum and abutilon to identify compounds responsible for attraction of p. interrupta to these plants in the field . We also screened olfactory receptor neurons (orns) on the sorghum chafer antenna with potential kairomones using ssr . The behavioral activity of several compounds active in gc - ead and ssr was tested in field trials, in search of potent attractants . Such attractants could be used in future control efforts, either in mass trapping or in monitoring, or as part of integrated pest management . Insects for electrophysiological experiments male and female sorghum chafers were collected at rasa (0955n, 4005e), located 255 km northeast of addis ababa, ethiopia . Adult beetles were sexed based on the presence of a ventral, abdominal groove in males (rigout 1989), and kept separately . After transport to alnarp, sweden, adults were kept in clear plastic boxes (30 12 22 cm, cofa plastics ab, stockholm, sweden) with a 1:1:1 mixture of planting soil (yrkesplantjord, weibull trdgrd ab, hammenhg, sweden), peat (vxa trdgrdstorv, econova garden ab, se, sweden) and composted cow dung (simontorps bas, weibull trdgrd ab). Boxes were kept at 25c, 70% relative humidity, and a l16:d8 h cycle . Headspace plant volatile collection volatiles were collected from the developmental stage of the plant most attractive to the beetles, during the time of day when the beetles feed intensively, i.e., 10 am to 4 pm . For abutilon, the top 20 cm of a single abutilon plant, including flowers, seed pods, and leaves, was enclosed for each collection . For sorghum, volatiles were collected from a single panicle at the soft dough stage (milky stage). Polyacetate bags (35 43 cm; toppits scandinavia ab, sweden) sealed with steel wire around the stem of the plant were used for aerations . 50 mm) packed with 25 mg superq, mesh 80/100 (alltech, deerfield, il, usa) with glass wool and teflon stoppers at both ends (birgersson and bergstrm, 1989). The filter was placed in the polyacetate bag and connected by pvc tubing to a small battery operated pump (pas-500 personal air sampler, supelco, bellefonte, pa, usa). The flow of the pump was 200 ml / min, and collections were made in the field for 2 h. immediately after collection, the columns were rinsed with 200 l of redistilled hexane into 1.1 ml tapered glass vials (1.1 stvg, chromacol ltd ., welwyn garden city, uk). Vials with extracts were kept in an icebox for transportation to the laboratory, and thereafter kept at 18c until analysis . Gas chromatograph - coupled electroantennographic detection (gc - ead) the response of p. interrupta antennae to volatiles was studied by gc - ead using an agilent technologies gas chromatograph (gc), model 6890, equipped with a fused silica capillary column (30 m 0.2 mm) coated with innowax (0.25 m film thickness) (agilent technologies inc ., santa clara, ca, usa). For each run, 2 l of sample were injected in splitless mode . Hydrogen was used as mobile phase at a linear velocity of 45 cm / sec . The oven temperature was programmed from 40c (5 min hold) to 230c at 5c / min . Compounds eluting from the column were split 1:1 in a four - way splitter, with nitrogen as make up gas (20 ml / min), and delivered to the fid and to the antenna . Compounds were carried to the antenna through a glass tube by a charcoal - filtered and humidified air stream at 0.5 m / sec . . The antenna was excised with fine forceps and placed in an antennal holder (hillbur 2001; joac, lund, sweden), and the signal was amplified (joac) and analyzed with gc - ead software (syntech, hilversum, the netherlands). Each extract was tested on five different antennae per sex, for a total of ten antennae per extract . Chemical identification samples of plant volatile collections were analyzed by combined gas chromatography and mass spectrometry (gc - ms) hewlett packard 6890 gc and 5973 ms (agilent technologies inc . ). The gc was fitted with the same column under the same conditions as for gc - ead, but usinig helium (35 cm / sec) as carrier . Identifications of compounds were confirmed by comparison of mass spectra in the nist 1998 and wiley 1998 commercial mass spectral databases, and with those of authentic gc standards, except for methyl anthranilate, which was not available at the time when gc - ms analysis was done . Synthetic compounds synthetic standards for all experiments were purchased from sigma - aldrich (for purity and cas number, see table 1). A total of 82 compounds were used in single sensillum recordings (table 1). 2006), volatiles from tropical fruit (macku and jennings 1987; ibes et al . 2007; pandit et al . 2009), and volatiles related to microbial degradation and fermentation (chatonnet et al . 1992 approximately half of the compounds used previously have been found to elicit behavioral or electrophysiological activity in the sorghum chafer or related scarab beetles (stensmyr et al . Table 1synthetic compounds used for single cell screening#compoundscas%*14-ethylphenola123 - 07 - 999 * 14-methylphenola106 - 44 - 599 * 2(e)-2-hexenalh6728 - 26 - 398 * 2(e)-2-hexen-1-olh928 - 95 - 096 * 2(e)-2-hexenyl acetateh2497 - 18 - 998 * 2(e)-3-hexen-1-olh928 - 97 - 298 * 2(z)-3-hexen-1-olh928 - 96 - 198 * 2(z)-3-hexenyl acetateh3681 - 71 - 8983hexanalh66 - 25 - 198 * 31-hexanolh111 - 27 - 398 * 3hexyl acetateh142 - 92 - 798 * 3nonanalh124 - 19 - 69531-nonanolh143 - 08 - 899,5 * 31-octanolh111 - 87 - 599,5 * 3()-3-octanolh589 - 98 - 099 * 3()-1-octen-3-olh3391 - 86 - 498 * 4anetholeh4180 - 23 - 899 * 4benzaldehydeh100 - 52 - 799,5 * 4benzylalcoholh100 - 51 - 699 * 4eugenolh97 - 53 - 098 * 4methyl benzoateh93 - 58 - 3994methyl anthranilateh134 - 20 - 399 * 42-phenylethanolh60 - 12 - 898 * 42-phenylethyl propionateh122 - 70 - 398 * 5()-acetoina513 - 86 - 097 * 5racemic 2,3-butanediola513 - 85 - 9995carvacrola499 - 75 - 2985cinnamic aldehydea104 - 55 - 298 * 5methyl cinnamatea103 - 26 - 499 * 5methyl salicylatea119 - 36 - 899 * 5phenylacetaldehydea122 - 78 - 190 * 5phenylacetonitrilea140 - 29 - 499 * 5thymola89 - 83 - 899,5 * 6butyric acidh107 - 92 - 699 * 6n - caproic acidh142 - 62 - 199,5 * 6isovaleric acidh503 - 74 - 2986valeric acidh109 - 52 - 499,8 * 7isoamyl alcoholh123 - 51 - 398 * 76-methyl-5-hepten-2-oneh78 - 70 - 6997tetradecaneh629 - 59 - 499,57tridecaneh629 - 50 - 599,5 * 8()-beta - caryophylleneh87 - 44 - 598,5 * 8(-)-trans - citronellolh106 - 22 - 995 * 8geraniolh106 - 24 - 198 * 8geranyl acetateh105 - 87 - 398 * 8()linaloolh78 - 70 - 697 * 8linalool oxideshn / a978methyl jasmonateh1211 - 29 - 6958nerolidolh7212 - 44 - 4989()delta - decalactoneh705 - 86 - 2989()gamma - decalactoneh706 - 14 - 9979()gamma - hexalactoneh695 - 06 - 798 * 9()gamma - nonanlactoneh104 - 61 - 0979()gamma - octalactoneh104 - 50 - 7979()gamma - undecalactoneh104 - 67 - 69910()ethyl 3-hydroxybutyrateh5405 - 41 - 497 * 10(z)-3-hexenyl butyrateh16491 - 36 - 498 * 10(z)-3-hexenyl isobutyrateh41519 - 23 - 798 * 10(z)-3-hexenyl tiglateh67883 - 79 - 897 * 11butyl butyrateh109 - 21 - 798 * 11ethyl butyrateh105 - 54 - 499 * 11ethyl hexanoateh123 - 66 - 09911ethyl propionateh105 - 37 - 399 * 11hexyl butyrateh2639 - 63 - 69811methyl butyrateh623 - 42 - 799 * 11methyl hexanoateh106 - 70 - 799 * 11methyl octanoateh111 - 11 - 59911methyl propionateh554 - 12 - 199 * 11propyl butyrateh105 - 66 - 89912butyl isobutyrateh97 - 87 - 09712hexyl hexanoateh6378 - 65 - 097 * 12isoamyl acetateh123 - 92 - 298 * 12isoamyl butyrateh106 - 27 - 49812isobutyl acetateh110 - 19 - 099,8 * 12isobutyl isobutyrateh97 - 85 - 89912isopentyl isobutyrateh2050 - 01 - 398 * 12isopropyl acetateh108 - 21 - 499,813acetic acidp64 - 19 - 79913acetonep67 - 64 - 199,913ethanolp64 - 17 - 599 * 13ethyl acetatep141 - 78 - 699,513propionic acidp79 - 09 - 499,5 *, compound active in single sensillum recordings #, screening blends, solvent used (a, acetone, h, hexane, p, paraffin oil)cas, chemical abstracts service number%, minimum purity in percent synthetic compounds used for single cell screening *, compound active in single sensillum recordings s, solvent used (a, acetone, h, hexane, p, paraffin oil) cas, chemical abstracts service number%, minimum purity in percent single sensillum recordings (ssr) single synthetic compounds were diluted to 1 g/l in acetone or hexane, depending on polarity (table 1). Blends of 210 compounds, with each component at the same concentration as in the single compound dilutions, were also prepared for screening purposes (see below; table 1). Stimuli were prepared by applying 10 l of 1 g/l solution to a 1.5 1 cm piece of whatman filter paper (no . 3, whatman, maidstone, united kingdom) that was placed in a disposable pasteur pipette (150 mm soda lime glass, vwr international, stockholm, sweden). For compounds diluted in hexane or acetone, solvent was allowed to evaporate before stimuli were used in experiments . After evaporation of solvent, 1 ml pipette tips were put on the wide end of the pasteur pipettes, to reduce any further evaporation of the test compound(s). Between trials, stimulus pipettes were kept at 18 c, to avoid evaporation . For comparison, stimulus pipettes containing only solvent as well as empty pipettes were prepared . To ensure that stimulus pipettes were not exhausted, new ones were prepared once per week (after having been used a maximum of ten times), except for screening pipettes, where new ones were prepared each day.insects were restrained with parafilm (pm-992, pecheney plastic packaging, menasha, wi, usa) and fixed on microscope slides (ca . 76 26 mm, menzel - glser, braunschweig, germany) using dental wax (surgident periphery wax, heraeus kulzer gmbh, hanau, germany), with the lamellae held open on a wax surface using 2 - 3 mm long pieces of thin tungsten wire . Sensilla were contacted with a tungsten electrode (diam 0.12 mm, harvard apparatus ltd, edenbridge, united kingdom) electrolytically sharpened in a saturated kno2 solution (hubel 1957), using a dc-3k rechts pm-10 piezo micromanipulator (mrzhuser wetzler gmbh, wetzler, germany). The signal from the orns was registered and amplified 10 times with a probe (inr-02, syntech), amplified 200 times with a syntech un-06 ac / dc amplifier, and transferred to a computer through an idac-4-usb (syntech), where it was visualized and analyzed with the software autospike v. 2.2 (syntech).a constant flow of 0.5 m / sec of charcoal - filtered and humidified air was delivered through a glass tube with its outlet approximately 15 mm from the antenna . Stimuli were presented to the insect by inserting the stimulus pipette through a hole in the glass tube, and blowing an air puff of 2.5 ml during 0.5 sec through the pipette into the air stream, using a stimulus controller (syntech sfc-1/b). Control stimuli were delivered first, followed by screening stimuli containing multiple compounds (screening blends listed in table 1). For all screening stimuli that elicited a positive response of approximately 40 hz, the pipettes loaded with all compounds in the blend(s) were brought from the freezer and tested individually after thawing at room temperature for 5 min.the net response to a stimulus was obtained by counting action potentials (spikes) during 0.5 sec starting from the time after the stimulation period at which the earliest response for the neuron was found, and deducting the number of action potentials during 0.5 sec immediately prior to the response . Each neuron was also subjected to blank stimuli (i.e., only solvent), and the net response to the blank was deducted from the response to the test compounds . The resulting value was doubled to obtain a value corresponding to spikes / sec (hz). The time between the start of the stimulation period and the onset of a response, i.e., increased number of action potentials, sometimes varied between different recording sessions, due to slight variations in the air flow . For each neuron, counting of action potentials was started from the time at which the earliest response in that neuron occurred . Field experiments related to gc - ead field experiments with the sorghum and abutilon compounds were carried out at rasa, ethiopia (see above). A complete randomized block design with n = 10 was used . The distance between traps was 10 m, and blocks were separated by at least 50 m. dispensers were placed in cardboard holders (78 37 mm, silvandersson ab, knred, sweden) fitted into a slot in the vanes of japanese beetle traps (trc, palo alto, ca, usa), which were suspended approximately 3 m above ground from wooden poles . The traps were emptied daily, and lures were replaced in the morning before the onset of activity for adult p. interrupta . Unbaited traps were used as a negative control.experiments were performed during two periods: july 1116 and october 713, 2006 . The latter tests were done during the cropping season, when the sorghum had seeds in the milky stage . The july experiments were carried out in a grazing area characterized by scattered acacia trees . In october, traps were placed along the borders of five sorghum fields located approximately 500 m from the july test sites . The individual sorghum compounds, (z)-3-hexen-1-ol, tridecane, 1-octen-3-ol, and 1-octanol, were applied at a dose of 100 mg each . In addition to the sorghum compounds, eugenol and methyl salicylate also were tested . Two of the sorghum - related blends were tested both in july and in october: a blend of the four sorghum compounds with the same total dose (100 mg) as for the individual compounds and in a ratio mimicking what was found in the sorghum headspace, i.e., 10 mg (z)-3-hexen-1-ol + 30 mg tridecane + 30 mg 1-octen-3-ol + 30 mg 1-octanol, and the same sorghum blend with the addition of 30 mg methyl salicylate . In addition to these, three more blends were tested in october: the sorghum blend with the addition of 30 mg eugenol, the sorghum blend with the addition of 30 mg eugenol and 30 mg methyl salicylate, and a blend of 50 mg eugenol and 50 mg methyl salicylate.the individual abutilon compounds were also tested at a dose of 100 mg: (z)-3-hexen-1-ol (the same traps as in the sorghum experiment), tetradecane, methyl anthranilate, and methyl salicylate . An abutilon blend at a total dose of 100 mg and with ratios mimicking the headspace collections was also tested as follows: 20 mg (z)-3-hexen-1-ol + 20 mg tetradecane + 5 mg methyl anthranilate + 55 mg methyl salicylate . Furthermore, in october, a blend consisting of the abutilon blend with the addition of 30 mg eugenol was added to the experiment . Field experiments related to ssr the materials and methods used in ssr - related field experiments were the same as those used for field experiments related to gc - ead, except n = 5 and treatments were moved one step within blocks each day to minimize any impact of possible position effects . Some previously untested compounds also were applied to new dispenser types (see below).six novel compounds selected by ssr were tested on 49 july 2008 on unused farmland with sparse vegetation near the village of embuay bad in ethiopia (0948n, 4000e), 1206 m above sea level, 265 km northeast of addis ababa, ethiopia . Five of the novel compounds tested (anethole, benzaldehyde, racemic 2,3-butanediol, isoamyl alcohol, and methyl octanoate) were selected on the basis that they elicited strong ssr response in separate orn classes that did not respond to compounds previously tested in the field . For comparison methyl benzoate was tested for reasons different from the other compounds it was included since it activated the same orn type as methyl salicylate, which previously had been shown to be highly attractive . Olfactory receptor neurons responding to eugenol did not respond to other compounds included in the screening process . Practical limitations forced us to forego testing of some compounds as it was not possible to acquire necessary quantities in suitable purity, and other compounds were not included since they are more commonly associated with foliage than fruit or flowers.a dose of 100 mg of pure compound (for purity and cas number, see table 1) was loaded onto a dispenser that was matched to the volatility (as indicated by boiling point) of the compound . 2 dental cotton roll, demedis gmbh, langen, germany) were used as dispensers for anethole, eugenol, methyl benzoate, methyl octanoate, and methyl salicylate . For dispensing benzaldehyde and 2,3-butanediol, cotton rolls were pushed into 4 ml vials (45 14.7 mm, clear, skandinaviska genetec ab, vstra frlunda, sweden) until the cotton was level with the rim of the opening of the vial . Compound was applied to the cotton roll after it had been placed in the vial . For isoamyl alcohol, a dispenser was made where a cotton roll was put inside a vial closed with a cap (black, closed top, 13 mm, skandinaviska genetec ab). The cotton roll was placed so that it was in direct contact with the cap when the cap was screwed tight to the vial . The chemical was not applied to the cotton roll directly beneath the hole in the cap, but instead towards the edge of the vial, before screwing on the cap . Statistical analysis for field experiments, data for total catch of p. interrupta (cumulative over the field testing period) per trap was square root - transformed ((+ 1)). Data was analyzed with a general linear model (glm), with treatment (type of lure) as a fixed effect, and block as a random effect (minitab 14 for windows). Trap catch data is presented in graphs as untransformed means with error bars denoting standard error of the mean . Gc - ead analyses and identification of host plant volatiles several compounds in both the abutilon (fig . The ead - active compounds collected from abutilon headspace extracts were identified as (z)-3-hexen-1-ol, tetradecane, methyl salicylate, and methyl anthranilate (fig . 1a), and in sorghum as tridecane, (z)-3-hexen-1-ol, 1-octen-3-ol, and 1-octanol (fig . Similar antennal responses were recorded from antennae of females (not shown). In abutilon headspace extracts, methyl salicylate was a major constituent, making up approximately 14.4% of the extract . In sorghum extract, however, it was present at low concentrations, and did not elicit any antennal responses . Despite a low concentration of methyl anthranilate in the abutilon extract, 1simultaneous response of flame ionization detector (fid) and male pachnoda interrupta antennae (ead) to volatile compounds collected from a abutilon and b sorghum . The upper traces in each figure represent the signal from the fid and the lower traces represent the signal from the ead . The compounds collected from abutilon headspace extracts were identified as (z)-3-hexen-1-ol (1), tetradecane (2), methyl salicylate (3), and methyl anthranilate (4), and in sorghum tridecane (5), (z)-3-hexen-1-ol (1), 1-octen-3-ol (6), and 1-octanol (7) simultaneous response of flame ionization detector (fid) and male pachnoda interrupta antennae (ead) to volatile compounds collected from a abutilon and b sorghum . The upper traces in each figure represent the signal from the fid and the lower traces represent the signal from the ead . The compounds collected from abutilon headspace extracts were identified as (z)-3-hexen-1-ol (1), tetradecane (2), methyl salicylate (3), and methyl anthranilate (4), and in sorghum tridecane (5), (z)-3-hexen-1-ol (1), 1-octen-3-ol (6), and 1-octanol (7) field experiments related to gc - ead during july and october, there were no significant differences in trap catches between the sorghum blend, which caught an average of 12 beetles per trap over the experimental period in july and 11 beetles in october (blend 8, fig . 2), and the individual sorghum compounds . In july, traps baited with methyl salicylate caught significantly more beetles (21 beetles / trap) than traps baited with tridecane or (z)-3-hexen-1-ol . Eugenol baited traps caught significantly more beetles (40 beetles / trap) than all other treatments, except the sorghum blend with the addition of methyl salicylate (40 beetles / trap, blend 9, fig . 2a). In october, the sorghum blend with eugenol (82 beetles / trap, blend 10) and the sorghum blend with eugenol and methyl salicylate (70 beetles / trap) attracted by far the highest numbers of beetles, followed by the sorghum blend with methyl salicylate, the two - component eugenol - methyl salicylate blend, and eugenol alone . In both july and october, the four - component abutilon blend caught significantly more beetles (22 beetles / trap in july and 17 in october, blend 7, fig . 3) than traps baited with the single compounds (z)-3-hexen-1-ol and tetradecane . While there were no significant differences in trap catches between traps baited with the abutilon blend and the blend without methyl salicylate (15 beetles / trap) in july, the complete blend caught more beetles in october . In both seasons, there were no significant differences in trap catch between the abutilon blend and methyl salicylate presented as a single compound or eugenol as a single compound . When methyl anthranilate was added as a single compound in october, it was as attractive as the abutilon blend and eugenol and methyl salicylate (20 beetles / trap, fig . The other treatment that was added, the four - component abutilon blend combined with eugenol, was equally attractive (42 beetles / trap, blend code 9, fig . 3b) as the binary mixture of methyl salicylate and eugenol (37 beetles / trap), and more attractive than any other treatment . During both seasons, all treatments caught significantly more beetles than the unbaited control traps . Male and female beetles followed the same pattern of attraction, with no clear differences between the sexes in which baits were most attractive (data not shown). 2number of pachnoda interrupta captured in traps baited with synthetic sorghum compounds in a july, 2006 and b october, 2006 . Glm: july, (n = 10, f = 78.1, df = 7, 63, p <0.001); october, (n = 10, f = 114.1, df = 10, 90, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b). 3number of pachnoda interrupta captured in traps baited with synthetic abutilon compounds in a july, 2006 and b october, 2006 . Glm: july 2006, (n = 10, f = 12.7, df = 5, 45, p <0.001); october 2006, (n = 10, f = 42.1, df = 8, 72, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b). Nt indicates that the treatment was not tested number of pachnoda interrupta captured in traps baited with synthetic sorghum compounds in a july, 2006 and b october, 2006 . Glm: july, (n = 10, f = 78.1, df = 7, 63, p <0.001); october, (n = 10, f = 114.1, df = 10, 90, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b). Nt indicates that the treatment was not tested number of pachnoda interrupta captured in traps baited with synthetic abutilon compounds in a july, 2006 and b october, 2006 . Glm: july 2006, (n = 10, f = 12.7, df = 5, 45, p <0.001); october 2006, (n = 10, f = 42.1, df = 8, 72, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b). Nt indicates that the treatment was not tested single sensillum recordings the antennal morphology of p. interrupta is similar to that of the closely related scarab p. marginata, as described by stensmyr et al . P. interrupta has a typical scarab antenna, where sensilla are present on the inner surfaces of the three lamellae at the tip of the antenna . Most sensilla are of the placodea morphological type, with a small minority of smooth peg and coeloconic sensilla . Our ssr recordings stem from sensilla placodea, as we only managed to get intermittent contacts with orns of the other morphological types.contacted sensilla typically contained two orns, which in most cases could be separated by differences in spike amplitude (fig . 4). In sensilla containing two neurons, both neurons sometimes fired simultaneously, resulting in double spikes with amplitudes greater than that of either neuron by itself . When subjected to synthetic stimuli, orns usually responded strongly with a train of action potentials (spikes) to a few compounds . Data for the sexes were pooled, as no types of orns were numerous enough for a meaningful comparison between the sexes . Out of the 156 sensilla, orn response to stimulation with control stimuli (blank) normally was below 10 hz (data not shown), but 3 orns gave a blank response of 50 hz or above and were excluded from analysis, since we deemed that there was a risk that the cells had either been injured and were not responding properly, or that some contamination had been present in the odor stimulation system . We also excluded 8 orns for which no compound elicited a net response of 40 hz or above, as these weak responses were unlikely to indicate key stimuli suited for evaluation in field trials . The remaining 97 neurons responded with a net response of 40 hz or above to at least one stimulus . Of the 82 test compounds, 57 elicited spike responses over 80 hz at least once (table 1). Not all responses could be assigned to a specific class, but 94 responding orns could be grouped tentatively into 26 classes (table 2). In most cases, orns responded to a single compound or a group of functionally or structurally similar compounds with one eliciting a stronger response than the rest . For some neurons, several compounds elicited strong responses, with no clear ranking between the 23 best compounds . For orn classes, the average response to ligands is shown down to 40 hz (table 2). 4the two orns present in this sensillum are distinguished by the amplitudes of their action potentials . The orn responding to linalool oxide (denoted a) has a higher amplitude than the orn responding to methyl salicylate (b). There are also double spikes, where both neurons fire simultaneously (d), that have a greater amplitude than either neuron by itselftable 2olfactory receptor neuron classes in the sorghum chafer, pachnoda interruptaseveral of the orn classes responded to stimulation with compounds that commonly occur in the volatile profiles of fruit and flowers (table 2). There also were classes that responded to stimulation with compounds associated with foliage, i.e., green leaf volatiles (glvs). No orn response was found to ethanol, acetone, acetic acid, or propionic acid, and there was only one response to ethyl acetate . However, orns responded to isovaleric acid, acetoin, racemic 2,3-butanediol, 4-ethylphenol, and 4-methylphenol . The two orns present in this sensillum are distinguished by the amplitudes of their action potentials . The orn responding to linalool oxide (denoted a) has a higher amplitude than the orn responding to methyl salicylate (b). There are also double spikes, where both neurons fire simultaneously (d), that have a greater amplitude than either neuron by itself olfactory receptor neuron classes in the sorghum chafer, pachnoda interrupta field experiments related to ssr traps baited with racemic 2,3-butanediol caught significantly more beetles over the experimental period than traps baited with other compounds (an average of 205 beetles / trap, fig . 5; f = 80.56, p <0.001), catching three times more beetles than the second best bait, eugenol (which caught 69 beetles / trap), and six times more than the third best, methyl salicylate (36 beetles / trap). Apart from 2,3-butanediol, traps baited with previously untested compounds did not catch significantly more p. interrupta than the unbaited control traps . Males and females follow the same pattern of attraction; there were no clear differences between the sexes for which baits were most attractive (data not shown). The type of bait had a significant effect on catch (glm, n = 10, f = 80.56, df = 8, 72, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b) average trap catch of pachnoda interrupta in july 2008 . The type of bait had a significant effect on catch (glm, n = 10, f = 80.56, df = 8, 72, p <0.001). Subgroups denoted by different letters are significantly different at =0.05 (n = 10, tukey s b) some insect species require specific blends of several compounds to be attracted to their host (bruce et al ., 2005), e.g., the apple maggot fly, rhagoletis pomonella (linn et al ., 2005), the grapevine moth, lobesia botrana (tasin et al . 2006, 2007), and the fruit fly, drosophila melanogaster (zhu et al . 2003; ruebenbauer et al . 2008). However, for many fruit and flower visiting scarabs, individual compounds constitute efficient attractants (donaldson et al . Relying on key compounds rather than specific blends could be an efficient general host detection strategy for a polyphagous herbivore such as p. interrupta . Such a strategy could be enhanced by the presence of reliable signals from plants that attract animals for pollination or fruit (seed) dispersal . Our results from field experiments with blends that mimic the hosts abutilon and sorghum indicated that p. interrupta is attracted to a few key components in the volatile profiles of these plants, rather than ratio - specific blends (fig . 2 and 3), and thus we focused our search for attractants on single compounds . As we also noted that the most attractive compounds were associated with fruits and flowers (e.g., eugenol and methyl salicylate), rather than foliage [e.g., (z)-3-hexen-1-ol], we primarily tested floral- and fruit - related kairomones in the single sensillum screening . Among the kairomones selected for screening, many have been found in several hosts of p. interrupta, e.g., banana (macku and jennings 1987; ibes et al . 1998; boudhrioua et al . 2003) guava (carasek and pawliszyn 2006), mango (clara et al . 2007; pandit et al . 2009), and various flowers (knudsen et al ., 2006). 2000; xiao and ping 2007), since the sorghum chafer has been reported to be attracted to residue from the fermentation of tella beer . The use of single sensillum recordings (ssr) for screening of potential kairomones enabled us to select compounds for field testing that activate separate olfactory receptor neuron (orn) classes . Regardless of how information from the olfactory system is interpreted at higher levels of the nervous system, the activation of additional components of the orn array should increase the likelihood of releasing a behavior, compared to redundant activation of the same orn classes with several different compounds . Behavioral redundancy between compounds that activate the same orn class has been observed in the bark beetle ips typographus, where compounds detected by the same orn do not cause any synergistic repellent effects when combined in field trapping experiments (andersson et al . Broad response spectra for orns could be an efficient solution for detecting several compounds that relay essentially the same information (baker et al . The best field attractants in our study (fig . 2, 3 and 5) and in a previous study (wolde - hawariat et al . 2007) were all detected by single orn classes no response to these ligands were found in other orn classes (table 2). Thus, activation of a single orn class seems to be sufficient for the release of attraction behavior . Results from i. typographus also indicate that several compounds may cause sufficient activation of a single orn class to release behavior (andersson et al . 2009). Methyl benzoate thus was included in the field experiment as it was a secondary ligand of methyl salicylate orns (table 2). Methyl salicylate was highly attractive to p. interrupta in the field (wolde - hawariat et al . 2007), and to p. marginata in a two - choice bioassay (larsson et al . 2003), even though it was detected by one of the least commonly found orns in p. marginata (stensmyr et al methyl salicylate is the primary odorant for a particular orn in the moth mamestra brassicae, and as in p. interrupta, this neuron also has a weaker secondary response to methyl benzoate (ulland et al . Egg - laying was inhibited by the presence of methyl salicylate on dispensers near the host plant (cabbage) while methyl benzoate was not tested (ibid . ). In our field experiments, catches of p. interrupta in traps baited with methyl benzoate were not significantly higher than that of control traps (fig . Testing of other compounds that elicit secondary responses in orn classes that respond to attractive compounds could shed light on their role in insect behavior . Despite the fact that p. interrupta is attracted strongly to fermentation products such as tella beer residue (ministry of agriculture and ethiopian agricultural research organization 1999), orns that respond to primary fermentation products such as ethanol, acetic acid, propionic acid, or acetone, appear to be absent or rare on the p. interrupta antenna . Some of these compounds are attractive to other insects that feed on fermenting substrates (e.g., fruit), such as d. melanogaster, which are attracted to acetic acid, acetone, and ethanol in lab trapping bioassays (zhu et al . We did, however, find orns in p. interrupta that responded to other substances related to fermentation and microbial degradation, such as racemic 2,3-butanediol, acetoin, 4-ethylphenol, 4-methylphenol, and ethyl acetate (one neuron). The orn class in p. interrupta responding to 2,3-butanediol also responded to acetoin . In the closely related p. marginata, acetoin was significantly more attractive than a blank control in a two - choice bioassay, while racemic 2,3-butanediol was not (larsson et al . This is interesting as both ligands are detected by the same orn, and none of the other orn classes found in the study respond to either compound (stensmyr et al . Acetoin and 2,3-butanediol are active in other beetle species as well, where certain isomers or mixtures of isomers are often needed to elicit activity . Meso-2,3-butanediol, (2r,3r)-2,3-butanediol, and (r)-acetoin are emitted by female rhizotrogus majalis and detected only by male antennae in ead (nojima et al . 2003). Male scapanes australis emit acetoin, 2-butanol, and 2,3-butanediol, with the first two being necessary and sufficient to attract both sexes in field trapping experiments (rochat et al . (r)-acetoin has been identified as a female - emitted sex pheromone in the summer chafer, amphimallon solstitiale (tolasch et al . The addition of either acetoin or plant matter is necessary to elicit high levels of attraction; the pheromone alone does not suffice (said et al . 2005). In the ssr screening, we found orns that responded to five of the seven compounds identified as active by gc - ead (methyl anthranilate, methyl salicylate, (z)-3-hexen-1-ol, 1-octanol, and 1-octen-3-ol), but we did not find any responses to tetradecane or tridecane (table 1, 2). It is possible that tetradecane and tridecane are detected by one or several rare orn types, or by orns present in smooth peg sensilla or sensilla coeloconica, to which we had only intermittent contacts . Discrepancies between electroantennographic and single sensillum methods have been observed in previous studies (blight et al . 1995; barata et al . 2002; wibe 2004). Wibe (2004) compared gc - ead and gc - ssr as tools for identification of active compounds for the pine weevil, hylobius abietis, in aerations of sawdust from norway spruce (picea abies), and found that gc - ssr led to the identification of a higher number of active compounds than gc - ead . Apart from racemic 2,3-butanediol, the most attractive compounds in the field were eugenol, methyl anthranilate, and methyl salicylate (fig . 2, 3, and 5). 1997), and is a common defensive compound in higher plants in response to herbivory (kessler and baldwin 2001). In field experiments, volatiles emitted from surrounding vegetation thus may include some of the compounds tested, or similar compounds, which could affect the results of field experiments . In the sites used, a salient example is sorghum, which has flowers and seeds in stages attractive to the beetles in october, but not in july . This also coincides with life cycle changes in the beetles, which may affect trap catch . Sorghum chafers mate and feed in july, while in october, the newly emerged adults feed only before going into aestivation until july the following year . Experiments evaluating the effects of these factors were outside the scope of our study . By using ssr to target a large fraction of the peripheral olfactory system, we identified racemic 2,3-butanediol as an efficient field attractant, without needing to test all of the 57 compounds in our screening that elicited response (table 1, 2). This compound could be useful in future control or monitoring, especially since it is highly attractive to both male and female p. interrupta . Future field experiments should clarify which isomers of 2,3-butanediol (or mixtures thereof) are responsible for attraction . Field tests also should include other fruit- or flower - related compounds that activate so far untested orn classes, as well as further compounds related to microbial degradation or fermentation, and blends of the best attractants . P. interrupta may serve as a useful model for future research on host searching in polyphagous fruit- and flower - feeding insects.
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Obesity is a growing problem in the united states (flegal et al ., 1998; kuczmarski et al ., 1994; mokdad et al . Among many determinants of obesity, frequency of meals, snacking, and eating - out have attracted research and practical interests because of their direct applicability to everyday life . Fabry and co - workers reported that eating small frequent meals (nibbling) was strongly associated with lower body weight than eating large few meals (gorging) (fabry et al ., 1964, 1966; hejda & fabry, 1964). But, the inverse relationship between meal frequency and body weight has not been consistently found in recent other studies in population studies (charzewska et al ., 1981; dreon et al ., 1988; edelstein et al ., 1992; metzner et al ., 1977; summerbell, 1996). Nibbling did not produce better results in weight loss than gorging in clinical studies, when the amount of total energy was restricted (finkelstein & fryer, 1971). (1997) concluded in their review paper that nibbling does not have energy metabolic advantages in terms of energy utilization over gorging . It has also been hypothesized that eating frequent meals may result in higher amount of energy than eating infrequent meals, however, data on this hypothesis is relatively limited . Kant (1995) reported that energy intake was positively associated with meal frequency in the national health and nutrition examination and survey i epidemiologic follow - up study . However, energy intake levels in the dataset were below the predicted minimal energy needs (1.4 * bmr) (goldberg et al ., 1991). With limited data availability and imprecision of dietary assessment methods, snacking has been discussed in relation to obesity in similar ways to meal frequency has . Snacking has sometime been studied in the context of meal frequency, because definitions of meals or snacking have not been fully established (gatenby, 1997). Booth (1988) proposed that snacking in addition to meals may lead to higher energy intake . High - fat, high - sugar and high - energy foods are often associated with snacking, possibly resulting in consuming " empty calories (drummond et al ., 1996). " Studies on whether frequent meals or snacking leads to higher energy consumption reported mixed results (kirk, 2000; lioret et al ., 2008). Eating - out is one of the significant phenomna in the industrialized and modernized society (finklestein, 1989). In the u.s ., about half of the food expenditures of adults are spent eating away from home (blisard et al ., 2002; clausen, 2000). In the u.k ., eating - out became much more commonplace at the end of the twentieth century (warde & martens, 2000). Eating - out is thought to be associated with obesity because foods eaten away home tend to be in larger portion size and higher fat and energy content (ma et al ., 2003; young & nestle, 2002). If and how frequent eating - out is related to body weight, however, has not been fully studied . Frequency of meals, snacking, and eating - out are determined by many factors . Immigrants and their children provide unique information on relationships among meal frequency, snacking, eating - out, and body weight, because people acculturate in different speed and patterns . Acculturation refers to a process of overall adaptation on both individual and group levels, including cultural, psychological, social, economic, and political aspects (berry, 1997). Since current international migration flows from undeveloped or developing countries to developed countries, immigrants and their children may be in different stages of showing changes from industrialization and modernization . This study examined whether acculturation was associated with meal frequency, snacking, and eating - out and whether meal frequency, snacking and eating - out were related to weight status using korean americans as an example . Specifically, we hypothesized that: 1) higher acculturation is related to lower meal frequency, more frequent snacking, and more frequent eating - out and 2) lower meal frequency, more frequent snacking, and more frequent eating - out are associated with higher prevalence of overweight . Data for this study were collected by a cross - sectional mail survey of a national sample of korean americans in the continental u.s . Adult (17 years old or older) korean americans were randomly sampled from u.s . Korean surnames were used to target ethnicity and the listing was screened to make sure that only people with korean surnames were included . The sample was stratified by four variables: 1) region (northeastern, midwestern, southern, and western following the national health and nutrition examination and survey (u.s . Department of health and human services . 1981)), 2) urban - rural area (standard metropolitan statistical area (smsa) and non - smsa), 3) age as a proxy for generation (under 35 years and over 35 years), and 4) gender (male and female). The pre - tested questionnaire was mailed to a total of 1113 people with korean surnames in the continental u.s . A personally addressed cover letter in both english and korean explaining the purpose of the study was sent along with two questionnaires (in english and korean). Out of the 1113 questionnaires mailed out, 259 (33%) were returned as undeliverable . Of the 854 deliverable questionnaires, 470 (55% of the total deliverable sample) responded, with 105 who responded that they were not korean americans and two who refused to participate . After careful screening, 16 questionnaires were determined to be unusable . This analysis used a total of 347 questionnaires, which was 42% of the total deliverable sample . The respondents did not show significantly different patterns from the sampling list distribution by region, area, or gender . There are no widely accepted acculturation scales for korean americans . In this study, a bidimensional model (berry, 1997) provided a framework and gordon's seven dimensions (gordon, 1964) were used to identify variables to measure overall acculturation in korean americans . Each dimension was independently measured in two axes: relation to american society and retention of korean ethnic society . Cluster analysis on acculturation resulted in three distinct groups: acculturated, bicultural, and traditional (lee et al ., 2000; 2003). Acculturated korean americans were either born in the u.s . Or immigrated to the u.s . When they were very young . Generally, they were most comfortable with american society compared to the other acculturation groups . The largest, traditional group was situated on the opposite end from the acculturated group . They were more likely to have limited english proficiency and felt most comfortable with korean culture and environments . The bicultural group appeared to be in between the acculturated and the traditional group, however, it was notable that the bicultural group had a wider social network and social participation with american mainstream than the acculturated group . More detailed information on acculturation scale and acculturation groups can be found elsewhere (lee et al ., 2000). Frequency of meal was estimated by asking how often respondents eat breakfast, lunch, and dinner . Response categories were never, once a week or less, 2~3 times a week, 4~6 times a week, and everyday . Response categories were re - coded because of low frequencies in some of the categories . " Never " and " once a week or less " category for regularity of lunch were combined together, leaving " once a week or less, " " 2~3 times a week, " " 4~6 times a week, " and " everyday . " Response categories for regularity of dinner were re - coded into three response categories with the first three categories (" never, " " once a week or less, " and " 2~3 times a week ") collapsed . Frequency of snacking was also asked using the same response categories . With responses for frequency of breakfast, lunch, and dinner, a new variable (daily frequency of meals) was created . Respondents who reported eating each meal everyday were given a score of 1 for each meal, while others received a score of 0 . Combining the scores for three meals produced a value for frequency of meals, ranging from 0 to 3; a value of 0 would mean a respondent did not have regularly established meal frequency, while a value of 3 would mean a respondent had a regularly established meal frequency of three . There were few respondents with a value of 0, therefore, they were combined with those with a value of 1 . Eating - out behaviors was determined by asking how often respondents eat out or take out from restaurants: never or rarely, 1~2 times a month, about once a week, 2~3 times a week, and almost everyday . Weight status was determined by calculating body mass index (bmi) with self - reported height and weight . Bmi is a good indicator of adiposity and self reported height and weight have adequate validity for population studies (bowman & delucia, 1992). Respondents were grouped by bmi into four groups: underweight (bmi<18.5), normal weight (18.5bmi<25), overweight (25bmi<30), and obese (bmi30) (who, 1998). In multiple logistic regressions, overweight individuals (bmi25) were compared to non - overweight individuals . Control variables included sex, age, size of place of residence, education, income, working status, and marital status . Size of place of residence was divided into smsa (0) vs non - smsa (1) according to zip code of residence . Income categories were re - coded to six categories because of low frequency in some of the categories . Working status was collapsed into two categories: currently working (1) and not working (0). Married people were treated as a reference category and all other categories were collapsed into unmarried . Chi - squares and analysis of variance with scheff test were used to examine bivariate relationships . Logistic regression (proportional odds model) was applied to simultaneously investigate the relationships of all study variables in multi - variable models . Adult (17 years old or older) korean americans were randomly sampled from u.s . Korean surnames were used to target ethnicity and the listing was screened to make sure that only people with korean surnames were included . The sample was stratified by four variables: 1) region (northeastern, midwestern, southern, and western following the national health and nutrition examination and survey (u.s . Department of health and human services . 1981)), 2) urban - rural area (standard metropolitan statistical area (smsa) and non - smsa), 3) age as a proxy for generation (under 35 years and over 35 years), and 4) gender (male and female). The pre - tested questionnaire was mailed to a total of 1113 people with korean surnames in the continental u.s . A personally addressed cover letter in both english and korean explaining the purpose of the study was sent along with two questionnaires (in english and korean). Out of the 1113 questionnaires mailed out, 259 (33%) were returned as undeliverable . Of the 854 deliverable questionnaires, 470 (55% of the total deliverable sample) responded, with 105 who responded that they were not korean americans and two who refused to participate . After careful screening, 16 questionnaires were determined to be unusable . This analysis used a total of 347 questionnaires, which was 42% of the total deliverable sample . The respondents did not show significantly different patterns from the sampling list distribution by region, area, or gender . There are no widely accepted acculturation scales for korean americans . In this study, a bidimensional model (berry, 1997) provided a framework and gordon's seven dimensions (gordon, 1964) were used to identify variables to measure overall acculturation in korean americans . Each dimension was independently measured in two axes: relation to american society and retention of korean ethnic society . Cluster analysis on acculturation resulted in three distinct groups: acculturated, bicultural, and traditional (lee et al . Acculturated korean americans were either born in the u.s . Or immigrated to the u.s . When they were very young . Generally, they were most comfortable with american society compared to the other acculturation groups . The largest, traditional group was situated on the opposite end from the acculturated group . They were more likely to have limited english proficiency and felt most comfortable with korean culture and environments . The bicultural group appeared to be in between the acculturated and the traditional group, however, it was notable that the bicultural group had a wider social network and social participation with american mainstream than the acculturated group . More detailed information on acculturation scale and acculturation groups can be found elsewhere (lee et al ., 2000). Frequency of meal was estimated by asking how often respondents eat breakfast, lunch, and dinner . Response categories were never, once a week or less, 2~3 times a week, 4~6 times a week, and everyday . Response categories were re - coded because of low frequencies in some of the categories . " Never " and " once a week or less " category for regularity of lunch were combined together, leaving " once a week or less, " " 2~3 times a week, " " 4~6 times a week, " and " everyday . " Response categories for regularity of dinner were re - coded into three response categories with the first three categories (" never, " " once a week or less, " and " 2~3 times a week ") collapsed . Frequency of snacking was also asked using the same response categories . With responses for frequency of breakfast, lunch, and dinner, a new variable (daily frequency of meals) was created . Respondents who reported eating each meal everyday were given a score of 1 for each meal, while others received a score of 0 . Combining the scores for three meals produced a value for frequency of meals, ranging from 0 to 3; a value of 0 would mean a respondent did not have regularly established meal frequency, while a value of 3 would mean a respondent had a regularly established meal frequency of three . There were few respondents with a value of 0, therefore, they were combined with those with a value of 1 . Eating - out behaviors was determined by asking how often respondents eat out or take out from restaurants: never or rarely, 1~2 times a month, about once a week, 2~3 times a week, and almost everyday . Weight status was determined by calculating body mass index (bmi) with self - reported height and weight . Bmi is a good indicator of adiposity and self reported height and weight have adequate validity for population studies (bowman & delucia, 1992). Respondents were grouped by bmi into four groups: underweight (bmi<18.5), normal weight (18.5bmi<25), overweight (25bmi<30), and obese (bmi30) (who, 1998). In multiple logistic regressions, overweight individuals (bmi25) were compared to non - overweight individuals . Control variables included sex, age, size of place of residence, education, income, working status, and marital status . Size of place of residence was divided into smsa (0) vs non - smsa (1) according to zip code of residence . Income categories were re - coded to six categories because of low frequency in some of the categories . Working status was collapsed into two categories: currently working (1) and not working (0). Married people were treated as a reference category and all other categories were collapsed into unmarried . Chi - squares and analysis of variance with scheff test were used to examine bivariate relationships . Logistic regression (proportional odds model) was applied to simultaneously investigate the relationships of all study variables in multi - variable models . About half of the sample's acculturation status was traditional, a third was bicultural and a sixth could be classified as acculturated . The respondents reported average age of 40 years, average education of 16 years, and average income of approximately $35,000 . The majority of them were working, while men were more likely working than women . Korean american men had significantly higher bmi than korean american women . While more korean american men (30%) were overweight than korean american women (8%), more korean american women were underweight (9%) than korean american men (4%). Only about a third reported that they ate three meals a day everyday (table 2). Breakfast was the least frequent meal of the day with less than half reporting eating breakfast everyday . Dinner was the most frequent meal of the day with 86% eating dinner everyday . About a quarter of the respondents reported having a snack everyday, and women tended to snack more often than men (=11.025, p<0.05). More than half (58%) reported that they usually eat out or get take - out food at least once a week . Since there were few significant and meaningful sex differences among acculturation, socioeconomic, meal frequency variables, further analyses were carried out with combined male and female respondents . In the case of analyses with weight status, the low prevalence of overweight and obesity among women forced the analyses done only in men . Relationships between acculturation and independent variables (frequency of meals, frequency of snack, and frequency of eating - out) were examined (table 3). Controlling for sociodemographic variables (age, sex, size of place of residence, income, education, working status, and marital status), relationships between acculturation and frequency of snacking and eating - out were significant . Acculturated korean americans were more likely to eat snacks more frequently than traditional korean americans; however, significant relationships were not found for any other acculturation group comparisons . Higher frequency of eating - out was also significantly associated with being men, younger age, higher income, and unmarried status . After controlling for age, size of place of residence, income, education, working status, and marital status, bicultural men were more than twice as likely to be overweight than traditional men (table 3). When frequency of each meal, snacking, and eating - out were entered into the model, only frequency of snacking was significant . Korean american men who snack more often were 1.3 times more likely to be overweight . When daily frequency of meals, rather than frequency of each meal, were entered to the model, only acculturation remained significant . This study examined how acculturation was related to frequency of meals, snacking, and eating - out behaviors in korean americans and whether frequency of meals, snacking, and eating - out were associated with body weight . Acculturation was not significantly associated with frequency of meals, but significantly associated with frequency of snacking and eating - out . Multiple regression models showed that only frequency of snacking was significantly related to the likelihood of being overweight in men . Acculturated korean americans tended to more frequently eat snacks and eat out than traditional korean americans . This relationship between acculturation and snacking and eat - out was also reported in japanese americans . Third - generation japanese americans were engaged in more frequent snacking and eating - out than second - generation japanese americans (kudo et al ., 2000). These results seem to support that higher acculturation may lead to changes in daily eating patterns, which includes more eating occasions outside of traditional meals at home . It is worth mentioning that frequency of meals was not significantly related to most sociodemographic variables . Older korean americans were more likely to eat breakfast and korean americans living in non - smsa areas to eat lunch . None of the variables examined in this study were significantly associated with frequency of dinner . These results seem to indicate that dinner is the most constant and stable meal a day . In contrast, frequency of eating - out was significantly associated with several sociodemographic variables as well as acculturation . Korean americans were more likely to eat out if they were acculturated, unmarried, educated men with higher income . . Both married and unmarried individuals appeared to engage in similar daily eating patterns, suggesting that culture and hunger structured the timing and frequency of consumption . However, the source of food differed substantially by marital status, with unmarried people much more often seeking food out of their household than those who are married . Marriage provides a commensal partner for eating occasions, and norms of marriage encourage eating at home (bove et al ., 2003). Many people are reluctant to or not able to prepare foods just for themselves, and this may lead many unmarried people to use commercially prepared foods and eat in restaurants or bring takeout food home . This study was limited in making causal inference because of the cross - sectional study design . We used the traditional and colloquial definition of meals (breakfast, lunch, and dinner) and snacking, therefore, information from people who do not fit into the traditional meal patterns may not have been fully obtained . Since meals and snacks are defined by respondents, results may have been affected by personal bias . However, since this study asked each meal separately then combined meals into one variable, personal bias on a definition of " meals " would have been less than asking " how many meals do you eat a day? " Definition of " meals " warrants more attention because consistent use of a well - defined " meal " variable will help clarify possible relationships and mechanisms between meal frequency and body weight (chiva, 1997; gatenby, 1997). This study did not examine mechanisms of the relationships between meal frequency, eating - out and body weight . One speculated mechanism on the relationship between meal frequency and body weight is that meal frequency affects energy utilization . Studies appear to indicate that there are no significant relationships between meal frequency and energy utilization, when energy intake is controlled (bellisle et al ., 1997). Therefore, meal frequency would not affect body weight in short terms, if isocaloric diets are consumed (bellisle et al . However, it is not conclusive if the same results will be found with free - living individuals where energy intake will not be controlled . Although it has been speculated that frequent meals may result in higher energy intake (booth, 1988), issues in dietary assessment (under - and over - reporting) have hindered to reach meaningful conclusion (bellisle et al ., 1997). Whether frequent meals and/or snacking lead to over consumption needs to be further studied with combination of dietary assessment and metabolic assessment . In addition to the relationship between frequency of meal and obesity, there is growing evidence on possible advantages of frequent meals on other health conditions such as maintaining better control of blood glucose (jenkins et al ., 1994), although some studies (franceschi et al ., 1992; gerhardsson de verdier & longnecker, 1992; young & wolf, 1990) reported that frequent meals may be related to colorectal cancer . Future research should examine relative risk and benefit of having frequent meals in order to make a recommendation to healthy and free - living population.
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Obesity is known as a risk factor for the development of metabolic disorders such as chronic inflammation, insulin resistance, dyslipidemia, arterial hypertension and atherosclerosis (1). The production of some of the adipiokins is resulted from obesity, type2 diabetics, and metabolic syndrome (3). Retinol binding protein 4 (rbp4) is a type of adipokine related to the family of lipocalin proteins which transports vitamin a (retinol) to serum (4). Adipose tissue may be a less important source for rbp4 concentrations in humans than animals (6). Although serum level of rbp4 in obesity, type2 diabetes, and metabolic syndrome is increased, the report is not accurate concentrations (7). Recently, higher rbp4 levels in obese and overweigh individuals have been reported (6). Besides, after adjustment for age, serum rbp4 had a positive relation with waist circumference, total abdominal fat and visceral fat . Cho et al (7) showed that human obesity is increased with rbp4 serum levels, and bmi is significantly higher than those of lean controls . In this study, there was not any relationship between plasma levels and the amount or percentage of body fat . Furthermore, the study by broch et al (3) concluded that there was no relationship among insulin resistance, rbp4, and anthropometric measurements such as waist circumference, hip circumference, whr, bmi, and fat mass . Rbp4 levels is related to systolic blood pressure, and obesity - related biochemical parameters (fasting glucose, fasting insulin, total cholesterol, ldl - c and insulin resistance index), even after adjustment for age and fat . It is likely that increase in rbp4 level, will lead to increased in insulin resistance, dyslipidemia, and cardiovascular diseases (8). Some studies in humans rbp4 levels have shown inversely correlated insulin sensitivity and hdl - c levels . It is a point to mention that with some components of metabolic syndrome including bmi, triglyceride levels and systolic blood pressure there was a positive relationship (7, 813). Aerobic exercise will reduce the rbp4 levels in menopause women with type2 diabetes, middle - aged women, and young women (14, 15). A study showed that in response to four week exercise training in a wide age range of patients, rbp4 level was reduced (15)., after three months of exercise training did not observe significant differences in the levels of rbp4 in korean women (16). As noted, obesity is one effective factor in increasing the levels of rbp4 in humans . Studies have also shown that the rbp4 level is much higher in obese people than in lean ones . Therefore, weight loss will reduce serum concentrations of rbp4 (3, 12). Studies have shown that a 5% weight loss, will lead to an increased insulin resistance index . Only slightly, the rbp4 gene will reduces fat tissue . And rbp4 level is not affected by this amount of weight loss (6, 12). Other studies have described that improvement in metabolism after exercise can reduce rbp4 levels (6). This also improves insulin sensitivity caused by exercise training in humans which are associated with lower levels rbp4 (17). Another study showed that a decrease in body fat caused by exercise, leads to a decreased level of rbp4 (14). According to the controversial studies on the exercise effect and rbp4 levels, few studies which have been done in this area, and because the exact mechanism of exercise effect on 4rbp reduction is not clear this study was designed . This study was confirmed in 2012 by isfahan university of medical sciences our institution ethics review board for human studies and participants signed an informed consent . Athletes, smokers, patients, and persons who used a weight loss method were excluded from the study . No change in body weight over 2 kg and with no endocrine disorders, diabetes, cardiovascular diseases and chronic conditions had been invited, and then justified . Then, participants were randomly assigned in to the experimental group (n=10) and controls (n=10) group . All the variables, including age, height (the stadiometer brand was seca, made in germany, with a sensitivity of 1 mm). Weight, body fat percentage, body mass index, waist - hip ratio (in body instrument, model 3, and the brand name of biospace, made in korea). The serum glucose level (glucose monitoring kit, colorimetric enzymatic method, pars azmoon trading company serum insulin levels (elisa sandwich kit, mercodia ab company, uppsala, sweden, with a sensitivity of lmu / l). Insulin resistance (based on using homeostasis model, and by implicating insulin and glucose concentrations were calculated). The level of hdl - c, ldl - c, tg, total cholesterol by the colorimetric kits from pars azmoon trading company made in iran was measured . Serum rbp4 levels were measured using the elisa sandwich kit from cusabio co., china, was used . The aerobic exercises consisted of warm - up exercises for 10 minutes (walking, stretching, and jogging) followed by principal aerobic activity . The task started by a maximum heart rate of 60 - 65% . In the first session, progress during the first week was based on 60 - 75% of maximum heart rate and timing reached to 25 - 30 minutes . In the third week, the heart rate reached to 75 - 80%, and timing to 35 - 40 minutes . This process evens lasted till the seventh week . In the seventh week, the heart rate was set to 80 - 85%, and timing to 45 - 50 minutes . This process lasted till the 12th week . At the end of each session, there was a slow cool - down time with stretching exercises which would last for 10 minutes . Blood samples were collected in two stages; 24 hours before the start of the first session (pre - test), and 24 hours after the last session (after 12th week), after overnight fasting and at rest, at 8 am . Each time, 10ml blood sample was collected in the sitting position, and from the left anterior vein . After 10 minutes samples incubation in room temperature, samples were centrifuged (10min, 3000rpm). The centrifugation was done to remove the serum from blood clots . Finally, the samples were frozen in -70c . Descriptive statistics was used to describe characteristics of the subjects, including age, height, weight, body mass index, body fat percentage, waist - to - hip ratio, serum glucose, and insulin, hdl - c, ldl - c, tg, tc and rbp4 . For variables normality kolmogorov - smirnov test was used . To evaluate within groups differences t - dependent test, and to calculate differences between groups the amount of weight, bmi, whr, and fat percentage was significantly reduced due to aerobic exercises (p<0.05). Individual characteristics of subjects in experimental and control groups change variables from pre - test to post - test in experimental and control groups the rbp4 level decreased significantly . Also, hdl - c, tg, insulin, and insulin resistance were also significant in comparison with control group (p<0.05). Ldl - c level, and total cholesterol did not significantly decrease after a 12-week aerobic exercise (p<0.05). Due to the results of this study, insulin, insulin resistance and rbp4 serum decreased significantly after aerobic exercise . Obesity increases the rbp4 level in human, while weight loss will reduce rbp4 serum concentrations (12). Studies have shown that rbp4 serum concentration is strongly related to waist circumference, body fat percent, and whr (17). It was also concluded that training exercises will reduce body fat or will keep the body weight stable (18). Based on the stated points, a period of aerobic exercise will decrease weight, bmi, whr, and body fat percentage . Lipolysis of triglycerides in upper body subcutaneous is much greater in comparison to adipose tissue triglycerides . Also, intra - abdominal adipose tissue lipolysis will increase during exercise (18). This was proved in the current study . According to the research results and relationship between rbp4 with fat percentage, and whr; after a period of aerobic exercise these variables showed significant differences . That is to say, the significant reduction in the concentration of rbp4 serum is depended on these variables . Endurance exercise increased hdl - c, but decreased triglycerides and individuals with higher levels of aerobic activity or a higher aerobic capacity, have a higher level of hdl - c, but a lower level of triglycerides (19). Because of the benefits of aerobic exercise; due to increased mitochondrial density, oxidative enzymes in muscle increase . Furthermore, the electron transport chain enzyme activity, the activity of enzymes involved in the oxidation of fats, especially oxidized beta - oxidation cycle enzymes, and also activity of lipoprotein lipase will increase . Increased capacity of fat consumption in the muscles that have been under endurance exercise, is because of their capability to transfer free fatty acids and their increased capacity to oxidize fats . The decreases in lipids that are because of aerobic exercises are related to weight loss, and decrease in subcutaneous fat . An example of change in metabolic system can be lipoprotein enzyme activity which creates lipase (16). In this study, hdl and triglycerides in the experimental group showed a significant difference that according to the above mentioned statements such results would be expected after a period of aerobic exercise . A study on triglycerides and hdl was carried out with lim et al . That showed consistent results in the young group . In other words, the reason for the difference between the older and younger groups in this research can be higher baseline levels of triglycerides and lower hdl level in the older group (15). Some of the mechanisms that can improve the insulin task after endurance exercises are: increased insulin signalling receptor, increased glucose transporter protein (glut4), glycogen storage capacity due to increased enzyme activity of synthase glycogen and hexokinase, increased release of glucose from the blood to muscle capillaries due to increased glucose uptake in muscle and changes in muscle composition, and decreased clearance and increased release of free fatty acids (21) in the current study aerobic exercises decreased glucose, while insulin level decreased significantly . We can conclude that insulin sensitivity increased, insulin resistance decreased and this increase and decrease were significant . Some of the studies have shown that a more decrease in abdominal fat will lead to insulin sensitivity . Fat (especially abdominal) with the production of inflammatory factors may play a key role in insulin resistance and metabolic problems associated with obesity (22). In the present study, whr, which reflects abdominal fat, was significantly lower in the experimental group, so this may be a factor to explain the reduction in insulin resistance . Exercise enhances insulin function by reducing the intracellular accumulation of triglycerides and fatty acid oxidation (23). In the present study, triglyceride levels were significantly reduced in the experimental group, so perhaps this is another reason for the decrease in insulin levels and insulin resistance in our study . It is important to note that the subjects in the study had a normal glucose level, and this can perhaps be a reason that it did not change significantly . The results with the findings of rose et al ., which worked on the effect of three to four months of aerobic exercise on fasting glucose levels in obese men and women with normal baseline glucose, was consistent . Rose et al . Showed that exercise had an opuscule effect on the fasting glucose level in healthy non - diabetic subjects (24). In one study, three months of combined activities (aerobic and endurance) showed no significant effect on glucose levels (16), which is consistent with the results of this research ., bloom and chang studies are inconsistent with the results of the present study (2527). Lack of a significant effect on insulin resistance perhaps was because of a small number of subjects in the study (25). This may be attributed to the intensity of exercise . In bloom and chang s investigation, perhaps the reason was intensity and duration of exercise (7 days of moderate - intensity aerobic exercise) (26). Furthermore, the results of the present study are consistent with previous studies (14, 28, 23). In one study it is also shown that the concentration of serum rbp4 is in relation with fasting glucose levels, and improved glucose metabolism after exercise relative to diabetes is the reason that rbp4 level was reduced (6). In the present study the fasting glucose level was not significant . But, because of the part that rbp4 level is related to insulin level and insulin resistance, perhaps the reason for the significant change in rbp4 level in this study is because of a decrease in insulin level and insulin resistance . The results of the present study are in line with what choi et al ., (2008) concluded . They investigated the relation of combined exercise (aerobic and endurance) in obese women . They did not observe any significant change in the subjects rbp4 level after three months . In the study of lim et al ., (2008), aerobic activity with 85 - 60% insensitivity of vo2 max, significantly decreased rbp4 level in the older and the younger group . The reason for the difference in rbp4 level among the younger groups could be related to the types of subjects . In the present study, et al . Study, (2010) the three groups naming control group, endurance training and aerobic training was existed . Compared to the others in the within group comparison the significant difference was in endurance group (29). These trainings will add to bulk but without adding any fat (14), and as it was stated rbp4 serum level has a strong association with bmi, body fat percentage, waist circumference and it will be decreased by weight loss (3, 8). The effect of aerobic exercises with the intensity of about 75 - 50% of pulse with rbp4 index was shown . The results of this study showed the beneficial role of aerobic exercise on metabolic syndrome factors, such as triglyceride, hdl - c, insulin, insulin resistance, and body composition . It is important to note that more control on some restrictions such as nutritional status of the subjects, emotions, and the number of subjects is necessary for more detailed assessments in further research studies . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
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, an estrogenic lack determines a condition characterized by the prevalence of bone reabsorption phase over the bone formation phase . Osteoporosis in postmenopause recognizes also low serum 1,25(oh)2d levels and a reduced intestinal transport of calcium, always due to the estrogenic lack . The reabsorption markers increase up to two times the values seen in premenopause, while values of bone formation markers increase only by 50% . The biochemical indicators of increased osteoblastic activity are represented by the serum levels of osteocalcin and of alkaline phosphatase, while the urinary levels of hydroxyproline are expression of an increased osteoclastic activity . In presence of an accelerated bone replacement, osteocalcin and alkaline phosphatase serum levels and urinary hydroxyproline excretion there is a wide debate concerning the effects of the treatment with l - thyroxin (lt4) on bmd . Thyroid hormones certainly determine an increase of both osteoblastic and osteoclastic activities over both cortical and trabecular bones [2, 3]. However, the data about the real impact that lt4 therapy exerts on bmd of women subjected to prolonged periods of treatment appear contrasting . In particular, an approach built on the preventive determination of the anamnestic profile considered at risk for bone demineralization in women undergoing lt4 use is lacking . Studies in vitro suggest that thyroid hormones increase more the re - absorption than bone formation, hence determining a loss of bone mass [8, 9]. It has also been reported how thyrotoxicosis increases the fracture risk in post - menopause women and in patients with low bone mass peak . Fractures are more frequent in those parts of the skeleton in which predominates the cortical bone, such as the hip and the distal part of the arm . A recent study has also shown that endogenous subclinical hyperthyroidism might be considered an additional risk factor for osteoporosis in postmenopausal women, especially for cortical bone, whereas exogenous subclinical hyperthyroidism has no effect on bmd . Post - menopausal women with nodular thyroid disease often undergo continuative and prolonged treatment with lt4 administered at suppressive dosages . Nodular thyroid disease and osteoporosis share common factors: (a) both are present with an elevated frequency in the general population; (b) they are more prevalent in the female sex; (c) the incidence increases with age . Since increased levels of thyroid hormones may contribute to bone demineralization . The aim of this study was to evaluate the effects of treatment with a fixed dose of lt4 on bmd in physiological post - menopausal women with normofunctioning nodular thyroid disease, compared to women, the same average age, who did not receive lt4 . To examine the effects of the treatment with lt4 on the bmd, 110 (range 5055 years, average age 53), natural post - menopausal patients with normo - functioning uninodular or multinodular benign thyroid disease were enrolled, after informed consent, if they: had last regular menstruation less than 5 years before;had a normal t - score (1 sd);did not have secondary causes of bone demineralization (genetic, endocrine - metabolic, osteoarticular, hematologic, neoplastic, gastrointestinal, drug administration, and/or prolonged immobilization);did not receive lt4 treatment in the previous two years;did not have a history positive for vertebral fracture . Had last regular menstruation less than 5 years before; had a normal t - score (1 sd); did not have secondary causes of bone demineralization (genetic, endocrine - metabolic, osteoarticular, hematologic, neoplastic, gastrointestinal, drug administration, and/or prolonged immobilization); did not receive lt4 treatment in the previous two years; did not have a history positive for vertebral fracture . Following preliminary evaluation, all patients were prescribed lt4 at the fixed dose of 1.6 g / kg / die . Exclusion criteria surgical menopause, hormone replacement therapy in the last two years and autoimmune thyroid disorders . Surgical menopause, hormone replacement therapy in the last two years and autoimmune thyroid disorders . Fifty women of the same average age (range 5055 yrs, average age 53.5), having their last menstruation less than 5 years before (last referred menstruation), with normal t - score (1 sd) and not receiving lt4 . All patients underwent evaluation of bmd by dual x - ray absorptiometry of the lumbar vertebrae (bone densitomiters gammadensit x - ray, l'acn scientific laboratories; cerro maggiore (mi) italy) before lt4 treatment was begun (t0) and 2 years after treatment (group a) and/or followup (group b) (t2). The serum levels of tsh (eclia, roche diagnostics, monza (mi), italy), ft4, ft3 (eclia, roche diagnostics, monza (mi), italy), abtg and abtpo (eclia, roche diagnostics, monza (mi), italy), were evaluated at t0 and at t2 . All the patients enrolled underwent measurements of calcium serum levels (colorimetric method; roche diagnostics, monza (mi), italy), 24 h urinary calcium concentration (colorimetric method, roche diagnostics, monza (mi), italy), serum alkaline phosphatase (ap) bone isoenzyme levels (enzymatic method; metra tm bap eia) and 24 h urinary hydroxyproline concentration (colorimetric method; lta; bussero (mi), italy) at t0 and t2 . The results are reported as mean sem throughout the study . According to lt4 therapy, patients were divided into 2 groups: a group which received lt4 therapy (group a) and group which did not receive any treatment (group b). The statistical analysis was performed using the student t - test for paired or unpaired data, as suitable . The odd's ratios of the risk factors in relationship to bone mineralization loss were determined . The patients enrolled had a mean age of 53.4 years (range 5055 yrs). As expected, the treatment with lt4 caused a significant reduction of tsh serum levels (p <0.0001) at t2 . At t0, the group a patients had a mean t - score of 0.22 0.07 which decreased significantly to 1.02 0.10 at t2 (p <0.001). The total calcium concentration in serum and 24 h urine after treatment with lt4 did not change significantly in comparison to the pretreatment values (tables 1 and 2). The serum levels of ap and the amount of hydroxyproline excreted with the 24 h urine increased significantly (p <0.0001) during treatment with lt4 (tables 1 and 2). The impact of some risk factors for osteoporosis was evaluated on the bmd of the patients treated with lt4 . The results of this analysis are shown in table 3 . A positive history for osteoporosis, smoking habit, and length of menopause did not give a significant odd ratio . In contrast, the following risk factors turned out to influence in a statistically significant manner the bmd: bmi <19 kg / m, the onset of menarche after the age of 15 years, a positive history for periods of prolonged amenorrhea, nulliparity . The results of this study showed that patients with nodular thyroid disease treated with a fixed dose of lt4 have a slight, but highly significant reduction of the bmd after two years of treatment . Forty - one out of 110 women (37.3%) had osteopenia at t2 (tra 1 e t - score 2.5 sd); in the control group osteopenia at t2 occurred in 6% of patients . The loss of bmd appeared mainly related to the following factors: (1) bmi <19 kg / m; (2) menarche onset after the 15th year of age; (3) a clinical history positive for periods of secondary amenorrhea; (4) nulliparity . In parallel with the modifications of the bmd after two years of lt4 treatment, it was also observed an increase in 24 h urinary hydroxyproline concentration, suggestive of increased osteoclastic activity, and increased serum ap levels, an index of increased osteoblastic activity . Altogether therefore these data suggest that lt4 treatment increases bone metabolic turnover, with prevalence of the re - absorption phase . The results of this study, therefore, favor the hypothesis that the treatment with suppressive dosages of lt4, frequent in the clinical practice of women in menopause with nodular thyroid disease, is a risk factor for the progression of bone demineralization . In particular, women with the following predisposing risk factors: thinness, delayed menarche, periods of secondary amenorrhea during the reproductive age, nulliparity, are likely to undergo a more profound negative impact of lt4 treatment on bmd . Contrasting results have been published about the role played by lt4 treatment on the bmd . The discrepancies between the various studies are determined by methodological bias, such as (a) progressive reduction of the lt4 dosage prescribed to women which by itself may justify a reduced risk for bone loss; (b) inclusion of patients with a clinical history positive for hypo- or hyper - thyroidism which has been reported to impair bmd; (c) differences in the duration of the treatment; (d) heterogeneous methods and site of bmd evaluation; (e) lack of data collection relative to the role of other possible interfering factors; (f) inclusion of pre - menopausal women; (g) unmatched controls (for body weight, age at menarche and at menopause, calcium dietary intake, smoking habits, alcohol intake, exercise, etc . ). A meta - analysis study, published in 1996, reviewed 41 controlled cross - sectional studies, including about 1250 patients, concerning the impact of thyroid hormone therapy on bmd . Suppressive lt4 therapy was associated with significant bone loss in postmenopausal women (but not in premenopausal women), whereas, conversely, replacement therapy was associated with bone loss in premenopausal women (spine and hip), but not in postmenopausal women . The detrimental effect of thyroid hormones appeared more marked on cortical bone than on trabecular bone . We will briefly review the major studies appeared in literature afterwards which can be divided into those showing a detrimental effect of lt4 on bmd and those showing no such an effect [1224]. A careful selection of a group of post - menopausal women with thyroid nodular disease under treatment with a fixed doses of lt4 allowed us to show that this treatment causes a slight but significant reduction of bmd . In addition, bmd reduction was associated with the following osteoporosis risk factors: bmi <19 kg / m, the onset of menarche after the age of 15 years, history positive for period of amenorrhea, and nulliparity . In consideration that lt4 treatment is effective in a low percentage of patients with benign thyroid nodules, estimated to be 1020%, a careful benefit / risk evaluation has to be taken into account before lt4 treatment is prescribed, particularly when other risk factors for bone demineralization are present . The present study confirms comments from our previous article, conducted on 99 post - menopausal women, all of them receiving lt4, prospectively evaluated after one - year treatment . In addition, to previous data, the present study consider an homogeneous control group, for average age and clinical features, including only physiological menopause limited to 5-year history (last menstruation). It also increase to two - year observation length of women receiving therapy, allowing a better understanding of the phenomenon . Moreover, this study excludes conditioning anamnestic factors such as: surgical menopause, hrt, andautoimmune thyroid disorders.
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The seemingly inert bone of vertebrates is, in reality, a living tissue that retains adaptive capability throughout the life - span . Transformation of bone in response to alterations of its mechanical environment is evident in numerous examples, e.g. From the bone loss observed during space flight and bed rest as well as their recovery . Such adaptive modifications of bone are often engendered by alterations in geometry, both during immobilization as well as in sport - specific responses to increased loading[8 - 10]. However, even when there is no net gain or loss in bone mass, it is evident that a continuous process of bone renewal takes place, which is often called remodelling . This process involves resorption of bone plus subsequent formation, and it is thought to be partly of random nature and partly specifically targeted to repair tissue damaged by micro - cracks11 . Disorders of these mechanisms are held responsible for bone disorders such as osteoporosis and osteogenesis imperfecta, and a deeper understanding will be a prerequisite for effective therapeutic strategies . It is conceivable that these mechanisms also relate to the bone loss of astronauts, and that understanding them will enable the design of effective countermeasures . In principle, mechanically driven adaptive responses of human bone had been observed and described early by wolff, later known as wolff s law of bone transformation, and it was darcy thompson who coined the principle form follows function. Pauwels explained quite well how load forces could determine certain characteristics of the geometry of human long - bones . Frost proposed the mechanostat theory to explain bone adaptation as a feed - back control mechanism . His three - way rule that proposes shifts of the bone surface depending on different on - off criteria explains the observations in principle but does not yet have a quantitative mechanistic background . Modelling and remodelling, with the former describing the shaping of bones and prevailing during growth, and the latter describing the self - renewal of bone that prevails during adulthood . It has been recognized that these processes do not occur simultaneously, and the idea therefore emerged that they are biologically distinct . Evidence suggests that osteocytes, living cells embedded in the bone matrix, play a crucial role in mechano - sensation and transduction of the mechanical signals . Another finding was that dynamic bone loading (i.e. Incorporating frequent load changes) is more effective at inducing bone adaptation processes than static loading . With this background in mind huiskes and co - workers created a finite element analysis (fea) based computer model for remodelling processes within trabecular bone that used a mechanistic approach with a strong cell - biological background of bone adaptation . In their remodelling law they assume the osteocytes in the bone matrix to be the main control element for the recruitment and bone generating activity of osteoblasts . They are postulated to take strain energy density as the leading regulatory parameter for the mechano - transduction . Early studies have looked upon the diaphysis of long bones and their optimal shape studying interspecies diversity and investigating the adaptive response of different load types like compression, bending, and torsion on the cross section with numerical methods[23 - 25]. However, these studies have accepted a tubular shape a priori . The influence of axial torsion for the adaptation of cross section parameters was in most cases found to be limited . It is a known fact in orthopaedic surgery that bones can remedy their misalignment after fracture (so - called flexure neutralization). However elegant the past studies on bone shaping are, they do not have a mechanistic explanation for it . It is not understood whether bones look the way they do because they are used the way they are used, or vice versa . Or, in other words: whether form follows function or function follows form, and to what macroscopic extent bone is a self - organizing tissue is yet to be established . Of course, there might be also a genetic background, but overwhelming evidence suggests that function can explain the form to a large extent . The goal in this study is to link tissue - level adaptive responses to whole - bone shaping in order to understand why bones are shaped the way they are . For this we are focussing on the basic principles responsible for reshaping of bone - like structures in response to loading . The present approach uses computer modelling to perform experiments of thoughts on a simplified level . The algorithm originally proposed by huiskes for the remodelling domain has been adapted in this study, in order to show that the principles are the same for modelling and remodelling . In order to foster that vision and to avoid confusion with the historical terminology we use the term transformation for both modelling and remodelling in this paper . As a first step, we describe a bone shaft analogue both in terms of physiologically - motivated boundary conditions as well as in terms of code implementation . We hypothesize that a bone shaft- analogue load pattern will generate a straight tube - like compact structure from different precursor geometries, showing that the straight tube in full three dimensions - that obviously seems to be the optimum structure - is supported by the adaptation laws on microbiological level, and that it is not only an adaptation of an existing tubular cross section . If the straight tube is the preferred geometry, then one should expect (as our secondary hypothesis), that flexed precursor geometries should be straightened as suggested by flexure neutralization in clinical observations . Both hypotheses presume that form follows the function of bone and bone parts, and therefore loads are one of the main drivers of bone anatomy . The in silico transformation cycle used here assumes that mechanic loads are guiding the transformation process . The cycle (figure 1) starts from a given model geometry, where a random load pattern comparable to those acting on the diaphysis of long bones is then applied . Next, the resulting stress distribution within the structure is calculated by means of finite element analysis (fea) using the commercial fea software ansys . The distribution of strain energy density rate in the volume elements of the model is the control parameter of this mechanostat and is the input for the transformation algorithm, which results in a modification of the model geometry . With this modified geometry the geometry models were defined within a design space, which is structured by a regular cubic voxel mesh with an edge length of 75 voxels . The housing mesh size was 4.5x4.5x4.5 mm, bringing the voxel length to 0.06 mm . The elements (voxels) in this mesh were either defined as isotropic bone material with a compact bone - like young s modulus, or of non - bone character with young s modulus two orders of magnitude less . Following huiskes example, structures similar to a primary spongiosa were defined as regular grid inside the cubic design space with a regular and homogeneous pattern of bone . In this approach therefore the design space was selected to be only slightly larger than the objects to be analysed . A total of four different starting geometries was used (see figure 2). In one direction (z - direction) the geometries extended over the entire design space, while in the other directions the geometries did not touch the design space edges, in order to leave space for apposition . The following starting geometries were used: starting geometries; for all four different starting geometries cl, bl, fl and ft a 3d - view (+ y/-z - view, x - axis horizontally) together with cross - section representations in the xy - plane (top) and in the xz - plane (bottom) are shown (x - axis horizontally): cl a cylindrically shaped bravais lattice, bl a cuboidal shaped cubic bravais lattice (box lattice), fl a cubic bravais lattice in form of a flexed cylinder and ft a flexed tube; find more explanations in the text . Cl: a cylindrical shaped cubic bravais lattice with the axial length of the full design space size of 4.5 mm and a width of 3.38 mm . Bl: a quadratic cuboid sized bravais lattice (box lattice) with the length of the full design space size of 4.5 mm and diameter of 2.25 mm . We intentionally chose smaller dimensions in comparison to geometry 1 in order to get an idea, if there might be an influence on the final dimensions . Also the unit cell of the lattice has been modified (see explanation below). Fl: a flexed cylindrical bravais lattice with the length of the full design space size of 4.5 mm and a diameter of 3.38 mm . The degree of flexion was sufficient to let the flexed geometry occupy the whole design space in a mediolateral direction . Ft: a flexed cylindrical tube with the length of the full design space size of 4.5 mm and a diameter of 3.38 mm . With this geometry we want to find out if flexure neutralization is also possible starting from flexed compact structures . The unit cell of our cubic lattices in cl and fl is a cube of an edge length of 8 voxels (= 0.48 mm) with beams of 2x2 voxels at all edges building a cubic frame . Putting together that kind of unit cells, a cubic lattice of quadratic beams of width 4 voxels (= 0.24 mm) is generated . The unit cell of bl is different in having a width of 6 voxels (0.36 mm) with one voxel sized beams at all edges building a lattice with thinner beams of 2x2 voxels (0.12 mm) width . In all cases we used a template lattice generated in matlab and matched that lattice into the mesh of our fe model using ansys standard functionality . The irregularity was assumed not to influence the simulation results, because for one type of starting geometry always the same lattice was used for all runs . The value for young s modulus of 15000 mpa for cortical bone was in line with our reference and similar to the observed range from 16000 to 19000 mpa for rat cortical bone . For rat cortical bone poisson numbers between 0.3 and 0.42 were found, so we took 0.36 as a mean for our simulations . For simulation of mechanical loads the geometry models were implemented in the finite element simulation software ansys (ansys germany gmbh, darmstadt) version 12.1 . In this numerical model each voxel is represented by a brick element with quadratic shape function . For the load calculations the geometry s bottom and top surfaces (with respect to the z - direction) the bottom plate was fixed in three directions, and the surface of the top plate was subjected to different types of loads such as axial compression, varying lateral bending and torque (details are given in table 1). The scripting capability of ansys workbench has been used to modify both the external force vector as well as the torque vector of the ansys-moment in subsequent calculation steps in order to simulate the variance of the load pattern (figure 3). Values for ktr are given as well as step number per run and starting geometry . Every run has got an identifier (i d) that indicates its starting geometry . See figure 2 for explanation of the acronyms cl, bl, fl and ft . We estimated the rat load conditions by scaling down known load conditions of the human case, because measurements of real loads on these animals in vivo are incomplete in the literature . From force measurements in smart knee prostheses there we find maxima of 350% body weight (bw) for axial load (knee load in z - direction ~ axial load on tibia), some 2% bwm torque around z - axis (can be taken as the axial torque on the tibia) as well as shear forces in the knee up to 40% bw (can be taken as bending forces for the tibia). For a rat of 1 kg we can scale that to 35 n axial load, 4 n bending load and 0.2 nm (200 nmm) axial torque . For running and jumping the loads will be much higher, especially the bending load, because higher dynamic forces will come into effect . Therefore, as depicted in figure 3, a constant major compressive force in axial direction with a magnitude of 100 n was applied on the rigid top surface and modulated by a smaller component in lateral direction . The lateral force was taken as random in direction with a constant magnitude (40 n in all cases, see details in table 1). In figure 3 the resulting force sum on the top plate is depicted as a black arrow, the dashed circle visualizing the random direction in 360. in order to study the influence of lateral bending components that are non - uniformly distributed we narrowed the possible random direction down to a 90 segment in two of the runs . The magenta arrow in figure 3 represents an additional random torque around the length axis of the model . At any step it can assume any value between a maximum clockwise and counter - clockwise magnitude (uniformly distributed). The absolute values of the maxima in both directions are identical, comparable in magnitude to values for rat long bones above . In all cases where torque was present, 180 nmm as the maximum magnitude (see table 1 for details) was taken . The transformation law has been adopted from huiskes et al . And simulates the modification of bone density m (value between 0 and 1) in terms of the bone - generating activities of the osteoblasts and the bone - resorbing activities of osteoclasts . Following huiskes et al ., strain energy density (sed) rate was taken as the leading mechanical signal that activates the osteocytes . In the following the algorithm proposed by this group is described: the modification of bone density mtot at time t and location x is given by positive contributions of osteoblasts mbl and negative contributions of osteoclasts mcl: the osteoblast contributions are given by: for p(x, t)> ktr, disappears for p(x, t) p is a trigger signal at a surface position and depends on the distances between osteocytes (given by f(x, xi)), the sensitivities of the osteocytes (given by i) and the sed rates (given by r(xi, t)). As can be seen from equation (2), there is a threshold parameter ktr that makes sure, that the system only reacts on p - values that reach a certain level . R(xi, t) calculates from s(xi, t) as given in equation (4), the sed and a load frequency of f, the latter taken as 1 for typical walking - like load pattern: this formula results if we look at a load e(t) oscillating with frequency f, taking values between 0 and a maximum value . If we follow the derivation of ruimerman we can calculate the maximum sed smax: the corresponding maximum sed rate can be calculated as assuming that a single step corresponds to several days in reality and our s(xi, t) is the smax of the oscillating load pattern during that time, we can take over formula 4a for s(xi, t) and analogous equation 4b for r(xi, t). Comparing both equations we calculated p(x, t) for up to seven shells of voxels neighboring a surface voxel . For this calculation we had to take into account the number of osteocytes per voxel that we took as 44000 per mm as for human cortical bone, the number of voxels and the number of osteocytes per voxel defining the total number of osteocytes (the n of equation 3) taken into account . From m(x, t) the young s modulus e(x, t) can be calculated as emaxm with in our case emax= 15000mpa and =3 (see reference). For the resorption give: where rcl is a statistical value, wherein the recruitment rate of osteoclast cavities (in [1/voxel / day]) on the surface (focl) and the resorbed volume per cavity (vr) play a role . We calculated the voxel resorption rate as: with vvox the voxel volume . For better comparability with the huiskes calculations we use a correction factor ccorr= (dx / dxhuis), where dx, dxhuis are the voxel sizes of our model and the huiskes reference model . Because only surface voxels are involved, we have to correct huiskes focl parameter (recruitment rate of bone modeling units bmu) for our differing voxel sizes in two dimensions (always looking at the surface). We did not take into account coupling between osteoclast and osteoblast - activity and assume a random distribution of osteoclast activity over the whole surface . The transformation cycle was implemented in matlab (mathworks inc ., natick, mass . Strain energy densities resulting from the simulated load applications were calculated using fea based on the commercial package ansys, using a preconditioned conjugate gradient (pcg) solver . Calculations were performed on a high performance computing cluster (ibm x3550 and x idataplex computers). For each run, between 200 and 400 iterations were calculated before we could be sure, that the forming process had converged . Each calculation step (a single loop of the transformation cycle) took around 60 minutes . In pre - calculations it had been found that the given threshold ktr had to be adapted significantly in order to achieve an activation of the process . When varying the magnitude of ktr it appeared that the value of this parameter has a big impact on the final geometry in dynamic equilibrium . To better understand the underlying systematics, we performed several additional computational runs in order to check the sensitivity for this parameter . As a result for further simulation experiments we selected a setting of ktr=0.05 nmol/(mmday), because this value provided a good tubular shape in the basic load configuration . A total of eight runs was performed with the cylindrical lattice (cl) as starting geometry (identifier clx with x=0 .7). In five of them (cl0 to cl4) in run cl5 we set the torsional load to zero with all other parameters unchanged . In run cl6 we kept the bending load in a narrow angle of 90 degrees (into the quadrant of positive x- and y - direction) in order to see any influence of directed bending . In run cl7 we combined directing bending forces as in cl6 with missing torsional loads . In all cl - runs 200 steps have been calculated except for cl4, where we extended to 400 steps in order to demonstrate the stability of the final geometry . In addition two runs with the box lattice (bl) as starting structure (bl1, bl2) have been performed to investigate the dependence on the starting geometry . Again one of them was without torsional load (bl2). In order to investigate the flexure problem four runs have been calculated, two starting from the flexed cylindrical lattice (fl), the other two starting from the flexed tubular structure (ft), in both cases once with torsion and once without . Table 2 shows an overview of the parameter settings in the huiskes algorithm that have been used in all calculations . Constant settings of important parameters of the huiskes model (equations 2 - 4); with given dimensions, the r(xi, t)- resp . The s(xi, t)-values from the fem results have to be entered in gpa . In order to be able to compare to earlier investigations on bone shaft geometries we calculated the radius r of the final shaft geometries as well as the parameter r / t, with t being the thickness of the corticalis . For convergence checking several indicators have been calculated for the mid - shaft slice (at 2.25 mm in z - direction) that give an idea of topological modifications with time . The indicators were: number of bone voxelsmean radiusstandard deviation of bone voxels from mean radiusthe two principle moments of areaoptically assessed shapedeviation index i d number of bone voxels standard deviation of bone voxels from mean radius the two principle moments of area optically assessed shape the deviation index i d was calculated using formula (8). With e(t1,x, y) and e(t2,x, y) being the young s moduli at two different times at the same position x, y of the mid - shaft slice . In the tables of figures 4a - c results of our computations are presented as 3d - visualizations and as silhouettes of a mid - shaft cross - section on the top right and an axial cross - section on the bottom right in the 3 column . For comparison the starting geometry is shown as well in 3d as in the cross - sectional silhouettes in the 2 column . Bone voxels that survived from the beginning, the blue ones being voxels that have been generated during the simulation . The two remaining columns show the outer radius and, for comparison with literature, the r / t value at mid - shaft only for cases where a regular tube can be identified at mid - shaft . Because of the rough surface of structures the measurement of diameters and wall thickness could not be very precise, thus we provided an estimate for the variation . Results for runs cl0 to cl4; same presentation as in figure 2 with starting geometries always a 3d view together with two crosssections; original voxels in green, new voxels in blue . Results for runs cl5 to cl7 and for runs bl1 and bl2; same color code as in figure 4a . Results for runs fl1, fl2, ft1, ft2; same color code as in figure 4a . Quick convergence was found after about 200 calculation steps for all lattice - type starting geometries . Figure 5 shows as an example the run cl4 to demonstrate that within about 100 to a maximum of 200 steps the system has reached its final geometry . The characteristic parameters have attained a level that will be kept more or less stable (radius, standard deviation of radius) or with a small drift (moments of area and bone voxel number). It can be easily seen, that the moments of area vary in phase with the voxel number . The id - value converges quickly to a quite low but non - zero value . Monitoring of parameters of slice 37 (mid - shaft) during an ultra - long run of 600 steps of experiment cl4 as a function of the step number; lower part: number of bone voxels (green), mean radius (blue) and its standard deviation (magenta); upper part: deviation index (black) and the both principle moments of area (light blue and brown); in addition silhouettes of cross - sections at step 0, 100, 200, 300 400, 500 are given . Looking into the results of the different runs, a strong shape - imposing influence was found for the threshold values ktr . As can be seen from equation 2, this threshold defines a minimum value, below which an integrated trigger signal on a surface position does not activate bone formation . First calculations with a high setting for this value provided truss - like final structures . Systematic variation in ktr in this study yielded a close relationship between this parameter and structural properties, which can be seen easily by comparing the final geometries of the runs cl1 to cl4 in the table of figure 4a . From a closed straight tube for ktr=0.05 nmol/(mmday) the structure opens up and broadens more and more at the ends of the tube with increasing magnitude of ktr and finally grows into the open truss - like form at ktr=1.2 nmol/(mmday). The beams of the truss seem to become more filigree with further increasing magnitude of ktr . Looking more closely now at cl4, where we used the low value for ktr, we observed the conversion of our spongious starting geometry to a closed and straight tubular structure with a clear corticalis . (see table in figure 4b) we run the same conditions without the axial torsional load component . As it can be clearly seen, the final geometry is no longer tubular, but has the form of a truncated cone . The mid shaft cross section has smaller diameter and an irregular shape compared to the torsional loaded run . Cl6 and cl7 (see table in figure 4b) show the effects of introducing a strongly directed bending load towards one of the quadrants . The quadrant in question is marked by a red angle in the mid shaft cross section representation . It can be seen that with torsional load in cl6 we got a geometry that is not far from that of uniform distributed bending in cl4 . There is a slightly increased thickness of the tube wall in this quadrant, but the effect is much less than could be expected . Here we have a completely different picture - no tubular geometry at all, indicating an important role of torsion for maintaining the tube . In bl1 (results in table of figure 4b) we used the same conditions as in cl4, but started from the quadratic (instead of a circular) cross section of starting scheme bl with quite a small diameter in comparison to the start setting of cl4 . From a cuboid starting lattice we again got a tubular geometry the final outer diameter was only slightly smaller (1.6 instead of 1.7 mm). The difference can be found in the much thicker tube wall resulting in a r / t value of 1.6 . Bl2 that started with same conditions as bl1 but, analogue to cl5, without torsional load showed a similar behavior as cl5 (see table in figure 4b). From our flexed starting geometries fl1, fl2, ft1, ft2 we extracted the following results shown in table 3c: fl1 (the flexed spongiosa as initial geometry), after 200 steps, results in a regular straight tubular structure that is very similar to that of the comparable cl4 run, which started in unflexed configuration . For ft1, which started from a flexed tube configuration, the effect and the resulting geometry is a straight tube as well, but a bit less regular as with the spongiosa based fl1 . When torsional loads are switched off we find, when starting from a spongy structure (example fl2), a final geometry very similar to the results of runs cl5 and bl2, namely a truncated cone . We do not get a truncated cone - shaped structure which we may have expected when comparing it to the flexed lattice case fl2 . There seems to be a trend to narrow the upper diameter . But it can be stated in any case: a closed straight tube does not develop . Different from the smooth surfaces of long bones in nature the surfaces of all of our final geometries show a considerable degree of roughness . It is the principle goal of this publication to establish an in silico platform for testing of bone transformation laws and to use it to identify possible mechanisms and laws on the mesoscopic level that are able to explain the phenomenological laws established by frost from a mechanistic point of view . As the first study in this context the present study tried to explain common geometrical transformations of bone (e.g. Spongy to compact as in the metaphysis during growth, or flexure neutralization) mainly on the basis of a transformation law that has been proposed for the mechanically - driven remodelling of trabecular bone . Investigating principles in an experiment of thought, we focus on a construct that has sufficient similarity to the shaft of a long bone of a rat from the viewpoint of load characteristics . Before discussing the results let us first come back to general aspects and some differences of the present model approach with respect to the frost mechanostat . Due to the bottom - up approach that we used in order to study bone as a self - organizing tissue, our simulations did not take into account a merit function or any other the fact that even in the absence of such constraining assumptions, our simulations found r / t - values that are very similar to those reported by currey and alexander for land mammals may add to the applicability of our results . We are aware that the transformation law under investigation has been proposed only for adaptation processes based on bone basic multicellular units (bmu) next to marrow at trabecular and endocortical surfaces . In this study it was applied generally for all surfaces including the periosteal ones, so that we reduced the two different mechanisms of frost (modeling and remodeling) to a single mechanism . The model relies on a steady bone absorption like a thermostat that only needs one set - point in a cold climate, because there is always heat dissipation and no active cooling is required . It is astonishing how already this single - set - point set - up can manage an effective mechano - adaptation . In our future investigations, it will certainly be important to have a closer look whether this set - up can also explain the findings of a lazy zone and if that is not the case to identify a set - point mechanism for the active cooling (meaning active reduction of bone mass in case of disuse) analogous to bone . Resuming the results we find the following: in line with our main hypothesis the typical load pattern assumed for long bones in combination with a generalized modelling / remodelling transformation algorithm based on reasonable assumptions on bone physiologic principles supports the formation and maintenance of regular and straight tubular structures.in line with our secondary hypothesis this kind of formation of regular straight tubular structures is independent of the starting geometry and therefore also holds for flexed geometries, leading to flexure neutralization.the findings 1) and 2) are valid under certain boundary conditions: a) the set - point - values used in the calculation are in a well - defined range, otherwise truss - like open structures arise; b) axial torsional loads have to be present; otherwise a variety of geometries form that are not straight or tubular.bending loads into a selected direction do not deform the tubular shape very much . The thickness of the wall is thicker on the loaded side . Again this statement loses its validity if torsion is missing.the independence of starting geometry of finding 2) holds for the tubular shape, but not for the dimensions of the resulting cross - sections with respect to radius and wall thickness.rough surfaces develop in our simulations in comparison to smooth surfaces in reality . In line with our main hypothesis the a generalized modelling / remodelling transformation algorithm based on reasonable assumptions on bone physiologic principles supports the formation and maintenance of regular and straight tubular structures . In line with our secondary hypothesis this kind of formation of regular straight tubular structures is independent of the starting geometry and therefore also holds for flexed geometries, leading to flexure neutralization . The findings 1) and 2) are valid under certain boundary conditions: a) the set - point - values used in the calculation are in a well - defined range, otherwise truss - like open structures arise; b) axial torsional loads have to be present; otherwise a variety of geometries form that are not straight or tubular . Bending loads into a selected direction do not deform the tubular shape very much . The independence of starting geometry of finding 2) holds for the tubular shape, but not for the dimensions of the resulting cross - sections with respect to radius and wall thickness . Thus, and in line with the initial hypothesis, mechano - adaption occurred from different starting conditions - spongy, non - cylindrical and flexed geometries, implying that load patterns typical for a bone shaft evoke the well - known tubular structure with closed compact bone in the cortical area . In the first instance other authors in the literature have taken this principal geometry as given and looked on dimensional variations, or investigated variations of the cross sectional geometry . In this study form follows function principle and assumed load as the main shape defining and maintaining entity . It is a more surprising outcome that the presence of axial torsion was crucial, not to say required in order to achieve realistic shaft structures . In the literature torsion normally plays a minor role . In the work of mittlmeier26 the authors found out in a computational study that torsion cannot explain deviations from the ideal tube . Indeed torsion does not explain deviations, but it seems to ensure that the base geometry is the tube . If one takes tubular shape as given and only looks on a thin cross section, as those authors did, then you will find no influence of torsion . This is underlined when looking on the cases of this study where directed bending is involved . Here we find only a minor impact on the thickness of the tubular wall when torsion is in place, but break - down of the tube without torsion . Others came to very similar results when they simulated the development of a long bone cross section when directed bending was effective . The tube shape was lost if not compensated by taking the degree of calcification of the bone tissue into account . We do not see other computational studies where the influence of torsion has been an issue . The reason for that might be that torsional loads in long bones could not be measured very well in vivo and therefore have been neglected or taken of minor influence . It is only recently that data have been obtained from smart prostheses after knee replacement that show that axial torques are present . These values have been taken as reference for our own simulations (see above). A recent experimental in vivo study from our group has yielded that also in healthy young people axial torsion of substantial magnitude is present during locomotion . If we assume that the anatomy of bone is optimized for the load it experiences and want to learn why it has this shape we have to look closely into the regimes in our model where we find deviations . Beside the missing torsion we find a high value of the threshold parameter ktr as a possible reason . At first glance that seems to be a more technical aspect of the simulation, but we have to be aware that the parameter ktr plays the role of the modeling set - point in the sense of the frost mechanostat because it rules the onset of bone adsorption . In our model it also seems to be an important determinant for bone shape building . The higher the set - point with respect to the magnitude of the loads or in reverse the lower the forces with respect to the set - point the more the system seems to prefer deviation from the ideal tube to truss - like open geometries . Such structures are often found in engineering and structural biology (e.g. Trabecular bone material), but are not typical for a complete bone . . If there are small local differences in the stress pattern, the slightly higher stressed areas will be very selectively reinforced . Reinforced areas draw additional load and reduce stress in other areas so that this differentiation is amplified . If we translate this virtual phenomenon into relevance for real bone adaptation processes we should consequently expect that disuse based osteoporosis in a long bone shaft should tend to be irregular in the sense that areas already weakened are candidates for enhanced bone reduction . But of course at this point we should not forget that we have simplifications in our models . For another open question our set - point phenomenon might be relevant as well, namely the question why spongy (resp . Is it only because other types of forces, especially shear forces, are becoming dominant there, or is it because we are in a lower force regime with respect to the set - point? The truss - structures that result from the structure optimizing process of remodeling strongly resemble principles that have been applied in engineering for many years as method of biologic growth in computer aided optimization (cao), the latter being derived from principles learned from tree growth . It is not really a surprise that what is good for nature in finding optimized structure in tree growth could also be a major principle in bone growth and adaptation . We have found that under the given load conditions, if torsion is present and the set - point is in the right range, straight tubular geometry is achieved independently of the starting geometries be it a cylindrical or a cuboid lattice or even a flexed lattice or tube . Especially, the straightening - or flexure - neutralization - could not be expected . In his classic experiment of thought, frost had argued that the mechanostat theory alone cannot explain flexure - neutralization, and he had therefore partly waived his theory and postulated a it is interesting, therefore, that taking the mechanostat idea to a truly quantitative level the mechanostat can be reconciled with flexure neutralization . With this finding we are in line with the outcomes of roberts et al . In their computational study using a different contour based simulation approach . This states that the straightening of askew healed bone in fast modelling and remodelling bones as seen in the bones of children can be explained as driven by load, thereby suggesting that the guiding influence is bone adsorption to bone surfaces near high strain energy areas . Looking at parameters like radius of the tube or thickness of the wall we find differences depending on starting conditions . While the radius is quite similar, the c / t value starting from cuboid lattice with smaller diameter is with 2.0 significantly smaller than the values of the other runs starting from other lattices lying around 2.4 . . Why do we see such rough surfaces in our final structures, rather than the smooth kind of surfaces seen in nature? This is equally true for the endocortical as well as for the periosteal surfaces, which is rather unsurprising as the mechanisms in our model are identical for both . This is related to the fact that the simulation can only adapt by adding and removing little cubes . By chance the volume of the voxel in the present simulation (2.2 * 10 mm) is quite similar to the vr, the typical size of a bmu cavity (1.5 * 10 mm). However, in reality the bmu cavities are not cubes, but flat and elongated pits . In addition the simulation finds its position completely randomly on the surface, so it might happen that a voxel is taken away where there is already a pit in existence, leading to ragged surfaces with pits up to 2 voxel sizes deep . In principle, this could be improved by much better resolution both in space and time, as both temporal and spatial resolution are quite coarse in these simulation experiments . In this sense a smoother surface can be expected when enhancing the grid resolution to below that of the bmu size . The temporal resolution of our model (30-days per step) only allows for resolving the delta in bone adsorption and resorption during an entire bmu activity . Modelling resorption in smaller time steps will undoubtedly lead to smoother surfaces, but would by far exceed current computing limitations . Thus, in conclusion, the present exercise, although initiated as a first approach, has yielded interesting results in that a remodeling law without any specific assumptions on surface - organization like in the three - way rule can explain mechano - adaptation in terms of compactization, corticalization, circularization, and flexure neutralization . Of the many variations possible in our computer model, we have identified appropriate setting of ktr, the threshold parameter of bone formation, as well as application of torsional loads as powerful stimuli . This outcome makes us believe that numerical implementation of mechano - adaption is a fruitful field of research, and it is intended to prepare a platform, where we can invite other investigators to use our tools and to join our efforts on this field . Having investigated a very narrow aspect of bone mechano - adaptation, a logical next step will be to study the epiphyseal region of long bones, too . First results reveal problems arising from the different scales of macroscopic shape forming and mesoscopic trabecular transformation, which we are currently trying to consolidate with a two scale approach looking at high resolution trabecular spots inside a matrix of coarser resolution structures at the macroscopic level.
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