Neuronal networks are balanced by mechanisms of homeostatic plasticity, which adjusts synaptic strength via molecular and morphological changes in the pre- and post-synapse. Here, we wondered whether the hyaluronic acid-based extracellular matrix (ECM) of the brain is involved in mechanisms of homeostatic plasticity. We hypothesized that the ECM, being rich in chondroitin sulfate proteoglycans such as brevican, which are suggested to stabilize synapses by their inhibitory effect on structural plasticity, must be remodelled to allow for structural and molecular changes during conditions of homeostatic plasticity. We found a high abundance of cleaved brevican fragments throughout the hippocampus and cortex and in neuronal cultures, with the strongest labelling in perineuronal nets on parvalbumin-positive interneurons. Using an antibody specific for a brevican fragment cleaved by the matrix metalloprotease ADAMTS4, we identified the enzyme as the main brevican-processing protease. Interestingly, we found ADAMTS4 largely associated with synapses. After inducing homeostatic plasticity in neuronal cell cultures by prolonged network inactivation, we found increased brevican processing at inhibitory as well as excitatory synapses, which is in line with the ADAMTS4 subcellular localization. Thus, the ECM is remodelled in conditions of homeostatic plasticity, which may liberate synapses to allow for a higher degree of structural plasticity. Destabilizing influences such as long-lasting deflection or attenuation of excitation can be counterbalanced by mechanisms of homeostatic plasticity. This form of plasticity preserves network stability, adjusting synaptic strength within the network by profound molecular changes within the pre- and the post-synapse –3. From It was hypothesized that the appearance of the mature ECM is functionally involved in the switch from developmental to adult modes of synaptic plasticity . This is2.(a)In vitro studies showed that one of the most efficient brevican-cleaving enzymes is ADAMTS4 [a), which have previously been reported to detect exclusively the N-terminal cleavage product of brevican but not the full-length protein [b). The 53 kDa N-terminal cleavage product was present in the supernatant of ADAMTS4/brevican co-transfected cells, but barely detectable in brevican-only-expressing cells (b). Immunoreactivity with the neo antibody proved that the 53 kDa N-terminal fragment was derived from ADAMTS4 cleavage. It is of note that this antibody did not detect the full-length protein (b). Thus, the neo antibody is a valuable tool to detect exclusively the ADAMTS4-derived cleavage fragment of brevican and thus to monitor ADAMTS4 activity.Brevican can be processed by a large number of extracellular proteases . In vitr ADAMTS4 . The cle ADAMTS4 ,18,19. S protein ,19,20. Fng cells b. Immuno protein b. Thus, (b)a,b). In accordance with previous reports, brevican was also found in the myelin (My), synaptosome (Syn) and light membrane (LM) fractions, although to a much lesser extent [a,c). However, when we compared the relative abundance of full-length brevican and its C-terminal 80 kDa fragment across the fractions (ratio 80/145 kDa band), we found the fragment enriched in the soluble fractions S1 and S2 , which is not the case for the 53 kDa portion . Taken together, this suggests a stronger association of the HA-binding N-terminal fragment to neural membranes compared with full-length or 80 kDa brevican.To check for effects of cleavage on membrane association of brevican, we analysed the distribution of proteolytic fragments in subcellular fractions from rat brain . As expected, we detected full-length brevican in all brain fractions, with the strongest intensity in fraction S2 where soluble components of the ECM are found  (electror extent . In addi(c)c and electronic supplementary material, figure S2a–d). Staining with the neo antibody appeared very similar to total brevican staining with some deviations. In the molecular layer of the hippocampus, the neo antibody immunoreactivity mainly had a diffuse appearance (figure 1c). This may represent labelling of axonal coats at perisynaptic sites as recently described in human tissue [To study the spatial distribution of brevican processing in mature rodent brain, we investigated the localization of the ADAMTS4-derived proteolytic fragment in relation to the distribution of total brevican in rat brain sections by immunohistochemistry. While brevican staining was observed as diffuse labelling in most brain regions including hippocampus and cortex, in addition it appeared as a dense net-like structure typical for PNNs around a subset of cells, which were mostly parvalbumin-positive interneurons in vitro in dissociated neuronal cultures, making them an excellent model by which to study ECM-linked molecular events during synaptic plasticity [in vitro) 21–24, a time point when the mature form of the ECM is fully developed in these cultures [d and electronic supplementary material, figure S1d). The largest population of neurons exhibited a moderate staining and represented most likely MAP2-rich excitatory neurons . However, the staining on a subpopulation of cells was more intense and had a net-like appearance as observed in brain slices . Indeed, cells bearing PNN-like structures were co-stained with the inhibitory cell marker anti-GAD65 and partially also with PV . Neo epitope labelling exhibited a weak, dotted pattern on most neurons . However, on a small number of GAD65-positive interneurons, the staining was intense and had a typical PNN-like appearance. The staining co-localized well with pan brevican antibody . High-resolution images reveal the net-like appearance of the neo epitope surrounding inhibitory as well as excitatory synapses . Thus, the ADAMTS4-derived N-terminal fragment of brevican exhibits a similar distribution in dissociated cultures as in vivo, and is found associated with PNNs as well as peri-synaptically.Others and we reported previously that PNN-like structures develop asticity –27. Ther(e)a). Next, we probed Western blots from cells lysates of dissociated high-density cortical cultures (b). It has previously been reported that ADAMTS4 undergoes a series of activation steps through proteolytic cleavage yielding a characteristic pattern of fragments [c,d). ADAMTS4 immunoreactivity was found in a punctate pattern in cell bodies and neurites. Surprisingly, co-staining with the excitatory post-synaptic marker Homer1 revealed that 48 ± 2.1% of all excitatory synapses and 54 ± 2.9% of inhibitory synapses contained ADAMTS4. Thus, ADAMTS4 is indeed expressed and activated in dissociated neuronal cultures and further associates to a large degree with synapses.The presence of the brevican fragment immunoreactivity in culture points to expression of active ADAMTS4 under culture conditions. To prove this, we analysed ADAMTS4 immunoreactivity with an antibody directed against the proteolytic domain of ADAMTS4. This antibody was shown to be specific, because on HEK293T cells overexpressing ADAMTS4 fused to EGFP it detected a band of approximately 125 kDa in the cell lysate, representing the full-length protein, whereas no band was detected in untransfected HEK293T cells a. Next, cultures b. It hasragments ,29. Inderagments . Finally(f)e shows, in all samples both the full-length and the N-terminal brevican fragment were present. Quantification revealed that all samples contained the same amount of total brevican, which was determined by summing the proteolytic fragment and the full-length protein (figure 2f). However, the amount of 53 kDa fragment was clearly increased after network silencing, suggesting higher proteolytic activity in treated cultures (figure 2f). To prove the involvement of ADAMTS4 in this processing of brevican, we used the neo antibody and indeed found a similar increase of the ADAMTS4-derived fragment in the TTX-treated samples, suggesting ADAMTS4 is the major responsible protease (figure 2f).The ECM of the brain has been implicated in reducing structural plasticity in the adult. Therefore, we wondered whether treatments, which are known to induce plasticity, would alter processing of brevican as a major constituent of adult brain ECM. It has previously been reported that prolonged network silencing induces structural and functional changes at synapses in dissociated neuronal cultures ,32. Therfigure 2d and electronic supplementary material, figure S2a). To our surprise, neo staining did not differ between the experimental groups, suggesting no overall change in brevican processing (g). Because the ECM enwraps synapses and thereby may contribute to their stabilization, and because we found ADAMTS4 to be present near synapses, we hypothesized that brevican is differentially processed in the immediate vicinity of synapses. To test this, we used Homer and GAD65 antibodies as markers for excitatory or inhibitory synapses, respectively, and measured fluorescence intensity of neo antibody staining in an area 1 μm around inhibitory or excitatory synapses. Interestingly, we found increased neo immunoreactivity around both synapse types after TTX treatment (g). Taken together, our results suggest that long-term inactivation of neuronal networks leads to increased brevican processing at inhibitory as well as excitatory synapses, which may be a prerequisite for synaptic rearrangements under conditions of homeostatic plasticity.Next, we examined in which cellular compartment proteolysis occurs. For this purpose, dissociated cortical cultures were treated with TTX for 48 h and subsequently stained with neo epitope antibody. We co-stained neurons with the somatodendritic marker protein MAP2 and used this staining as a mask to determine neo epitope fluorescence intensity as a measure of proteolytic cleavage Subcellular fractionation suggested that emerging proteolytic fragments are differentially associated with cell membranes. While the C-terminal 80 kDa fragment, which contains the CS side chains, is mainly found in the soluble fractions, the 53 kDa fragment is more tightly associated with membranous fractions such as synaptosomes and light membranes . This suggests that brevican cleavage may not only loosen the ECM structure by degrading one of its major components, but also locally removes the non-permissive cue for structural plasticity, the chondroitin sulfates. However, brevican is not the main CS-bearing molecule in the ECM. In fact, it has been suggested this may be the closely related lectican aggrecan . ADAMTS4(b)Recent work on matrix metalloprotease 9 (MMP9) and the serine protease neurotrypsin suggests that ECM proteolysis may expose new epitopes that exert signalling functions important for synaptic plasticity . ProteolThe serine protease neurotrypsin is secreted from the pre-synapse during high synaptic activity . Its onl(c)Indeed, the mature form of the ECM is not only a plasticity-restricting structure, but as a series of works have shown it is important to maintain LTP, also a measure for synaptic plasticity . In fact4.(a)For a complete list of used antibodies and dilutions, see the electronic supplementary material, section Methods.(b)2. Cells were plated in polystyrene six-well plates (300 000 cells per well) for immunoblots and on poly-l-lysine-coated glass coverslips (50 000 cells per coverslip) for immunocytochemistry. Cell cultures of HEK293T cells stably expressing brevican were cultured as described [Primary cultures of cortical neurons for WB and ICC were prepared as described previously . All celescribed . A descr(c)Subcellular fractionation of adult rat brains was performed as described previously .(d)Find a detailed description of Western blotting in the electronic supplementary material, section Methods.(e)Immunocytochemistry and immunohistochemistry were performed as described ,47. Find(f)isiview software (version 2.1.1). For each set of coverslips (treatments versus control), the same exposure time was taken. All images were analysed using NIH ImageJ 1.47v or OpenView software (written by N. Ziv and co-workers [rism v. 5 software (GraphPad Software).Images were acquired with Zeiss Axio imager A2 (Zeiss Microimaging), a CoolSNAP MYOcamera and V-workers ) with ap


The International Headache Society classification of primary headaches in the International ClassificationI of Headache Disorders (ICHD) is almost universally accepted by researchers and clinicians. It is highly unlikely that reputable journals will accept submissions for publication if the cohorts have not been selected strictly according the ICHD. Likewise, in the clinical setting the appropriate treatment is prescribed according to how the patient’s headache is classified. But how reliable is the ICHD? As it does not appear to be based on data, its scientific validity is questionable. If indeed it cannot be scientifically validated, then the results of research based on the ICHD would be inaccurate. The practical implications of this are that the research data on the efficacy of migraine drugs is inaccurate. As a result, patients diagnosed according to this classification often receive the incorrect treatment.No conflict of interest.


Based on three decades of citation data from across scientific fields of science, we study trends in impact factor biased self-citations of scholarly journals, using a purpose-built and easy to use citation based measure. Our measure is given by the ratio between i) the relative share of journal self-citations to papers published in the last two years, and ii) the relative share of journal self-citations to papers published in preceding years. A ratio higher than one suggests that a journal’s impact factor is disproportionally affected (inflated) by self-citations. Using recently reported survey data, we show that there is a relation between high values of our proposed measure and coercive journal self-citation malpractices. We use our measure to perform a large-scale analysis of impact factor biased journal self-citations. Our main empirical result is, that the share of journals for which our measure has a (very) high value has remained stable between the 1980s and the early 2000s, but has since risen strongly in all fields of science. This time span corresponds well with the growing obsession with the impact factor as a journal evaluation measure over the last decade. Taken together, this suggests a trend of increasingly pervasive journal self-citation malpractices, with all due unwanted consequences such as inflated perceived importance of journals and biased journal rankings. It is well known that the impact factor–arguably the single most important measure for assessing the quality or impact of scholarly journals–is vulnerable to all sorts of manipulation  or) or8]) oThis paper studies the phenomenon of ‘impact factor biased journal self-citations’. We use this term to refer to the phenomenon of journals having a disproportionally large proportion of journal self-citations to the past two years, which are the years that determine the impact factor of a journal, relative to their proportion of journal self-citations to earlier years. Impact factor biased journal self-citations may result from coercive citation practices and authors’ strategic response to such (anticipated) practices. They may also result from journal self-citations in manipulated editorials or simply from a tendency of editors to be more inclined to accept submissions that include many references to papers published recently in their journal. However, as we will discuss, there are also legitimate mechanisms that may result in impact factor biased journal self-citations.Our study contributes to the growing literature on impact factor manipulation in the following two ways. First, we present an easy to use measure of impact factor biased journal self-citations. As we will show, by means of an empirical comparison of our measure with recently reported results of an author survey into coercive journal self-citation malpractices, the measure provides a useful tool to identify  a small subset of journals that are relatively likely to engage in self-citation malpractices. As such, it provides a practical tool for a first diagnosis of, for example, the coercive journal self-citation phenomenon. Second, we apply our measure in a large scale study of long term trends in impact factor biased journal self-citations, based on three decades of citation data for thousands of journals. Our empirical analyses show that impact factor biased journal self-citation practices have become much more prevalent over the past decade. This corresponds well with the time frame during which the impact factor has gained widespread attention among researchers and journal editors . It thery to papers published in impact factor years , with the share of journal self-citations to papers published in the five preceding years. The IFBSCP of a journal equals 1 if the share of journal self-citations to papers published in impact factor years equals the share of journal self-citations to papers published in preceding years. An IFBSCP that is (much) above 1 signals a disproportional share of journal self-citations to papers published in impact factor years. Although there may be perfectly legitimate reasons for a high IFBSCP (see Consider a measure of Impact Factor Biased Self-citation Practices (from here on: IFBSCP). This measure  between 1987 and 2004, has since rapidly increased. Our data allow us to explore if there are any eye-catching differences in (trends in) IFBSCP between the Physical, Life, and Social Sciences. More specifically, Inspection of Figs Journals identified as coercers by survey respondents. Number of coercive observations represents the number of times a journal was identified by independent survey respondents as requesting self-citations that (i) give no indication that the manuscript was lacking in attribution, (ii) make no suggestion as to specific articles, authors, or a body of work requiring review, and (iii) only guide authors to add citations from the editor’s journal.” The Table contains 175 journals, from which we selected those 64 journals which met the following conditions: i) the number of coercive observations (as defined directly above) was larger than one, ii) the journal is indexed in the Web of Science database, iii) an IFBSCP value could be computed for the year 2011, going back to 2004 . We focus on the year 2011 because journals may have altered their practices in response to the publication of ), recently published in a particular journal is a signal of that journal’s current interest in the topic; in line with this consideration, she goes on to submit her work to that journal as opposed to some other journal. This scenario too would lead to an increase in the journal’s IFBSCP. A third potential mechanism which would cause high but legitimate IFBSCP values is as follows: in first instance, the spreading of new ideas is likely to take place in the community where the ideas were originally presented. Over time, the most influential ideas then gradually become noticed and gain traction in other scientific communities. This scenario would imply a relatively large number of journal self-citations in years directly after publication of an idea, compared to more distant (future) years.However, it cannot be claimed that a high IFBSCP provides proof of unethical behavior of editors or authors. In fact, there are other, perfectly legitimate reasons why a journal may have a relatively high IFBSCP. A recent paper by one of us  presentsTaking into account the above-mentioned legitimate reasons for high IFBSCP values, we stress that a high IFBSCP does not in itself prove wrongdoing by the associated journal. Nonetheless, we do believe that the IFBSCP measure has strong potential as a tool for first diagnosis: it can be used to quickly sift through a large set of candidate journals, and to identify the subset which is relatively likely to include potential culprits. This subset then should be subject to further scrutiny before journal-specific conclusions can be drawn. Our empirical comparison between the IFBSCP measure and the number of reported observations of coercive citation practices in a recent survey  providesWe apply the IFBSCP measure to analyze citation patterns for all scientific fields combined since 1987, as well as for the particular domains of Life, Physical, and Social Sciences. We believe that, notwithstanding the caution that should be exercised when interpreting the IFBSCP measure, two over-all conclusions are warranted based on our results: first, between 1987 and the early 2000s, the mean IFBSCP and the share of journals whose IFBSCP surpasses a given threshold has been quite stable; this suggests that there is a stable underlying set of legitimate mechanisms that together result in a mean IFBSCP of around 1.4 at the aggregate level. Second, since about 2004, there has been a pronounced rise in mean IFBSCP and particularly in the share of journals whose IFBSCP surpasses relatively high thresholds ; this suggests that during the last decade, and on top of the legitimate mechanisms discussed earlier, there have increasingly been other influences driving up the IFBSCP, the practices of coercive journal self-citation and strategic responses by authors being obvious candidates.Although it cannot be ruled out that this steep recent increase in IFBSCP is due to profound changes in the legitimate mechanisms discussed earlier, it should be noted that the importance of the (two year) impact factor has increasingly gained attention among researchers, policy makers, and managers of scientific institutions over the past decade. For example,  show thaTogether, it seems fair to consider the strong rise in mean IFBSCP and in the share of journals with a (very) high IFBSCP, as circumstantial evidence for a trend of increasingly pervasive journal self-citation malpractices, with all due unwanted consequences such as inflated perceived importance of journals and biased journal rankings. We recommend that future studies into such malpractices use the IFBSCP measure to zoom in on a small subset of journals whose IFBSCP is suspiciously high, before embarking on the collection of much needed case study evidence for or against the actual malpractice at the level of particular journals.y represents the year for which the IFBSCP is computed; y to papers published in the same journal in year y – 1; y to papers published in the same journal in year y − 2; note that years y − 1 and y − 2 constitute the two years that are used for computing the journal’s impact factor. total number of citations from papers published in any journal in year y to papers published in the focal journal in year y – 1; Y stands for reach of the analysis .Assuming that journal self-citation malpractices are aimed at inflating a journal’s impact factor, one would expect that a journal which actively engages in such a malpractice, has a share of journal self-citations to papers published in that journal in recent years that is relatively large compared with the share of journal self-citations to papers published in that journal in earlier years. This is the underlying rationale for the IFBSCP measure, which is formally denoted for a given journal as follows:y in the analyses, which generates overlap between the years represented in the numerator and denominator. Third, the IFBSCP measure as defined above assumes that journal self-citation malpractices typically refer to papers from the past two years, which are the years that determine the traditional two year impact factor. Of course, following the same ideas as above, a variant of the IFBSCP measure for the five year impact factor can be easily derived.Three comments need to be made on the above definition of the IFBSCP measure. First, the idea of analyzing the time trend in the ratio of self-citations to total citations is also used in recent studies on journal self-citations and journal citation cartels . SecondY = 7, so in effect we compare the share of journal self-citations to papers published in ‘impact factor-years’ with the share of journal self-citations to papers published in the five years preceding the ‘impact factor-years’ . To focus our analyses on journals for which sufficiently reliable statistics can be computed, we exclude those journals that–in a given year–cite their own papers in the past seven years fewer than 50 times. This results in the exclusion of 88,842 out of 164,757 cases, where each case represents a combination of a journal and a year). The total number of journals considered increases from 1520 in 1987 to 4767 in 2015; the total number of cases in the period 1987–2015 being 75,915. For the analyses reported in We extract citation data from the Web of Science database for all fields in the Sciences and the Social Sciences. We do not consider fields in the Arts and Humanities. Journals in the Arts and Humanities do not have an impact factor, and presumably editors of these journals therefore have less incentive to engage in coercive self-citation practices. We compute the IFBSCP for every journal and for every year in the period 1987–2015. We set Y at 7 (implying a comparison between self-citations for the two impact factor years and the five preceeding years) is somewhat arbitrary. We therefore report the results of a series of stability analyses which we performed to investigate the impact on IFBSCP of setting Y at different values. Specifically, we consider the year 2015 , and select all journals that continuously published during the previous decade, i.e., in each of the years 2005–2015. This results in 7223 journals. For each of these journals, we compute IFBSCPs for Y ∈ {4,5,6,7,8,9,10}. We report three different stability metrics: first, we report the Pearson correlation between the IFBSCP with Y set at 7 and each of the alternative specifications. These correlations range from 0.71 (Y = 4) to .95 (Y = 8); note that it was expected that the highest correlations would be found for values of Y close to 7, since the underlying citation data have large overlap. To study stability in rank ordering , we compute Kendall’s tau correlations as well. These correlations range from 0.69 (Y = 4) to 0.90 (Y = 8). Finally, we compute–for each journal–the ratio of the IFBSCP for Y = 7 and the IFBSCP for Y = 4 and Y = 10; i.e., we compare Y = 7 with the two most extreme values of Y in our data. The ratio (IFBSCP for Y = 7) / (IFBSCP for Y = 4) has a mean  of 1.16; the 10-th percentile IFBSCP equals 0.84, and the 90-th percentile IFBSCP equals 1.51. The ratio (IFBSCP for Y = 7) / (IFBSCP for Y = 10) has a mean  of 0.94; the 10-th percentile IFBSCP equals 0.78, and the 90-th percentile IFBSCP equals 1.07. In combination, these analyses indicate that the IFBSCP is stable across different values of Y.The decision to set S1 FileContains citation data from which (XLSX)Click here for additional data file.S2 FileContains the IFBSCPs reported in (XLSX)Click here for additional data file.


Acute renal failure due to ureter compression after a mesh-plug inguinal repair in a kidney transplant recipient has not been previously reported to our knowledge. A 62-year-old man, who successfully underwent kidney transplantation from a deceased donor 6 years earlier, was admitted for elective repair of a direct inguinal hernia. The patient underwent an open mesh-plug repair of the inguinal hernia with placement of a plug in the preperitoneal space. We did not observe the transplanted ureter and bladder during dissection of the inguinal canal. Immediately after surgery, the patient became anuric, and a graft sonography demonstrated massive hydronephrosis. The serum creatinine level increased rapidly, and the patient underwent an emergency reoperation 8 hours later. During surgery, we did not identify the ureter but, immediately after plug removal, urine output increased progressively. We completed the hernia repair using the standard technique, without plug interposition, and the postoperative course was uneventful with complete resolution of graft dysfunction 3 days later.Furthermore, we reviewed the clinical features of complications related to inguinal hernia surgery. An increased risk of urological complications was reported recently in patients with a previous prosthetic hernia repair undergoing kidney transplantation, mainly due to the mesh adhesion to surrounding structures, making the extraperitoneal dissection during the transplant surgery very challenging. Moreover, older male kidney transplant recipients undergoing an inguinal hernia repair may be at higher risk of graft dysfunction due to inguinal herniation of a transplanted ureter.Mesh-plug inguinal hernia repair is a safe surgical technique, but this unique case suggests that kidney transplant recipients with inguinal hernia may be at higher risk of serious urological complications. Surgeons must be aware of the graft and ureter position before proceeding with hernia repair. A prompt diagnosis with graft sonography and abdominal computed tomography scan and emergency surgery may avoid the need for nephrostomy and may resolve graft dysfunction more rapidly. Impressive improvements in surgical techniques and the routine use of intraoperative ureteric stenting have significantly reduced the rate of major urological complications after kidney transplantation.We report a unique case of acute renal failure due to ureteral obstruction after a mesh-plug inguinal hernia repair in a kidney transplant recipient. The patient involved in this study gave his written informed consent authorizing all procedures, and ethical consent for this study was not necessary as it did not involve any experimental treatment, and does not reveal any confidential information about patient that could violate their privacyWe also review the clinical features of urological complications caused by inguinal hernia surgery.® stitches. We placed an unsecured onlay mesh patch with the ends draped around the cord structures at the level of the internal ring. We did not note the donor transplanted ureter and bladder during dissection, neither medial nor superior to the right-sided inguinal hernia, and we completed the surgical intervention without intraoperative complications.A 62-year-old man with an end-stage renal disease secondary to polycystic kidney disease underwent successful deceased donor kidney transplantation. Six years after transplantation, the patient presented with vague abdominal pain and an inguinal hernia. His renal function was stable (SCr 1.5 mg/dL), and preoperative abdominal ultrasonography demonstrated a well-functioning kidney with no hydronephrosis and the presence of a direct inguinal hernia. An elective, open mesh-plug repair of the inguinal hernia was performed, by opening the external oblique muscle in the direction of the muscle fibers and reinserting the direct sac into the abdominal cavity, without opening the sac. We subsequently inserted an umbrella-shaped plug into the posterior wall defect and secured it with 3 interrupted Prolene® stitches without plug interposition. We then placed an onlay mesh and completed the hernia repair using the standard technique. The postoperative course was uneventful and there was a progressive improvement in graft function. Three days after reoperation, the serum creatinine level decreased to 1.5 mg/dL and graft sonography demonstrated significantly reduced hydronephrosis. Magnetic resonance imaging (MRI) of the lower abdomen, performed 2 days after surgery, demonstrated fluid collection in the inguinal region at the site of previous plug implantation, near the passage of the ureter, confirming that the plug was responsible for ureteral compression  and totally extraperitoneal (TEP) endoscopic techniques, are currently the methods of choice for the treatment of primary unilateral inguinal hernia. Open Lichtenstein and mesh-plug techniques provide similar outcomes in terms of hernia recurrence and chronic pain,23 although recent evidence suggests a lower likelihood of reoperation for mesh-plug repair.24 Compared with the Lichtenstein repair, endoscopic TEP and TAPP repairs have the advantages of lower rates of wound infection as well as earlier resumption of normal working activity, and, most importantly, a lower risk of chronic pain.26To our knowledge, this is the first reported case of acute renal failure in a kidney transplant recipient after inguinal hernia mesh-plug repair. Inguinal hernia repair is one of the most common surgical interventions performed worldwide. By some estimates, ∼0.8% of the general population in developed countries undergoes surgical repair in a 5-year period.27 described a kidney transplant recipient who underwent laparoscopic TAPP repair of a symptomatic bilateral inguinal hernia 2 years earlier: at surgery, the fundus of the bladder and the pubis were completely encased in fibrous tissue compounded with mesh, making the extraperitoneal dissection of the bladder exceptionally challenging. More recently, Tse and Clancy10 reported a patient who developed stenosis of the renal transplant ureter after a laparoscopic repair of a bilateral recurrent inguinal hernia. The transplanted ureter was obstructed and adhered to the mesh. After dissection from the mesh, the stenotic segment was resected and the ureter was reanastomosed over a ureteric stent to the bladder. Similar findings were encountered in patients undergoing an open mesh-plug repair before transplantation. Weale et al28 reported surgical technical difficulties in 4 patients who underwent kidney transplantation on the same side of previous mesh-plug hernia repairs because of adherence of surrounding structures to the mesh and fixation of the cord. Ortiz et al29 reported ureteral necrosis in a kidney transplant recipient with a previous mesh-plug hernia repair. In this patient, the ureteral necrosis may have been related to an inflammatory reaction near the transplanted ureter, which was proximal to the implanted prosthetic mesh. Taken together, these studies suggest that kidney transplantation should be performed in the contralateral site of a previously implanted, unilateral mesh for inguinal hernia repair, and, in cases of bilateral hernia, laparoscopic repair with mesh should be avoided whenever possible.29An increased risk of urological complications was reported recently in patients with a previous prosthetic hernia repair undergoing kidney transplantation. Nadalin et al19 Factors that may contribute to inguinal herniation of the transplant kidney include the existence of a redundant long ureter, placement of donor ureter over the spermatic cord, obesity, and stricture of the ureterovesicular anastomosis leading to twisting and kinking of the ureter into the inguinal canal.19 Older male patients, with a median age of 56.9 years, were more commonly affected. Inguinal herniation of the ureter is a late cause of obstructive uropathy and presented at a mean of 10.6 years  after transplantation. Almost all patients presented with graft dysfunction together with inguinal pain. Graft sonography usually demonstrated various degrees of hydronephrosis, and, in most cases, the diagnosis was confirmed by computed tomography or MRI, which allowed a clear picture for surgical planning. Although conservative treatment has been described,9 most patients required a nephrostomy procedure before elective surgical repair. The definitive treatment was inguinal herniorrhaphy without the need for ureter reimplantation. However, in the case of ureter necrosis,12 long redundant ureter17 or stricture of the ureter,10 a ureteral resection with reanastomosis, was required.Inguinal hernia after renal transplantation may present as a urological complication because of herniation into the inguinal canal of the transplanted ureter. A total of 13 cases of inguinal herniation of a transplanted ureter have been reported in the literature (Table 30 reported a case of anuria in a renal transplant recipient following inguinal herniorrhaphy due to accidental ligation of the ureter, and successful treatment included a nephrostomy and ureteral reimplantation. In the present case, we attributed acute renal failure to ureteral compression by the plug that was placed in the preperitoneal space (Figure Acute renal failure after inguinal hernia repair is reported rarely among kidney transplant recipients. Selman et ale Figure . ImmediaIn conclusion, although mesh-plug inguinal hernia repair is a safe surgical technique, this unique report highlights that kidney transplant recipients presenting inguinal hernia may be predisposed to a higher risk of urological complications and graft dysfunction. Surgeons must be aware of graft and ureter positions before proceeding with hernia repair. A prompt diagnosis with graft sonography and abdominal computed tomography scan, followed by emergency surgery, may avoid the need for nephrostomy positioning and may improve the resolution of graft dysfunction.


Morning stiffness is a major symptom of juvenile idiopathic arthritis (JIA) and it is usually associated with active disease. However, it is common view that children with disease quiescence may have some degrees of residual morning stiffness. The 2004 preliminary criteria for inactive disease (ID) in JIA did not include the assessment of morning stiffness, whereas the 2011 revision of the criteria has allowed the presence of morning stiffness lasting ≤ 15 minutes. However, it is still unknown whether the disease status of children with ID who have or do not have morning stiffness is comparable.To compare the disease status of children with JIA who meet the 2011 revised criteria for ID and have or do not have a morning stiffness lasting ≤ 15 minutes.A database at the study center including 785 patients who had undergone a total of 2957 visits, which included a parent report of the presence and duration of morning stiffness, was analyzed to identify all visits in which patients met the criteria for ID. In each visit, the duration of morning stiffness was categorized as follows: ≤15 min, 15-30 min, 30-60 min, 1-2 hr, >2 hr. Clinical assessments included demographic features, and parent-reported outcomes. In case a patient met the ID criteria in more than 1 visit, only the first visit was retained.A total of 460 visits in which the patient met the criteria for ID were evaluated. Absence of morning stiffness was reported in 390 (84.8%) visits, whereas in 70 visits 15.2%) there was morning stiffness. Among the visits with morning stiffness, in 41 (8.9%) duration was ≤15 min, and in 29 (6.3%) duration was >15 min. Table 5.2% therAmong patients who met the 2011 criteria for ID, those with morning stiffness ≤15 min had worse parent-reported outcomes than those without morning stiffness. This finding suggests that parents may not perceive their child’s disease state as true remission when lower degrees of morning stiffness are present. Notably, a sizeable proportion (6.3%) of children meeting the 2004 ID criteria had morning stiffness lasting > 15 min. The change of the criterion “Duration of morning stiffness of ≤ 15 minutes” to “Absence of morning stiffness” in the definition for ID should be considered.None declared.


Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator.Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species.Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance.Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic. Plants produce and store energy reserves for various purposes. A major use of these energy reserves is to facilitate growth and propagation of the next generation and they are laid down in sink tissues, e.g. seeds and tubers. The plant storage reserves, starch, oil and sugars, are supplying mankind with the majority of calories but have also important industrial applications. The type of storage compound and in which tissue of the plant the storage product is located varies among plant species. Generally, the biosynthesis of storage compounds, starch, oil and sugars, is known in quite detail but the knowledge of why a certain type of these products accumulates and the underlying mechanisms are largely lacking ,2. With Beta vulgaris ssp. altissima) and related subspecies which produce no starch but sucrose during tap-root development. The reason why beets exclusively have sucrose as a storage compound is not known. However, one factor that might have been of importance are the saline growth conditions where the wild ancestor, sea beet (Beta vulgaris ssp. maritima), grows. Sea beet is growing, as the name implicates, by the sea, and can have both an annual and biennial life cycle and has a similar cell organization and storage accumulation as sugar beet [In general, tap-roots have starch biosynthetic and deposition capacity and starch granules can readily be found in cells of parsnips, carrots and swedes. An exception among tap-roots is sugar beet . Sugar beet and parsnip root have similar behaviour and morphology but the main storage compounds of the two tap-roots differ. Sugar beet and parsnip were grown in a greenhouse and samples were taken at two different developmental time points. At these two time points samples were taken both at the end of the light- as well as at the end of the dark period of the day to monitor potential diurnal changes. Roots from the two plant species were comparatively studied with focus on carbon allocation as well as expression of genes essential for starch accumulation and activities of the main starch biosynthetic enzymes.The aim of this study was to, during a developmental cycle, investigate the nature of the storage compounds and to what extent genes and enzymes central to starch biosynthesis are manifested in tap-roots of sugar beet and parsnip .The leaves of parsnip and sugar beet appeared to behave as any other photosynthetic source tissue. Leaf tissue displayed diurnal changes with excess sucrose produced during photosynthesis stored as starch during the light period and subsequently degraded with sucrose resynthesized and transported to other parts of the plant during the dark period  and starch were measured in sugar beet root and parsnip tap-root and calculated as percentage of dry weight matter. No difference was found between samples of light and dark sampled tap-roots from the same developmental time point. Therefore the results were combined to sugar beet 41 DAP, sugar beet 54 DAP, parsnip 48 DAP and parsnip 61 DAP. The measured starch of sugar beet was found to be insignificant at around 1% of the dry weight at both time points. Sugar content increased from 48% at 41 DAP to 56% of dry weight matter at 54 DAP. More than 98% of the sugar in sugar beet tap-root was sucrose and only very small amounts of fructose and glucose could be detected Figure . The parIn addition to sucrose, hexoses were found at significant levels in parsnip. Among sugars the proportion of sucrose increased from 56% to 72% at the second time point. As a result hexose proportions decreased with development, glucose from 19% to 12% and fructose from 25% to 16% of total sugars.Soluble proteins were extracted from sugar beet and parsnip tap-roots for further analysis of enzyme activities involved in starch biosynthesis. Protein concentrations of fresh weight were approximately twice as high for parsnip samples compared to sugar beet samples with small fluctuations between harvest time points (results not shown).Activities of the main enzymes in the starch biosynthetic pathway were investigated in tap-root crude protein extracts from parsnip and sugar beet. Four enzymes are critical in the building of branched α-glucans in amyloplasts; phosphoglucomutase (PGM), ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS) and starch branching enzyme (SBE). Activities could be detected for all four enzymes in both parsnip and sugar beet tap-root samples Table . Due to Phosphoglucomutase activity can be found in the cytosol and the plastid. PGM activity was detected in both sugar beet and parsnip tap-root at similar activities per protein level Table . It coulThe AGPase enzyme activity was similar between the parsnip and sugar beet samples with regards to enzyme activity per protein level Table . No alteParsnip and sugar beet upheld comparable levels of starch synthase per protein level leading to precipitable α- glucans Table .All samples of respective species displayed starch branching enzyme activity. SBE activity levels per μg protein ranged for sugar beet from 38% to 61% of the levels found in parsnip. The late parsnip harvests displayed in general a higher starch branching enzyme activity level than sugar beet  and parsnip (61 DAP), were compared between sugar beet and parsnip. This analysis showed that all major genes coding for starch biosynthetic enzymes or genes coding for hexose-phosphate conversion were expressed in sugar beet tap-root even though no starch was produced  exists in both cytosolic and plastidic forms which are derived from different genes, where the plastidic form has been shown to be of importance for starch synthesis of dicots ,22. The The aim of this investigation was to study carbon storage accumulation in developing sugar beet and parsnip tap-roots. Furthermore, the concomitant presence or lack of activity from starch biosynthetic enzymes or expression of their corresponding genes was studied, which potentially could explain the differential storage strategies among the two species. As parsnip partially stores starch while sugar beet stores sucrose, a comparison between these two species will provide a better understanding of carbon allocation in these underground storage tissues and also an understanding of the genetic and enzymatic factors governing the accumulation of the two different carbon storage compounds.The two time points for sampling were chosen when the roots were assumed to be in an early accumulating phase, reassuring that the filling of the sink cells was ongoing. Sugar beet is stated to be fully active in a quite early stage . ParsnipIn the early phase of root and tuber storage organ development, cells consist mainly of a large vacuole. The vacuole contains sugars which are used as energy and building blocks for cell proliferation and expansion. The small starch granules that are present in the juvenile root cells are at this stage displaced towards the cell walls. During development, cells change from structural expansion of the organ to storage accumulation and most cells switch to filling up storage reserves such as starch granules or oil droplets and the vacuole is gradually compressed -26. SugaInvestigation of tissue samples provided visual evidence for presence or absence of starch granules in the tap-root cells. The observation of enlarged starch granules and reduced size of the vacuoles in parsnip tap-root cells during growth corroborates with active starch biosynthetic enzyme activities. Sugar beet, however, maintained their relative vacuole size whereas cell walls thickened with no visible starch granules formed.Sucrose produced in photosynthetic source cells is transported to the sink cells where sucrose cleaving enzymes (sucrose synthase and invertase) convert the sucrose to hexoses in different subcellular compartments. The hexoses in the sugar beet tap-root are thought to be resynthesized to sucrose by sucrose phosphate synthase and sucrose phosphate phosphatase to be transported into the vacuole ,27-29. OIn a typical starch accumulating plant, such as potato, the hexoses are transported as hexose phosphates into the amyloplast where it is utilized for the synthesis of starch. Starch accumulation is a response to sucrose availability and thus, no hexoses are stored in the potato tuber . In our Several genes involved in starch biosynthesis have been isolated and studied in sugar beet although this has been performed with regards to photosynthetic structures. Additional expressed genes in sugar beet that are involved in starch biosynthesis can be found from searching Expressed Sequence Tag (EST) databases. Structural studies of sugar beet leaf tissue support the presence of starch biosynthetic enzyme activities by illustrating the common diurnal ability of photosynthetic tissue to produce starch. However, according to our knowledge, until now there has been no study directed to the presence or lack of expressed genes or activity of enzymes involved in starch biosynthesis in sugar beet tap-root that could explain the complete absence of starch in these structures. The aim of our enzymatic studies was primarily to determine presence or absence of starch biosynthetic enzyme activity and, surprisingly, our studies showed that sugar beet root had good activities of major starch biosynthetic enzymes, but no starch was accumulated. The four enzymes PGM, AGPase, SS and SBE, which are taking part in synthesis of branched glucans, were all active in sugar beet tap-root and within the same order of magnitude as in parsnip tap-root. When comparing expression levels of genes encoding these key enzymes in starch synthesis in sugar beet and parsnip tap-root, it is evident that most of these genes were also expressed at levels within the same order of magnitude. Exceptions to this were the genes coding for plastidic phosphoglucomutase and the large subunit of ADP-glucose pyrophosphorylase. However, this does not explain the complete lack of visible starch granules in sugar beet tap-root. For example, mutants or silencing of these two genes as well as transgenic silencing studies of Arabidopsis and pea display low starch content, but not a complete loss of starch accumulation -33. SimiA number of studies of genes and enzymes of importance for synthesising the branched polymeric structure of α-1,4 linkages with α-1,6 branches have been published. Starch granule formation is more complex than production of long branched glucose polymers. To organize the long and branched glucose molecules into well-organized granules, debranching activities are needed for trimming the glucans and thus structuring the granule -36. The Even in root crops considered as non-starchy, such as carrot, starch is accumulated . Indeed In conclusion, gene expression and enzymatic activities could be found for the major participants in starch biosynthesis in sugar beet, despite that structural analyses and chemical analysis failed to indicate any presence of starch. Even though some genes were found to be less expressed in sugar beet tap-root, a complete lack of starch granules cannot be explained by these results. Thus, there must be another mechanism or mechanisms which prevent sugar beet from producing starch in the tap-root, a default storage compound for underground sink organs. Starch is an energy-efficient storage form due to the insoluble starch granule compared to the soluble sucrose. During the storage phase from one year to the next, sugar beet tap-root needs to maintain an energy potential in order to keep the high concentration of sucrose in the vacuole, 500 mM sucrose in comparison to the 75 mM sucrose in the cytosol . This apBeta vulgaris ssp. altissima, “Balder”) and sea beet (Beta vulgaris ssp. maritima) were kindly provided by Nordic Genetic Resource center, Alnarp, Sweden.Sugar beet seeds (Pastinaca sativa “White Gem”) were purchased online from Impecta Fröhandel, Julita, Sweden, http://www.impecta.se.Parsnip seeds . Sugar beet was sampled at 41 DAP and 54 DAP. The primary root fresh weight was measured. Dry weight determination was performed by freeze drying the roots (n > =3) until no weight change was noted (≈72 hrs). Plant tissues were frozen in liquid nitrogen and stored at −80°C for further studies.Sea beet was cultivated in an aeroponic system  and sampFixation and plastic embedding of fresh roots and leaves of parsnip and sugar beet was performed as previously described .Triple staining methylene blue-azur A-safranin O (MAS), visualising proteins, lipids and starch, was performed to obtain an overview of the embedded tap-root tissue ,42.In order to stain starch, the leaf sections were covered with 50% Lugol’s solution , for 1 min, rinsed with water, air-dried, and mounted with Biomount .MAS and Lugol’s stained sections were studied in a light microscope .Homogenized sea-beet tap-root tissue was spread on a microscope slide and Lugol’s solution was added. The stained tissue was studied in a light microscope .2O and mounted with Mowiol 4–88  [The fluorochrome Calcofluor White  was used to visualise β-glucans at 420 nm ,44. SectCA, USA)  to be stSince the measurements were made on whole root homogenates a minor part of the obtained values derives from non-storage parts of the cell such as cell-walls or non-storage cellular compartments. Also, when measuring starch there is a possibility that other α-glucans, for instance phytoglycogen, are included in the assay by the method used.The analysis is a standardized method, SCAN-P 91:09, recommended to be used by Scandinavian pulp, paper and board industry.2O and hydrolysed to glucose in a two-step enzymatic process [Sugar beet and parsnip root tissue were freeze dried and homogenized by grinding in a mortar. For the starch assay the homogenate was dissolved in an appropriate volume ddH process . The gluFor the sugar analysis, 100 mg freeze dried homogenate was dissolved in 1 ml 80% EtOH and extracted at −20°C for two weeks. Analysis of sugars was made on an ion exchange chromatograph  using the same setup and procedure as the starch analysis. The analysis was performed with sugar solutions of known concentration and composition as standards.Crude protein extracts were obtained by homogenizing root tissue in a mixer mill  in a stainless steel container, pre-chilled in liquid nitrogen to keep the tissue frozen and the enzyme activity intact. Protein was extracted from the fine powder according to a modified protocol which excludes BSA from the extraction buffer . The extPGM activity was determined in a spectrophotometric coupled assay. Conversion of glucose-1-phosphate (G1P) is catalyzed by PGM and the resulting glucose-6-phosphate (G6P) is subsequently catalyzed by glucose-6-phosphate dehydrogenase to 6-phosphogluconate. In parallel with the second reaction, NADP is reduced to NADPH and the reaction is measured at 340 nm . ExtractAGPase activity was determined  on 20 μg2PO4 with Mg-Am stain and following the measuring procedure as in the assay. Phosphate content in crude protein extract was measured by inactivating the crude enzyme extract at 60°C for 10 min and then measuring the samples as described for the standard curve. The background content of pyrophosphate was measured by incubating the inactivated crude extract with inorganic pyrophosphatase and then assaying phosphate content same procedure as the standard curve. Enzyme activity was calculated as produced ADP-glucose (nmol) per soluble crude protein (μg) per minute.AGPase catalyzes the reaction conversion of ATP and G1P to ADP-glucose and pyrophosphate (PPi). The assay measures phosphate after splitting produced PPi by inorganic pyrophosphatase. A standard curve for phosphate was made by mixing various concentrations of KHThe specificity of the assay was examined by excluding G1P and ATP from the substrate both separately and in combination to determine and exclude the cytosolic UDP-glucose pyrophosphorylase activity.2O. Five ml scintillation mix  was added to 0.5 ml of the dissolved starch product and radioactivity was measured in a liquid scintillation counter . The starch synthase activity was calculated as the amount ADP-glucose converted to starch per minute and μg total protein.Crude root protein extract (10 μg) was assayed for starch synthase activity. Activity was calculated by measurements after 0, 30, 90 and 120 minutes. The starch synthase assay was performed as previously described but with a small modification, where amylopectin in the substrate solution was exchanged to glycogen . The reaCrude protein extract (10 μg) was assayed for starch branching enzyme activity  with somSamples were chosen from the second sampling of sugar beet (54 DAP) and parsnip (61 DAP). Total RNA was extracted from a homogenate of three pooled individuals of root or leaf tissue respectively with Plant RNA Reagent . Concentration was measured on a NanoDrop  and quality was confirmed on a 1.2%, E-gel .DNA sequencing and data processing was provided by Eurofins as a service. Two normalised random primed cDNA libraries were produced from pooled leaf and tap-root mRNA from sugar beet and parsnip respectively. These were subsequently subjected for sequencing using Roche GS FLX Titanium series chemistry at a scale of ½ segment of a full run for each cDNA library.After quality analysis, passed reads were assembled into contigs and contigs collected in one reference file each for sugar beet and parsnip respectively. 531,058 passed reads were used for sugar beet and 563,841 reads were used for parsnip.Two 3'-fragment cDNA libraries with bar-coded adaptors were produced from tap-root mRNA from sugar beet and parsnip respectively. These were subsequently subjected to sequencing using Illumina HiSeq 2000 technology utilizing one channel in total for both samples. 24,586,598 reads passed quality analysis for sugar beet and 39,749,856 reads for parsnip. As a next step Illumina reads were assembled and used to improve the reference files produced after the Roche GS FLX Titanium sequencing and assembly resulted in new reference files for both parsnip and sugar beet where contigs consisted of data from both sequencing sets.Passed Illumina reads were mapped to the final reference files produced for sugar beet and parsnip. The number of reads mapped to each contig yielded an estimate of gene expression corresponding to the particular contig in comparison to number of reads mapped to other contigs. As only 3'-fragments were used for mapping, this by itself resulted in a normalization of the reads for each transcript. Gene expression for each transcript is thus expressed as the number of mapped reads to a specific contig per million reads (RPM).ESTs: Expressed sequence tags; DAP: Days after planting; PGM: Phosphoglucomutase; AGPase: ADP-glucose pyrophosphorylase; SS: Starch synthase; SBE: Starch branching enzyme.This study has partially been financed by Lyckeby Stärkelsen Research Foundation, which is the research foundation of a commercial entity. This relation has not affected our interpretation of data or presentation of information.HT designed and conducted the majority of the experimental work. MA contributed in designing and coordinating the project, edited and revised the manuscript, SM participated in the structural work and edited the manuscript, IT performed the sugar and starch analysis, PH contributed in designing and coordinating the project and conducted the transcriptome analysis. HT and PH wrote the manuscript. All authors read and approved the final manuscript.Sections of leaves illustrating diurnal changes. Sections of leaves stained with Lugol’s solution illustrating diurnal changes. a. Sugar beet leaf sampled 12 hours after sunrise. Dark spots, indicated by arrows, show accumulated starch. b. Sugar beet leaf sampled in dark, no starch is detected, c. Parsnip leaves sampled 12 hours after sunrise. Starch is detected. d. Parsnip leaf sampled in dark, No starch is detected but chloroplasts are shown clearly. Scale bar 50 μm.Click here for fileIllustration of cambium rings. Cambium rings of green house grown sugar beet roots 41 days after planting (a) and 54 days after planting (b). Sections are stained with Lugol’s solution. Scale bars 5 mm.Click here for file


In vivo results suggested that this can have acceleratory or inhibitory influences, depending on direction and magnitude of motion, indicating that some complications in fracture treatment could be avoided by optimizing the fixation stiffness. However, general statements are difficult to make due to the limited number of experimental findings. The aim of this study was therefore to numerically investigate healing outcomes under various combinations of shear and axial fixation stiffness, and to detect the optimal configuration. A calibrated and established numerical model was used to predict fracture healing for numerous combinations of axial and shear fixation stiffness under physiological, superimposed, axial compressive and translational shear loading in sheep. Characteristic maps of healing outcome versus fixation stiffness  were created. The results suggest that delayed healing of 3 mm transversal fracture gaps will occur for highly flexible or very rigid axial fixation, which was corroborated by in vivo findings. The optimal fixation stiffness for ovine long bone fractures was predicted to be 1000–2500 N/mm in the axial and >300 N/mm in the shear direction. In summary, an optimized, moderate axial stiffness together with certain shear stiffness enhances fracture healing processes. The negative influence of one improper stiffness can be compensated by adjustment of the stiffness in the other direction.The stiffness of fracture fixation devices together with musculoskeletal loading defines the mechanical environment within a long bone fracture, and can be quantified by the interfragmentary movement.  Fractures typically heal successfully, however five to ten per cent of all fractures show complications such as healing delays or non-unions To stabilize long bone fractures against these loading influences, surgeons use either external fixation, plate fixation, or intramedullary nailing Based on animal experiments in sheep, it was found that both magnitude and direction of IFM perform important roles; while small and moderate axial compressive IFM are widely accepted to stimulate healing in vivo experiments and investigated fixation device samples, a general statement of the correlation between fixation stability and healing outcome is hard to achieve from in vivo data.This indicates that some of the occuring healing complications might be the result of inhibitive mechanical conditions arising from improper fixation stiffness. Thus, finding an optimal fixation stiffness was aspired by means of sheep experiments in vivo results, in silico simulations have large potential to provide insights into the mechanical influence on the healing outcome.Numerical fracture healing simulation, however, has the abilities to freely define the fixation stiffness independent of the design of the fixation devices used, to define and control the acting loading situation, and to simulate large numbers of arbitrarily defined fixation scenarios. Given a proper corroboration by available and suitable Hence, the aim of the present study was to numerically simulate healing outcomes for sheep diaphyseal fractures under physiological loading, being treated by fixations with various combinations of different axial and translational shear fixation stiffness. Thereby, optimal as well as detrimental configurations of fixation stiffness should be identified.in vivo studies Bend = EIk is defined asL is the overall length of the bone model, bendu is the applied displacement, and bendF is the corresponding reaction bending force. In a previous study For the present computational study, a three-dimensional finite element model of an idealized mid-diaphyseal, transversal, osteotomy geometry in the ovine tibia  and its healing region was created and meshed in ANSYS  using tetrahedral elements. Material properties were obtained in a previous study fix,axialk = 1, 50, 500, 1000, 1500, 2000, 2500, 3000, 3500, 5000, 7500, 10000 N/mm) and translational shear  fixation stiffness for two gap sizes: most in vivo studies in sheep apply gaps of 2–3 mm, therefore a gap size of 3 mm was chosen, additionally, a small gap size of 1 mm was investigated. For each combination, the bending stiffness was reported relatively to the bending stiffness of the respective intact tibial bone  at several healing time points . We used a surface-fitting tool implemented in MATLAB  to create the continuous characteristic maps from the single data points.To create characteristic maps of healing outcome resulting from fixation stability, healing processes were simulated for a total of 96 different combinations of axial .As another indicator for the healing progress, the extracortical bony callus volume was calculated as a percentage of the numerically predefined healing region, where callus formation can take place. This is reported at different healing time points for several exemplary simulations in order to describe the progress of callus development and correlate it to their respective healing success. A further parameter for the callus size is the callus index, which is defined as the ratio between callus and cortex diameters  although it has developed only a moderate CI of 1.6 after 6 weeks. Furthermore, the CI is not directly correlated to the actual callus size, since it only accounts for the largest callus diameter, but not for the total callus volume. This is visible when comparing cases D and E in in vivo findings in vivo findings Review of the literature on fracture healing experiments in sheep revealed only a limited number of studies which could be applied to corroborate the obtained computational results, as listed in Besides systemic, biological factors, the mechanical conditions within a fracture gap influence the bone healing process decisively. The present study focuses on these mechanical effects. Thus, characteristic maps were created which show the bending stiffness of an osteotomized sheep tibia as a function of superimposed translational shear and axial compressive fixation stiffness at several healing time points under physiological loading conditions. To evaluate the validity of the obtained simulation results, numerous outcomes of appropriate experiments were compared to the generated characteristic maps.in vivo experimental conclusions of Epari et al. in vivo studies, our findings could only be corroborated for 3 mm gap situations, nonetheless we also investigated a small gap size of 1 mm. We found that small gaps result in desirable healing almost independently of the stiffness configuration. We assume that this is the effect of direct gap ossification, as was found in other experiments applying minimal IFM on small defects in sheep The findings presented go beyond the The characteristic maps presented indicate areas where mechanical stimulation is insufficient for callus formation for the 3 mm gap. According to Perren et al. An examination on the ability of the callus size  to serve as an indicator for the success of fracture healing revealed that no dependable prognosis of the healing outcome can be made solely based on the callus size at early healing time points. This is according to Marsh The results of this study show that the optimal healing outcome is reached with a moderate callus volume. For very small calluses, we predict unstable healing due to insufficient mechanical stimulation, whereas large calluses can result in healing, delayed healing or even non-unions despite the large callus volume. The latter can be characterized as hypertrophic non-unions, where large calluses are developed which will not result in bony bridging due to large persisting IFM.in vivo experiments, which themselves provide only punctual information due to their limited number. Furthermore, the corroboration of the numerical simulations depends on the availability of adequate in vivo data, which is still limited. Although for numerous cases the respective shear stiffness could only be estimated and was assigned with 20%-error bars, the underlying algorithm was calibrated on various loading conditions in sheep in vivo data points. Thereby, the applied model reaches a relatively high validity to properly predict fracture healing processes in sheep.There are two major drawbacks which influence the clinical relevance of the present study: (1) it is a numerical study which is accompanied with several modeling assumptions and limitations. First, we excluded endosteal healing regions; this was reasonable since it represents the situation for intramedullary nailing, and pre-investigations indicated that it has no remarkable effect on the simulated healing results. Furthermore, revascularization after fracture is mainly derived rather from surrounding soft tissue and periosteum than from the bone marrow (2) This study simulated the fracture healing processes in sheep. Apart from differences between sheep and humans in the loading situation In summary, this study was able to simulate the influence of fixation stiffness on fracture healing processes and revealed the optimal fixation stiffness configuration for rapid fracture healing. The presented findings provide numerous insights into desirable or disadvantageous mechanical conditions which help to optimize fracture treatment by adjustment of the fracture fixation stability in both axial and shear directions. We conclude that an optimized, moderate axial stiffness together with certain shear rigidity is essential for enhanced fracture healing. Furthermore, inhibiting influences of one loading direction can be compensated by adjusting the other directional stiffness appropriately.


Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer. However, de novo and acquired resistance to these antibodies presents a serious limitation to successful HER2 targeting treatment. The identification of novel epitopes of HER2 that can be used for functional/region-specific blockade could represent a central step in the development of new clinically relevant anti-HER2 antibodies. In the present study, we present a novel computational approach as an auxiliary tool for identification of novel HER2 epitopes. We hypothesized that the structurally and linearly evolutionarily conserved motifs of the extracellular domain of HER2 (ECD HER2) contain potential druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our initial hypothesis. Considering that structurally and linearly conserved motifs can provide functional specific configurations, we propose that by comparing the two types of conserved motifs, additional druggable epitopes/targets in the ECD HER2 protein can be identified, which can be further modified for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available. ErbB2 gene, is observed in approximately 20% of breast cancers, and amplification of HER2 significantly correlates with increased disease aggressiveness and thereby with poor patient outcome Human epidermal growth factor receptor 2 (HER2) is one of four members of the EGF receptor family of receptor tyrosine kinases that mediate cell proliferation, differentiation and survival de novo or acquired resistance to trastuzumab has developed Trastuzumab, a monoclonal antibody directed against the extracellular domain of HER2, which consists of four domains  Pertuzumab is another humanized monoclonal antibody that binds to the extracellular domain II of HER2, the dimerization arm Another therapeutic strategy to block HER2 makes use of small molecule tyrosine kinase inhibitors (TKIs), such as the dual EGFR/HER2 TKI lapatinib In the present study, we report a novel computational process used to predict functional motifs of HER2, which matched very well with epitopes of trastuzumab and pertuzumab (HER2's antibodies).http://www.uniprot.org/) with default setting.The human HER2 amino acid sequence with accession number P04626 was compared to the UNIPROT database using BLAST via the web service (http://prosite.expasy.org/scanprosite/). The databases used for structurally conserved motifs scanning included UniProtKB/Swiss-Prot, splice variants and UniProtKB/TrEMBL databases. The searching results with low level scores were also allowed to show in the output. The other parameters used the default setting.According to the domain organization of the ErbB family, the extracellular domain region of HER2 has ∼620 residues http://www.bioinf.man.ac.uk/cgi-bin/dbbrowser/fingerPRINTScan/FPScan_fam.cgi) with default settings.The amino sequence of the extracellular domain of HER2 (ECD HER2) was aligned for fingerprints in web service (http://www.ncbi.nlm.nih.gov/structure/?term=HER2). Using Cn3D software, the structurally conserved fragments of ECD HER2 were superposed and labeled on the two crystal structures. Furthermore, as supplementary reference to our prediction, FoldX (http://foldx.crg.es/) were introduced with in silico mutagenesis 'repair module', which allowed to predict the amino acids involved in the binding of HER2 to the antibodies by alanine mutation based on 'force field' statistical considerations The crystal structure of the extracellular domain of the human HER2 complexed with trastuzumab Fab (PDB ID: 1N8Z) and the structure of HER2-pertuzumab complex (PDB ID: 1S78) were obtained from NCBI , pig (Sus scrofa), mouse (Mus musculus), rat (Rattus norvegicus), and cow (Bos taurus)  . This inTo determine the structurally conserved motifs in the ECD HER2 protein, the protein sequence from M1 to E620 was uploaded in Prosite ExPASy. We obtained three hits .The first hit, Fragment 1 (F1), was predicted as a type of Sushi domain by the presence of four conserved cysteine residues (C246-C293 and C277-C309), forming two disulfide bounds The second hit, Fragment 2 (F2), was predicted as a NHL domain, defined by amino acid homological presenting in three proteins: NCL-1, HT2A, and LIN-41In order to explore the superposition of those structurally conserved fragments with functional domains of ECD HER2, the predicted fragments were superposed on the crystal structure of ECD HER2  [33]. F1To investigate whether the predicted structurally conserved motifs/fragments could be key regions for the biological function(s) of HER2, they were compared with epitopes of HER2 antibodies based on the HER2: trastuzumab The structural conserved F1 was completely embedded in the center of domain II in blue, , which hAll these structural comparisons results between the predicted conserved fragments and epitopes of HER2's antibodies implied that the structurally/functionally conserved fragments of HER2 probably contain more potentially powerful epitopes/target sites.For purpose of combination of linear feature and structural characterization of epitopes to reduce the prediction error, the PRINTS database, another deterministic pattern, was introduced. And then, 9 top fingerprints were significantly screened from the ECD HER2 sequence with p-value<0.01 . Among tTo further explore the match between the linear and structural conserved regions and epitopes of HER2's antibodies (trastuzumab and pertuzumab), they were compared. Some linear and structural conserved overlapped regions, including fingerprints ALPHATUBULIN 600, ANTRNSFRASE 280, 4FE4SRDOXIN 595, BACE1 290 and TRNASYNTHGLU 316, did not present in the interface of HER2:antibodies , implyinGreat achievements have been obtained with antibody-based therapies in the treatment of cancer The identification of the most efficient epitopes/target sites, as the first step for epitope-based vaccine/drug design, is still an unsolved problem. In the present study, we hypothesized that evolutionary structurally conserved fragments of the ECD of HER2 contain efficient druggable epitopes/targets.We used the PROSITE database that can detect biologically meaningful inter-domains, based on manually derived alignments and extensive manually curated documentation Furthermore, considering that the linear conserved motifs most probably have a meaningful role in the formation of druggable epitopes, we combined the two kinds of motifs and compared the results generated. Some of these segments were not presented in the epitopes of trastuzumab and pertuzumab, but included in the structurally conserved fragments. The result implies that these segments could be potential new epitopes/targets of ECD HER2 for peptide-based vaccine and drug design. Hence, this novel computational process provides a new complementary approach for efficiently selecting critical regions/sites as epitope for drug targeting.Numerous studies have shown that HER2 is involved in many fundamental cellular processes, including cell migration, cell survival and cell proliferation and differentiation  functional bypass of blockade by either up-regulation of downstream signaling or equivalent alternate pathways, (2) failure to elicit immune-mediated killing of tumor cells, and (3) hindrance of antibody binding to HER2. With respect to the latter, our study may provide cues for novel molecular mechanisms of resistance by pinpointing important biological epitopes of ECD HER2. Conversely, the approach presented here may provide the means to identify potentially druggable epitopes/targets that can bypass known impediments to antibody binding to HER2, such as expression of a constitutively active, truncated form of HER2 that lacks the ECD, and consequently the binding site of trastuzumab, or masking of trastuzumab cognate epitopes by steric hindrance of HER2 by cell surface proteins In conclusion, by comparing the structurally conserved motifs of ECD HER2 protein with epitopes of trastuzumab and pertuzumab, we confirmed our hypothesis that evolutionally structural conserved motifs of the ECD HER2 protein contain potentially druggable epitopes/targets. Furthermore, on the basis of a comparison between the structurally conserved motifs and the linearly conserved motifs, we proposed that some segments being potentially more important biological epitopes of ECD HER2 can be further modified for potential therapeutic application. In the present study, we also provide a novel procedure to predict or search for potential epitopes or key target sites in proteins, which may contribute to the design of epitope-based vaccines and drugs. In particular, when there is no X-ray crystal structure available, it is easy to narrow the searching region by means of this process, sharply reducing the interesting residues.


Opioid maintenance treatment (OMT) is the most widely used treatment for opioid dependence. The opioid maintenance treatment (OMT) programme represents an opportunity for people who are opioid users to minimize the many negative health and societal outcomes associated with opioid use through meeting the physiological need of their bodies for opioids. The purpose of this study is to shed some light on how clients in the Norwegian OMT programme see their level of influence on their own treatment.It is a qualitative enquiry using semi-structured interviews of seven OMT clients living in various locations in Norway. The analysis of the material utilized a grounded theory-inspired approach.This study show that the clients who were part of the OMT programme had better lives than people with untreated addictions did. However, the participants experienced having to play by the rules of the OMT programme if they wanted to have successful treatment. This resulted in varying degrees of dissatisfaction with the treatment.The results indicated that the clients felt objectified and disenfranchised in the OMT programme, and points out the low level of influence on their own treatment felt by the OMT clients. People who have used short-acting opiates, such as heroin or morphine-like drugs (opioids), for a long time will develop neurobiological changes to their endorphin system . The opiAt the end of 2012, 7038 of Norway’s approximately 5 million inhabitants had received OMT treatment. The number of people on OMT treatment has increased every year since 1998, with the total number of people who had received OMT in Norway up to the end of 2012 being 9404 , whereasThe aim of the present study was to develop a deep and detailed knowledge of the experiences of OMT clients and how this influences their own treatment.It is widely recognized that clients in need of health care, at all levels, should be empowered . InvolveThis exploratory descriptive study utilizes a grounded theory-inspired approach . GroundeIt was an open recruitment of informants through a local charity organization and a Facebook group. Fifteen people agreed to an interview. However, conducting the interviews was difficult for many of the persons who agreed to be interviewed. Therefore, it was in all seven OMT clients who were interviewed. The empirical material from the interview gave a sufficient for analysis and publication although saturation cannot be invoked. Three of them were recruited via an OMT user group on Facebook (‘LAR Nett’ in Norwegian), and were interviewed via Skype. Four of the interviewees were recruited via a NGO for people who have opioid dependence and other dependences in a city in eastern Norway. There was a good spread of geography (four different districts in Norway), gender and age, and many of the interviewees had been on the OMT programme for several years. Four women and three men were interviewed, giving a total of seven, as mentioned above. The age ranged from 26 to 49 years, with an average of 41 years. The demographic data are displayed in Table The interviews were carried out using qualitative, semi-structured interviews. In each interview, some new questions were used to give the richest possible empirical material and come as close as possible to saturation .In grounded theory, data collection and data analysis are carried out in parallel and combine an inductive and a deductive process , 36, 37.People on the OMT programme are in a vulnerable situation. Vulnerability and competence to consent were specifically assessed for each participant through a conversation especially to assess whether participants were in satisfactory mental state before the actual interview and throughout the interview situation of the interviewer, whom have training in mental health. The interviewees were recruited through an open invitation in a Facebook group for OMT interested individuals and a NGO to avoid any pressure to join. They received information about the study, and were sure that it was voluntary to participate and all data would be anonymized. All interviewees gave their written, informed consent. The study’s research ethics were evaluated by the Data Protection Officer . The research ethics of the World Medical Association’s Helsinki Declaration were followed. This pertained particularly to the principle of no harm and to protect the participants’ privacy .A better life – if you follow the rules of the game’, which can be related to all categories and subcategories, and was expressed by all interviewees. All the respondents indicated that life on an OMT regimen was better than life before inclusion on the OMT programme. They were all excited to go to the pharmacy and pick up their OMT drug, and thus stay healthy, avoid withdrawal and be able to use their energy for their everyday lives. As they received a free OMT drug they did not have to spend time and effort raising money for drugs. The relationship between the categories can be seen in Table The seven interviewees/clients had many stories to tell about the OMT regimen. The main category was ‘For me who wants to become totally drug free it is positive that with OMT you have a certain supervision. That there is a certain control and meetings with the responsibility group.On the whole, several of the interviewees were satisfied with their lives, and expressed an experience of freedom. They all said that the OMT programme saves lives and can provide a good opportunity for rehabilitation. Through the OMT programme clients have the opportunity to regain the kind of life that most people have, such as going to a restaurant with your partner, relaxing at home or engaging in leisure activities. Via the OMT drug, the illegal drug became more distant and participants felt more physically relaxed. The quote below shows the importance of experienced support:Five of the seven interviewees were working or have other daytime activity: One had a paid job, whereas the others were engaged in voluntary work. They talked about the experience of building trust through engaging in work. Interviewees stressed the importance of the drug substitution regimen in order to be able to function at work, and that for each individual the drug should be the one that works best. Some interviewees felt that their opinion was taken into consideration when it came to changing both the type of drug  and its dosage. The building of trust was important.And then I got the necessary self-confidence and I dared to go out … and now I actually apply for proper jobs.Two of the interviewees emphasized that it is not solely the substitution drug that enables them to function well in everyday life and work. These two were very satisfied with the attention that they had received. The OMT programme was much more than just receiving a drug. It was important to have frequent coordinating team meetings with a doctor, nurse, social worker and OMT programme consultant. They had meetings every 2–3 months. One interviewee said that the ability to keep a job – an internship in a shop – boosted her self-esteem. The ability to function in a job gave her self-confidence:The OMT programme is a life-saving programme. One interviewee said that he would have been dead if he had not been included in the programme. He was a heavy multi-drug user and had had several overdoses before he was included in the OMT programme.Before it was the first thing you thought about when you woke up in the morning and the last thing you thought about before you went to bed, and the whole day centred around that. But now you wake up in the morning, put a pill under your tongue and get going, and the day is just like any other day. You don’t think about it 24 h a day, like you did before.The OMT programme helped the participants to stay clean. It made it easy to abolish the craving for drugs. To be included in the OMT programme and receive drug substitution meant that they could concentrate on getting their lives back on track. One of the interviewees said that he regarded himself as a better human being after joining the OMT programme and starting a Subutex (buprenorphine) regimen. He said that the OMT programme had helped him distance himself from drugs, and consequently he did not have to lie and steal. The craving for drugs was gone when he was using Subutex:Interviewees indicated that the OMT programme had given them more freedom, but that they were obliged to follow the OMT system to carry on with their collection schemes and their drug dose. Overall, they had improved quality of life. The interviewees were happy to get replacement drugs, but their satisfaction over dealing with the OMT framework varied. Even those who were most critical of the OMT programme – despite a feeling of powerlessness caused by the OMT system and its conditions – appreciated that it saves lives and that life without it would have been worse.There they are sitting, the three people that somehow have power over me, sitting there and go through the things I’ve done and how they perceive me. I also feel that if there is something I want to say in the meeting, it is not always taken fully into account. As if they have decided in advance what will be said.You have no influence over the treatment.The OMT programme has power over the clients’ lives, and the strength and opportunity to build them up but clients were afraid that they could use the power to the contrary. The participants experienced a lack of co-determination in the OMT programme and thought that their opinions were not taken into account. Several interviewees feared sanctions if they were honest about their use on the side or if they did not deliver their urine samples on time. The OMT regimen that the clients encountered, the attitudes that were projected and the extent to which the programme locally followed the OMT guidelines or made its own rules depended on what part of the country they lived in. One of the interviewees felt that it had been decided in advance what was to be discussed in the coordinating team meetings. The fact that he had subjects that he wanted to discuss was not so interesting to the other participants in the meeting. Another interviewee described such meetings as being for ‘reprimand by the principal’. Another dreaded every meeting and every phone call, even though she knew that it would most probably go well every time:There is a reason why it is heroin that we use. It’s because it works best. It cures both anxiety and nerves and abstinence. It’s somehow the ultimate drug. I think that many of those who don’t succeed with OMT may have managed if they had received heroin instead.Several interviewees emphasized that the OMT programme should provide several types of drugs. In addition to the current medications methadone, Subutex and Suboxone, there are other drugs that are used in some countries, e.g. Substitol and legal heroin , 40:TherIn some places, suddenly everyone must use Suboxone. You feel that you are part of a group that some people feel they can do as they please with.The OMT guidelines are interpreted differently in various districts, and this could have consequences if you have to move to another part of the country, or you lose your GP with whom you have a good relationship:You are afraid of punishment. So my attitude has always been: ‘Say as little as possible, and certainly nothing that can be used against you later on.’Another difficulty for patients on the OMT programme was that use on the side might have consequences for them taking their OMT drugs with them when on holiday. The result may be that you adapt to the OMT system in a way that minimizes the negative consequences for yourself:… they even threaten to take away my methadone, so I immediately began to step down the pills … . You can’t call that collaboration … I don’t have much choice. If they take my methadone away from me, it is very likely that I will end up in my grave.The general perception was that the OMT staff were kind and nice, but were well aware that they had all the power:That’s a little special with OMT, they are very drug focused. It is not so important that you have bought an apartment or go to school or … . It’s like ‘Yeah, but have you been taking drugs?’ You can sit all alone at home and have anxiety and a terrible life, but clean urine samples. And then you are sort of clever!There was a perception of unsatisfactory follow-up. The OMT clients experienced that the personnel focuses on the negative as use of legal drugs rather than the fact that all goes well in other areas. Some pointed out that they should give acknowledgment to the OMT clients who dared to sit in a coordinating team meeting and admit to taking other drugs. With a response like this, more people may dare to be honest:Even if interviewees were doing well in various arenas such as housing and social ones, the OMT programme tended to focus more on clean urine samples, the motivation to stay away from drugs and to what extent clients stick to the agreement. According to the clients, the follow-up was unsatisfactory.In the responsibility groups, that everyone is a member of, there should be someone who has stopped abusing drugs and who has participated in OMT. This person may be called an employee with personal experience. Because those who have never been stoned don’t know what we’re talking about! They pretend that they do, but they don’t, you know!One of the interviewees claimed that the OMT programme is not so good at providing information about what the client is entitled to. The interviewees meant that this could partly be explained by the fact that some of the staff had little or no relevant training for their job as an OMT consultant. Furthermore, it was pointed out that several of the OMT employees have little or no experience with drugs. One of the interviewees wished that the OMT employees were people with personal experience within the programme:You feel like a criminal. It is as if they assume that you will try to trick and cheat. As if they think that after all you are just a junkie, so they cannot entirely trust you!Despite several years in the OMT system, the clients expressed disappointment about the fact that the control measures and meetings for the treatment system never ended. They felt that suspicion never stopped. Even if it is the clients who have requested treatment to get clean, the interviews revealed that interviewees have the impression that the OMT staff think that clients have a hidden agenda:There is not much that makes me angrier than to hear their condescending attitude!I wish they would see us as individuals rather than to think that all OMT clients are dirty drug addicts who will do anything to get something to get high on.The interviewees pointed out that in the public debate one occasionally hears terms such as ‘state drugs’ used about narcotic drugs funded by the state and ‘state addicts’ about people who receive these narcotic drugs  in a rehabilitation programme funded by state. It can be particularly hurtful for OMT clients when employees have condescending attitudes. Some clients expressed a feeling of stigmatization. The following quotes from two interviewees illustrate this:We’re still a bit like second-rate citizens. There are not many other patient groups who have to come on certain days and fetch their medication, and at any time may get a phone call and be ordered to submit a urine sample while someone watches them pee.Several clients had felt hurt and distrusted by the staff on the OMT programme. One reason is that the OMT client may have the feeling of not being a regular client, but a drug addict – with the negative connotations that this term implies. You are defined as a client, but still have a sense of being treated differently from clients who are not addicts. This feeling is caused by several conditions described as degrading by the OMT clients. Several mentioned feelings of inferiority and stigmatization. Only clients who are addicts must deliver urine samples under supervision and pick up their medication at certain times. Several of the interviewees thought that it was unreasonable to bring in urine samples a couple of times a week, and one of them did not understand why for the rest of her life she would be expected to deliver urine samples at short notice:This study summarizes the experience of being a patient within the Norwegian OMT regimen. It has shown that clinicians have taken decisions without client involvement. Clinicians are under increased pressure to focus on dialogue and to be the client’s advocate – in addition to establishing a more equal relationship. Professional capacity is based on the clinician’s ability to listen to and interact with the client in ways that give space for the client’s agenda. This kind of new professionalism may appear devoid of paternalism . How do Two of the interviewees stated that their opinion was taken into account as far as both choice of the OMT drug and the drug’s strength were concerned. This was part of the reason why they functioned relatively well both mentally and physically. Thanks to the OMT programme, clients could spend their energy on something sensible rather than trying to get hold of drugs, and this was seen as meaningful. These interviewees considered it meaningful (compare Antonovsky ) that thSeveral interviewees felt that they got a lot of support from their OMT consultant, that they had a say and were able to participate in their own treatment. As a result, the OMT system was perceived as being more comprehensible. According to Antonovsky , one hasDetoxification of the OMT medication is not the prime objective of treatment. The prime objective is continued physiological and social stabilization. As yet, there is no validated medical cure for opiate addiction. Until a curative medication or a safe curative procedure has been developed, many of the patients may have to remain in treatment for the duration of their lives to avoid relapses, increased criminality, subsequent overdoses and death . Why do The dependability of the present study was ensured by following recognized scientific methods of data collection and analysis . The conThe findings in the present study show that clients of the OMT programme have a better life if they follow the rules. It is clear that those clients who do not have a final say in decisions affecting them, could feel insulted that control never stops. Clients of the OMT programme would probably have experienced a higher quality of life if, in addition to receiving the OMT drug, they had felt that they could have more influence on their treatment. This would have increased their sense of coherence . The fin