Evidence in the medical literature suggests that trial registration may not be preventing selective reporting of results. We wondered about the place of such information in the peer-review process.We asked 1,503 corresponding authors of clinical trials and 1,733 reviewers to complete an online survey soliciting their views on the use of trial registry information during the peer-review process.1,136 authors (n = 713) and reviewers (n = 423) responded (37.5%); 676 (59.5%) had reviewed an article reporting a clinical trial in the past 2 years. Among these, 232 (34.3%) examined information registered on a trial registry. If one or more items  differed between the registry record and the manuscript, 206 (88.8%) mentioned the discrepancy in their review comments, 46 (19.8%) advised editors not to accept the manuscript, and 8 did nothing. The reviewers' reasons for not using the trial registry information included a lack of registration number in the manuscript , lack of time , lack of usefulness of registered information for peer review , lack of awareness about registries , and excessive complexity of the process .This survey revealed that only one-third of the peer reviewers surveyed examined registered trial information and reported any discrepancies to journal editors. Another study found that for 62% of trial publications, at least one primary outcome was changed, introduced, or omitted as compared with the submitted protocol Several authors have shown evidence of selective outcome reporting in published results of randomized controlled trials (RCTs) In 2005, the International Committee of Medical Journal Editors (ICMJE) initiated a policy requiring investigators to record basic information about their RCT into a clinical trial registry before participant enrollment as a pre-condition for publishing the trial's findings i.e., registered before the study ended with a clearly defined primary outcome). Among articles for adequately registered trials, 31% (46/147) showed discrepancies between the registered and published outcomes. These data suggest that registered information is not sufficiently being consulted to identify unacknowledged changes to primary outcomes during the manuscript review process.Previous studies have shown that selective outcome reporting remains prevalent among registered trials We surveyed the journal reviewers and corresponding authors of recently published reports of RCTs to determine their views on the use of trial registries during the peer-review process.The present research is not considered as biomedical research according to the French legislation We included reviewers of manuscripts of RCTs, including corresponding authors and journal reviewers. To identify the corresponding author of RCT articles recently indexed in PubMed, we used the search term “randomized controlled trial” on 19/12/2011 to identify reports of RCTs published in 2010 and 2011. We also applied the highly sensitive search strategy described by Robinson and Dickersin New England Journal of Medicine (NEJM), Journal of the American Medical Association (JAMA), and Annals of the Rheumatic Diseases (ARD) from the last journal issues for the years 2010 or 2011, which thank reviewers for their contributions. The e-mail address search was the same as for corresponding authors, namely, first a PubMed search, then a search on the Internet via Google. We included all reviewers of these three journals, whether they reviewed clinical trials or not.We obtained the list of reviewers of If a corresponding author or reviewer appeared more than once in the participant search, only the most recent e-mail address was retrieved. From April to May 2012, the participants were sent e-mails inviting them to complete an online survey  and S3, The survey asked participants about their knowledge, current practices, and opinions regarding trial registration and peer review of manuscripts . The queWe recorded the peer reviewers' research affiliations, along with the number of annual peer reviews they completed and the number of trials they had joined as investigators. The survey comprised two parts: 1) the reviewer's experiences based on the most recent manuscript reviewed over the previous two years, and 2) whether they compared registered information with manuscript information during the review process, and if so, whether they reported any discrepancies. For participants who responded that the reviewer should verify trial registration or check for discrepancies, we asked them to rate the effectiveness of various methods to facilitate the comparison of registered and reported information during the peer-review process. Ratings of 4 and 5 on a scale from 1 to 5 were considered effective .a priori response rate to be about 30%, assuming that 5% of e-mails would not reach their intended recipient and that some trialists would not have reviewed an article within the previous two years. We sought about 500 survey responses to ensure a sufficient sample size. Data are represented descriptively as proportions. Categorical variables were analyzed by chi-square test. Statistical analysis involved use of R software .Web-based surveys produce variable response rates NEJM (n = 227), JAMA (n = 773), and ARD (n = 777). The flow of participants is in We surveyed 1,503 corresponding authors of recently published RCTs indexed in PubMed, and 1,777 peer reviewers for Of the 1,136 respondents, most  were affiliated with a university hospital . Only 11The percentage of trial investigators and the average number of published articles reviewed per year from 2007 to 2011 were higher in the reviewer group than in the author group .Of the 676 respondents who had peer reviewed an article in the previous two years, 58 responses were missing or not useable for analysis. Overall, 232 respondents (34.3%) had examined the information registered on a trial registry; most were corresponding authors  and 78 were journal reviewers. The most commonly reviewed items in the registration record were the primary outcome (94.4%), eligibility criteria (83.2%), planned sample size (83.2%), secondary outcomes (81%), and posted results (47.8%). When one or more of the above methodological components differed between the registry record and the manuscript, 206 respondents (88.8%) mentioned the discrepancy in their review comments, 46 (19.8%) advised editors not to accept the reported manuscript, and 8 did nothing. Four respondents indicated that their recommendations to journal editors differed depending on the importance of the discrepancies.For 386 respondents who did not look at registry information during the peer review, the most common reasons included a lack of registration number in the manuscript , lack of time , lack of useful registered information , lack of awareness of the availability of registered information , excessive complexity of the process , lack of registration record  and not remembering to check the registry record . Overall, 13 participants reported not having looked at the trial registry because they thought that the journal had done so before sending the manuscript to review.For the 676 participants who had peer reviewed an article in the previous two years, 317 (46.9%) and 182 (26.9%) felt that the managing or academic editor, respectively, was responsible for checking that the trial was registered. Only 50 respondents (7.4%) believed that the peer reviewer should have this responsibility. Other proposed responsible parties were the editor-in-chief (n = 21), investigator (n = 13), research ethics committee (n = 4), and sponsor (n = 3). Ten participants thought that it was not useful to check whether a trial was registered. By contrast, respondents considered a more evenly divided responsibility for checking for consistency between the registered information and the manuscript .The 177 respondents who felt that the peer reviewer should verify that a trial was registered or discrepant with the manuscript were asked to rate the effectiveness of certain options for facilitating the peer reviewer's ability to compare registries and manuscripts. The three most effective options were providing peer reviewers with a direct Web link to the corresponding trial record on the registry website , providing a list of the registered information to accompany the full manuscript , and providing the registration number with the manuscript .We surveyed corresponding authors and peer reviewers of medical journals about the role of registered trial information in the peer-review process. Of the 676 respondents who had peer reviewed a manuscript reporting the results of a clinical trial in the past two years, only one-third examined the registered information for the trial. When discrepancies were identified, most respondents (88.8%) mentioned them in their review comments, and 19.8% advised editors not to accept the manuscript.Despite measures to improve trial transparency and prevent selective outcome reporting, recent studies reveal that these issues remain prevalent but are identifiable using registered trial information Alternatively, as several of our respondents suggested, managing or academic editors could themselves verify that the trial had been registered before patient recruitment and could check that there are no discrepancies between the registered and published items  before sending the manuscript to peer reviewers.One-quarter of respondents who did not examine the registry record when they peer reviewed a manuscript stated that the reason was a perceived lack of usefulness of registered information. Previous studies have found that the completeness and quality of information in registries is variable. Zarin et al. assessed ClinicalTrials.gov data that were publicly available for a sample of 3,284 registered trials. Of the 2,324 posted results entries that had been posted publicly, 14% were linked to a PubMed citation through an indexed ClinicalTrials.gov registration number. Of 2,178 clinical trials with posted results records, 20% had more than two reported primary outcomes measures and 5% more than five. For some studies, posted results include more than 100 primary and secondary outcome measures According to previous studies, using the Internet to conduct surveys is associated with a variable response rate (from 9% to 94%) and a lower rate than with a traditional mailed survey This study has several limitations. Our sample of authors who published trials in PubMed-indexed journals should be broadly generalizable, but we also surveyed a sample of reviewers of medical journals with high impact factors, which could differ systematically from reviewers of other journals. Indeed, these reviewers may have been more aware of the issues of trial registration and discrepancies between the registered protocol and the published results. Our results may thus overestimate the use of trial registration information by peer reviewers. Another limitation might be that we provided the respondents with a list of pre-defined options for improving the peer-review process. However, we added an open-question to solicit any other suggestions.We reveal that only one-third of peer reviewers of articles of RCTs use the information recorded on the trial registry website, and most report any discrepancies to journal editors. The scientific and medical communities could emphasize the value and role of trial registration to facilitate peer review of trial manuscripts and could introduce concrete measures to facilitate the access to and use of trial registry information.Figure S1Flow chart of participants in the study.(TIF)Click here for additional data file.Table S1Respondent characteristics.(DOCX)Click here for additional data file.Appendix S1Web questionnaire.(DOCX)Click here for additional data file.Appendix S2Mail for corresponding authors.(DOCX)Click here for additional data file.Appendix S3Mail for reviewers.(DOCX)Click here for additional data file.


Sodium thiosulfate (STS) reduced calcium stone formation in both humans and genetic hypercalciuric stone forming (GHS) rats. We sought to measure urine chemistry changes resulting from STS administration in people.STS was given to healthy and hypercalciuric stone forming adults. Five normal non-stone forming adults (mean age 33 years), and 5 people with idiopathic hypercalciuria and calcium kidney stones (mean age 66 years) participated. Two baseline 24-hour urine collections were performed on days 2 and 3 of 3 days of self-selected diets. Subjects then drank STS 10 mmol twice a day for 7 days and did urine collections while repeating the self-selected diet. Results were compared by non-parametric Wilcoxon signed rank test. The primary outcome was the resulting change in urine chemistry.3 concentration after the STS period: it did not change. The net effect was an increase in supersaturation of uric acid, and no change in supersaturation of calcium oxalate or calcium phosphate.STS administration did not cause a significant change in urinary calcium excretion in either group. In both groups, 24 hour urinary ammonium (P = 0.005) and sulfate excretion (P = 0.007) increased, and urinary pH fell (P = 0.005); citrate excretion fell (P<0.05) in hypercalciuric participants but not in non-stone formers. Among stone formers with hypercalciuria, 3 of 5 patients had measurement of serum HCO3 did not change, urine tests suggested an acid load in both non-stone forming and hypercalciuric stone-forming participants. The long term safety of STS needs to be determined before the drug can be tested in humans for long-term prevention of stone recurrence.The basis for studies demonstrating that STS prevented stones in rats and people was not reflected by the changes in urine chemistry reported here. Although serum HCO Sodium thiosulfate (STS) has a number of uses in medicine, the most well known of which is its role as an antidote for cyanide poisoning. It acts via the conversion of cyanide to the more water-soluble thiocyanate which is readily excreted in urine Two studies, one in genetic hypercalciuric stone-forming (GHS) rats and one in humans, have shown a decrease in the incidence of kidney stones following STS administration Our study sought to investigate whether urine chemistries of humans on STS therapy would suggest efficacy in reducing urinary lithogenicity, and whether evidence for an associated acid load would be present.The study population consisted of two distinct arms. 2 (range 60.0- 91.0 ml/min/1.73 m2). Three patients had calcium stones composed of calcium oxalate and calcium phosphate, the latter component constituting 20, 40, or 50% of analyzed stones. The other 2 patients did not provide stones for analysis but had radio-opaque stones seen on plain abdominal radiography. Patients over the age of 80 were excluded from the study. Patients who had hypercalciuria in the past but did not have hypercalciuria on baseline collections done immediately prior to STS administration were not excluded. All patients were prescribed a diet demonstrated in a randomized controlled trial to reduce the incidence of recurrent stones in participants with hypercalciuria . The second group consisted of an equal number of participants with a mean age of 66 years. This group included 4 men and 1 woman all of whom had been seen and evaluated as outpatients in the Metabolic Stone Clinic and who were documented calcium stone formers with a history of hypercalciuria, specifically with 24 hour urine calcium measurements of greater than 300 mg/d on collections done within a year prior to the study, while following a self-selected diet. We identified 10 eligible patients seen in the Metabolic Stone Clinic in recent months and the first 5 who were contacted and agreed to participate were enrolled. The participants were recruited between January-April, 2011. The mean serum creatinine concentration was 1.1+0.1 mg/dl (range 0.9–1.2 mg/dl) and the mean estimated glomerular filtration rate was 74.2+12.4 ml/min/1.73 mThe patients were selected at random and solicited for participation. The study was approved by the institutional review board of the New York Harbor VA Healthcare System and registered with Clinicaltrials.gov as NCT01088555. The study was performed under US Food and Drug Administration Investigational New Drug number 106,424. All subjects provided written consent to participate in the study.Each participant undertook 2 baseline 24 hour urine collections over two consecutive days during which they were instructed to keep detailed food diaries while eating their self-selected diets. Oral doses of STS were then taken by the subjects for 7 days after the completion of the baseline urine collections. STS was dispensed by the research pharmacist to the patients and taken home for self-adminstration. The dose was 10 mmol (5 ml of a 2 M solution) twice a day, replicating the protocol described by Yatzidis. The taste and smell of STS precludes blinding the study. On days 5, 6 and 7, subjects replicated the diets they had documented during the pre-STS baseline urine collections. On days 6 and 7 of the medication administration period, participants undertook a second set of two 24 hour urine collections. Food diaries were not collected; they were simply used to allow subjects to replicate diets. Three of the 5 stone forming participants also had basic metabolic panels, including serum bicarbonate levels, checked prior to STS administration and on day 7 of taking STS; the other 2 did not have post-STS serum measurements because of logistical problems.During the urine collections, the urine was maintained at room temperature. An antimicrobial and a urine volume marker were added to each urine container and then a 50 ml aliquot of urine was obtained. The participants performed the urine collections at home, and then mailed their urine collections to Litholink Corp  for analysis In each 24 hour urine sample, we measured calcium, chloride, creatinine, magnesium, sodium, potassium, phosphate, ammonium, and uric acid concentrations by standard laboratory technique using a Beckman Synchron CX5 . pH was measured by glass electrode. Oxalate was measured by enzyme assay using oxalate oxidase . Citrate was measured by enzyme assay using citrate lyase . From these analyses supersaturation (SS) was calculated with respect to calcium oxalate, calcium phosphate, and uric acid using the iterative computer program EQUIL 2 Thiosulfate and sulfate were measured by ion chromatography using a Dionex ICS 2000 system . Samples were loaded into a 25-µl loop using an autosampler and injected onto an AG-11 guard column and AS-11 analytical column in series, with KOH as the mobile phase. Ion peaks were detected using a conductivity meter with the eluent background conductivity suppressed using an anion self-regenerating suppressor. We have found thiosulfate to be stable in urine under the collection conditions used in this study.P<0.05.Results of baseline and post-STS urine collections were compared by non- parametric Wilcoxon signed rank test and the effects of STS in controls and hypercalciuric stone forming participants were compared by Mann Whitney U test. Statistical data were generated and analyzed with a commercially available software package, Systat . Results were expressed as mean±SD and differences were considered statistically significant at All 10 patients enrolled completed the study without any lost to follow-up. The results of all participants were included. The results of STS administration in both the normal participants, the hypercalciuric participants and the 2 groups combined are presented in There was no evidence to suggest the development of metabolic acidosis based on the pre- and post-STS serum bicarbonate levels in the 3 stone forming patients from whom these data were collected . All 3 hUrine volume, creatinine, urea, and sodium excretion remained unchanged in both arms of the study pre- and post-STS, and in the combined data, confirming replication of diets over the course of the collections. Administration of the drug was well tolerated with no patients stopping therapy due to adverse events. All patients were questioned about their experience with the drug: 2 patients had transient episodes of loose or watery stools which resolved despite continued STS administration and 2 patients described foul smelling stool. No deviations from the protocol occurred.3]. Similar results occurred in the control non-stone-formers, with the exception that citrate did not fall. These effects on urine chemistry, with no net change in lithogenicity, would not explain remarkable reductions in stone activity reported in a non-controlled, non-randomized clinical study In the first study to examine a full set of urinary analytes after administration of oral STS, neither healthy controls, nor hypercalciuric calcium stone formers, experienced statistically significant increases in urine calcium excretion or calcium oxalate or calcium phosphate supersaturation. In stone-formers, there were decreases in urine pH and citrate, and increases in ammonium excretion without a change in serum [HCOOur urine results are also not consistent with the findings of Yatzidis et al 3] measurements while taking STS, all 3 had clear evidence of having received an acid load based on urine chemistry while maintaining normal serum [HCO3]. The lack of change in serum [HCO3] in the stone formers and the lack of change in urine citrate in the controls (in whom serum [HCO3] was not measured) suggest that ammoniagenesis was adequate to compensate for the presumed acid load. The difference in citrate excretion between controls and stone-formers despite similar increases in urine ammonium excretion, is interesting but not explained and suggests that perhaps stone-formers have greater renal citrate reabsorption in response to acid loads, contributing to relative hypocitraturia. The decrease in urine pH caused a significant increase in supersaturation of uric acid and could be associated with an increase in uric acid stone formation in some susceptible patients.The present study is consistent with thiosulfate presenting a net acid load, suggested by increases in urine ammonium excretion, and decreases in citrate excretion (in stone-formers) and urine pH. The etiology of this acid load is not clear. Stool losses of bicarbonate or potential base could be an explanation, though most patients had no diarrhea and those who did, experienced it only transiently. Previous reports of metabolic acidosis after parenteral administration of STS for cyanide poisoning and calcific uremic arteriolopathy exist a’s of that strong acid are 0.6 and 1.7 and the common knowledge that the sodium salt of a strong acid is not itself an acid. More likely, the acid load is due to the oxidation of thiosulfate to sulfate by the liver, producing protons Some recent literature has claimed that sodium thiosulfate is a strong acid The urine findings are similar to those noted in GHS rats given STS 3] did not change in the 3 in whom it was measured despite their significant changes in urine chemistry, we do not believe this small sample is an important limitation to knowing whether an acid load was presented. The urine findings clearly show that it occurred. We cannot speculate regarding the generalizability of these findings to a broader population of calcium stone formers with these or other urinary risk factors for stone formation.One limitation of the study is the small sample size of the 2 groups and that the groups are not matched for age. Since the primary outcome was not a comparison of the effects of the drug on the two populations we do not consider the age difference to be important. In fact, combining the results of the 2 groups demonstrated that the results were highly consistent between them with the only difference being a fall in urine citrate in hypercalciuric stone-formers but not in the non-stone-forming controls. Finding healthy people willing to take an investigational drug and do multiple urine collections among a hospital population was more difficult than finding stone formers interested in advancing the knowledge of their disorder and its potential treatment. Only after the results in the healthy group did we realize that it would be desirable to measure serum chemistry after drug administration and because of logistical issues could not get blood while participants were still taking the drug in 2 of the 5 stone formers. Since serum [HCO3] does not necessarily confirm the safety of long-term STS administration, as the finding is potentially related to two limitations of the study: its small sample size and relatively short duration of STS administration. STS therapy for the prevention of nephrolithiasis would likely need to continue for many years, and as such, studies of at least months to years would be necessary to sufficiently rule out development of sustained metabolic acidosis. A larger study with a longer duration more similar in scale and length to that of Yatzidis The absence of a significant change in serum [HCOThe appropriate therapy for prevention of recurrent calcium phosphate stones is unclear as no randomized controlled trials with this outcome have been completed


Existing mouse models of Roux-en-Y gastric bypass (RYGB) surgery are not comparable to human RYGB in gastric pouch volume for a large or absent gastric volume. The aim of this study was to develop and characterize a mouse RYGB model that closely replicates gastric pouch size of human RYGB surgery of about 5% of total gastric volume. We established this model in diet-induced obese (DIO) mice of C57BL/6J. This surgery resulted in a sustained 30% weight loss, entirely accounted for by decreased fat mass but not lean mass, compared to sham-operated mice on the high fat diet. Compared to sham-operated mice, energy expenditure corrected for total body weight was significantly increased by about 25%, and substrate utilization was shifted toward higher carbohydrate utilization at 8 weeks after RYGB when body weight had stabilized at the lower level. The energy expenditure persisted and carbohydrate utilization was even more pronounced when the mice were fed chow diet. Although significantly increased during daytime, overall locomotor activity was not significantly different. In response to cold exposure, RYGB mice exhibited an improved capacity to maintain the body temperature. In insulin tolerance test, exogenous insulin-induced suppression of plasma glucose levels was significantly greater in RYGB mice at 4 weeks after surgery. Paradoxically, food intake measured at 5 weeks after surgery was significantly increased, possibly in compensation for increased fecal energy loss and energy expenditure. In conclusion, this new model is a viable alternative to existing murine RYGB models and the model matches human RYGB surgery in anatomy. This model will be useful for studying molecular mechanisms involved in the beneficial effects of RYGB on body weight and glucose homeostasis. Among bariatric surgeries, Roux-en-Y gastric bypass (RYGB) is most effective in the treatment of obesity, type-2 diabetes, and other comorbidities of the obese state The aim of our study was, therefore, to develop and verify a mouse model of RYGB that is based on a pouch size of about 5% of the total stomach volume, similar to typical RYGB surgery in humans, which could then be used in the mechanistic studies using genetically altered mice. By taking particular care to preserve the vasculature near the cardia of the stomach, we were attempting to reduce acute surgical trauma to facilitate intake of solid food soon after surgery. Male C57/BL6J mice were made obese by feeding them a high-fat diet for 12 weeks and then used in the surgery. To verify the effectiveness of the surgery, we monitored body weight for 8 weeks after surgery and measured body composition, food intake, energy expenditure, body temperature response to cold exposure, locomotor activity, and insulin tolerance at specific time points after surgery.All experiments were performed in compliance with and were approved by the Institutional Animal Care and Use Committee (IACUC) of Pennington Biomedical Research Center. Animals were housed under conventional conditions at the Pennington Animal Facility. C57BL/6J mice from Jackson Laboratory were fed a high fat diet  at 8 weeks-old for 12 weeks. At the time of surgery, they weighed 46±5 g. All animals were fed ad libitum with HFD before and after surgery, except in the energy expenditure experiment where mice were fed the high fat diet for the first 3 days and then chow diet for the subsequent 3 days in the metabolic chamber. In addition, mice were given access to chow and high-fat diet for about 2–3 days prior to surgery.Animals were fasted 4 to 6 hours before operation. Anesthesia was induced and maintained with isoflurane. Standard aseptic procedures were used throughout. For RYGB, a midline incision starting from the xiphoid process was made and the perigastric ligaments were ligated and cut to release the stomach.As illustrated schematically in The second step is to make a small gastric pouch with a volume of about 5% of the total gastric volume, the standard pouch size in human RYGB. To this end, a titanium clip  was applied across the area just below the cardia, from which the left gastric vessel bundles had been lifted or removed , Fig. 2DThe third step comprises the jejuno-jejunostomy that determines the lengths of the Roux and common limbs. In pilot experiments, we determined the length of the small intestine in vivo in chow-fed and high fat diet-induced obese mice, by using the 1 cm long tip of a pair of angled forceps. We found that the average length of the small intestine from the ligament of Treitz to the cecum is about 22 cm in chow-fed mice and about 20 cm in long-term DIO C57/BL6J mice. From this, we determined that in obese mice, the optimal lengths are about 5–6 cm for the Roux limb and 5–6 cm for the biliopancreatic limb. Therefore, the jejunum was transected about 2 cm distal to the ligament of Treitz, and the cut ends were sterilized with 5% povidone iodine. The distal end was brought up to the stomach pouch for end-to-end anastomosis as described above. For the jejuno-jejunostomy, a longitudinal slit was made on the antimesenteric side of the jejunum at 6 cm distal to the site of gastrojejunostomy, and the proximal end of the jejunum was joined in an end-to-side anastomosis using 11-0 nylon suture in an interrupted fashion. This resulted in a common limb consisting of the distal jejunum and the ileum of about 12 cm . Before For the sham operation, the perigastric ligaments were cut, and then a 3 mm incision was made in the stomach wall and closed with a titanium clip. In addition, the jejunum was transected 2 cm distal to the ligament of Treitz, and the two cut ends were anastomosed using 11-0 nylon suture in an interrupted fashion. These procedures are designed to inflict at least some of the tissue damage and similar surgical trauma as by the real RYGB, but not to change the nutrient flow through the GI-tract, which is hypothesized to cause its beneficial effects.For the first 24 hours after the operation, the mice were put in regular shoe box cages without bedding on a heating pad set at 35°C. The mice were given 0.7 ml of 5% dextrose subcutaneously and carprofen  for analgesia, immediately after surgery. Water and normal chow was made available after recovery from anesthesia. On the day after surgery, mice were moved to shoe box cages with regular bedding, and high-fat diet was provided 3 days after surgery.Body weight was measured daily, and body composition was measured at the end of the observation period at 8 weeks by NMR. Food intake was measured at 5 weeks after surgery. Pre-weighed high-fat diet was given directly into the shoe box cages and the remaining diet was measured 3 days later. Average daily intake was calculated with taking spillage into account.Ten DIO mice were used in this study after surgery. Body composition (NMR), energy expenditure, and cold response were tested between 6–9 weeks after surgery. Those metabolic tests were conducted using protocols described elsewhere Insulin tolerance was performed at 4 weeks after surgery to determine insulin sensitivity. The test was conducted with peritoneal injection of insulin at 0.7 U/Kg after 4 h fasting.In this study, values are presented as mean ± SEM. Student's t test was used in most data analysis for body weight, insulin tolerance, energy expenditure, substrate utilization, and body temperature. Two-way ANOVA was used in data analysis for diet and surgery effects on energy expenditure. P values<0.05 were considered significant. All statistical tests were two-tailed.After establishing the basic surgical approach in carcasses and non-survival surgeries, RYGB was carried out in an additional 34 mice to determine optimal postsurgical dietary requirements and verify the intended pouch size. Fifteen (44%) of these mice died or had to be euthanized within the first week after surgery, mainly due to complications caused by mechanical obstruction at the end-to-side jejuno-jejunostomy. To reduce this risk, we used 11-0 nylon suture with 16–20 interrupted stitches and employed as little bowel wall as possible (<0.5 mm on each side) in the anastomosis .RYGB was then performed in a total of 5 mice, and sham surgery in 6 mice in this study. One of the five RYGB mice exhibited lack of appetite and rapid weight loss and was euthanized 5 days after surgery. Part of the Roux limb had formed adhesions to the gastrojejunostomy leading to mechanical obstruction. The remaining 4 RYGB mice recovered without problems and were followed for 8 weeks after surgery, yielding the results reported here. The operating time for each mouse was 80–90 minutes, and the mice were able to eat some solid diet within 24 hours after surgery without complications. All 6 sham-operated mice recovered quickly.RYGB resulted in rapid loss of body weight over the first 3 weeks . RYGB miInsulin sensitivity was examined in the mice at 4 weeks after surgery with the insulin tolerance test. The result suggests that RYGB mice exhibit significant improvement in insulin sensitivity . The sigSomewhat unexpectedly, we found that RYGB mice increased intake of high-fat diet by almost 80% compared to sham-operated mice, measured 5 weeks after surgery  . IncreasIn addition, we found that energy expenditure normalized to total body mass was significantly enhanced by about 26% in RYGB mice compared to sham-operated mice  . Energy The respiratory exchange ratio (RER) was measured to obtain information regarding substrate utilization for mitochondrial ATP production, with high RER values indicating preferential carbohydrate utilization and low RER values indicating preferential fat utilization. At 8 weeks after surgery, when all excess fat mass had been lost and body weight was stable, RER during the dark phase was significantly higher in RYGB compared to sham-operated mice , suggestThe physical activity level (locomotor activity) was examined to determine its possible role in elevated energy expenditure. Locomotor activity was significantly higher in RYGB compared to sham-operated mice during the light phase , probablTo measure energy expenditure and physical activity in mice on low-fat diet, we switched animals in the Oxymax system after 3 days of high-fat to 3 days of regular chow diet and analyzed the data for the last two days after the mice had adapted to the new diet. Similar to the period of high-fat feeding, energy expenditure corrected for total body mass was significantly higher by about 23% in RYGB compared to sham-operated mice under conditions of low-fat feeding, and the effect was present during both the light and dark periods .However, unlike under high-fat feeding, the increase in RER was much more robust in RYGB mice under low-fat (high-carbohydrate) feeding and extended throughout day and night , suggestRYGB mice had a higher energy metabolism on both HFD and chow diet. It is not known if brown fat contributes to energy metabolism. Brown fat produces heat from glucose and fatty acids in the maintenance of body temperature. To address this question, we conducted a cold response experiment to test the brown fat function at 6 weeks after surgery. In the cold room of 4°C, the body temperature dropped significantly over 2 hours in the sham group . The droExisting mouse models for RYGB surgery have either no gastric pouch at all In our pilot studies, we found that after RYGB mice often died or had to be euthanized because of intestinal obstruction at the jejuno-jejunostomy, resulting in a mortality rate of more than 50%. Using 11-0 nylon sutures in an interrupted fashion, significantly reduced such obstruction and mortality. Failure to properly arrange the Roux limb in an “S” fashion appeared to increase the risk for the intestinal obstruction. Obstruction usually manifests itself on the first day after surgery with secretion from the eyes and lethargy. If there are no postsurgical complications, the mice look healthy and alert, and are able to eat solid diet within 24 hours. In most other mouse and rat models, liquid diet has to be used for up to 10 days to prevent obstruction, and animals have to be carefully adapted to solid foods Another postsurgical complication often observed in mice and rats is foamy saliva smeared over the snout. The saliva often contains traces of blood, and the affected animals exhibit excessive mouth wiping. We believe that this is mainly due to excessive stretch of the esophagus when preparing the gastric pouch and gastro-jejunal anastomosis.Additional recommendations according to our experience are as follows: 1) Providing regular chow for 2–3 days before surgery appears to reduce the risk of complications. This beneficial effect may be due to the higher fiber content of chow, which is known to stimulate gastrointestinal motility We show here that the model replicates some, but not all, physiological changes reported after RYGB in humans The mouse model also differs from human RYGB, in that the weight loss is much more rapid in our and other mouse models, with about 20–30% weight lost within two weeks after surgery, whereas weight loss in humans is much more protracted over about 12 months. This rapid weight loss makes it difficult to study the weight-loss-independent mechanisms for improvement of glucose homeostasis. We found evidence for improved insulin-sensitivity by measuring the ability of insulin to lower plasma glucose at 4 weeks after surgery, when maximal weight loss had already occurred. We are not able to ascribe it to weight-independent mechanisms for insulin sensitization in rodent models, and pair-feeding/weight-matching strategies will be necessary in future experiments.In conclusion, we describe and verify a novel model of RYGB in diet-induced obese mice with the size of the gastric pouch closely matching human surgery. The model is characterized by increased energy expenditure in the face of increased food intake and resulting in sustained loss of excess fat and improvement in insulin sensitivity. The model will be valuable to test candidate mechanisms in genetically altered animals.


The large inter-patient and inter-study variability in forced expiratory volume in 1 second (FEV1) with bronchodilators makes determination of optimal doses difficult in conventional dose-ranging studies. We considered alternative methods of analysis.Indacaterol is a once-daily long-acting inhaled βWe utilized a novel modelling approach to provide a robust analysis of the bronchodilatory dose response to indacaterol. This involved pooled analysis of study-level data to characterize the bronchodilatory dose response, and nonlinear mixed-effects analysis of patient-level data to characterize the impact of baseline covariates.1 response. Indacaterol 75 μg achieved 74% of the maximum effect on trough FEV1, and exceeded the midpoint of the 100-140 mL range that represents the minimal clinically important difference , with a 90% probability that the mean improvement vs placebo exceeded the MCID. Indacaterol 150 μg achieved 85% of the model-predicted maximum effect on trough FEV1 and was numerically superior to all comparators (99.9% probability of exceeding MCID). Indacaterol 300 μg was the lowest dose that achieved the model-predicted maximum trough response.The study-level analysis pooled summary statistics for each steady-state visit in 11 placebo-controlled studies. These study-level summaries encompassed data from 7476 patients at indacaterol doses of 18.75-600 μg once daily, and showed that doses of 75 μg and above achieved clinically important improvements in predicted trough FEV1, significantly affects the dose response, indicating that patients with more severe COPD require higher doses to achieve optimal bronchodilation.The patient-level analysis included data from 1835 patients from two dose-ranging studies of indacaterol 18.75-600 μg once daily. This analysis provided a characterization of dose response consistent with the study-level analysis, and demonstrated that disease severity, as captured by baseline FEVComprehensive assessment of the bronchodilatory dose response of indacaterol in COPD patients provided a robust confirmation that 75 μg is the minimum effective dose, and that 150 and 300 μg are expected to provide optimal bronchodilation, particularly in patients with severe disease. The efficacy and safety of indacaterol was evaluated in an extensive Phase III clinical programme in which patients received doses of up to 600 μg once daily for up to 52 weeks [1) vs placebo up to 26 weeks [1, the higher dose was associated with incremental benefits in terms of symptomatic relief, such as dyspnoea [Indacaterol is the first long-acting inhaled β1 for evaluation of bronchodilators in COPD), a dose response is established and a target dose can be selected as the smallest dose that differs from placebo and has both a clinically relevant effect and an acceptable safety profile [Conventional dose-ranging trials rely on hypothesis testing and use placebo corrected mean responses to compare dose levels and determine the existence of a dose response. If at least one dose achieves a statistically significant difference compared with placebo for an appropriate endpoint  and includes a locally weighted scatterplot smoothing (LOESS) curve to highlight the main trend. While the overall FEV1 in the population varied from about 0.5 to 3 L, the maximum drug response vs placebo is under 200 mL as depicted in the figure inset. This is indicative of the low signal-to-noise ratio of the bronchodilatory response in COPD. The impact of this issue on the interpretation of study results is best illustrated by considering the variability of a single dose level within and between trials. Figure 1 response to indacaterol 150 μg across six different studies. Each panel represents the results from one trial. The data points are the least square means (LSM) for each study visit. The grey area within each panel provides a visual representation of the range of responses observed within each trial. The panels are ranked by the median response observed in each trial. This figure shows that the intra- and inter-study variability in mean trough FEV1 may be as high as 50 mL, whereas the inter-study variability in median response may be about 60 mL. The implications of this observation is that relying on single LSM values does not provide adequate precision to easily differentiate between dose levels.The potential of indacaterol as a bronchodilator is best appreciated when the responses across all the tested dose levels are expressed together in a dose-response relationship. However, given the inherent variability in measurements of lung function relative to the drug-induced change achieved by bronchodilators, accurate characterization of the dose response relationship is difficult. Figure To overcome this inherent difficulty in using conventional methodology to accurately establish dose responses for bronchodilators in COPD, two alternative approaches were explored. The first approach focused on the study level results typically reported for bronchodilator assessment. The aim of this approach was to use study level LSM from COPD studies in the indacaterol development programme to provide estimates of the dose response and of the precision of typical study level data used for the purpose of dose selection. The second approach focused on individual patient data from two studies. The aim of this approach was also to determine the dose response while exploring individual patient characteristics that may affect the drug response and hence dose selection. To our knowledge, this is the first time that these novel modelling methods have been used to characterize dose response in COPD patients receiving a bronchodilator.1 that exceeded the midpoint of the 100-140 mL range considered to represent the minimum clinically important difference (MCID) for FEV1 in COPD  [Two approaches were used: 1) an integrated analysis of study-level data, and 2) an integrated analysis of patient-level data. The objectives of both analyses were to provide a precise quantitative characterization of the dose-response relationship of indacaterol and the responses to comparators used in some trials. The key metrics of interest were: the minimum effective dose (MED), defined as the lowest dose that achieved a median trough FEV≥120 mL) ; the opt1 and had a duration of at least 14 days. Indacaterol was compared with formoterol in two studies, with salmeterol in four studies and with tiotropium in one study. Note all comparator data was at steady-state and assessed at the same study visits in the respective studies. LSM contrasts to placebo and associated standard errors were collected from individual study reports to create the study-level pooled analysis dataset. The LSM estimates were obtained after various covariate adjustments in the original statistical analyses of each individual study  maximum possible response, and ED50 characterizes drug potency and corresponds to the indacaterol dose producing 50% of the maximum effect. The model included between-study (δi) and within-study, between-visit (γij) variability on Emax and was analysed using a Bayesian methodology.where i is an index for study and j for study arm, EAs the summary data used in this analysis are contrasts to placebo, the model was constrained to have a null response with placebo (dose = 0). Summary information on formoterol, salmeterol and tiotropium, collected in the studies included in this pooled analysis, was also added  model was used , based o0 is the response to placebo, and E0i and Emi are random effects to account for inter-patient variation in response. NLME models are often used for the purposes of pooling individual patient data as they allow the differences between patients to be accounted for in an unbiased manner as fixed effects (e.g. patient characteristics such as age and disease status) and random effects (e.g. the remaining random differences that cannot be accounted for by patient characteristics).where i is an index for patient and j for study day (14 or 15), E0i and Emi) to account for within-patient correlation of the observed responses, as well as covariate adjustments . A transform-both-sides approach was used, with the logarithm transformation applied to both the response and the model. An additive residual error term was specified after log transformation. The primary goal was to derive an estimate of the dose response for the improvement over placebo in trough FEV1 based on individual measurements in each patient.The base model included inter-individual variability , COPD severity  use of 1 are shown in figure The data used in the study-level analysis of trough FEV1 responses for each of the comparators included in this analysis. The most striking feature of the plot is that the response to indacaterol at the plateau exceeds the response of all comparators. In other words, doses of indacaterol 150 μg or greater provide greater average trough bronchodilation than the comparators.The outcome of the study-level analysis of the 24-h trough values is also presented in figure 50 was 28 μg, with a 95% CI ranging between 12 and 52 μg . In other words, 75 μg is the most likely lowest tested dose that exceeds the MCID.A key advantage of a comprehensive quantitative characterization of the dose response is that it allows generation of precise probabilistic statements about the relative responses. Figure Figure Since this analysis relied on study-level summaries (LSM), it is possible to assess the predictive performance of these data. This is important, as study-level summaries are often used to support dose-selection decisions for bronchodilators. In figure max) and ED50 were respectively 185 mL  and 19 μg . This translated into indacaterol 18.75 μg providing 49% of the maximum trough FEV1 effect, compared with 66% for indacaterol 37.5 μg, 79% for indacaterol 75 μg, 89% for indacaterol 150 μg, 94% for indacaterol 300 μg and 97% for indacaterol 600 μg.The patient-level analysis, although restricted to the two dose-ranging studies, provided a characterization of dose response that was similar to that obtained in the study-level analysis. The final NLME model used for the patient-level analysis produced a slightly steeper dose-response for the typical COPD patient representative of the population in the two studies. The estimated maximum effect  and the sensitivity (ED50) to a bronchodilator are strongly influenced by the baseline FEV1. In other words, as disease severity increases (i.e. baseline FEV1 decreases), patients require higher doses to obtain an optimal response. This relationship can be seen in a three dimensional display , there is a decrease in the relative improvement across all doses. However, this decrease is strongest for doses of 75 μg or lower. Doses of 150 μg or higher provide sustained bronchodilation that is largely independent of disease severity.To better understand the relationship between dose and baseline FEVFinally, it is instructive to place the findings of this analysis in the context of the GOLD classification of COPD severity . For thi1 and the poor precision of the conventional methodologies. Simply increasing trial size is not a viable option because the patient numbers required to attain sufficient precision to differentiate active treatments over the dose range would be prohibitively large. To overcome this methodological limitation, alternative approaches were explored using the large indacaterol database to provide a rigorous evaluation of the indacaterol dose response in COPD.Conventional dose-ranging trials for bronchodilators, such as those used to evaluate tiotropium, salmeterol and formoterol ,10-12,201 (observed peak or area under the curve over 0-4 h post-dose).The study-level analysis provided a precise characterization of the dose response using study level data. Data from 11 studies, ranging from 2 to 52 weeks over a dose range from 18.75 to 600 μg were available, including data from 7476 patients and with treatment arm sizes ranging from 65 to 437 patients. Despite the large within- and between-study variability, the analysis was able to meet the requirements of a dose-ranging analysis, namely to precisely differentiate doses over the effective range. Although not shown, a similar pattern was seen in an analysis of peak FEV1, it is not possible to precisely determine the metrics such as the MED or differentiate active treatments using pairwise comparisons in typically sized trials. The implication is that the conventional approaches to dose-ranging of bronchodilators cannot easily meet their quantitative objectives. Only through pooling information in a model based approach is it possible to attain the precision necessary to draw robust quantitative conclusions on treatment responses.Beyond the characterization of the indacaterol dose response itself, the study-level analysis provides unique insights into the precision of the conclusions that may be drawn from typical trials that investigate the efficacy of bronchodilators. Given the typical variability in FEVWhile the overall objective of the patient level analysis was also to characterize the dose response, it had the further aim of quantifying the impact of patient characteristics on dose response and ultimately dose selection. The patient level analysis dataset was restricted to the two dose-ranging studies as these were most relevant to the question at hand. Although restriction of the analysis to 2-week data contrasts with the study-level pooled analysis (which pooled data between Week 2 and Month 6), the similar outcomes from the two analyses reinforce the overall conclusions while providing further insights into the impact of patient characteristics on dose response.1, as a marker of disease severity, is the most important patient characteristic that influences the dose response. As disease progresses (baseline FEV1 decreases) the shape of the dose response changes. However, with doses of 150 μg or greater, the relative response becomes more or less independent of baseline. In other words, doses of 150 μg or greater are required to ensure that patients can achieve optimal benefit. This finding is particularly pertinent to the 25% of the studied COPD population with baseline FEV1 less than 1 L. To our knowledge, this analysis is the first to demonstrate and quantify a relationship between COPD severity and dose response.The key finding of the patient level analysis was that baseline FEV1 and patient-reported outcomes such as TDI [1 with indacaterol doses of 150 μg and higher seen in our analyses would therefore be expected to result in greater improvements in these patient-reported outcomes. In support of this, indacaterol doses of 150 and 300 μg have been shown to result in significantly greater improvements in TDI than placebo in patients with moderate-to-severe COPD, with the 300 μg resulting in numerically  greater improvements than indacaterol 150 μg [1 and TDI support the concept of identifying the minimum indacaterol doses that provide near maximum bronchodilation so as to optimize the clinical benefit.A number of measures are available for quantifying dyspnoea . TDI is widely used to assess dyspnoea  and was h as TDI . The higl 150 μg . This co1 this is the case even in very large trials. The key difference between the approaches presented in this manuscript and conventional methods is the use of an explicit model, in this case the Emax model, to pool information across dose levels. It is the pooling of information that provides the greater precision compared to the conventional method, which relies simply on each independent point estimate. In terms of overall efficiency, the patient level analysis of the dose response provides the greatest level of insight for the least number of patients studied. However, a key prerequisite for such an analysis is that data on an adequate dose range is available. In the current analysis, it was necessary to combine two studies to achieve this goal. While this requirement for a wider dose range and larger study population may be considered a drawback of model based methods, it has been suggested this is the price necessary to pay for adequate and robust characterization of the dose response [It is worth briefly commenting on the presented methods in the context of the original dose selection. Conventional dose-ranging trials rely on hypothesis testing and use contrast statistics to compare dose levels and determine the existence of a dose response. Using placebo corrected means to characterize the dose response and distinguish between doses is not robust if the CIs overlap; for FEVresponse .While the conventional approach originally selected the 150 and 300 μg doses, uncertainty remained about their location on the dose response and, in particular, the efficacy provided by these doses relative to the MCID. The current analyses support the selection of 150 and 300 μg as the lowest doses that ensure optimal response across the spectrum of disease severity, while identifying 75 μg as the MED. The direct clinical benefit of this analysis is that it confirms the selection of doses of indacaterol that provide incremental benefit over other bronchodilators at levels that are at the threshold of the maximum trough response.In conclusion, thorough analysis of dose response is critical to the successful evaluation of drug treatments in COPD. Model-based approaches such as those described here should allow more informed decisions to be made regarding doses for further evaluation by complementing the results from more classical dose-ranging studies. These comprehensive analyses of the dose response of indacaterol in COPD, showed that 75 μg is the MED of indacaterol and confirms that indacaterol 150 and 300 μg are expected to provide optimal bronchodilation, particularly in patients with severe disease.(CI): confidence interval; (COPD): chronic obstructive pulmonary disease; (FEV1): forced expiratory volume in 1 second; (LOESS): locally weighted scatterplot smoothing; (LSM): least squares mean; (MCID): minimal clinically important difference; (MED): minimum effective dose; (NLME): nonlinear mixed effects; (TDI): transition dyspnoea indexThe authors declare that they have no competing interests.All authors were involved in the conception and design, or acquisition of data, or analysis and interpretation of data; reviewed each draft of the manuscript and revised it critically for important intellectual content; and approved the final version of the manuscript.Appendix.Click here for file


Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively.Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type.We have now characterized all 11 hexokinase encoding genes in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.We conclude that the hexokinase gene family is more diverse in  Saccharomyces thus has a glucokinase, ScGlk1, and two dual specificity hexokinases, ScHxk1 and ScHxk2. The eukaryotic hexokinases are all related to each other, but are unrelated to prokaryotic glucokinases and hexokinases. Plants also have a fructokinase which is unrelated to the hexokinases [Hexokinases catalyze the first step in hexose metabolism, the phosphorylation of glucose and fructose. Hexokinases that show a higher specificity for glucose than for fructose are sometimes called glucokinases. The yeast okinases .Physcomitrella hexokinase PpHxk1, a soluble protein with a transit peptide [Hexokinases are found in several different intracellular locations. The three yeast hexokinases are cytosolic, but ScHxk2 can also enter the nucleus . Animal  peptide  that loc peptide -14.In addition to their metabolic roles, eukaryotic hexokinases have also been implicated in signal transduction. Mitochondria-associated hexokinases have thus been shown to negatively affect programmed cell death in both animals and plants, by preventing the release of cytochrome c from mitochondria -17. It sArabidopsis [An important question is where the enzyme exerts its signaling function. Early work in yeast focused on the cytosol, since the yeast hexokinases are cytosolic. However, further studies have shown that ScHxk2 also can translocate into the nucleus, where it forms a complex with the Mig1 repressor ,24. Simibidopsis  and ricebidopsis ,27 suggePhyscomitrella patens is unique among plants in that gene targeting by homologous recombination works in it with frequencies comparable to yeast [Physcomitrella a powerful model system for studies of plant gene function [Physcomitrella genome has further strengthened it as a model plant [Physcomitrella hexokinase PpHxk1, which by gene targeting was shown to account for 80% of the glucose phosphorylating activity in protonemal tissue [Physcomitrella like other plants possesses several hexokinases, which makes it difficult to draw conclusions about gene function from the knockout of a single gene.The moss to yeast . This hafunction ,30. The el plant . We havel tissue . Furtherl tissue . Part ofPhyscomitrella genome. Seven of the genes predict proteins that clearly belong to the previously described types A and B [We here report the characterization of all eleven genes encoding putative hexokinase proteins in the  A and B . However-2 s-1. Subculturing of Physcomitrella patens protonemal tissue was done on cellophane overlaid 0.8% agar plates containing BCD media  at 30 μmol melements ), supplePpHXK1 we also found several other hexokinase encoding sequences [PpHXK2 and PpHXK3  where we isolated equences . From thata base  for morevailable , we searNOS1 terminator [BamHI or BglII sites were designed to facilitate sticky end ligation of PCR products into the BamHI site between the 35S promoter and the rsGFP coding region  from the vector psmRS-GFP, a pUC118 based plasmid with the 35S promoter in front of a soluble modified red shifted GFP followed by the ® Orange was detected with Zeiss filter set number 20 . The nucleic acid stain 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to visualize the nucleus and detected using a DAPI/Hoechst filter .GFP fusion constructs were transiently expressed in wild type protoplasts after PEG-mediated transformation . The traHXK1, HXK2 and GLK1 genes in the W303-1A background [Physcomitrella were cloned into the high copy number 2 μm URA3 shuttle vector pFL61 [PGK promoter  was used for sequence editing, sequence analysis and building of contigs. The sequence of PpHxk1 differs in one position leucine-55) from the published sequence 5 from th as previPhyscomitrella, PpHxk1, is responsible for most of the hexokinase activity in protonemal tissue extracts. Thus, 80% of the total glucose phosphorylating activity, including almost all of the activity in the chloroplast stroma, disappears when the PpHXK1 gene is disrupted [PpHXK1 disruption [Physcomitrella and we found that they can be grouped into four different types that show some variation in their exon-intron organization  and rice (ten). It has previously been noted that metabolic enzymes are overrepresented in Physcomitrella, possibly reflecting a more diverse metabolism in mosses than in seed plants [We have previously shown that the major hexokinase in isrupted . Howeversruption . We thersruption  revealedd plants .PpHXK6, for which no transcript has been found. However, for four of the genes, PpHXK5, PpHXK9, PpHXK10 and PpHXK11, only aberrantly spliced transcripts causing premature termination have been sequenced. It should be noted that two other genes, PpHXK3 and PpHXK7, had both correctly and incorrectly spliced transcripts. This suggests that alternative splicing is common, and that correctly spliced products therefore could exist also for the four aberrantly spliced genes. A sequence analysis of the genes does not suggest that any of them is a pseudogene, since both predicted protein sequences and other important features such as consensus sites for splicing are well conserved. The only possible exception is PpHxk11 which has a few amino acid substitutions in positions suggested to be important for catalytic activity (see below).The well-conserved protein sequences and the presence of cDNAs for most of the genes among our PCR products and in public EST data bases ,42 suggePhyscomitrella hexokinases belong to the two previously described types. Thus, in addition to PpHxk1, two more type A hexokinases are encoded by PpHXK5 and PpHXK6. Based on the sequences, PpHxk6 appears to be more closely related to PpHxk1 than PpHxk5. Four of the predicted Physcomitrella hexokinases, PpHxk2, PpHxk3, PpHxk7 and PpHxk8 have N-terminal membrane anchors similar to the N-termini of type B hexokinases from other plants.We have previously classified plant hexokinases into two types  dependinPhyscomitrella hexokinases do not conform to the criteria that we used to define types A and B. One hexokinase, PpHxk4, has a truncated N-terminus without either a membrane anchor or an organelle import peptide. We will refer to this novel type as a type C hexokinase. Interestingly, no hexokinase with a truncated N-terminus is encoded by the Arabidopsis genome. The rice genome predicts two hexokinases with truncated N-termini, the OsHXK7 and OsHXK8 gene products [However, some of the Physcomitrella genome also predicts three additional hexokinases, PpHxk9, PpHxk10, and PpHxk11, which we will refer to as type D. Like the type B hexokinases, they possess N-terminal membrane anchors, but these anchors differ in sequence from the type B hexokinases  and rice (OsHxk4) are more similar to each other than to the other Arabidopsis and rice hexokinases  sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinases in Physcomitrella have undergone concerted evolution.We have characterized all 11 hexokinase encoding genes in the moss Physcomitrella hexokinases and constructed various plasmids. AN, TO, MU and MT transformed Physcomitrella protoplasts and analyzed the GFP expression. AN and MT analyzed the transformed Physcomitrella protoplasts treated with the mitochondria specific dye. All authors participated in the design and coordination of the study. All authors have read and approved the final manuscript.AN, TO, MU and MT carried out the experimental work. TO performed the yeast complementation study. All authors were involved in the sequencing and in the phylogenetic analysis. AN, TO, MU and MT cloned the Genomic and cDNA clones encoding hexokinases. Physcomitrella hexokinase genes and cDNA clones and the primers used for cloning them into the pCR®2.1-TOPO vector.Click here for fileOligonucleotide primers. Oligonucleotide primers used. Most of the primers are named after the gene to be amplified, whether it binds to the 3' or 5' part, and whether it is followed by a BamHI, BglII or SmaI site. Primers whose names end with a T were used to clone inserts where the membrane anchor or chloroplast transit peptide was removed. The primer combinations used in the various cases are listed in Tables S1 and S3.Click here for fileHexokinase-GFP fusion and yeast expression plasmids. The first column lists the plasmids used for intracellular localization and yeast complementation studies. The primers and templates used to make these plasmids are listed in the last two columns. The amino acid residues of the different hexokinases that are predicted to be expressed after cloning into the vectors psmRS-GFP (GFP fusions) and pFL61 (yeast complementation) are also listed.Click here for fileSequence Identity matrix for the N-terminal region of type B and D hexokinases. Comparison of N-terminal regions containing the membrane anchor of type B and D hexokinases, illustrated as a two-way sequence identity matrix. The two or three most similar hexokinases in each comparison are in highlighted in bold.Click here for fileArabidopsis, rice, and Physcomitrella genomesAlignment of the hexokinases and hexokinase-like proteins that are predicted by the . The protein sequences shown are those predicted by the annotated genomes. The most common residues in each position are enclosed within boxes. The 20 most conserved residues identified by Kuser et al. [et al. [S. cerevisiae, the fission yeast S. pombe, the nematode C. elegans and human hexokinase IV, were included as an outgroup in order to root the tree.r et al.  are mark [et al. ,46 are aClick here for fileIntracellular localization of truncated hexokinase-GFP fusions. Fluorescence microscopy pictures of wild type moss protoplasts transiently expressing different truncated versions of the Physcomitrella hexokinases fused to GFP. The hexokinase codons that were fused in frame to GFP are indicated for each hexokinase. GFP fluorescence is shown in green, with chlorophyll auto-fluorescence in red serving as a chloroplast marker. Protoplasts expressing GFP alone were also included as a control.Click here for filePpHXK3 promoter and alternative splicing of the PpHXK7 transcriptSequence polymorphism in the . a. Microsatellite repeat in the PpHXK3 promoter that shows evidence of rapid evolution. The two sequence variants are shown.b. The two splice variants of PpHXK7 with (PpHXK7b) and without (PpHXK7a) an N-terminal membrane anchor. The nucleotide sequences and predicted encoded peptide sequences of the two splice variants are shown. The splice sites and the start codons are underlined, and the methionines are highlighted by a black background.Click here for filePhyscomitrella hexokinase transcriptsAlternative splicing of . The different Physcomitrella hexokinase transcripts showing alternative splicing. The plasmids names and the type of alternative splicing within these transcripts are listed together with the predicted effect on the expressed protein.Click here for filePhyscomitrella hexokinasesAccession numbers for . The accession numbers of the PpHXK2-pPHXK11 transcripts and the corresponding GeneIDs are listed.Click here for file


Soft tissue artifact is known to be one of the main sources of errors in motion analysis by means of stereophotogrammetry. Among many approaches to reduce such errors, one is to estimate the position of anatomical landmarks during a motion with joint angle or displacement of skin markers, which is the so-called compensation method of anatomical landmarks. The position of anatomical landmarks was modeled from the data of the so-called dynamic calibration, in which anatomical landmark positions are calibrated in an ad hoc motion. This study aimed to apply the compensation methods with joint angle and skin marker displacement to three lower extremity motions  in ten healthy males and compare their reliability. To compare the methods, two sets of kinematic variables were calculated using two different marker clusters, and the difference was obtained. Results showed that the compensation method with skin marker displacement had less differences by 30–60% compared to without compensation. In addition, it had significantly less difference in some kinematic variables (7 of 18) by 25–40% compared to the compensation method with joint angle. Skin marker-based stereophotogrammetry is the most commonly used technique to analyze motions, despite significant errors due to the deformation of soft tissues such as skin and muscle. The displacement of skin markers relative to the underlying bones is called soft tissue artifact (STA), and it is responsible for errors in motion analysis. Skin marker displacement can be as much as 40 mm in the lower extremities , 2. ErroMethods proposed to reduce STA errors are based on either one of two principles: (1) treating the STA as an independent noise irrespective of motor tasks and (2) modeling a systematic pattern of STA in relation to motor tasks. Representatives of the first category are the studies of Challis , Ball anThe second category includes methods that assessed task-related patterns of STA by obtaining the positions of anatomical landmark—which indicates the skeletal pose—and skin markers at multiple postures or in an ad hoc motion. Cappello et al.  and CappAs an alternative to anatomical landmark position compensation with joint angle, Ryu et al.  proposedThe present study applied the two anatomical landmark position compensation methods in real lower extremity movements of healthy people. This involved motion analysis of the hip, knee, and ankle joints in three lower extremity motions, walking, sit-to-stand/stand-to-sit, and step up/down, in 10 healthy males. The performance of the compensation method with skin markers was compared to the method with joint angle.A motion measurement system with six cameras  was used to measure lower extremity motions . The accuracy of the system was assessed by comparing the measured distance between two marker positions to the known distance, such that the variation of the distance indicates error, as described by . Mean erTen healthy young males with no previous history of musculoskeletal or neurological disorders related to the lower extremities participated in the experiment. The mean height, weight, and age of the participants were 1.75 m (SD = 0.03), 69.3 kg (SD = 5.8), and 26.2 years (SD = 3.0), respectively. All the participants signed informed consent forms. The anatomical coordinate frame defined by anatomical landmarks (ACF) of the pelvis, thigh, shank, and foot of the participants was defined according to . Left/riTwenty reflective markers (20 mm diameter) were placed on the right lower limb segments of the participants . Four maThe participants performed six motor tasks: (1) standing static posture; (2) flexion/extension, abduction/adduction, and internal/external rotation of hip joint; (3) hip joint swing motion with fixed knee joint; (4) sitting static posture; (5) knee joint motion with fixed ankle; (6) walking, sit-to-stand/stand-to-sit, and step up/down. Standing and sitting static postures were held for 1-2 minutes for anatomical landmark calibration, and hip joint flexion/extension, abduction/adduction, and internal/external rotation were performed to identify the center of the hip joint. Hip joint swing motion with fixed knee and knee joint motion with fixed ankle were conducted as ad hoc motions for the dynamic anatomical landmark calibration. Walking, sit-to-stand/stand-to-sit, and step up/down were performed as target motions for analysis. Anatomical landmark calibration was performed in both standing and sitting static postures using a pointer on which two markers with a known distance were mounted see . In the a, b, c and an anatomical landmark p, a TCF is defined by the skin markers, and the position of p relative to the TCF is computed like Geometric calculations were used to determine the positions of thigh and shank anatomical landmarks relative to the TCFs on the corresponding body segments and on neighboring segments. For example, if there are three skin marker 1 by T1, T2, T3, and TCF2 by T4, T5, T6) and two shank TCFs  were defined to compare the reliability of the two compensation methods. The difference between two values of kinematic variables, which is calculated with two sets of TCFs, was used as reliability measure. The local coordinates of each anatomical landmark of the thigh and shank were fixed in each TCF. The local coordinates of each thigh anatomical landmark were also fixed in a shank TCF and that of each shank anatomical landmark were fixed in the TCF of the foot by markers F1–F4. Two thigh TCFs , t: translation vector (3 × 1), and ε: random error (3 × 1).The poses of all the TCFs and ACFs during ad hoc motions and three target motions were calculated using the Singular Value Decomposition algorithm of . The metR & t to minimizeFind n: Number of markers (≥3). where a: local coordinate of anatomical landmark, b: global coordinate of anatomical landmark, R: rotational matrix (3 × 3) of shank TCF, and t: translation vector (3 × 1) of shank TCF.The displacements of the anatomical landmarks and skin markers on the thigh during the hip joint swing motion with fixed knee were obtained in reference to the two thigh TCFs. The positions of anatomical landmarks  were reconstructed using a shank TCF and the relevant anatomical landmark local coordinates (3)b=Ra+ls: local coordinate of anatomical landmark, which is reconstructed from shank TCF relative to thigh TCF and lt: local coordinate of anatomical landmarks relative thigh TCF, which is computed from static posture. b: global coordinate of thigh anatomical landmark, sn: position of one marker of skin marker cluster of thigh, n = i, j, and k, and o: the origin of a thigh TCF, which can be selected appropriately.Anatomical landmark displacements were calculated as the difference between the local coordinates of the reconstructed anatomical landmarks and those fixed in the standing static posture for the two thigh TCFs as in (4)DisplLikewise, the displacement of skin markers T4 (for thigh TCF1) and T3 (for thigh TCF2) was calculated to model the anatomical landmark displacement with the displacement of a skin marker, which is not a member of a marker cluster. They were computed by subtracting the local coordinates in each thigh TCF fixed in the static posture from the measured ones during motion. In the same way, the displacements of anatomical landmarks  and skin markers S4 (for shank TCF1) and S3 (for shank TCF2) during the knee joint motion with fixed ankle were obtained in reference to the two shank TCFs.The relationship between the displacements of anatomical landmarks and skin markers was represented in a linear model using a simple regression form because of its simplicity and repeatability. Each axial component of an anatomical landmark displacement was plotted with the three axial components of the relevant skin marker displacement. The skin marker component with the highest correlation coefficient with anatomical landmark displacement was identified. It is possible to use multiple regression models with three components, but in this case, there will be multicollinearity in the model and the developed model will be much varied by analyzers. Anatomical landmark displacement with joint rotation in the sagittal plane was modeled to compare the alternative method of  with theTarget lower extremity motions  were analyzed using three methods: the compensation method with skin markers , with joReliability of the three methods was determined by the difference between two sets of kinematic variables estimated using two marker clusters, as described by the method of . The effTwo-way analysis of variance (ANOVA) was conducted to determine if motion analysis differences are affected by the type of analysis method and the type of target motion. For each kinematic variable, time series differences were obtained. Then, two-way ANOVA was performed with the type of analysis method and type of motion as independent variables. Differences found to be significantly affected by analysis method or motion type were further examined using the Student-Newman-Keuls (SNK) test to determine if the differences are statistically different from one another. rLE1, ΔrME1, ΔrFH1 in TCF1, and ΔrLE2, ΔrME2, ΔrFH2 in TCF2) and the thigh skin marker displacements  for a participant are shown in y and z components of ΔrLE1 had a weak dependency with the displacement of T4, and this is similar with the y component of ΔrME2 and z component of ΔrFH2. Likewise, most of the shank anatomical landmark displacements  had a high dependency with the shank skin marker displacements (S4 and S3). This study identified one axial component of the skin markers that was highly correlated with each component of anatomical landmark displacements.There was a dependency between the displacements of the anatomical landmarks and the skin marker in the corresponding TCFs in the ad hoc motions. The plots between the thigh anatomical landmark displacements . The effect of motion type , analysis method , and their interaction with the mean differences for 18 kinematic variables are shown in The student Newman-Keuls (SNK) test of the mean differences for the three methods showed that compensation with skin marker displacement was more effective than without compensation for most kinematic variables and more effective than compensation with joint angle for some of them. For most angular joint motions, compensation with skin marker displacement had significantly smaller (33–60%) mean differences than without compensation, except for ankle flexion/extension see . CompensMoreover, compensation with skin marker displacement had significantly smaller (27–52%) mean differences than without compensation for five of the nine linear motions see . It alsoBoth anatomical landmark compensation methods (with skin marker displacement and joint angle) showed good reliability in real lower extremity motions. In the study, differences between two marker clusters for the hip and knee kinematic variables  were significantly reduced by 30–60% by anatomical landmark compensation with skin marker displacement compared to that without compensation. Reduction of the differences by anatomical landmark compensation with joint angle ranged from 10 to 60%. Of the two anatomical landmark compensation methods, the one using skin marker displacement showed slightly better reliability in analyzing lower extremity motions. Results showed that the differences of five kinematic variables  were significantly reduced by compensation with skin marker displacement by 30–40% more than joint angle compensation regardless of the target motion. The former method also significantly reduced the differences of knee flexion/extension and ankle internal/external rotation in sitting and stepping than the latter method by 25–30%. Compensation with joint angle was 35–50% more reliable than with skin marker displacement for only one variable  in some target motions. Compensation with joint angle had some limitations in analyzing the kinematics of the ankle joint. While compensation with joint angle was as good as with skin marker displacement in analyzing hip and knee joint motions, it had larger mean differences than without compensation for some variables of the ankle joint. This seems to be because of the relatively large inaccuracy of the joint angle used in anatomical landmark compensation. Without compensation, the mean difference of knee flexion/extension was as large (4.0°) as that of hip flexion/extension (3.8°); the joint angle estimated without compensation was used to estimate anatomical landmark positions in compensation with joint angle. In contrast, anatomical landmark displacements of the shank (5–20 mm) were small relative to those of the thigh (15–40 mm). Therefore, the unreliable knee joint angle seems to have a large effect on the anatomical landmark position estimation of the shank relative to the thigh. The compensation with skin marker displacement and with joint angle still had some residual differences. Even in a stationary posture, mean differences between two sets of kinematic variables estimated using two marker clusters, which represent the instrumental errors in this study, were 0.1–3° and 1–16 mm for angular and linear motions, respectively. During target motions, mean differences for compensation with skin marker displacement and compensation with joint angle were 1–5° and 5–30 mm for angular and linear motions, respectively. This study had a limitation that it only evaluated the reliability of compensation methods, but not accuracy. To compute the accuracy of the compensation methods, tracking the position of underlying bones is necessary, and errors in kinematic variables should be compared to validate the compensation methods. But this study could not do it due to practical reasons, thus further study will be needed to validate the methods.Moreover, the relationship between the displacement of the anatomical landmarks and those of skin markers reflected STA partially. STA in the thigh can occurr with hip and knee joint motions and that in the shank with knee and ankle joint motions. However, this study obtained the relationship in the thigh from only hip joint motions and those in the shank from only knee joint motions. Therefore, the relationship would not consider the whole STA of real lower extremity motions in which both the proximal and distal joints move together. A further study will be necessary to analyze and use the relationship using additional motions such as ankle joint motion with knee fixed and knee joint motion with hip fixed. This study applied the compensation methods in some lower extremity motions and compared their reliability. Compensation with skin marker displacement was more reliably than with joint angle, although both methods were superior to without compensation. For hip and knee motions, both anatomical landmark compensation methods reduced differences between marker clusters by half in the three motions than without compensation. Compensation with joint angle had some weaknesses in analyzing ankle motion, whereas compensation with skin marker displacement consistently showed less difference between marker clusters than without compensation.