There is a growing patient interest in being involved in the decision-making process. However, little information is provided on how this information should be structured.Does it make a difference, in patient treatment decision-making, whether information is given based on the benefits or on the side effects in rotator cuff disorders?It is a prospective randomized study that includes patients diagnosed with rotator cuff tears.Patients were randomly allocated to either group A (benefit-inform) or group B (side effect-inform) and were asked to answer the following questions based on their assigned group:Group A: Your doctor informs you that you have a rotator cuff tear and states that if he/she surgically repairs your cuff tear you will improve and that the cuff remains healed at the 2-year follow-up in 71% of the cases where surgery is done.Would you choose surgery? Yes or NoGroup B: Your doctor informs you that you have a rotator cuff tear and that if he/she surgically repairs your cuff tear you will improve and that the cuff is torn again at 2-year follow-up in 29% of the cases where surgery is done.Would you choose surgery? Yes or NoAge, gender, the shoulder affected and the functional status assessed through the Constant score were also recorded.P = 0.000). In group A, 36 of 43 (84%) accepted surgery, compared to 17 of 37 (46%) in group B.80 patients were randomized (43 to group A and 37 to group B). The patients assigned to group A (benefit) accepted surgery significantly more frequently than those assigned to group B (complication) (The way that information on rotator cuff disorders is provided strongly influences patients’ treatment decisions.NCT03205852. Registered 29 June 2017. Retrospectively registered.ClinicalTrials.gov,  The paternalistic approach to treatment decision-making based on the doctor’s preferences has largely been superseded by involving patients throughout the decision-making process where possible. Moreover, it has been shown that patient involvement results in more cost-effective services and improves health outcomes, quality of life and patient satisfaction –7.Most health conditions can be approached with different strategies that have implicit advantages and disadvantages. Rotator cuff disorders often have various conservative and surgical approaches, each with their own risks and benefits . Many stIt seems crucial to provide the patient with information about the pros and cons of different treatment strategies and share treatment decision-making. However, little information is provided on how this information should be structured. Should we give the patient information based on the benefits or should we give the information based on the complications of the treatment?The objective of this study was to evaluate the effect on patient treatment decision-making if information was given based on the benefit or on the complications in rotator cuff disorders. A secondary objective was to examine the association of socio-demographic variables and functional impairment in the patient decisions.A prospective randomized study was conducted of patients diagnosed with rotator cuff tears who had been recruited at their first clinical visit. The rotator cuff tears were documented with magnetic resonance imaging (MRI) in all patients. Patients were excluded if they had had previous shoulder surgery, worker’s compensation, or were unwilling to participate.Patients were recruited from July to December 2014 in a single tertiary referral hospital. One hundred and eleven patients attended the Shoulder Unit for their first clinical visit during the recruitment period. Twenty-seven were excluded due to previous shoulder surgery and 4 were excluded as recipients of worker’s compensation. Eighty patients ultimately met the inclusion criteria : Your doctor informs you that you have a rotator cuff tear and states that if he/she surgically repairs your cuff tear you will improve and that the cuff remains healed at the 2-year follow-up in 71% of the cases where surgery is done.Would you choose surgery? Yes or NoGroup B (complication): Your doctor informs you that you have a rotator cuff tear and that if he/she surgically repairs your cuff tear you will improve and that the cuff is torn again at 2-year follow-up in 29% of the cases where surgery is done.Would you choose surgery? Yes or NoThe patients were randomly allocated to either group A (benefit) or group B (complication) and were asked to answer the following questions based on their assigned group:The data used to construct the question was obtained by arbitrarily choosing the study by Boileau et al. in which the authors declared that they obtained complete healing of the rotator cuff in 71% of the patients who underwent arthroscopic repair of full-thickness tears of the supraspinatus .Age, gender, the shoulder affected, and the functional status assessed using the Constant score were also recorded . The ConU test. P values of < 0.05 were considered statistically significant. Analyses were performed using the SPSS 18.0 software package .The power calculation for the sample size was based on an estimated reduction of 25% in the agreement to accept surgery, accepting an alpha risk of 0.05 and a beta risk inferior to 0.2. Forty patients were needed in each group to detect a significant difference between the two groups if the presence of the effect in group 1 was 0.6 and was 0.85 in group 2 using an ARCSINUS approximation test. Categorical variables are described with frequencies and percentages, and continuous variables are expressed as mean and standard deviations (SD). Comparisons of categorical variables between groups were assessed with the chi-square or Fisher exact tests, as appropriate. Quantitative variables were compared via the Mann-Whitney The study was approved by the Ethics Committee and all included patients provided signed informed consent.There were 44 females and 36 males. The mean age was 50.8 years (ranging from 23 to 80). In 49 patients, the right shoulder was affected and the left shoulder in 31 patients. Forty-three patients were allocated to group A (benefit) and 37 to group B (complication). The mean age of the patients in group A was 50.8 years old and 54.5 years in group B. In group A, there were 29 females and 14 males while there were 25 females and 12 males in group B. The mean Constant score total was 62.3 for the patients included in group A and 58.2 for the patients in group B.P = 0.000) between groups relative to their acceptance of surgery. Thirty-six out of the 43 (84%) of patients included in group A (benefit) accepted surgery compared to 17 out of the 37 (46%) in group B (complication).The overall results showed a significant difference  as well as the development of decision-making support tools that aid in choosing whether to do surgery or not for a rotator cuff disorder.


To evaluate the effectiveness of corticosteroid ointment in hypertrophic scars prevention following Cesarean section.th day), three months and six months. The treatment group received corticosteroid cream every other day for three months. Comparative evaluations and time-bound changes were evaluated in both groups.This study was conducted between June 2017-May 2018 in Acıbadem Kozyatagı Hospital. Sixty-one patients (31 treatment and 30 control patients) took part in the current study which evaluated wound outcomes and patient satisfaction. All patients’ wound characteristics were assessed via the modified Vancouver Scar Scale (MVSS) score  at baseline , while 12.9% (n=4) of the patients were satisfied in the control group. Two patients reported itching after treatment.The clinical outcomes in both groups were similar. Although vascularity and height parameters improved in three months, similar results were also observed in the group that did not receive treatment after the end of six months. This may have been due to the fact that treatment was stopped after three months. We recommend that the hypothesis be tested in larger series in future studies. Post-operative surgical scars such as keloids and hypertrophic scars (HS) generally occur among persons with abnormal wound healing.2Cesarean section (CS) is one of the most common major surgical interventions carried out on the female population and postoperative scar development is not rare after the procedure. Scars do not only create problems from an esthetic point of view, they also increase the risk for infection and may also cause adverse symptoms such as itching, pain etc. These problems in post-natal women may lead to serious psychological problems in a patient group which is known to be predisposed to distress and depression.Several treatment and prophylactic modalities including surgical excision, radiation, pressure therapy, cryotherapy, topical silicone gel, intralesional corticosteroid injections, interferons, fluorouracil, laser treatment, and many other medications have been used in the management of HS and keloids.13The aim of our study was to determine the prophylactic efficacy of topical corticosteroid ointment as a non-invasive form of therapy for post-operative HS and keloids following Cesarean section.The present prospective study was conducted between June 2017-May 2018 in Acıbadem Kozyatagı Hospital, Istanbul, Turkey. This study was in line with the Declaration of Helsinki and was approved by the hospital Ethics Committee. All patients were informed about the study and written consent was obtained.Sixty-two patients who underwent a cesarean section through a primary Pfannenstiel incision were included. The patient cohort included were aged between 21–42 years and were determined to have type II-IV Fitzpatrick skin types. All patients’ wound closures were performed via the same procedure performed by the same surgeon. Subcutaneous tissues were closed with interrupted sutures (2/0 Caprosyn™ [polygytone 6211]). Skin was closed in continuous manner with 4/0 Monocryl™ (poliglecaprone 25). The exclusion criteria for the study were as follows: having a history of HS or keloids, having previously undergone surgeries which involved abdominal incisions, having a systemic infection, suffering from wounds with discharge or without visible signs of normal epithelialization, having chronic medical illnesses known to affect wound healing , and finally, those with a known hypersensitivity/allergy to corticosteroids were excluded.th postoperative day, patients who met the selection criteria were randomized to the experiment group or control group. Randomization was performed with SPSS 21 program. Patients were numbered and recorded in the program. Random number assignment was made for each patient using the RV Bernoulli (0.5) method. Zero codes were assigned to the control group and 1 codes to the experimental group. At the end of the analyses, 31 patients were included in the control group and 30 patients were included in the experimental group. In the experiment group (n=30), methylprednisolone cream  was applied as a thin layer to the incision site twice a day, starting from postoperative day 10. However, in order to prevent skin atrophy, the applications were performed every other day. The treatment duration was three months. The control group received no treatment. A blinded dermatologist performed the follow-up examinations before treatment , after three months of treatment, and three months after discontinuation of the treatment (at the end of the 6th month). During the follow-up period, one patient from the experiment group was excluded because of wound dehiscence which occurred at the end of post-op 2nd month. Therefore, a total of 61 patients (30 patients in the experiment group and 31 patients in the control group) were analyzed at the end of the 6-month follow-up period.After confirming normal epithelialization and wound healing at the incision site on the 10pigmentation , vascularity , pliability , and height .General Electric Voluson730 Pro Ultrasound, 5-17MHz, USA) was used to precisely measure scar thickness.Scar assessment was performed using the modified Vancouver Scar Scale (MVSS) by assessing scar Side effects experienced by the patients were also assessed every two months. At the end of the 6-month follow-up period, patients in both groups were asked to rate their satisfaction using a 4-point grading scale .All analyses were performed with the SPSS v21 program. The normal distribution of numerical variables was checked with the Shapiro-Wilk test. Variables that conformed to normal distribution were calculated and reported as mean ± standard deviation (SD), while the remaining variables were calculated and reported as median (minimum – maximum). Categorical variables were given in frequency of occurrence and percentage. The Student t-test was used for cross-group comparisons of non-repeating measurements of continuous numerical data. Friedman’s two-way variance analysis was used to assess repeating measurements for the MVSS variable and this was compared with the Mann Whitney U test by obtaining the amount of change over time. In the evaluation of the categorical data measured repeatedly, Generalized Estimating Equations/GEE) and Chi-squared analyses were utilized. The cases where P values were 0.05 and below were considered statistically significant.Sixty-two patients were included in the study. One patient in the experiment group was excluded due to wound dehiscence and subsequent suspicion of infection at two months. A final group of 61 patients (30 patients in the experiment group and 31 patients in the control group) completed the planned duration of six months in the study. The mean age of subjects was 31.28 ± 3.95 years. There was no significant difference between the control and experiment groups (p=0.816).th day), 3rd month and 6th month scores revealed a significant decreased at the 3rd month and 6th month evaluations as compared with the baseline evaluation. There was no significant difference between the 3rd and 6th month evaluations. When the experiment and control groups were compared, height scores were significantly lower in the experiment group at the 3rd month assessment (p=0.001), while there was no significant difference between the groups at the 6th month assessment (p=0.163).As regard to height score, comparison of baseline . However, there were no significant difference between the two groups at 6th month evaluation (p=0.097). Evaluation of pigmentation scores showed that 6th month scores were significantly lower than baseline and 3rd month scores in both groups; however, no significant differences were observed when groups were compared with each other.In both groups, scar vascularity showed a trend of significant reduction over time. Vascularity decline in the experiment group was significantly more than the control group at 3rd month and 6th month total MVSS scores revealed that all scoring parameters  significantly decreased across the board as compared to baseline evaluation in both groups. There was no significant difference between the 3rd and 6th month scoring values . After three months of using the methylprednisolone cream, 20% (n=6) of the experimental group and 12.9% (n=4) of the control group reported “high satisfaction” for scar-healing. In terms of adverse effects, itching was reported by two patients following the application of the cream , or in patients who have recurrent HS.24Studies have shown that the topical use of corticosteroid cream is promising in HS management, with higher rates of satisfaction when compared to silicone-based controls.We aimed to evaluate a novel approach where corticosteroid cream was used in the early post-operative period during which tissue absorption is favorable. To the best of our knowledge, this is the first study to determine the efficacy of topical methylprednisolone cream in the early post-operative period for the prevention of scarring after cesarean section.th month evaluation in both groups, with no significant differences between the groups. The most prominent changes were recorded in the height and vascularity score subsections of the MVSS at the 3rd month assessment of the treatment group. Patient satisfaction was also higher in the experimental group which received methylprednisolone treatment.We have demonstrated a lower score on all MVSS parameters  at the 6Although intralesional corticosteroid injections have been preferred in some studies,We had relatively few participants which may have compromised statistical power. Furthermore, this is a study of wounds inflicted by a single operating surgeon; therefore, the specialty and experience of the surgeon is not evaluated. In addition, when allocating an experimental group with control group it should be considered that there are other potential methods to create the groups other than including age-matched counterparts. Repeating the methodology on a larger series of patients and trying different topical corticosteroid formulations could be considered in future studies.The clinical outcomes of individuals who did not receive any treatment and those who received corticosteroids were seen to be similar. Although positive effects were seen for certain outcomes at three months, similar final results were observed in the group that did not receive treatment at six months. This may have been due to the fact that treatment was ceased after three months, as the use of topical steroids is limited to 12 weeks in order to prevent side effects. We recommend that the hypothesis be tested in larger series in future studies.


Cinnamomum cassia and Rosa Laevigata, both of which have been used to treat symptoms typically associated with BPH in traditional Asian medicine. The aims of this trial are to assess whether HT080 can alleviate LUTS/BPH in middle-aged and elderly men, and to investigate the safety of HT080.Lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH) are one of the most common conditions seen in middle-aged and elderly men and threaten their quality of life. Since conventional pharmacotherapy for LUTS/BPH can lead to sexual side effects, herbal therapies are widely used as complementary and alternative treatments worldwide. HT080 is an herbal extract of A double-blind, randomized, placebo-controlled, two-arm parallel group trial will be conducted in men with moderate LUTS/BPH. A total of 100 eligible men aged 40 to 75 years with an International Prostate Symptom Score of 8 to 19 will be randomized in a 1:1 ratio and receive either HT080 (500 mg) or placebo twice a day for 12 weeks. All participants will be evaluated for efficacy and safety at baseline and weeks 6 and 12. The primary endpoint is the change in International Prostate Symptom Score between baseline and week 12. The secondary efficacy variables are uroflowmetry parameters , serum prostate-specific antigen, testosterone, and dihydrotestosterone levels, the International Index of Erectile Function score, and participant-reported global response assessment scores. The safety assessments include adverse events, laboratory tests results, vital signs, and physical examination.This is a first-in human trial designed to investigate the efficacy and safety of HT080 among middle-aged and elderly men with LUTS/BPH. This prospective study with a double-blind randomized design will provide high-quality evidence supporting the use of HT080 for LUTS/BPH.Korean Clinical Research Information Service (KCT0004286) Registered September 6, 2019. LUTS in men generally results from benign prostatic hyperplasia (BPH) which is characterized by hyperproliferation of epithelial and stromal cells of the prostate. The global prevalence of BPH has rapidly increased with aging in the population, thus becoming an economic burden. In Korea, the prevalence of BPH in 2016 increased 2.2-fold that in 2012 and approached 37.5% and 53.8% among men in their 60s and in those above 80 years of age, respectively.Lower urinary tract symptoms (LUTS) are common in middle-aged and elderly men and significantly impair the quality of life. Two classes of drugs, α-adrenergic receptor blockers and 5α-reductase inhibitors, are used for pharmacotherapy. Unfortunately, these drugs are associated with sexual side effects, including ejaculatory disorders for α-adrenergic receptor blockers and erectile dysfunction, ejaculatory dysfunction, and decreased libido for 5α-reductase inhibitors. This has led a to an increased demand of herbal therapies  as complementary and alternative treatments among numerous patients worldwide.,7 In Europe and the USA, about half or more of BPH patients use herbal therapies alone or with other drugs.,8 In Asian countries including China and Korea, traditional herbal medicine has been used to treat BPH for hundreds of years, and is still commonly used to this day.–11Treatment options for LUTS associated with BPH (LUTS/BPH) include watchful waiting, lifestyle modification, medications, and surgery.Cinnamomum cassia bark and Rosa laevigata fruit. It was developed by screening various herbs used in traditional Asian medicine for the treatment of LUTS, with the aim of reducing prostate smooth muscle tone and prostate growth. These two herbs have been traditionally prescribed in combination to treat symptoms typically associated with BPH including frequent urination and nocturia in China.,13C cassia, commonly called Chinese cassia or cassia cinnamon, is an evergreen tree that originated in southern China and is now cultivated in eastern and southern Asia for its bark, cinnamon. In traditional Korean, Chinese, and Ayurvedic medicine, the bark of C cassia has been widely used to treat frequent urination, nocturia, difficulty in urinating, and a weak urine stream in middle-aged and elderly men.,10,14,15 A systematic review reported that C. cassia is the most frequently used single herb in Chinese herbal medicine for BPH and was used in 17 (54.8%) of 31 studies. Concurrent with these clinical applications, cinnamic acid, the major component of C cassia, serves as a potent antagonist of the α1A-adrenoceptor in the rat prostate. Furthermore, C verum, which is used as a substitute for C cassia and vice versa, has reportedly reduced testosterone-induced BPH by downregulating 5α- reductase and androgen receptor expression.R laevigata is a climbing shrub originating from South China and Southeast Asia, and its fruit has been used to treat frequent urination, urinary incontinence, and sexual dysfunction in traditional Chinese and Korean medicine.,19R Laevigata reportedly decreases the voiding frequency and increases the inter-micturition interval and urine volume by inhibiting smooth muscle contraction in a mouse pollakiuria model. Furthermore, R laevigata has been shown as one of the most potent 5α-reductase inhibitors in a screening assay for anti-5α-reductase, including 162 plants. Although traditional use and preclinical findings support the applications of HT080, a well-designed prospective clinical trial has yet to be conducted to evaluate the efficacy and safety of HT080.HT080 is an herbal extract of 1.1This trial primarily aims to determine whether HT080 is superior to the placebo for reducing LUTS, as measured on the basis of the International Prostate Symptom Score (IPSS), in middle-aged and elderly men with moderate LUTS/BPH. The secondary objectives are to investigate the effects of HT080 on uroflowmetry parameters and androgen levels, and to assess the safety of HT080. The exploratory objective is to evaluate whether HT080 affects sexual function.1.2This study is a double-blind, randomized, placebo-controlled, superiority trial with two parallel groups. Eligible participants will be randomly assigned to either the HT080 or placebo group with a 1:1 allocation ratio and will receive the investigational product for 12 weeks.22.1Participants will be recruited from Department of Urology, Yonsei University Severance Hospital . The estimated recruitment period will be 1 year beginning from October 2019. The recruitment will be advertised on hospital bulletin boards and websites. Monthly newsletters will be sent to encourage enrollment until the required sample size is achieved. The first participant has not been enrolled yet.2.2Men aged 40–75 yearsAn IPSS of 8–19Prostate size ≥ 20 gAvailability of written informed consent to participate in the study.Following will be the inclusion criteria for the study:Current treatment for clinically significant acute or chronic cerebrovascular, immune, respiratory, hepatobiliary, kidney and urinary, neurological, musculoskeletal, psychiatric, infectious, or hemato-oncological diseases. .Serum prostate-specific antigen level ≥ 10 ng/mL. .Maximal urinary flow rate < 5 mL/sPost-void residual urine volume > 200 mLBladder tumors, urinary stones, urethral strictures, bladder neck contracture, inflammation of the lower urinary tract (bladder and urethra), or urinary tuberculosisDiagnosis of prostate cancerHistory of genitourinary surgery, including prostate surgery or other invasive procedures for treatment of prostatic conditions Uncontrolled hypertension (systolic blood pressure ≥ 160 mmHg or diastolic blood pressure ≥ 100 mmHg measured after a 10-min rest)Uncontrolled diabetes (fasting blood glucose level ≥ 180 mg/dL or administration of drugs for diabetes within the preceding 3 months)Thyroid diseasesSerum creatinine level > 2-fold the upper limit of normalAspartate and alanine aminotransferase levels ≥ 3-fold the upper limit of normalAdministration of drugs (antimuscarinics and/or others) for benign prostatic hyperplasia (BPH) or intake of functional foods within the preceding 4 weeksParticipation in other clinical trials within the preceding 3 months or plans to participate in other trials during the present study.Unsuitability for study participation based on the investigator's discretion.Following will be the exclusion criteria for the study:2.3This trial is designed to provide treatment with either HT080 or placebo in subjects with LUTS/BPH and assess symptom improvement during 12 weeks of treatment. The flow diagram is shown in Figure 2.4C cassia extract and 190 mg of R laevigata extract). To ensure blinding, the placebo tablet will be of the same size, shape, and color as the active tablet. Participants will be orally administered 2 active or placebo tablets twice daily  for 12 weeks. HT080 will be administered at a dose of 1000 mg daily. The investigational product will be packaged as a 6-week supply and will be prescribed to participants at baseline and week 6. To improve the adherence to the treatment, participants will be encouraged to adhere to the prescribed dosage regimen, and compliance will be evaluated by counting the number of unused tablets returned at 6 and 12 weeks.The investigational product will be provided by COSMAX BIO Inc. . The active product is a 500 mg tablet containing 250 mg of HT080  trial discontinuation is deemed necessary by the investigator.Criteria for discontinuing intervention will include the following:2.5Eligible participants will be randomized in a double-blind fashion to HT080 or placebo (1:1 ratio) using a block randomization method. The randomization sequence number will be generated by an independent statistician using the SAS system . Participants will be assigned a randomization number based on the order of enrollment and will receive the investigational product labeled with the corresponding randomization number during the study period. Block size will not be disclosed to the research personnel for allocation concealment. Packaging of the active product and the placebo will be identical to maintain double-blinding. The randomization sequence will be sealed in opaque envelopes and will not be disclosed until the end of the study, except for cases in which participants develop serious adverse events . If the investigator concludes that it is necessary to break the code, he/she will notify the contract research organization and the study sponsor regarding premature unbinding, and they will discuss and decide together whether the situation warrants breaking the code. In cases of a code break, the date and reason will be recorded in the case report form, and the corresponding participant will discontinue the study.2.6 Each item is scored 0 to 5, giving a maximum total score of 35. A total score of 0 to 7 indicates mild symptoms, 8 to 19 indicates moderate symptoms and 20–35 indicates severe symptoms. Participants will complete the IPSS questionnaire at screening, baseline, and weeks 6 and 12. The primary efficacy endpoint is the change in IPSS at week 12 from the baseline.The Korean IPSS questionnaire will be used to assess the primary endpoint. The IPSS, which has been used for decades to assess the severity of LUTS/BPH, contains seven questions on voiding symptoms  and storage symptoms . Participants will complete the International Index of Erectile Function questionnaire, a multi-dimensional self-report instrument widely used to evaluate male sexual function,,25 at baseline and weeks 6 and 12. The safety outcome measures include adverse events, laboratory tests  results, vital signs, and physical examination.As the secondary outcomes, uroflowmetry parameters  and serum prostate-specific antigen, testosterone, and dihydrotestosterone levels will be measured at screening and weeks 6 and 12. Global response assessment will be completed by participants at weeks 6 and 12 using a five-point scale . It has been reported that 70% of BPH patients has an associated erectile dysfunction.2.7Ganoderma lucidum improves IPSSs. Because the standard deviations for changes in IPSSs are not present in this literature, they have been approximated using the confidence intervals and significant probabilities of pre- and post-treatment values for IPSSs. The difference in the mean change in IPSSs between the HT080 and control groups is hypothesized to be 1.18 and the standard deviation is hypothesized to be 1.88. A total of 80 participants, 40 in each group, are required to achieve 80% power with a two-sided significance level of 5%. Considering a dropout rate of 20%, a total of 100 participants (50 in each group) will be enrolled.This trial aims to determine whether HT080 is superior to placebo for reducing the change in IPSSs from baseline to week 12. The sample size has been estimated based on the results of a previous study that had shown an extract of 2.8Efficacy outcomes will be assessed in both full analysis and per-protocol sets. The full analysis set will consist of all randomized participants who are subjected to at least one efficacy evaluation after being administered at least one dose of the study product without failing major inclusion criteria. The per-protocol population will include participants in the full analysis population who complete this trial without major protocol deviations that affect the study results. Safety outcomes will be analyzed in the safety set, which will include all randomized participants who receive at least one dose of study product. Missing values will be handled by the last observation carried forward method.t test or Wilcoxon rank sum test, depending on the normality of distribution, and the differences before and after treatment in each group will be analyzed using a paired t-test or Wilcoxon signed-rank test. Categorical variables will be compared using the chi-square test or Fisher exact test. For primary and secondary efficacy variables, analysis of covariance will be performed to compare the change from baseline to weeks 6 and 12 between the groups while adjusting for baseline values. If necessary, subgroup analysis stratified by baseline characteristics  will be performed. Adverse events will be coded by System Organ Class and Preferred Terms from the Medical Dictionary for Regulatory Activities. Probability values of <.05 with two-tailed tests will be considered significant. The statistical analysis will be performed with SPSS Statistics software version 25 . There will be no interim analyses.Continuous variables will be presented with mean, median, standard deviation, and range (min and max), and categorical variables will be presented as frequency and percentage. Continuous variables between the two groups will be compared by the two-sample 2.9To ensure data quality, regular monitoring by a contract research organization will be conducted in compliance with standard operating procedures until the end of the study. Experienced clinical research associates will verify source documents and case report forms. All documents related to this trial will be kept confidential in a separate facility, and access to the documents will only be allowed for the purposes of trial management. Data quality will be promoted using double data entry and range checks for data values. A data monitoring committee will not be necessary because the study product is low-risk and no interim analyses will be performed. Auditing is not scheduled for this study.2.10All adverse events will be recorded in the case report form regardless of causality. The investigator will evaluate the severity of symptoms and determine causal relationship between the study product and the adverse events. If a serious adverse event occurs, the investigator will report to the Institutional Review Board at the earliest and determine whether to continue or stop the trial. Participants will receive financial compensation for injuries found to be due to the study product or procedure.2.11Participants may withdraw from the trial at any time for any reason. The investigator may also withdraw participants from the trial due to medical or administrative reasons . Reasons for withdrawal and dropout will be recorded in the case report form.2.12Declaration of Helsinki and the Korean Good Clinical Practice guidelines. All participants will be provided detailed information about the trial and the principle investigator will obtain handwritten informed consent from each participant before proceeding with the trial. To protect participants’ identity, subject identification codes will be used instead of participants’ names in all documents. The sponsor and principal investigator will have access to the final dataset. The full protocol and datasets will not be publicized but available from the corresponding author on reasonable request. A model consent form is also available from the corresponding author. Results from this study will be presented at an academic conference or published in a peer-reviewed journal.This protocol  has been approved by the Institutional Review Board of Yonsei University Severance Hospital  and is registered with Clinical Research Information Service (Identifier: KCT0004286). The trial will be conducted in accordance with the 3 There is an unmet need to investigate the efficacy and safety of herbal medicines in well-designed and rigorous studies. Herein, we designed the study protocol of the first double-blind, randomized, placebo-controlled trial of HT080 as a single intervention for Korean men with LUTS/BPH.BPH is a common disease affecting the overall quality of life in industrial countries. Although traditional herbal medicines are widely used to treat LUTS/BPH in Asian countries including Korea, their efficacy remains unclear owing to the poor quality of clinical trials.,27 We intend to use not only subjective measures  but also objective measures, such as maximal urinary flow rate and post-void residual volume, to yield outcomes on voiding dysfunction. In addition, our inclusion criterion of an IPSS of 8–19 reflects men with moderate symptoms commonly observed in clinical practice.One of the limitations of this trial is that the treatment duration is 3 months, thus providing short-term outcomes. Furthermore, this study has a relatively small sample size, albeit yielding a statistical power of 80%. The present results will warrant confirmation with further long-term studies with larger sample sizes. Nevertheless, this study has several strengths; it is a prospective study with a double-blind randomized design. In addition, the IPSS questionnaire, the primary efficacy measure of this trial, has been established worldwide as a useful tool to determine the extent of LUTS/BPH, and the validity and reliability of its Korean version has been confirmed.In conclusion, this is a first-in-human trial of HT080 in men with LUTS/BPH. The results of this trial will provide high-quality evidence of therapeutic benefit of HT080 for the treatment of LUTS/BPH.We would like to thank all the participants and research personnel for their support of this study.Conceptualization: Hocheol KimMethodology: Jungbin Song, Seung Hwan LeeProject administration: Seung Hwan LeeSupervision: Seung Hwan LeeWriting – original draft: Jungbin SongWriting – review & editing: Seung Hwan Lee, Hocheol KimHocheol Kim orcid: 0000-0002-6690-0226.


Non-specific Lipid Transfer Proteins (nsLTPs) are widely distributed in the plant kingdom and constitute a superfamily of related proteins. Several hundreds of different nsLTP sequences—and counting—have been characterized so far, but their biological functions remain unclear. It has been clear for years that they present a certain interest for agronomic and nutritional issues. Deciphering their functions means collecting and analyzing a variety of data from gene sequence to protein structure, from cellular localization to the physiological role. As a huge and growing number of new protein sequences are available nowadays, extracting meaningful knowledge from sequence–structure–function relationships calls for the development of new tools and approaches. As nsLTPs show high evolutionary divergence, but a conserved common right handed superhelix structural fold, and as they are involved in a large number of key roles in plant development and defense, they are a stimulating case study for validating such an approach.In this study, we comprehensively investigated 797 nsLTP protein sequences, including a phylogenetic analysis on canonical protein sequences, three-dimensional structure modeling and functional annotation using several well-established bioinformatics programs. Additionally, two integrative methodologies using original tools were developed. The first was a new method for the detection of (i) conserved amino acid residues involved in structure stabilization and (ii) residues potentially involved in ligand interaction. The second was a structure–function classification based on the evolutionary trace display method using a new tree visualization interface. We also present a new tool for visualizing phylogenetic trees.Following this new protocol, an updated classification of the nsLTP superfamily was established and a new functional hypothesis for key residues is suggested. Lastly, this work allows a better representation of the diversity of plant nsLTPs in terms of sequence, structure and function. Since the work of Arabidopsis , and one soybean hydrophobic protein. In the RCSB Protein Database . According to the CATH database : C1–C6 and C5–C8 in type I structures; C1–C5 and C6–C8 in type II and IV structures. However, C2–C7 and C3–C4 bridges are common to all known nsLTP structures and the overall fold is conserved among the whole family.From a structural point of view, the nsLTP family belongs to the all-alpha class in the SCOP database , as thesDatabase  we listedatabase , nsLTPs From a functional point of view, plant nsLTPs are classified into different families depending on the scope of interest and their properties . Plant nnsLtp gene expression following pathogen infections, overexpression in transgenic plants, or their antimicrobial properties , Phytozome (http://www.phytozome.net) and NCBI databases (http://www.ncbi.nlm.nih.gov), using either Blast or keyword queries . Original data obtained on the Theobroma cacao genome were also investigated (Arabidopsis sequences previously identified by DOI 10.18167/DVN1/UNKLA6).A first pool of plant nsLTPs sequences was retrieved from the UniProtKB (Swiss-Prot + TrEMBL) , a restricted analysis was carried out using only the 10 nsLTPs for which at least one structure had previously been experimentally determined. Two new 10-sequence alignments were calculated, one by MUSCLE and one by MAFFT. Using the ViTo program , each alBased on the best alignment, a phylogenetic tree was calculated using PhyML . Lastly,http://www.rcsb.org/pdb). Theoretical structures were calculated for the other 787 proteins using the @tome2 suite of programs to perform homology modeling (http://atome.cbs.cnrs.fr). The quality of each final structure model was evaluated using Qmean  and organized in a dedicated database, together with the bibliographic references when available. The database was also enriched with additional information, such as methods used for gene expression studies , protein purification, in vitro or in planta antifungal and antibacterial activity, lipid binding or transport (fluorescence binding assay or in vitro lipid transfer). Information about tissues and organs used in cDNA libraries was collected from the NCBI databases (http://www.ncbi.nlm.nih.gov). A dataset with all the annotations is available online (DOI 10.18167/DVN1/1O5UAK).Extensive bibliographic work was carried out to collect and classify functional information available in the literature about the nsLTPs of the dataset. Gene ontology, plant ontology and trait ontology terms were collected from the Gramene Ontologies Database  are represented in gray/black, while specifically conserved positions among proteins of the same subfamily (specificity determining positions) are represented in saturated colors corresponding to the family ones. The tool enabled scrolling down of the alignment to easily identify both types of conserved positions and two distant parts of the alignment could be displayed together to compare distant phylogenetic subfamilies.Contacts with ligands, solvent accessibility and other parameters could also be displayed above the alignment. Using the Jmol interface, conserved amino acid residues could be projected on nsLTP representative 3D structures, so that the potential role of each position could be interpreted geometrically.Structural Trace Display is a method, based on evolutionary trace display ETD, , that seAs in a phylogenetic tree, nsLTPs in the structure tree were clustered according to their similarity. In the case of this particular tree, the similarity between nsLTPs was measured by a spatial distance in angströms . Decreasing distance cutoffs ranged from 11.5 Å  to 0.5 Å. Each cutoff application caused a division of the tree into one or more sub-trees that contained leaves  whose structural proximity altogether (represented by the pairwise RMSDs) was up to the value of the applied cutoff.We developed an integrative tree visualization tool called InTreeGreat in order to display the whole or some parts of either sequence or structure distance trees.InTreeGreat was implemented using PHP and Javascript, in order to generate and manipulate an SVG graphical object.The main objective of this tool is to graphically highlight correlations between 3D structures, evolution, functional annotations or any available heterogeneous data. In the context of this study, the interface was able to retrieve information from the nsLTP database to annotate the tree.InTreeGreat includes functionalities such as tree coloration, fading and collapsing. Heterogeneous data related to sequences  can be displayed in colored boxes, aligned to the tree.Using InTreeGreat to investigate our annotated structure tree, we looked for clusters of nsLTPs sharing the same kind of functional annotations. We focused our attention on one interesting functional role highlighted in several nsLTPs: the implication in plant defense mechanisms against pathogens (bacteria and/or fungus). In order to highlight structure–function relationships, we studied three groups of nsLTPs (see Results section for details): (i) the “defense cluster” ; (ii) the cluster containing all type I fold proteins ; (iii) a group manually composed of all type I fold nsLTPs for which a functional role in defense and/or resistance against pathogens had been reported in the literature (28 proteins).Within each of these three clusters, the protein structure showing the shortest RMS distance from all the others was selected as the representative structure of the cluster for the structural trace calculation.A structure-based sequence alignment was carried out on the nsLTP structures by Mustang software .http://mammoth.bcm.tmc.edu/ETserver.html) together with the representative structure of the cluster (selected as described in the previous paragraph).For each previously selected structural cluster, the corresponding set of protein sequences was extracted from the multiple structural alignment of the nsLTPs. The evolutionary trace (ET) method  was applhttp://www.pymol.org/).The ETs based on the structural alignments corresponding to the three nsLTP clusters were then compared to each other. To that end, the 30% top-ranked residues of the defense cluster trace were considered as constitutive of the reference trace  and their ranking and scores in the two other traces were analyzed. The results were compiled in a table and graphically visualized using PyMOL  was carried out twice, using the MUSCLE program on the one hand, and the MAFFT program on the other hand. The resulting alignments obtained with standard settings are shown on In both cases, cysteine residues of the 8CM aligned quite well among the three represented types of nsLTPs , except for the Cys5-X-Cys6 (CXC) pattern region (where X stands for any amino acid residue). MUSCLE did align type I Cys5 with types II and IV Cys5′, as well as type I Cys6 with types II and IV Cys6′ , just asTo determine which of these two alignments better aligned nsLTP type I and II CXC patterns, we used ViTO program to compare the effects on the 3D structures of both sequence alignments.The 3D projection of MUSCLE sequence alignment  showed tOn the contrary, according to the 3D projection of MAFFT sequence alignment , type I This compound approach allowed us to sort the 797 sequences unambiguously into two main families.Our dataset was mainly composed of nsLTPs from angiosperm species (19 monocotyledonous species and 83 eudicotyledonous species) plus five gymnosperm species (35 sequences), one lycophyte species (34 sequences) and two bryophyte species (17 sequences). The monocot sequences were mainly represented by Poales nsLTPs (256 out of 270 sequences) whereas Rosid nsLTPs were the most abundant (364 out of 436 sequences) within eudicots.Arabidopsis thaliana, Triticum aestivum and O. sativa nsLTP sequences, in nine types . This result mostly confirmed Boutrot’s classification, defined on ne types . The maine types  were notPhyscomitrella patens nsLTPs and type XI (23 sequences) was mainly composed of nsLTPs from eudicot species. A total of 12 nsLTPs were not included in any of the identified types: these were mainly Physcomitrella patens (six sequences) and Selaginella moellendorfii (four sequences) proteins (Type I nsLTPs formed a well-supported monophyletic group (branch support of 0.879) and predominated over the other types, as they accounted for more than half of our dataset (417 out of 797 sequences). This was also observed by proteins .Type XI were grouped in a cluster of 23 sequences in the phylogenetic tree, fairly well supported by a branch of 0.879 aLRT SH-like score. Type XI appeared between type I and the other types, but even though type XI and I were grouped together in the tree, it remained unclear which of the three groups  diverged first.Physcomitrella patens nsLTPs were type IX, the other six remained un-typed, and the only liverwort Marchantia polymorpha nsLTP was a type VI. The 34 Selaginella moellendorfii sequences were mainly types V and VI  and seven nsLTPs belonged to the new type XI. The Physcomitrella patens and Selaginella moellendorfii nsLTPs formed independent branches or were located at the same branch as type V in Wang’s phylogenetic tree  and XI were not identified in monocot species. Within the lycophyte and bryophyte species, no type II, III, IV nor VIII nsLTPs were identified. In the same way, no type III, VIII, IX or XI were identified within gymnosperm species. A total of 10 out of the 16 moss tic tree  and weretic tree . HoweverAllium nsLTPs may constitute a novel type of nsLTPs harboring a C-terminal pro-peptide localized in endomembrane compartments. In the prolamin superfamily tree of Allium cepa nsLTP (192_ALLCE) is closed but not included in the type I nsLTPs. In our phylogenetic tree, the three nsLTPs from Allium species were classified as type I. The 501_MEDTR medicago nsLTP was suggested to belong to a new nsLTP subfamily involved in lipid signaling (Arabidopsis DIR1 (151_typeIV_ARATH). In our phylogenetic tree, both proteins were identified as type IV nsLTPs.According to ignaling  like AraTheobroma cacao genome contains at least 46 nsLtp genes distributed across the 10 chromosomes. Several Theobroma cacao nsLtp genes are organized in clusters, as observed in the rice, Arabidopsis and sorghum genomes (Theobroma cacao nsLTPs were classified within the previously identified types and belonged mainly to type I (14 sequences), type VI (seven sequences) and type V (six sequences).The  genomes . Apart fIt is worth noting that all the nsLTPs identified as allergens (IgE binding) were type I, except one type II nsLTP (545_BRACM). The 501_MEDTR nsLTP was also suggested to play a role in the root nodulation process . Lipid sThis analysis was the most extensive so far and confirmed most of Boutrot’s classification, but complements it due to a larger dataset and a more detailed phylogeny analysis.Given the nsLTP fold conservation and the quality of the available experimental structures, reliable models could be obtained for all nsLTPs using the comparative modeling method, although the sequence identity observed among all nsLTP sequences was only in the range of 25%.Models deduced by fold-recognition using the @TOME-2 server displayed overall good quality, as shown in For 121 theoretical structures, the polypeptide chain could not be fully built and the resulting models were lacking at least one of the eight cysteine residues. Such models were discarded and only the complementary pool of 677 structures was kept for further analysis.http://atome.cbs.cnrs.fr/AT2B/SERVER/LTP.htmlAll the structural alignments and 3D models are available at: In order to challenge the structure–function relationship analysis on such a big set, we decided to develop a new tool called FAST, which builds consensus sequences for each family, and highlights the sequence conservation and specificities on the alignment and the associated 3D structures.Many residues specifically conserved in type I nsLTP1 corresponded to important folding differences between type I nsLTPs on the one side and all other LTP types on the other side. In the following sections, we list type I nsLTP-specific residues whose differential conservation was supported by structural or experimental data.First, Gly37, which was specifically conserved in type I nsLTPs, allowed very tight contact of helix 1 and helix 2, which were connected by the disulfide bridge Cys17-Cys34. The closest backbone distance between position 13 of helix 1 and position 37 of helix 2 was 3.34 Å in a type I nsLTP structure (PDB code 1mid) while it was 6.45 Å in a type II nsLTP structure (PDB code 1tuk). These increased helix distances closed the ligand tunnel, which was opened in type I nsLTPs between helix 1 and helix 3, and created two distinct cavities separated by a septum in type II nsLTPs . Larger Arginine and lysine residues at position 51 and bulky hydrophobic residues at positions 87 and 89 were two other conserved specificities among type I nsLTPs. The side chains at position 51 had type I-specific polar interactions with the ligand at the cavity entrance near the C-terminal loop, which were not found in other nsLTP types, as detailed later in In addition, in type I nsLTPs, the 5th and 6th cysteine residues belonged to helix 3 and were bridged with the first and 8th cysteines, respectively. These two-disulfide bridges tightened both sequence termini to the protein core. Conversely, in types II and IV nsLTPs, the 5th and 6th cysteines showed permuted bridging partners . The intermediate residue connecting the 5th and 6th cysteines was exposed to solvent in type I nsLTPs, while it was replaced by a bulky hydrophobic residue interacting with the ligand in the type II and IV nsLTP core at position 54 of the alignment. It was shown by site-directed mutagenesis that the replacement of this intermediate residue by an alanine residue perturbed folding, ligand binding and lipid transfer activity in type II nsLTPs . The posThe mutation to alanine of the residue at position 63 (residue 45 in Cheng’s article) was also shown experimentally to be destabilizing in type II nsLTPs . This poOther residues specifically conserved in type I nsLTPs were helix N-capping Thr6 and Thr47, whose side chains formed stabilizing hydrogen bonds with the protein backbone, and Tyr20, which was the center of a conserved hydrophobic cluster with Pro30 and Leu/Ile79. The interaction of Tyr20 with Pro30 was experimentally confirmed by the large up field shift of Pro30  protons . This coWe then analyzed the atomic interactions observed between type I nsLTPs and their associated ligands in 19 PDB structures . Most contacts involved hydrophobic side chains of the type I nsLTP proteins and carbons of the ligands. Marginally, the most frequent polar contacts involved the side chains of conserved arginines at position 46 of the type I nsLTP alignment, lysines at position 54, aspartic acids at position 90, and various polar atoms of histidines, lysines and asparagines at position 37. It should be stressed that none of these polar interactions were shared by more than 31% of the protein-ligand complexes (fewer than 6/19 PDB structures) although the least similar protein pair from the 19 structure set shared 67% sequence identities. This low level of polar contact conservation in homologous proteins with very similar sequences clearly indicated that no specific polar interactions anchored the protein-ligand complexes in particular conformations. From this statistical analysis of protein-ligand polar contacts that did not exhibit a preferential cavity region for the interaction with the ligand polar heads, it could not be concluded that there was a preferred ligand orientation in the type I nsLTP tunnel. This observation was supported by recent protein-docking simulations and protein binding evaluations, which also concluded on a lack of preferred orientations of the ligand in the cavities of type I nsLTPs, and clear dominance of hydrophobic interactions in the protein-ligand interface .Lastly, positions 82 to 94, which corresponded to the C-terminal loop, included some more residues specifically conserved in nsLTPs. This loop was much longer in type I nsLTPs than in other types, and had a major impact on the orientation of the ligand in the cavity, as shown in The potential impact of variability within the nsLTP family on the tree dimensional structure of the proteins was further investigated. As shown in The structural differences observed between type I nsLTPs vs. types II and IV can be generalized to other nsLTP types by looking at the alignment of consensus sequences in The fold of type I nsLTPs will be hereafter referred to as “Type-1 fold” and the alternative fold of Types II to XI will be referred to as “Type-2 fold”. The preceding analysis of the evolutive conservations specific to type I nsLTPs revealed many residues whose role could be explained by local structural differences with the available types II and IV nsLTP structures. This comparative structure analysis confirmed the clear separation between type I and all the other nsLTP types initially observed in the phylogenetic tree inferred from a multiple sequence alignment of the 797 available proteins. The key residues were usually present in type I nsLTPs only and suggested that many structural differences observed when comparing type I vs. types II and IV nsLTPs should also be observed vs. other nsLTP types, particularly regarding ligand orientation and cavity entrances. This observation should guide the choice of templates when nsLTP types with unknown structures are modeled by homology.In order to correlate the evolution of protein sequences and the impact on the corresponding structures, we produced a circular tree according to structural distances . WhereasSeveral studies also showed that type I and type II nsLTPs differed through the characteristics of the residue standing between Cys5 and Cys6, being, respectively, hydrophilic in type I and apolar in type II proteins . Based oDealing with big datasets can be cumbersome and requires a very efficient interface. To address this challenge, we developed InTreeGreat, a Javascript/PHP interface, compatible with every standard web navigator. It is able to display and explore any tree and to deal with branch and leaf coloring, branch lengths, branch support (or any other branch labels), and can aggregate heterogeneous data . Among the annotated nsLTPs (433 out of 797), we focused on those that had been reported for their role in plant defense and/or resistance against pathogens (bacteria and/or fungi). To simplify, we shall hereafter refer to them as “defense nsLTPs” in the present discussion. By investigating structural similarities between the 31 identified defense nsLTPs in our annotated dataset, we attempted to identify key amino acid residues that would be good candidates for SDM experiments as they may bestow their functional properties on these proteins.Looking at the distribution of the defense nsLTPs in our structural classification  we obserThe cluster containing all Type-1 fold defense nsLTPs corresponded to the whole type I part of the tree (402 members). The corresponding structural trace was calculated, but it could not be linked to the defense function, as the proportion of annotated nsLTPs with a defense function was too low .In order to obtain a meaningful trace of the potential defense function, we needed to select a cluster with a higher proportion of annotated defense nsLTPs. The best cluster we could find was a relatively small cluster (43 members) of proteins with a structural distance no greater than 1.5 Å , which contained 33% of the defense nsLTPs . This cluster will be referred to as “defense cluster” in the further discussion.The structural trace of the defense cluster showed several differences in comparison with the structural trace of the Type-1 fold cluster . Apart fThree other residues located at positions 137, 154 and 266 of the structural alignment were differently conserved in the three clusters. Interestingly, these three positions showed good conservation ranking, but the variability of the three corresponding residues was notably higher in the Type-1 fold cluster. Indeed, in the defense cluster trace, position 137 was occupied either by a valine or by an alanine residue  and position 154 was occupied either by a leucine or by a valine residue (Leu11 in protein 525). Thus, both positions were occupied by hydrophobic residues in defense proteins, which was not always the case in Type-1 fold proteins . In the The fact that these three positions of the structural alignment belonged to the top 30% most conserved among all Type-1 fold nsLTPs suggested their importance in these proteins. However, because the variability at these three positions was very small among defense nsLTPs and because the physico-chemical property was strongly conserved, we suspected that residues located at positions 137, 154 and 266 of the structural alignment might bestow functional specificity, at least in the case of defense/resistance proteins.Non-specific lipid transfer proteins sequence–structure analyses using either FAST or STD revealed some key residues or key positions . The structural trace analysis highlighted other amino acid residues . It is important to note that these two complementary analyses by FAST and STD were not meant to lead to the same kind of conclusions. Indeed, using sequence information projected on the 3D structure, the first method revealed nsLTP-type-specific amino acid residues that could be involved in structure stabilization and/or ligand interaction, given their structural context. The second method however, considered a set of functionally close nsLTPs sharing a very similar structure and highlighted over-representatively conserved amino acid residues that might thus bestow functional specificity on these proteins. These two approaches took inverse directions in the path sequence–structure–function. The “sequence-to-function” method would lead to more precise conclusions if more data about the inner structural mechanisms of lipid binding were available . The “function-to-sequence” method would give us a better overview of the range of nsLTP activities if the functional data were not so rare and heterogeneous.However, we assumed that this combination of approaches (i) allowed structure–sequence analysis for large multigene families, (ii) could reveal structural patterns related to functions that were not revealed so far, as alignments would have been limited to primary sequences only and (iii) allowed a comparison of groups composed of proteins with an evolutionary connection with groups displaying structural similarity.We combined two powerful alignment algorithms (MAFFT and MUSCLE) together with a 3D projection of the impact of alignment on the structure of proteins (VITO). Real-time monitoring of the impact of gap positions and lengths on the resulting 3D model offered the possibility of discriminating between various alignment possibilities. This allowed us to provide definitive insight into the old debate about the CXC pattern and its implication for the structure of LTPs . The resThe phylogenetic analysis was the most extensive to date, including 797 nsLTP sequences. This was a much more complete description than the previous one  led to the identification of amino acids involved in the functional specialization of some nsLTPs.STD allowed us to highlight amino acids specific to certain functions. One of the limiting points of this analysis remained the publication bias. Indeed, the annotations were not evenly distributed among available sequences, nor was it possible to distinguish between an unsearched function and a function not found. It seemed difficult to propose a solution to circumvent this bias .The structure tree clearly showed that all Type I ns-LTPs adopted the same folding (Type-1 fold), while all the other proteins adopted the second fold (Type-2 fold). This approach seemed very interesting but did not offer the same level of detail and the same analytical power as the phylogenetic approach. This was understandable, because phylogeny compares the different proteins with a much larger number of parameters  while the structure tree only uses the RMSD of the structures taken 2 by 2. While this innovative information was very interesting, it could potentially be improved if we had templates from each sub-family for the generation of molecular models . Indeed, at this level of analysis, it is conceivable that models obtained from experimental structures for the other types  would provide improved models allowing the detection of other key residues.Plant non-specific lipid transfer proteins constitute a complex family of proteins whose biological functions are far from well understood. However, it has become clear for years that they are of increasing interest for agronomical and nutritional issues.Experimental approaches are irreplaceable for accessing their inner functional mechanisms. However, such methods are expensive and time-consuming. Furthermore, they produce a large amount of heterogeneous data. For all these reasons, resorting to bioinformatics methods has long become necessary to organize and analyze existing data, and/or model and hypothesize new data.This paper presented a new methodology based on the combination of either classical or original bioinformatics approaches, using various computational tools to extract information and suggest new hypotheses from a large pool of experimental data about the plant nsLTP superfamily of proteins.In this paper, we:(1) suggested a new definition of the nsLTP superfamily, with a set of criteria based on sequence length, sequence composition  and structure (monodomain);(2) confirmed and enriched Boutrot’s phylogenetic classification of plant nsLTP sequences;(3) demonstrated the need for a small shift in the CXC alignment that reflected the existence of two main distinct nsLTP folds;(4) calculated 666 good quality theoretical 3D structures of nsLTPs;(5) developed an original alignment tool to detect conserved and specific positions among the different phylogenetic types of nsLTPs;(6) used the latter tool to reveal some key residues;(7) suggested a new structure-based classification of the 676 nsLTP structures now available , which that allow clustering by structural similarity;(8) annotated all available information about the function;(9) developed an original interface allowing quick visualization of several types of annotations on any phylogenetic tree;(10) revealed, using structural trace analysis, potential specific amino acid residues involved in plant defense and/or resistance against pathogens.Our work was made more difficult by the problems of annotation bias for which we did not expect a practical solution. However, it seemed that some of our results could be improved if we had additional experimental structures for all types of nsLTPs.To researchers who may not grasp the importance of the protein structure–function relationships we would like to insist on three main contributions of the methods presented in this work:- The structural classification agrees with the sequence classification by phylogenetic types.- The sequence–structure analysis highlights key-residues explaining the specificities of the different folds.- The structure–function analysis based on the ET of the aligned sequences can show the functional signature of a subfamily of proteins.Furthermore, the structural dichotomy between type I nsLTPs and all the others may go unnoticed by anyone who would focus on one sequence and who would not conduct a combined analysis on a large set of sequences. We encourage researchers studying nsLTPs to use this approach combining sequence alignment, phylogeny and structural biochemistry. This would enhance the power of the analysis by drawing a connection between primary sequence, 3D structure and function.More broadly, we consider that this type of combined approach should be favored for any study involving a multigenic family.


Aims: Physical activity (PA) has many benefits in type 1 diabetes mellitus (type 1 DM). However, PA levels in people with type 1 DM have not previously been measured accurately. We aimed to compare objectively measured PA in adults recently diagnosed with type 1 DM and healthy adults.Methods: Accelerometer data from 65 healthy adults  were compared with data from 50 people with type 1 DM , time since diagnosis <3months, HbA1c 76 ± 25 mmol/mol) in the EXTOD (Exercise for Type 1 Diabetes) pilot study. Briefly, EXTOD investigated the feasibility of recruiting recently diagnosed adults with type 1 DM into a yearlong exercise intervention. Multiple-regression models were used to investigate the association between diabetes status and activity outcomes.Results: Adults recently diagnosed with type 1 DM spent on average a quarter less time in moderate-to-vigorous-physical-activity (MVPA) per day than healthy adults  37.4 mins/day (9.1) Healthy adults: 52.9 mins/day (11.0)]. No difference in MVPA between the groups was seen at the weekend, but adults with type 1 DM spent more time in light physical activity (LPA), and less time in sedentary behavior. Time spent in sedentary or LPA during weekdays did not differ between groups.Summary: Adults recently diagnosed with type 1 DM do less MVPA. Health care workers should encourage these people to engage in more PA. Further studies are needed to assess PA in people with type 1 DM of longer duration. This is the first study to compare objectively measured physical activity in adults newly diagnosed with Type 1 diabetes mellitus (type 1 DM) with people without type 1 DM.Adults with newly diagnosed type 1 DM spent over a quarter less time in moderate-to-vigorous-physical-activity (MVPA) than adults without type 1 DM.Clinicians should assess activity levels of adults with newly diagnosed type 1 DM and encourage those who are not reaching guideline targets to do more.Regular physical activity (PA) plays a key role in the management of type 1 diabetes mellitus (type 1 DM) . As a reMost studies investigating physical activity (PA) levels in adults with type 1 DM have been based on self-reported data rather than objective data. A retrospective analysis of the Diabetes and Complications Trial found 19% of participants 271/1,441) were not achieving recommended PA levels . In the 1/1,441 wIn people with established type 1 DM many of the barriers, motivators and facilitators to PA are similar to the general public, such as lack of time, work pressures and bad weather –11. TheyNo studies have objectively measured the PA levels or patterns of people recently diagnosed with type 1 DM, a time when exercise habits may be greatly influenced. This study aimed to compare objectively measured PA levels in recently diagnosed adults with type 1 DM to healthy adults.This is an observational cross-sectional study comparing objectively measured PA data of adults newly diagnosed with type 1 DM with that of healthy adults. The EXTOD study was approved by the Birmingham East, North and Solihull Research Ethics Committee (0/H1206/4), UK, and the study to measure activity in healthy adults was approved by the Centre for Exercise, Nutrition and Health Science Ethics Committee, University of Bristol (EAN 001-15) and was part of RIBM MRes project. All participants provided written informed consent in accordance with the Declaration of Helsinki.Objectively measured PA in adults with type 1 DM came from participants in the Exercise in type 1 diabetes (EXTOD) study . This waHealthy adults were recruited from the University of Bristol and Taunton and Somerset NHS trust during March-June 2016. A global e-mail was sent out to all members of these two institutions explaining briefly about the study. In addition, flyers were put out in public areas. Participants had to be between 18 and 65 years old and on no medication. Upon request from potential participants, they were sent the participant information sheet, and if interested they were invited to attend an appointment at either the Centre for Exercise, Nutrition and Health Sciences at the University of Bristol or the Diabetes research unit at Taunton and Somerset NHS trust.Following informed consent, participants completed a questionnaire to confirm their health status, smoking status, and alcohol consumption. Weight and height were measured using standard procedures. BMI was calculated as weight (kg) divided by height (m) squared. Participants were provided with an accelerometer and instructions were given on how to wear the accelerometer for the next 7 days. Participants returned the accelerometer 1 week later either by appointment or by post. Brief feedback was given to the participants on their levels of activity over the 7 days they wore the accelerometer; a graphical interpretation of their physical activity over the 7 days was delivered via email after they had returned the accelerometer and the data had been downloaded.Participants wore the accelerometer  on a belt around the waist during waking hours, apart from swimming or bathing, for 7 days. The accelerometers were set to record data at 30 Hz and data were summarized for every minute. Accelerometer data were downloaded using Actilife software  and processed to generate outcome variables using KineSoft . Non-wear time was defined as a period of 60 min or longer with continuous zero values, with a spike tolerance of 2 min. Thresholds of ≥1,952 counts per minute (cpm) and <100 cpm were used to derive MVPA and sedentary time respectively , with liOutcome measures were time spent in sedentary behavior (minutes per day), time spent in LPA (minutes per day), and time spent in MVPA (minutes per day). Each of these variables were then summarized by weekday, weekend and all week to investigate the pattern of PA throughout the week. The number of bouts of MVPA ≥10 min was also used as a comparison between groups. We used bouts of over 10 min as guidelines suggest that the bouts of exercise that count toward the 150 min a week of moderate or intense activity should be of 10 or more minutes [3.4].t-tests , Mann-Whitney U-tests , and Chi-squared tests .Means/standard deviations and percentages were used to describe the characteristics of the participants. Differences between means of the two groups were tested by two-sample Multiple linear regression was used to assess the association between diabetes status and activity intensity . Model A was adjusted for sex, age, wear time, and model B was further adjusted lifestyle factors .To investigate the difference in the total number of bouts accumulated by both groups, logistic regression was used. Adjustment for confounders (model A and B) was made by creating a binary outcome  and fitting multiple logistic regression models. Due to not every participant wearing their accelerometer for 7 days, a sensitivity analysis was performed, which restricted the analysis to only those with 7 valid days of accelerometry.post-hoc power calculation showed that with an alpha level of 0.05, a minimum sample size of n = 50 in each arm, means of 52 and 37 min/standard deviations of 21 and 28 min of MVPA/day for healthy adults and adults with type 1 DM respectively, this study had 85% power to detect differences between the groups.It had not been appropriate to perform a power calculation for the original study (EXTOD), as it was a pilot. However, a Stata Statistical Software: Release 15. StataCorp LLC, College Station, TX).All analyses were performed using Stata v15  and 65 healthy adults (82%) left for analysis. Demographic information for those included in analysis is shown in Table Table For time spent in sedentary behaviors and LPA, there was no evidence of a difference on weekdays and over the whole week, but there was evidence for the adults with type 1 DM spending more time in LPA and less time in sedentary behaviors at the weekend.p = 0.06], over a quarter less time in MVPA [Predicted values [Mean (SD)] type 1 DM adults: 37.4 mins/day (9.1) Healthy adults: 52.9 mins/day (11.0)].For MVPA, statistical evidence was found for a difference on weekdays and across the whole week, but not on weekends. On average the type 1 DM group spent 10.9 min less in MVPA per day than the healthy adults after adjustment for confounders [ We investigated the difference in number of bouts of MVPA ≥10 min further by logistic regression, using the binary variable based on the median number of bouts as the outcome. No statistical evidence was found for a difference in numbers of bouts between the groups for either models  measured the activity of 75 Canadian adults with established type 1 DM and 75 Canadian adults without type 1 DM . They foIn the second study, by Finn and colleagues, PA was measured objectivity using an accelerometer (Actigraph) in 72  Irish adults with type 1 DM . SubjectIt is interesting that the lower MVPA in adults with type 1 DM appears to be driven by changes in weekday activity rather than weekend activity. During the week activity is influenced by work patterns and the type of job a person does whereas at the weekend people tend to have more control over their activity patterns. It might well be that the difference we are seeing in this study are due to a difference in the way that people travel to work  . We wereMore time spent being sedentary is associated with poorer metabolic health. In a systematic review of 29 observational studies, accelerometer-measured total sedentary time was detrimentally associated with both glycaemic control and lipid profile . People The lack of difference in sedentary time could be due to the fact that although their worries about hypoglycaemia, and lack of knowledge and/or confidence in managing their glucose around exercise prevents them from doing moderate and high intensity activities it does not stop them doing low intensity activities. Alternatively, it could be that a reduction in sedentary time is only seen after people have had type 1 DM for a number of years. Brazeau and colleagues did not report sedentary time in their study . In the In our data set, women were less active than men (data not shown). This is in keeping with an American study that objectively measured PA in 6,329 participants and found men to be more active than women  but at oA major strength of this study is the use of an objective and reliable method to assesses the physical activity levels . In addiIn conclusion, our results indicate that adults newly diagnosed with type 1 DM spend more than a quarter less time doing MVPA over the week compared to healthy adults. This suggests that steps need to be taken to try and improve the activity levels of adults newly diagnosed with type 1 DM. To do this clinicians will need to be trained in how to assess and encourage activity and also be furnished with knowledge about how to advise patients to manage their glucose around exercise. In addition, people with type 1 DM will need to be provided with knowledge and skills to safely manage their glucose around exercise. We and others are working on programmes to help support these changes.RM, SL, AC, and RA had full access to the primary data. All authors contributed to data analysis and interpretation, and the writing and editing of the report. All authors approved the final version of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.RA has received honoraria from Novo Nordisk, Sanofi-Aventis, and Merck Sharp & Dohme, and travel expenses from Sanofi-Aventi. PN has received honoraria and travel expenses from Novo Nordisk, Sanofi-Aventis and Lilly. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.