Sickle cell disease (SCD) is a common haematological disorder, affecting millions of people worldwide. It is most prevalent in malarial endemic areas in the tropics where outcomes are often poor due to resource constraints, resulting in most children dying before reaching adulthood. As increasing progress is made towards reducing under 5 mortality from infectious causes, non-communicable diseases (NCDs) including SCD have risen to the forefront of the global health agenda. Despite this, the global mortality burden of SCD remains poorly understood. This study aimed to estimate the incidence and mortality of SCD in children under 5 years of age in order to inform policy and develop sustainable strategies to improve outcomes.We performed a systematic literature search of Medline, EMBASE, Journals@Ovid, and Web of Science for studies on the incidence and mortality of SCD in children under 5, with search dates set from January 1980 and July 2017. We conducted random effects meta-analysis to obtain pooled meta-estimates of birth prevalence and mortality rates globally, and for each World Health Organization (WHO) region.67 papers were found with relevant data. 52 contained data on incidence and prevalence and 15 contained data on mortality. The overall pooled estimate of mortality from the limited data available was 0.64 per 100 years of child observation (95% CI = 0.28-1.00) with the highest rate seen in Africa 7.3 (95% CI = 4.03-10.57). The global meta-estimate for the birth prevalence of homozygous sickle cell disease was 112 per 100 000 live births (95% CI = 101-123) with a birth prevalence in Africa of 1125 per 100 000 (95% CI = 680.43-1570.54) compared with 43.12 per 100 000 (95% CI = 30.31-55.92) in Europe.There were a number of limitations in the depth and breadth of available data however it is clear that both the highest prevalence and highest mortality of SCD is in Africa. In order to address this burden, there is a need for national comprehensive newborn screening to identify patients, and the development of holistic SCD care programmes to provide therapeutics and education for families and children with SCD. This targeted funding should form part of a broader increased global focus on NCDs in childhood. Sickle cell disease (SCD) is the most common genetic haematological disorder, accounting for over 305000 births in 2010, with millions of people currently affected across the globe [The global distribution of sickle cell disease is closely linked to the natural protection against malaria afforded to individuals who are heterozygous for the sickle cell mutation. This selective advantage throughout human history has resulted in the distribution of HbS mutations closely reflecting the global malaria incidence, focused around the tropics . UnfortuAs increasing progress is made towards reducing under 5 mortality from infectious causes, non-communicable diseases (NCD) have risen to the forefront of the global health agenda. SCD is recognised as a significant cause of NCD-related childhood mortality and has been identified as an area requiring specific focus in order to meet the sustainable development goals ,6. DespiWe conducted a systematic review of the literature in accordance with the PRISMA guidelines . The seaData were extracted on study duration, location, WHO region, sample size and birth prevalence, and mortality from SCD. Where studies had overlap in data, the most recent study was used. Data extraction was also performed by two independent reviewers to minimise bias.Homozygous sickle cell disease was defined as being homozygous for HbSS or heterozygous with HbS-βth or HbSC; heterozygous sickle cell trait was defined as having the genotype HbAS. Genotype was ascertained by high performance liquid chromatography or iso-electric focussing.For each full text selected, we checked for sampling , statistical analysis , and case ascertainment . Studies were graded as high (4-5), moderate (2-3), or low quality (0-1). All low quality studies were excluded from the review.A random effects meta-analysis was performed on the extracted crude data to obtain pooled estimates of mortality and of birth prevalence for both homozygotes and heterozygotes for sickle cell. When estimating mortality, where studies did not provide mortality rate or child years of observation, child years of follow up was estimated by multiplying the sample population by the duration of the study. Global meta-estimates were obtained as well as meta-estimates for each world region as defined by the WHO. Due to limited regional data, we did not conduct sub-group (sensitivity) analysis based on the assessment of study quality. All statistical analyses were conducted on STATA .The results of the literature search are shown in the flowchart in From the 52 papers reporting on the incidence or prevalence of SCD, 44 papers reported on incidence and were prospective or retrospective cohort studies of live births. Most studies (n = 22) were from the region of the Americas, followed by 12 from Europe, 7 from Africa, 2 from South East Asia and 1 from the Eastern Mediterranean Region. The duration of the study ranged from 3 months to 26 years with a median duration of 4 years. There were also eight cross-sectional studies reporting on sickle cell disease prevalence in children under five years. Five of these were from Africa, one from the East Mediterranean Region, one from South East Asia and one from the region of the Americas.There were 15 studies which reported on mortality from sickle cell disease, which were prospective or retrospective cohort studies on groups of children with homozygous sickle cell disease. The sample size was between 52 and 2576, and the duration was from 6 months to 27 years with a median duration of 8 years. There was one from African region, two from Eastern Mediterranean Region, two from European region and 10 from the Americas.Online Supplementary Document(Online Supplementary Document).From all 66 studies 42 were rated as high quality and remaining 24 rated as moderate quality. All studies were conducted between 1996 and 2016. Details of all selected studies are in Tables S1, S2 and S3 in The global meta-estimate for the birth prevalence of homozygous sickle cell disease was 111.91 per 100 000 live births (95% CI = 100.77-123.05)  with the highest birth prevalence in Africa: 16 121.91 per 100 000 (95% CI = 11 853.32-20 390.49) and the lowest in Europe 803.57 (95% CI = 535.74-1071.39)  (From our pooled meta-estimates, we found the overall mortality rate to be 0.64 per 100 years of child observation with the highest rate in Africa at 7.3 per 100 years of observation. The confidence in these figures however is low due to limited available data. We estimated the global birth prevalence of homozygous SCD to be 0.11%. In 2015, this would amount to about 150 000 births with SCD. This is much lower than the estimates reported by Piel and colleagues .There were several limitations in data contributing to our estimates. There is a paucity of mortality data available for children under 5 globally, likely a reflection of the fact that in HICs under 5 mortality from SCD is now very low whereas in LMICs many children with SCD die before diagnosis . Only fiMost incidence and prevalence data were from regions with the lowest incidence of SCD – North America and Europe – in countries where there are reliable birth registries and mandatory neonatal screening. Only six studies from the African region could be included in the meta-estimate for birth prevalence and there was wide variation in estimates from these countries. Because there are few comprehensive national screening programmes in LMICs, the quality of data from these regions was lower, often coming from single centre studies or cross-sectional studies. The lack of national birth registries also meant that much of the data from LMICs, particularly in the African region, only captured births within health centre/hospitals, resulting in selection bias as in Sub-Saharan Africa only half of births occur within health care facilities . TherefoIt is clear from our results that SCD is an important contributor to under 5 mortality in LMICs, particularly sub-Saharan Africa. The wide discrepancy between mortality in these settings compared with HIC suggests that much of this mortality is largely preventable. There is considerable evidence for comprehensive SCD care programmes as a way to improve outcomes for children with SCD, providing penicillin and malarial chemoprophylaxis as well as family education ,13. TherEnhanced diagnostics to enable early case-identification will also be essential in improving SCD outcomes. Currently neonatal screening is not widely available in many LMICs and many children die having never been diagnosed . One impSCD is an under-recognised and under-funded cause of under 5 mortality, particularly in Africa. There are several well recognised and well evidenced ways to improve outcomes and the development of integrated treatment pathways for SCD sufferers would be a powerful way to tackle this excess mortality and morbidity. Comprehensive newborn screening programmes are a crucial part of attaining this and this may become more achievable with the promise of a rapid-diagnostic test on the horizon. The current evidence base is limited and there is a need for large scale prospective cohort studies to accurately assess the SCD burden. For any headway to be made in tackling SCD, funding needs to be reprioritised to bring NCDs to the fore, particularly genetic disease such as this one which have received little global investment to date.


The sternal wire code records details of coronary artery bypass surgery (CABG) inside patients, based on the orientation of wires used for sternal closure. Visible on X-ray, the code overcomes the problem of missing graft-notes needed before repeat angiography. We determined (i) the potential value (ii) acceptability and (iii) accuracy of the code in practice.(i) Consecutive coronary angiogram reports  were reviewed to identify patients with previous CABG and those with and without graft-notes before angiography. (ii) UK surgeons were surveyed on whether they would insert the code during CABG. (iii) A clinician, blinded to operative details, interpreted 16 post-CABG X-rays, 8 with the code and 8 without.p < 0.001).(i) Of 6483 angiography patients, 559 had previous CABG (9.2% (8.5–10%)). Graft-notes were missing in 91/559 (15.1% (12–18%)); almost all (88/91) among patients with acute myocardial infarction. (ii) In the survey, 66/71surgeons (93% (84–98%)) were willing to use the code. (iii) In the accuracy test, all coded X-rays were identified and 28/28 grafts correctly interpreted (About 1 in 6 patients with previous CABG, who require emergency coronary angiography, undergo this procedure without graft-notes and would benefit from the sternal wire code which appears clinically acceptable and accurate. BypasThe sternal wire code is a method for permanently recording CABG details inside the patient . It overThe code is based on the orientation of the wires used to close the sternum after CABG . It requThere is uncertainty over the proportion of patients who would benefit from such a code and the feasibility of introducing it into routine use. This prompted us to determine the prevalence of missing graft-notes in patients undergoing angiography and to assess the acceptability and accuracy of using the sternal wire code in practice.22.1We reviewed the electronic hospital records of 6454 consecutive patients undergoing coronary angiography at the Barts Heart Centre, London over a 1 year period (January 2015 to January 2016). From the procedure reports we identified patients who had undergone CABG and determined whether the graft-notes were available at the time of angiography. We determined whether the angiogram was required as an emergency procedure to treat a myocardial infarction or whether it was an elective investigation for stable angina. We also determined the number of patients who required additional imaging  coronary angiography or repeat invasive angiography) because of uncertainty over whether all grafts had been seen during the initial angiogram.2.2The willingness of surgeons to use the sternal wire code during CABG was assessed by questionnaire among cardiac surgeons attending the United Kingdom's Society for Cardiothoracic Surgeon's 2017 annual meeting. A poster describing the code was presented and consecutive surgeons were asked whether they would, in principal be willing to orientate their sternal wires to encode the graft information for future doctors. Completed questionnaires were returned and the positive and negative responses counted.2.3The accuracy of interpreting the sternal wire code was assessed by examining the post-operative X-rays of 16 patients following CABG; 8 consecutive patients in whom the code had been used and 8 in whom it had not been used. A cardiologist, who was unaware of the operation details, was shown the 16 post-operative X-rays in random sequence and asked to identify the X-rays displaying the code and among them the number and origin of bypass grafts used. The observed record was compared with details from the operation notes.p values were calculated using the binomial distribution. STATA was used for all analyses. The project was registered with the Clinical Effectiveness Board at Barts Health NHS Trust. Written consent was obtained from all patients whose X-ray images were used.Data were presented as absolute numbers and proportions together with 95% confidence intervals. 3Among 6484 patients undergoing coronary angiography, 599 had previous CABG (9.2% (95% CI 8.5–10%)) and 91/599 had no graft-notes available at the time of the angiogram (15.1% (12–18%)). In 87 of these 91 patients (96% (89–99%)), the angiogram was required as an emergency for an acute myocardial infarction. In 20/91 patients (22% (14–32%)) a second imaging investigation was required  before the graft information obtained was judged to be complete.In the survey of cardiothoracic surgeons, 71 questionnaires were handed out and all were completed and returned. There were 66/71 (93% (84–98%)) surgeons who were, in principal, willing to use the sternal wire code in CABG surgery and 5 who were not willing to use it.p < 0.001). Out of a total of 28 bypass grafts encoded (3.5 grafts per patient), 28 were correctly interpreted .4Our results show that about 1 in 10 patients undergoing coronary angiography have had previous CABG and 1 in 6 of these patients did not have graft-notes available before their angiogram; the proportion who would benefit from use of the sternal wire code. Almost all of the patients without graft-notes (88/91) presented as emergencies with acute myocardial infarction, with little or no time to search for records that were not immediately available. Prior knowledge of the number and origin of surgical grafts is necessary before any angiogram  and is pIf our results are representative of those in other centres, then an estimated 4000 post-CABG angiograms are undertaken in the UK each year without grafts-notes  . The steThe prevalence of missing graft-notes and therefore, the potential value of the code in practice, may have been underestimated in our study, since it relied on operators specifically recording in the angiogram reports, that graft-notes were missing and this documentation may have been incomplete. It is also possible that increased use of electronic health records will, in the future, improve graft-note availability prior to angiography. Neither approach alone is likely to be sufficient; use of electronic records and the sternal wire code are complementary rather than alternative strategies.4.1There are practical limitations to use of the code. Most surgeons use 8 wires to close the sternum, sufficient to encode up to 6 bypass grafts, but some operators (1 in 12 in our institution) use 4 longer double-loop wires sufficient for only two grafts. These variations in surgical practice will therefore limit the clinical application of the code. Also, between 0.2 and 5% of patients require wire removal because of discomfort or infection . It is uThe code does not provide information on the distal course of a bypass graft  or its anastomosis  but these variations do not limit the precision of the code in indicating the graft origin (subclavian or aorta) which is the most important information to the angiographer because this indicates how many grafts there are and where to find them.False interpretation of the code is a concern. In the accuracy assessment, use of an up-pointing wire (which is non-standard) to signal the start and end of the code avoided the potential problems of falsely interpreting sternal wires when the code had not been used or miscounting the number of grafts implanted when it had been used. Our initial experience is therefore encouraging but the sample size is small, and a larger audited demonstration project would confirm whether the benefits of the code outweigh the risks of false interpretation. Most UK cardiothoracic surgeons (93% of the sample we surveyed) agreed, in principle, to use the code were it to be introduced. However, what is acceptable in principle may not translate into practice, and a demonstration project would also help to determine actual clinical uptake.5Missing graft-notes following CABG in patients undergoing coronary angiography is a significant problem, affecting about 1 in 6 patients, almost all of whom require emergency procedures. The sternal wire code is a simple solution that provides a permanent surgical record inside the patient. Early experience indicates that the code is accurate. A demonstration project is now needed to see whether surgeons will use it. The benefit to patients depends on wide clinical uptake, which is a significant implementation challenge.BJW and DSW took part in the conception and design of the work. BJW, DSW, AR and AS took part in the acquisition of data, in the analysis/interpretation of data and in drafting the manuscript. All authors read and approved the final manuscript.DSW is a founder of Polypill Ltd. BJW, AR and AS declare that they have no competing interests.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


Atypical vascular pattern is one of the most important features by differentiating between benign and malignant pigmented skin lesions. Detection and analysis of vascular structures is a necessary initial step for skin mole assessment; it is a prerequisite step to provide an accurate outcome for the widely used 7-point checklist diagnostic algorithm. In this research we present a fully automated machine learning approach for segmenting vascular structures in dermoscopy colour images. The U-Net architecture is based on convolutional networks and designed for fast and precise segmentation of images. After preprocessing the images are randomly divided into 146516 patches of 64 × 64 pixels each. On the independent validation dataset including 74 images our implemented method showed high segmentation accuracy. For the U-Net convolutional neural network, an average DSC of 0.84, sensitivity 0.85, and specificity 0.81 has been achieved. Vascular structures due to small size and similarity to other local structures create enormous difficulties during the segmentation and assessment process. The use of advanced segmentation methods like deep learning, especially convolutional neural networks, has the potential to improve the accuracy of advanced local structure detection. Melanoma is the deadliest form of skin cancer which develops when skin cells multiply rapidly as a consequence of mutations in their DNA caused by the sun's ultraviolet (UV) radiation . MelanomAccording to the World Health Organization about 132,000 new cases of malignant melanoma are diagnosed worldwide each year. In some parts of the world, especially in New Zealand and Australia, melanoma is becoming more common every year and has more than doubled in the past 30 years ..4].Recently, the most commonly used diagnostic method is the 7-point checklist algorithm. The 7-point checklist is a diagnostic method that requires the identification of only 7 dermoscopic criteria, thus enabling even less experienced clinicians to perform the skin mole examination. One of the most important dermoscopic criteria is the atypical vascular pattern which has the second highest odds ratio of 7.42 and is among the 3 most important features that indicate the malignancy of the skin mole. Atypical vascular pattern appears as linear-irregular or dotted vessels not clearly combined with regression structures and associated with other local structures , 7. DermBased on the information above exact detection and classification of vascular structures is a crucial step in early and accurate diagnosis of malignant melanoma. In this paper we present a new and of the first approaches to the detection of vessels in dermoscopic colour images. In this study, deep learning methods (CNNs) have been used to fully automatically localize and segment vascular structure. To evaluate the performance of deep learning segmentation, we compared the outcome with the manual segmentation. Deep learning technologies have the potential to improve the accuracy and speed up the diagnosis of skin structure in clinical settings.To the best of our knowledge only few attempts have been made to detect vascular structures in dermoscopic colour images. Recently, Kharazmi et al. in work  proposedArtificial intelligence research has been around for more than half a century but in recent years a huge progress is observed in widely understood machine learning . AdvanceAs illustrated in  et al. and developed with TensorFlow [In this research we use the U-Net convolutional network architecture introduced by Ronneberger et al. in 2015 . This mensorFlow , 19.The first step in every medical image processing system is the image acquisition, which aims at obtaining an image of the highest quality. After dermoscopic image is acquired, it may not have the expected quality to perform the diagnostic analysis. Dermoscopic images are inhomogeneous and complex and furthermore include extraneous artifacts, such as skin lines, air bubbles, and hairs, which appear in virtually every image. The preprocessing stage consists of two parts. The first step is the removal of black frame that is introduced during the digitization process. The second step is a hair-removal algorithm which comprises two parts: hair detection and inpainting. These steps have been precisely described in our previous works , 21.N small patches xm,n of size 64 × 64 from the dermoscopy image I and the corresponding annotation G at the same position. The so-called ground-truth mask contains zeros and ones, where  = 1 informs that there is a vessel area at this location. Patch extraction can be performed in few different ways: nonoverlapping, overlapping, and randomly extracted. Each of these solutions has its advantages and disadvantages. To avoid the problem of class imbalance, patches have been extracted randomly around pixels pointing to vessel area both from dermoscopy image as well as from the accompanying masks. For 74 dermoscopy images with different resolution we have obtained 146516 patches. The size of the patches has been chosen experimentally and concatenated with the U-Net architecture. Larger patches require more max-pooling layers that reduce the localization accuracy, while small patches allow the network to see only little context [After preprocessing we extract  context .U-Net convolutional network is a popular architecture in the class of encoder-decoders, where the encoder reduces the spatial dimension of objects with pooling layers while decoder recovers the object details with upsampling layers. U-Net is a modified and extended version of fully convolutional network. x is the input to a neuron. Our first layer has 64-feature map. Afterwards the max-pooling operation is applied. The convolutional layers downsample the spatial dimension from 64 × 64 to 8 × 8. The expansive path consists of an upsampling of the feature map followed by upconvolutional and convolution layers with ReLU [x 3) pixels, respectively. We have chosen the Adam optimization algorithm that is an extension to stochastic gradient descent that has recently seen broader adoption for deep learning applications in computer vision and natural language processing.U-Net consists of contracting path (left side) and an expansive path (right side). U-Net is like a combination of convolutional and deconvolution layers. Contracting path structurally repeats a typical 3 × 3 convolutional layer (unpadded convolutions) followed by a Rectified Linear Unit and a 2 × 2 max-pooling operation with stride 2 for downsampling. On the expansive path, information is merged from layers of contracting path of appropriate resolution and layers of expansive path of lower resolution, so that a whole network recognizes patterns at several scales. The final layer of a 1 × 1 convolution is used to map each 64-component feature vectors to the desired number of classes . Input iith ReLU . As we hPH2 database [The proposed segmentation method based on the U-Net neural network architecture has been tested on colour dermoscopic images from a widely used Interactive Atlas of Dermoscopy . Images database , and imadatabase , 24. TheThe database set was divided into training set (80%) and test set (20%). The training set was used for train the U-Net, while the test set was used to analyse the training versus test error in case of overfitting. In the training stage the input image (patch) and the corresponding ground-truth mask are used to train the implemented U-Net network. The softmax layer at the end of the network creates a probabilistic two-channel output, just like a binary segmentation problem. However, the ground truth here is a probabilistic map, not a binary segmentation map. Training is performed for 100 epochs. As training continuous (seen by epoch) we can see that the generated mask becomes more precise . GrayscaFor the U-Net architecture the patch-size, the batch-size, and the weighted pixel-wise cross entropy were proved to be of high importance. The patch-size proved to be best at 64 × 64 pixels with a large batch-size. When the batch-size is too low, it might be unable to learn, thus negatively impacting total computation time. The predictions were thresholded at 0.5 and are displayed as a binary masks . Sørensen index also known as dice similarity coefficient which is a statistic used for comparing the similarity of two samples [The performance of the U-Net neural network vascular structure segmentation approach can be assessed based on the analysis of samples .X and Y, the Sørensen's formula is defined asGiven two binary sets, The DSC is a statistical measure that calculates the degree of overlapping between the experimental segmentation and the manual segmentation and is frequently used to compare segmentations.Furthermore, sensitivity and specificity are calculated using following equation:thy skin . Related Works presented the state of the art of previous studies concerning the segmentation of vascular patterns in dermoscopy images. In paper [SectionIn paper  authors In this research we have obtained accurate and comprehensive results showing that the applying of U-Net neural networks for local structure detection in dermoscopy colour images brings a valuable alternative to vascular structure detection. We believe that this solution can be implemented as part of a vascular pattern classification algorithm or furthermore a computer-aided diagnostic system for early detection of melanoma. Our technique shows a clear advantage over other implemented and stated in the related works section algorithms including detection accuracy, insensitive to different dermoscopy image acquisition methods.Starting from the described framework, further research efforts will be firstly addressed to compare and integrate the very promising approaches reported in the most recent literature, in order to improve the neural network and optimize layers and parameters. We will also conduct a follow-up study after collecting more data with different vascular patterns. Future research will concentrate on the possibility of vascular structure classification.


Although the effects of osteoporosis on the skeleton are well studied, site-specific and long-term studies on the mandible are still lacking. This study investigated the time-course changes of the bone microarchitecture in the mandibular condyle in comparison to the corresponding changes in the alveolar bone, body of the mandible, and femur. Thirty-six 11-week-old female Sprague-Dawley rats were divided into ovariectomized (OVX) (24 rats) and sham (12 rats) groups. The right femur and mandible were obtained from 6 OVX rats and 3 sham rats at 8, 12, 26, and 36 weeks after surgery, respectively. The histomorphometric analysis was performed using micro–computed tomography and histologic assessments from the (1) distal femur; (2) the alveolar bone and (3) the body of the mandible; (4) the subchondral and (5) the central region of the condyle. The Brown-Forsythe test was used to verify the assumptions for statistical analysis, and the Mann-Whitney U test was then performed. The mandibular condyle showed increased trabecular bone in both the OVX and sham groups, while the bone density was reduced in the distal femur and the mandible interradicular septum and body. When comparing the OVX group to the sham group, only the central condyle showed a significant reduction in bone density at 36 weeks. Osteoporosis behaves in different manners in different parts of the skeleton, and clinicians should be aware that patients displaying osteoporotic changes in the mandible are expected to show severely advanced bone mineral density reduction in other bones, such as the femur. A recent study reported that although bone mineral density (BMD) has been found to decrease in both the femur and the lumbar spine in patients with osteoporosis, the amount of the decrease in each region did not show a significant correlation2. Previous research has concluded that it is difficult to estimate the risk of hip fracture due to osteoporosis based on other body parts, such as the spine or forearm3.Osteoporosis is a skeletal disorder caused by disruption of the mineral density and microstructure of the bone4. In this context, each part of the mandible is expected to show completely different bone microarchitectural changes in response to osteoporosis. In particular, the mandibular condyle is a morphologically and functionally unique joint structure5. Despite its distinctive features, the impact of osteoporosis on the mandibular condyle has scarcely been studied.Mandibular bone affected by osteoporosis is also known to behave differently from other body parts. However, site-specific evaluations within the mandible and long-term investigations of changes in the mandible have rarely been performed. The mandible is a very complex structure, from both developmental and functional perspectives, compared to other skeletal structures. It is composed of joint, tooth-bearing, and muscle-attached areas, and each region shows a distinct trabecular bone pattern due to differences in function and mechanical stress7, because panoramic radiography is a commonly used modality and regular examinations are performed periodically on patients with little cost. However, panoramic radiography has limitations in assessing osteoporosis of the jaw, since the image presents the mandible and maxilla superimposed with various head and neck anatomic structures, such as the cervical spine or ghost shadow of the opposite side of the mandibular angle6. In addition, studies on osteoporosis in the jaw have been based on the premise that osteoporosis in the mandible corresponds to systemic osteoporosis elsewhere in the body. It is difficult to investigate the effect of systemic osteoporosis on the jaw in human beings. In addition, there is no secure evidence for a link between mandible BMD and estrogen deficiency–induced osteoporosis, as Nicolielo and colleagues stated in their review paper5. The degree to which BMD decreases due to osteoporosis in the mandible and how it is related to other skeletal bones should be investigated first, before finding a method of predicting systemic osteoporosis from mandibular bone changes on panoramic radiography.For assessing osteoporosis in human beings, many researchers have attempted to predict systemic osteoporosis based on the mandibular cortex width shown in dental panoramic radiographset al.8 reported that the microarchitecture of trabecular bone in mandible showed alterations at 12 weeks after ovariectomy, while Tanaka et al.9 reported that the alveolar bone of rats started to show changes a year after surgery. A recent review article also stated that limited evidence still exists regarding condyle resorption in response to estrogen deficiency–induced osteoporosis, with a paucity of controlled studies5.Furthermore, the study results that have been reported do not show good agreement. Hsu 10. Previous studies have confirmed that trabecular bone volume of the tibia, femur, and lumbar spine decreased within 12 weeks after surgery in this animal model12.Therefore, it is important from a clinical perspective to clearly characterize the associations of individual mandibular regions with systemic osteoporosis. For studying estrogen deficiency–induced osteoporosis, ovariectomized (OVX) rats are a generally accepted model, approved by the Food and Drug AdministrationThis study was conducted to investigate the timing of changes in the microarchitecture of the mandibular condyle in rats due to osteoporosis, with periodic post-ovariectomy observations. Furthermore, bone changes in multiple other regions of the mandible, including the mandibular alveolar bone and body, were compared to the corresponding changes in the distal femur.Thirty-six 11-week-old female Sprague-Dawley rats , were obtained from a breeder . After 7 days of acclimatization, 12-week-old female rats were housed in a temperature of 25 °C with a 12-hour light and 12-hour darkness cycle and allowed free access to water and a standard rodent diet. The experiment was conducted in the Laboratory Animal Unit, Yonsei Biomedical Research Institute, Yonsei University College of Medicine. Prior to the experiments, the animal experiment protocol was approved  by the Institutional Animal Care and Use Committee of Yonsei University Health System, and all experiments were performed in accordance with the University’s Guidelines for Animal Experimentation and ARRIVE guidelines.13. The procedure was performed by qualified veterinary surgeons. In brief, inhalation anesthesia was induced via 3–4% isoflurane and 1–2 L/min of oxygen in an induction chamber. Then, the rat’s hair was shaved from the back to both flanks, from the femur to rib cage. A 2-cm ventral incision was made, the fat pad was moved, and the ovaries were excised around the perinephric region. After removing the ovaries, the muscle layer was closed with a single-stitch suture  and the skin layer was closed with black silk . The control group underwent a similar standard procedure, except the ovaries were kept intact. Meloxicam  was given to rats postoperatively for 3 days for pain relief. Likewise, enrofloxacin  was given to prevent infection.The 36 rats were divided randomly into 2 groups: 24 rats underwent a bilateral ovariectomy (the OVX group) and the remaining 12 rats underwent sham surgery (the sham group) to simulate surgical stress based on a standardized procedure from a previous publicationSix rats in the OVX group and 3 rats in the sham group were euthanized at 8, 12, 26, and 36 weeks post-surgery, respectively. Euthanasia was done by carbon dioxide gas with 3% isoflurane with pure oxygen as the carrier for isoflurane anesthesia. The mandible and femur were harvested and the remaining soft tissue was carefully removed. The mandibles were split in half and all excised samples were fixed in 10% neutral buffered formalin solution for 2 days.The right side of the split mandible and the right side of the femur samples were scanned with a micro–computed tomography (CT) system  using the following settings: total rotation, 180°; step size, 0.3°; source settings, 130 kV and 60 μA; and exposure time, 0.5 s per step. Raw images were acquired with pixel dimensions of 15 μm × 15 μm, and were used to reconstruct three-dimensional images in the Skyscan software package , which were analyzed using CTAn version 1.12.0 .14. ROIs on the right side of the distal femur were selected, and a longitudinal extension of 1 mm was performed to avoid involving the primary spongiosa and new bone growth15   were limited to trabecular regions; cortical bone was discarded due to its particular sensitivity and lack of comparability to osteoporosis in humansh15 Fig. . ROIs ofM1) Fig. . The borM1) Fig. . The manM1) Fig. .Figure 1The following parameters of bone density and trabecular structure were carefully evaluated: (1) BMD; (2) bone volume/tissue volume , which refers to the ratio of the segmented bone volume to the total volume of the ROI; (3) trabecular thickness , which is a measure of the mean thickness; (4) trabecular separation , which is a measure of the average distance of the marrow cavities between the trabeculae; and (5) trabecular number , which indicates the average number of trabeculae per unit length.After obtaining micro-CT scans, the samples were fixed in standard formalin solution with 10% neutral buffer for 2 weeks. The fixed sample then underwent a decalcification process using Calci-Clear Rapid . Furthermore, dehydration using a graded ethanol series of 70%, 95%, and 100% was performed, and the samples were soaked in xylene and then embedded in paraffin. The mandibular body and molar region were sectioned in the mesial-distal direction, and the distal femur and the condyle were sectioned in the longitudinal direction in series of 3-µm thickness using a microtome . The samples were stained with Masson trichrome to discriminate bone from soft tissue. The series of sections were observed using a light microscope  and examined using a slide scanner .20.All parameters are reported as the mean ± standard deviation (SD) of each group. The homogeneity of variance between the control and experimental group was confirmed using the Brown-Forsythe test. The Mann–Whitney U test was performed to compare the distribution of the trabecular bone microarchitectural parameters of the mandibles and distal femur between the OVX and sham groups. A P-value of less than 0.05 was considered to indicate statistical significance. Data were analyzed using SPSS version 23.0 . We determined the sample size based on the effect size of BMD measurements in previous research with a power (1- β error) of 0.8 and an alpha error of 0.05 using G*Power 3.1.3 statistical softwareMicroscopy and micro-CT images revealed that the mandibular condyle showed increased trabecular bone in both the OVX and sham groups, unlike the femur and mandible areas Figs  and 3. OThe quantification of bone microarchitectural changes (mean ± SD) in the OVX and sham groups is shown in Figs All regional sites of the mandible showed delayed or absent BMD reduction compared to the distal femur. The mandibular condyle did not show noticeable changes in trabecular bone in either the subchondral or central region. Only the central region of the condyle at week 36 showed a significant difference in BMD, with a 14.0% difference between the OVX and sham groups Fig. .In the M1 interradicular region of the OVX group, significant bone changes were observed starting at week 26 post-surgery. By week 36, the BMD of the M1 interradicular region in the OVX group was reduced by 23.0% and Tb.Th was reduced by 26.7% compared to the sham group. The mandibular body also showed reductions in BMD, BV/TV, and Tb.Th  compared with the sham group. Tb.N and Tb.Sp did not show significant differences between the OVX and sham groups in either the mandibular body or the M1 interradicular area , a muscle-attaching basal bone area (the mandibular body), and a joint component , were precisely established as individual ROIs. These results were compared with those of the femur, which has been relatively thoroughly investigated in many previous studies. Thus, the findings of this study are noteworthy in that we performed an analysis at several time points over a relatively long period at multiple sites of the mandible.23. The bone mass reduction in the interradicular region was somewhat more prominent than in the body area. Johnston et al.24 also reported that the first molar interradicular septum is known to be the most sensitive site to osteoporosis due to estrogen deficiency.This study confirmed that the mandible, especially the interradicular and body areas, were less sensitive to systemic osteoporosis than the femur, which is consistent with several previous studies25. More specifically, bone microarchitecture, such as trabecular thickness, number, and distribution, affects the stability and long-term success of dental implants26. As Johnston and Ward stated, the deterioration of the alveolar bone in OVX rats resembles osteoporotic changes in postmenopausal women24. It should be kept in mind that animal studies cannot directly reflect biological changes of the human body, but the present study implies that patients diagnosed with osteoporosis through a femoral examination may not display as poor bone quality as expected in the mandible. In addition, the tooth-bearing area was the first to show osteoporotic changes, and showed the greatest extent of osteoporotic changes, in comparison to the mandibular body and condyle areas.The bony area surrounding the molars in the mandible has been commonly used as an ROI in previous studies due to its importance during dental implant surgery. An implant fixture may not be loaded until successful osseointegration is confirmed, and the duration of osseointegration is closely related to the bone qualityet al.17 reported that the subchondral area was under the transient influence of estrogen deficiency. This discrepancy might be due to the use of different time points in the analyses. The last time point after sacrifice was much earlier in their study (8 weeks postoperatively) while the current study traced OVX rats for a longer period. Since bone changes after 36 weeks post-ovariectomy have not been studied, it may be useful to perform further examinations with a longer follow-up period.It is interesting that the mandibular condyle showed a completely different response to estrogen deficiency from the alveolar bone and mandibular body. The subchondral region was completely protected from osteoporotic changes until week 36 post-ovariectomy. In contrast, Tanaka et al. suggested that ovariectomy suppressed bone gain, rather than accelerated bone resorption, in the central condyle area17. This study also showed that the BMD of the central condyle in the OVX group was slightly decreased at the last follow-up period, 36 weeks, while the sham group showed increase in the value. Jiao et al. reported that the trabecular bone density of the condyle in healthy female rats continuously increased even after the growth spurt27. These previous reports are somewhat consistent with the present study in that the trabecular bone pattern became noticeably denser on microscopy and micro-CT in the condyle area.In the central condyle region, BMD was the only factor that showed a statistically significant difference between the OVX and sham group at week 36. Other microarchitectural parameters did not show significant differences between the OVX and sham groups. Interestingly, the difference occurred due to an increase in BMD in the sham group, rather than a decrease in BMD in the OVX group. Tanaka 5, although the authors acknowledged that there was insufficient evidence to support this hypothesis. Few studies using a rat model have focused on condylar bone changes induced by estrogen deficiency and even those studies reported inconsistent results27. It is suspected that the follow-up period for those previous experiments was not sufficient for the condyle to present a certain degree of bone change, as was demonstrated in the current study. We found that 36 weeks post-ovariectomy was the initial point of a significant change in trabecular microarchitecture in the condyles of rats with estrogen deficiency–induced osteoporosis, compared with the normal group. It is necessary to conduct microarchitecture analyses at more advanced time points to confirm the time course of mandibular condyle resorption.The progression of condylar resorption stimulated by estrogen deficiency has not been clearly characterized, and has been assumed to reflect a dysfunctional remodeling process according to a systematic review of the previous literature in humansIn conclusion, osteoporosis behaves in different manners in different parts of the skeleton. The mandible presents a substantially delayed and less intense response in terms of BMD loss compared to the femur in a rat model of osteoporosis. The subchondral region of the condyle was the least influenced by osteoporosis in the mandible, while the first molar interradicular septum was a more sensitive area in terms of showing microarchitectural changes. Clinicians should be aware that patients displaying osteoporotic changes in the mandible are expected to show severely advanced BMD reduction in other bones, such as the femur.


LTBP2) segregating with the phenotype. Both these variants are predicted to replace evolutionary conserved amino acids, have a pathogenic effect on the encode protein, and have very low frequencies in the control databases. Mutations in LTBP2 are known to cause the Weill-Marchesani syndrome and a Weill-Marchesani-like syndrome, which include glaucoma in their clinical presentation. However, to our knowledge, this is the first published case of a JOAG subject associated with recessively inherited variants of LTPB2 and, thus, expands the repertoire of the known genetic causes of JOAG and the phenotypic spectrum of LTBP2 alleles.Juvenile open angle glaucoma (JOAG), which is an uncommon form of primary open angle glaucoma, is a clinically and genetically heterogeneous disorder. We report on a family with a recessively inherited form of JOAG. The proband has a superior and an inferior never fiber layer thinning in both the eyes and the nasal visual field (VF) defects in the left eye, which are clinical findings consistent with glaucomatous optic neuropathy. Whole exome sequencing revealed two novel compound heterozygous variants  in latent transforming growth factor-beta-binding protein 2 ( MYOC), cytochrome P450, family 1, subfamily B (CYP1B1), and optineurin (OPTN)  is found in ExAC and 1000 genome with allele frequency as high as 0.005512 and 0.0056, respectively, but has not been previously associated with glaucoma and is absent in the ClinVar database  in  pattern A. The madatabase . The secdatabase . Both of the identified missense variants  are predicted to replace evolutionary and highly conserved residues B and areLTBP2. Parents of the proband were not related and happened to be carriers of two distinct variants of LTBP2. The proband is atypical in his clinical presentation. His intraocular pressure was never measured above 21 mmHg and he had documented the progression of his glaucoma with intraocular pressures in the mid-teens. This is unusual for juvenile glaucoma, which is almost always associated with high intraocular pressures [LTBP2 has been associated with primary congenital glaucoma, pseudoexfoliation glaucoma, and POAG in older individuals . We presressures  with theressures ,29. The ressures . This paressures .LTBP2 variants have been found to cause the Weill-Marchesani syndrome, which is associated with glaucoma and severe myopia [LTBP2 have been identified according to the Human Gene Mutation Database (HGMD). Functional studies have suggested that variants in LTBP2 affects both the trabecular meshwork as well as scleral collagen and may obstruct the fluid outflow and results in increased IOP and glaucoma [LTBP2 cause phenotypes at different developmental stages and with variable severities. One possibility is that the genetic background modifies the phenotypic outcome of LTBP2 mutations. Alternatively, differences in mutant alleles of LTBP2 may directly account for the different phenotypic outcomes. Knock-in alleles of mouse LTBP2 engineered to model human variants could aid in addressing these questions. Our clinical and molecular analysis of LTBP2 alleles may prove useful for a future genetic diagnosis and counseling as well as for molecular epidemiology studies of JOAG.LTBP2 is an extracellular matrix microfibrillar protein with 20 EGF-like domains containing calcium-binding sites to carry out protein-protein interactions and is involved in sustaining the ciliary muscle trend as well as normal development of the eye anterior chamber . LTBP2 ve myopia . As of Aglaucoma . Since tglaucoma . It’s in


Vessel noise represents a relatively recent but rapidly increasing form of pollution, which affects the many organisms that use sound to inform their behavioural decisions. Recent research shows that anthropogenic noise can lead to reduced responsiveness to risk and higher mortality. The current laboratory experiment determined whether the playback of noise from motorboats powered by two- or four-stroke outboard engines affected the kinematics of the fast-start response in a juvenile coral reef fish, and the time scale over which the effects may occur. Results show that the two engine types produce slightly different sound spectra, which influence fish differently. Playback of 2-stroke engines had the greatest effect on activity, but only for a brief period (45 s). While noise from 4-stroke outboard engines affected fast-start kinematics, they had half the impact of noise from 2-stroke engines. Two-stroke engine noise affected routine swimming more than 4-stroke engines, while 4-stroke noise had a greater effect on the speed at which fish responded to a startle. Evidence suggests that the source of the noise pollution will have a major influence on the way marine organisms will respond, and this gives managers an important tool whereby they may reduce the effects of noise pollution on protected communities. Currently, there are over 9.3 million registered small (<8 m) motorboats in the USA3, 6 million in Europe4 and over 90,000 recreational motorboats on the Great Barrier Reef, Australia5. The impact of small motorboats has not received as much attention as the 94,000 merchant ships greater than 100 gross tonnes that traverse the oceans of the world6. However, small motorboats can potentially have a significant impact on communities of marine organisms, as motorboats use much of the shallow waterways and coastline that form the most productive habitats and sustain important fisheries.Vessel noise is recognised as an important source of pollution that is ubiquitous in aquatic environments within populated areas around the world8. These organisms often use sound as an important source of information and have well-developed ways of producing and hearing sound10. Many fishes, such as various species of perciform fishes , use sound for communication during territorial defence, aggression, mating, and as a method of indicating alarm12. Noise from vessels can mask, disrupt or distract organisms that use sound as a source of information1. While there has been concern at the level of some governments to limit anthropogenically-produced noise within inshore areas through legislation13, the empirical foundation on which these policies are based is generally lacking16.Anthropogenically-produced marine noise has been found to have significant effects on the distribution, behaviour, reproduction and survival of marine invertebrates and fishes18). This is particularly the case at the end of the larval phase when reef fishes, like other demersal marine organisms, are particularly vulnerable to predation. This life stage represents a population bottleneck, with high and variable mortality rates, averaging 50% within 2d of settlement19. Anything that alters the mortality trajectories at this early life stage may have a marked influence on the numbers entering subsequent less vulnerable life stages. Recently, Ferrari et al.20 found that 2-stroke motorboat noise affected the ability of settlement-stage fish to learn the identity of novel predators, leading to a 70% decrease in survival in the wild over 72 h. Simpson et al.17 also found that noise from motorboats reduced the likelihood and initiation distance of appropriate escape responses in juvenile fish. Short-term effects of anthropogenic noise on cognition and behaviour could therefore have a critical influence on survival via impacts on risk assessment, decision-making and anti-predator behaviour. However, detailed investigation of the kinematics of anti-predator responses in response to noise are still lacking.One of the most disruptive effects that anthropogenic noise can have on fishes is through its effect on their ability to assess risk and make choices concerning anti-predator behaviour  controlled by large Mauthner neurons, followed by the rapid contraction of the muscles on the opposite side leading to a rapid burst (~0.1 s) away from the original position23. To date however, few studies have examined the relative merits of 2-stroke and 4-stroke outboard engines as sources of marine noise and how their noises may differentially affect marine organisms.Knowing whether particular types of motors, which produce noise with specific characteristics , have different effects on fish performance is useful as it gives marine managers options for the regulation of marine noise. Two-stroke outboard engines power much of the inshore fleet of small motorboats used by recreational boaters around Australia and the Pacific nations due to their low cost and high power-to-weight ratio. Some countries have effectively outlawed their use due to the high smoke emissions  in preference for 4-stroke engines18) was that noise from 2-stroke outboard engine would be more detrimental to the fish than 4-stroke engines due to differences in the sounds produced.The goal of the current study was to determine whether noise from motorboats powered by two- or four-stroke outboard engines affected the routine swimming and kinematics of the fast-start response in a juvenile coral reef fish, and the time scale over which the effect may occur. Our prediction, based on previous research , was chosen as the target species for the study because it has life-history characteristics that are typical of many reef fishes, and young-of-the-year were caught in sufficiently large numbers for a well replicated experiment. It is a rubble-associated planktivore commonly found across the Indo-Pacific28. Newly metamorphosed whitetail damselfish were collected with light traps moored at least 50 m off the fringing reef of Lizard Island , northern Great Barrier Reef, Australia in February 2017. On the morning of capture, whitetail damselfish were transferred to 30 L tanks and fed Artemia in excess of requirements twice daily. Tanks did not have aeration stones and fresh seawater entered the tank through a pipe below the surface to reduce noise .Playback tracks of ambient reef noise and 2- and 4-stroke engines were composed from recordings made in October 2016 using a calibrated omnidirectional hydrophone  and a digital recorder  which was also fully calibrated using pure sine wave signals, measured in line with an oscilloscope, and produced by a function generator . The hydrophone was held 2 m from the surface by a researcher working from a small kayak to eliminate the noise of waves slapping on the hull of a research boat. Ambient reef sound was recorded next to a reef in 5 m water depth. The boat noise treatments were recorded at the same location with five 2-stroke and four 4-stroke engines on 5 m long dinghies each with a 30 hp motor driving past the hydrophone (20 to 100 m away). Outboard engines were 30 hp Suzuki DT30 (2-stroke) and DF30A (4-stroke).29, was also measured using an accelerometer  and a digital recorder (Zoom). Sound recordings of both the field and tank recordings were analyized with paPAM acoustics analysis package .Sound recordings were made for all parts of the experimental process from transport from the light traps, and maintenance within holding tanks Fig. , through32. Ten separate playback recordings for 2- and 4-stroke engines were collected (5 different engines for 2-stroke and 4 different 4-stroke) and analysed with MATLAB 2013a using paPAM analysis package and r packages “Soundecology” (v1.3.3) and re-verified with “seewave” (v2.1.0). To examine the noise within a biological context, a bandwidth between 0–2 kHz was applied to the analysis because pomacentrids are known to detect sounds within this range33.Differences between acoustic pressure emitted by the two engine types were explored using five recommended acoustic metrics: Root-Mean-Square sound pressure level (dB re 1 uPa) (RMS); Peak level (dB re 1 uPa); Consistency (percent time sound level exceeded 150 dB re 1 uPa); 90% energy envelope (time for 90% of the sound energy to be emitted); and Acoustic Complexity Index Two days after capture in light traps, fish were placed into individual 1 L aquaria and left in a water bath with subdued light for 30 to 60 min prior to their use in the test arena. Individual fish were then transferred to the fast-start arena Fig.  and allo1The fast start arena consisted of a thin (1.5 mm) transparent plastic circular arena (diameter 20 cm) within a 60 L white-sided container  off the wall of the arena.Total distance moved was calculated by tracking an individual’s position every 1 s for a period of 120 s. To examine if this variable changed with time, the data was separated into 15 s intervals.At the start of the second 5 min period within the fast-start arena, fish were videoed at 30 fps for 2 min to obtain an estimate of their space use and behaviour while under ambient or boat noise (2- and 4-stroke engines). The following variables were recorded:Response latency (s) was measured as the time interval between the stimulus onset and the first detectable movement leading to the escape of the animal.21, which is the period considered crucial for avoiding ambush predator attacks34.Response distance (m) is a measure of the total distance covered by the fish during the first two flips of the tail  was measured as the distance covered within a fixed time (25 ms). This fixed duration was based on the average duration (22.8 ms) of stage 1 and 2 (as defined above).Maximum response speed (m/s) was measured as the maximum speed achieved at any time during stage 1 and stage 2.Kinematic variables associated with the fast-start response were analysed using the image-analysis software Image-J, with a manual tracking plug-in (imagej.nih.gov/ij/). The centre of mass (CoM) of each fish was tracked for the duration of the response. The following kinematic variables were measured:In addition, distance of the fish’s centre of mass to the stimulus at the point of contact with the water surface was recorded to be used as a covariate when applicable. Most variables measured have been previously posited to be related to fish fitness Table .Table 2V35. The strength or importance of each of the original variables in discriminating among groups is displayed graphically as the length and direction of these vectors.A multivariate analysis of variance (MANOVA) was undertaken to determine whether there was a difference in the routine swimming or fast start kinematics among the playback of ambient sound, 2-stroke and 4-stroke engine noise treatments. To allow the routine swimming measures and kinematics to be incorporated into the same analysis, only the fish exposed to chronic sound treatments Table  were usea priori hypotheses: (a) does boat noise, regardless of source, affect fish behaviour? ; (b) is there a difference between fish affected by the playback of 2-stroke engine noise compared to 4-stroke engine noise? (regardless of whether acute or chronic); (c) is there a difference between fish affected by the playback of acute or chronic 2-stroke engine noise? ; (d) is there a difference between fish affected by the playback of acute or chronic 4-stroke engine noise? .One-factor analysis of variance was used to determine whether the acoustic treatments affected the routine swimming, thigmotaxis or kinematics of the white tailed damselfish. When significant, the nature of the differences was examined using planned comparisons to address four 10 transformed to satisfy the assumptions of the test.To determine the nature and duration of the effect of boat noise on routine swimming, total distance moved was measured in 15 s intervals for the 120 s routine swimming period. This was analysed using a multivariate repeated measures ANOVA, with factor ‘acoustic treatment’ , and time . One-factor ANOVAs were used to determine the nature of the significant interaction between treatment and time. Data were log12.5df = −5.7, p < 0.001; Table There was a marked difference between ambient and boat noise power spectra in both the pressure and particle acceleration domains for both the field measurements and recordings made within the experimental arena of the playback of all tracks Fig. . An exam10,304 = 3.945, P < 0.0001). A CDA displayed the nature of the differences found among treatment centroids . Planned comparisons indicated that fish under ambient reef sound had significantly faster response latencies  than fish under either boat noise treatment (P < 0.0001), however, fish exposed to 2-stroke noise had faster latencies than fish exposed to 4-stroke engine noise .Acoustic treatment affected the total distance moved during routine swimming, but this effect was influenced by time . This is particularly useful to management bodies because noise sources, particularly from vessels, can be modified and managed relatively easily compared to other forms of pollution.Few studies have examined the effect of noise from different engine types on fish behaviour, and this is the first study to examine the effect of noise with differing acoustic characteristics on routine behaviour and escape response kinematics. Our study shows that the playback of boat noise had a marked influence on the routine swimming, latency to respond and escape kinematics of a common juvenile damselfish. Moreover, the noise from playbacks that originated from 2- and 4-stroke outboard engines was sufficiently different to result in different impacts on both general space use, activity and escape response. This information forms part of a growing literature that allows us to better understand how fishes are affected by anthropogenically produced noise from different sources (e.g.Pomacentrus amboinensis), in response to real boat noise in the field. Holmes et al.38 found that the level of boldness exhibited by juvenile fish on habitat patches was immediately reduced in the presence of 2-stroke noise but recovered to pre-noise levels after 20 min of continuous motorboat noise. It is unclear why the time frames of habituation to noise are so different between these studies, but it may be due to factors including the artificiality of experiments, species-specific differences or difference in the metrics of activity. These differences between studies, and between engine types within our study, emphasise that the capacity of fishes to habituate to sound is a field that warrants further research39.The playback of noise from motorboats affected both the routine swimming and escape response of the white-tailed damsel, but this effect differed depending on the source of the noise. Noise from 2-stroke powered boats affected routine swimming, in particular their activity, as measured by the total distance moved. Fish were less active when in the vicinity of noise from 2-stroke powered boats, compared to ambient reef sound controls. However, this effect was only evident for the first 45 seconds after which routine swimming returned to levels comparable with controls. This result suggests that, at least with respect to routine swimming, fish may be able to habituate to noise over a relatively short time period. Similar potential to habituate to 2-stroke noise was found for another damselfish, the Ambon damselfish  was over three-times slower in the presence of 4-stroke noise compared to ambient reef sound controls. This compared to a doubling of speed to initiate a fast-start in the presence of noise from 2-stroke powered boats. This slowing of response times has the potential to greatly affect survival of an aware prey in response to a predator strike. A recent field study that assessed a broad range of performance criteria for newly settled damselfish found that latency to respond to a drop stimulus best predicted survival in the field out of 18 measured variableset al.18 examined the ability of a newly settled damselfish to assess and respond to risk in the presence of noise from passing 2- or 4-stroke boats over a shallow reef. Risk assessment was quantified as the ability of fish to respond with an antipredator response to odours released through the damage of conspecifics, while their ability to mount an effective startle response was assessed with an in situ looming stimulus test. In perciforms, which make up the majority of coral reef fishes, damage to the epidermis releases chemicals that elicit an antipredator response in conspecifics and closely related heterospecifics (e.g.40), which is similar to the chemical alarm odours released by freshwater ostariophysan fishes upon damage of club cells within the epidermis41. For both the odour and startle tests, fish were found to be incapable of mounting effective antipredator responses in the presence of noise from passing boats powered by 2-stroke outboard engines. Moreover, in the presence of 2-stroke noise, fish actually elicited a feeding response to the alarm odour, which contrasted to their risk averse response to alarm odours in the presence of 4-stroke engine noise. Given that alarm odours are only released upon damage of the skin of conspecifics they represent a reliable indicator of a nearby threat44, and so the feeding response found represents a maladaptive response. These findings, together with other studies in the laboratory that used playback of boat, ship or pile driving noise , add weight to our findings of a marked effect of engine noise on the startle response of fishes.To date, only one previous study has compared the effects of the noise from 2- and 4-stroke powered boats on fishes. McCormick et al.47 experimentally examined the fright response of hermit crabs (Coenobita clypeatus) under the influence of boat noise playback and concluded that distraction was the most likely mechanism underlying this response.Our experiment also found that the decrement in the efficacy of the fast-start response when exposed to the playback of noise from either 2- or 4-stroke engines was not due to the initial shock of an abrupt change in the soundscape. There was no difference in the fast-start kinematics between fish startled immediately upon the imposition of boat noise, and those that had been exposed to noise for up to 6 minutes prior to the drop-stimulus. This suggests that the noises affect the sensory system of the small fish in such a way that they cause an ongoing distraction over an ecologically relevant time frame. While routine swimming activity appeared to either not be affected (4-stroke noise) or recover quickly (2-stroke noise), the sensory inputs that govern the initiation  and speed of the C-start burst response could not adjust to the discombobulating effects of noise. Chan 18. The impacts of the subtle acoustic differences between engine types found in the present study suggest that other types of marine noise pollution  of the noise in relation to their capacity to hear sound. Frequency spectra in both the pressure and particle acceleration domains were consistently higher for the two-stroke noise compared to noise from 4-stroke engines. This made for a greater sound exposure in both domains for fish exposed to noise from 2-stroke engines. Moreover, the acoustic complexity was higher for noise from passing boats powered by 4-stroke engines, which is because boats powered by 2-stroke engines produced noise over a broader range of frequencies for a longer period of time during a boat pass. Interestingly, while both engine types produce noise spectra that are markedly different from ambient reef noise, the relatively subtle differences between engine-noise types has substantial influences on the ability of fishes in the vicinity to use other sensory cues, such as vision and smell14. The role that the auditory history of an individual plays in its physiological and behavioural response to recurrent and new noise sources is largely unknown (e.g.50). The spatial scale of these effects, the extent to which fish may use topography as barriers to sound, and the importance of the duty cycle of noise all warrant further research. The current study is supported by less-detailed field studies and emphasises the impact noise may have on a fundamental behavioural response – the avoidance of risk. Our findings underscore that all marine noise will not have the same impact on fishes, and this gives managers tools whereby they can actively reduce a key stressor on endangered or prioritised communities they decide to protect.Our study underscores the impact of anthropogenic noise on important aspects of the dynamics of fish populations. We have limited knowledge as to how long lasting these effects are, and the extent to which fish can habituate to this source of pollutionSuuplementary information


Heterogeneity in outcomes measured across trials of glucose-lowering interventions for people with type 2 diabetes impacts on the ability to compare findings and may mean that the results have little importance to healthcare professionals and the patients that they care for. The SCORE-IT study  has addressed this issue by establishing consensus on the most important outcomes for non-surgical interventions for hyperglycemia in type 2 diabetes.A comprehensive list of outcomes was developed from registered clinical trials, online patient resources, qualitative literature and long-term studies in the field. This list was then scored in a two-round online Delphi survey completed by healthcare professionals, people with type 2 diabetes, researchers in the field and healthcare policymakers. The results of this online Delphi were discussed and ratified at a face-to-face consensus meeting.173 people completed both rounds of the online survey , 20 of these attended the consensus meeting . Consensus was reached on 18 core outcomes across five domains, which include outcomes related to diabetes care, quality of life and long-term diabetes-related complications.Implementation of the core outcome set in future trials will ensure that outcomes of importance to all stakeholders are measured and reported, enhancing the relevance of trial findings and facilitating the comparison of results across trials. A systematic review of active clinical trials registered with ClinicalTrials.gov identified marked heterogeneity in the outcomes measured in trials of glucose-lowering interventions for people with type 2 diabetes.This inconsistency in outcomes impacts on the ability to compare findings and may mean that the results have little importance to healthcare professionals and the patients that they care for.Eighteen outcomes have been included in the SCORE-IT  core outcome set, across five domains, which reflect outcomes related to diabetes care, quality of life and diabetes-related complications.This core outcome set has been developed with input from all stakeholders including people with type 2 diabetes, healthcare professionals, researchers in the field and healthcare policymakers/payers, and has ensured that all participants had an equal voice when deciding the most important outcomes.Implementation of the SCORE-IT core outcome set in future clinical trials, of glucose-lowering interventions, will increase the relevance of research to all stakeholders and will allow results from different trials to be more easily compared and combined.This increased potential for meta-analysis will enable new, effective treatments to be made available to people with type 2 diabetes more quickly.Type 2 diabetes is a global pandemic. Current estimates indicate that 623 million people aged 20–79 will be affected by diabetes by 2045, with the majority of these cases being type 2 diabetes.Treatment for type 2 diabetes is targeted at the hyperglycemia arising due to a resistance to insulin action and an inadequate insulin secretory response.et alet alA recent review of open (actively recruiting or in follow-up period), phase III and IV trials registered with ClinicalTrials.gov identified considerable variation in the outcomes measured and reported for glucose-lowering therapies in people with type 2 diabetes.The aim of the SCORE-IT study  was to address this gap in outcomes research and develop a COS for use in clinical trials of non-surgical therapeutic interventions for the treatment of hyperglycemia in adults with type 2 diabetes that includes input from all stakeholders.The development of the COS involved three stages : (1) theet al.et al,The list of outcomes was generated from a number of sourcesThe long list of outcomes was used to populate an online Delphi survey, delivered using the DelphiManager platform.10.1136/bmjdrc-2019-000700.supp1Supplementary dataThe Delphi process was completed using two rounds (termed R1 and R2). In each round participants were presented with the list of outcomes and asked to score each outcome on how important it was to include in the COS, using a 9-point Likert scale presented in formats 1–9, with 1–3 labeled “not important,” 4–6 labeled “important but not critical,” and 7–9 labeled “critically important.”During R2, participants were shown the distribution of scores for each stakeholder group for each outcome together with their own score from R1 and asked to score the outcome again, using the same 1–9 Likert scale, taking this information into consideration.A face-to-face consensus meeting was held in Liverpool, UK, and the results of the online Delphi survey were presented. Participants who had completed both R1 and R2 of the Delphi were invited to attend. Prior to the meeting, participants received a copy of their scores from the online survey and a consensus matrix detailing the results of R2 by stakeholder group  and whic10.1136/bmjdrc-2019-000700.supp2Supplementary dataThe meeting was chaired by an independent non-clinical researcher with expertise in COS development methodology. Outcomes that had reached consensus “in” after R2 in all four stakeholder groups were presented first, followed by outcomes that had reached consensus “out” after R2 in all four stakeholder groups. Meeting participants were asked if they disagreed with the inclusion or exclusion of these items from the COS, respectively. All outcomes with two or more stakeholder groups reaching consensus “in” were discussed, outcomes with one stakeholder group reaching consensus “in” were shown to meeting participants, and participants asked if any of these should be discussed. Outcomes with no consensus and no group scoring consensus “in” were not discussed.Views for and against inclusion in the COS were sought by the meeting chair, who also ensured that participants had equal opportunity to comment prior to voting. Voting took place anonymously using the TurningPoint software and handsets . Following voting the results were displayed to participants by stakeholder group. For the purpose of the consensus meeting, stakeholder groups were condensed to healthcare professionals  and people with type 2 diabetes. Healthcare policymakers were present to provide their perspective and contribute to the discussions. The definition of consensus used in the Delphi survey  was applAttrition bias between R1 and R2 of the online Delphi was assessed by comparing the distribution of mean R1 scores for participants completing R1 only and participants completing both R1 and R2. Satisfaction with the consensus meeting process, organization and outcome was assessed using a questionnaire .10.1136/bmjdrc-2019-000700.supp3Supplementary dataThe SCORE-IT study was prospectively registered with the COMET Initiative .An overview of the SCORE-IT COS development process and final COS is shown in The systematic review of clinical trials and the rapid review of qualitative studies have been presented in detail elsewhere.The 223 outcomes were mapped onto a 38-category system and grouped under five domains .10.1136/bmjdrc-2019-000700.supp4Supplementary dataOne hundred and seventy three participants completed both R1 and R2 of the online survey. Participants comprised 37 healthcare professionals, 116 people with type 2 diabetes or their carers, 14 researchers in the field and 6 healthcare policymakers .At the end of R1, 10 outcomes met the predefined criteria for inclusion in the COS across all four stakeholder groups. Fifty-one responses were received to the free-text question asking participants if there were any additional outcomes they would like to add. These outcomes were reviewed by the SSC and one outcome “gut microbiome - the type/number of bacteria in someone’s digestive tract” was added and scored by participants in R2. A further three outcomes  were modified based on the free-text response to clarify the outcome.At the end of R2 of the Delphi, nine outcomes had reached consensus, for inclusion in the COS, across all four stakeholder groups, and nine outcomes had reached the definition for exclusion from the COS .Six outcomes reached the definition of “consensus in” in both R1 and R2 and have been included in the final COS.The overall attrition rate between R1 and R2 was 25%, with the highest attrition rate observed for specialist/practice nurses .The impact of attrition between rounds was assessed by comparing the average R1 scores of those who did not complete R2 against the distribution of scores for those completing both R1 and R2. Overall the distribution of average scores of those who did not complete R2 was similar to those completing both R1 and R2 for all stakeholder groups, suggesting that attrition bias was not present between rounds .10.1136/bmjdrc-2019-000700.supp5Supplementary dataTwenty participants attended the consensus meeting ; in addition to the 20 voting participants, there were 3 healthcare policymakers/payers who contributed to discussion along with members of the SSC. In the consensus meeting a further nine outcomes met the definition for inclusion in the COS, in addition to the nine outcomes that had reached the definition of consensus for inclusion at the end of R2 of the Delphi . Of thes10.1136/bmjdrc-2019-000700.supp6Supplementary data10.1136/bmjdrc-2019-000700.supp7Supplementary dataFeedback forms from the meeting were completed by 4 (57%) healthcare professionals and 12 (92%) people with type 2 diabetes. All participants were satisfied with the way the meeting was facilitated, felt able to contribute to the meeting and felt comfortable expressing their views. In terms of the consensus meeting producing a fair result, one health professional felt that voting may have been influenced by a dominant participant, and one person with type 2 diabetes neither agreed nor disagreed with the statement.The SCORE-IT study has developed patient and health professional consensus on outcomes for trials of the treatment of hyperglycemia in people with type 2 diabetes. The process to achieve consensus has ensured that all stakeholders including people with type 2 diabetes, healthcare professionals, researchers in the field and healthcare policymakers/payers have been able to contribute to the final COS. We recommend that future trials of interventions to treat hyperglycemia in people with type 2 diabetes use the SCORE-IT COS. This COS does not prevent other outcomes being measured, as appropriate to the intervention, but rather represents the minimum that should be measured.Particular strengths of the SCORE-IT COS include the use of methods meeting the COS-STAD (Core Outcome Set - STAndards for Development) recommendations,Although this study has had some international input, engagement both in the UK and on an international level was challenging, with only one patient and a fifth of healthcare professionals and researchers combined based outside of the UK. We sought to improve international input from people with type 2 diabetes through engagement with patient organizations and translation of the Delphi into the appropriate local language. However, despite Polish and Brazilian Portuguese versions being distributed, via direct email to members of patient organizations, participation was low, with only one person completing R1 of the Delphi in Polish. Choosing appropriate outcomes to measure is a top methodological priority for trialists working in low-income and middle-income countries (LMICs).et al.et alet al, suggesting that while there are some similarities the priorities of the stakeholders vary depending on the type of diabetes and the age group of participants.The SCORE-IT COS is specific for type 2 diabetes, yet there is overlap with outcomes of importance to young adults with type 1 diabetes identified by Byrne et alet alMurad The International Consortium for Health Outcomes Measurement (ICHOM) has recently reported a standard set for diabetes in adults. The development of this standard set has included international input from 26 experts (3 patients and 23 clinicians) and includes 26 outcomes. Fifty-four percent of the ICHOM standard set is reflected in the SCORE-IT COS, with some subtle differences. Most notably the life impact outcomes between the two outcome sets differ. Psychological well-being, diabetes distress and depression are included in the ICHOM, while these outcomes, scored collectively in SCORE-IT as “Emotional wellbeing,” did not reach the definition of “consensus in” in any round of the Delphi survey. Instead participants of the SCORE-IT study rated “global quality of life” and “activities of daily living” as the most important life impact outcomes. Two outcomes in the ICHOM standard set, “periodontal health” and “emergency room utilisation,” were not included in the SCORE-IT Delphi list of outcomes. Periodontal health was not measured/identified from any of the sources used to generate the long list of outcomes, while the need to attend the emergency room was identified in the systematic review but measured only in a single study and not taken forward to the Delphi survey. Neither outcome was added to the list by Delphi participants completing R1 of the Delphi survey.While there is substantial overlap between the ICHOM standard set and the SCORE-IT COS, differences may reflect the scope of the projects, clinical practice versus clinical trials, respectively, and may also be influenced by the methods used and the type, number and geographic location of the stakeholders involved. Nevertheless the overlap between studies is positive, and if outcomes are captured routinely in clinical practice then this may help improve the efficiency of clinical trials and reduce the burden to trial participants.The COS developed in the SCORE-IT study can be applied to future clinical trials of non-surgical interventions to treat hyperglycemia, and its use will allow comparisons to take place across trials, thereby reducing waste in research. The next steps will include seeking consensus on how these outcomes should be measured and to provide this guidance to researchers.