RCM is a microscopic imaging technique, which enables the users to rapidly examine tissue in vivo. Providing resolution at cellular-level morphology, RCM imaging combined with mosaicking has shown to be highly sensitive and specific for non-invasively guiding skin cancer diagnosis. However, current RCM mosaicking techniques with existing microscopes have been limited to two-dimensional sequences of individual still images, acquired in a highly controlled manner, and along a specific predefined raster path, covering a limited area. The recent advent of smaller handheld microscopes is enabling acquisition of videos, acquired in a relatively uncontrolled manner and along an ad-hoc arbitrarily free-form, non-rastered path. Mosaicking of video-images (video-mosaicking) is necessary to display large areas of tissue. Our video-mosaicking methods addresses this need. The method can handle unique challenges encountered during video capture such as motion blur artifacts due to rapid motion of the microscope over the imaged area, warping in frames due to changes in contact angle and varying resolution with depth. We present test examples of video-mosaics of melanoma and non-melanoma skin cancers, to demonstrate potential clinical utility.We describe a computer vision-based mosaicking method for  Approaches for mosaicking of a two-dimensional matrix of individual still images have been developed for bench-top work on tissue specimens in vitro, and ex vivo16. In bench-top settings, still images are typically acquired in a highly controlled manner along a specific predefined raster path. However, mosaicking for the newer, increasingly powerful and widespread implementation of video-imaging on living tissue in vivo is significantly more challenging. In this situation, video-images are acquired in a relatively uncontrolled manner and along an ad-hoc arbitrarily free-form, non-rastered path on living humans and animals. Approaches for mosaicking of a sequence of images from a video are called “video-mosaicking17”.Imaging over large areas of tissue is often necessary in clinical and research settings. However, high-resolution microscopy is limited to a small field of view in a single image acquisition. To extend the field of view, mosaicking techniques – acquiring a sequence of adjacent images and stitching or blending them together at the boundaries – have been developedin vivo are (i) deformations - i.e. warping or distortion in images - due to physical changes  at the microscope lens-to-tissue contact, (ii) abrupt motion artifacts - i.e. “jumps” or discontinuities between consecutive frames that are caused by sudden changes in depth or topography of tissue motion and/or manual and variable operator control of the microscope during imaging, and iii) optical sectioning and resolution may vary between consecutive images due to scattering and aberrations induced by changes in depth and morphology. In the presence of these difficulties, spatially overlapping images need to be “combined” to produce a video-mosaic.Three problems commonly occur when video-mosaicking in vivo. RCM imaging non-invasively acquires nuclear- and cellular-level detail with thin optical sectioning (1 – 3 μm) and high resolution (0.5 – 1 μm)19,Although these challenges, and our solutions presented in this paper are generic for any setting in which large field of view video-mosaics are needed from deep, high-resolution microscopic imaging in living tissue, we are, in this paper, specifically focused on the use of video-mosaicking for reflectance confocal microscopy (RCM) imaging in human skin 22. Recently, the Centers for Medicare and Medicaid Service in the USA granted current procedural terminology (CPT) codes, which means that RCM imaging is now a billable and reimbursable procedure23. The imaging is advancing toward routine implementation for guiding diagnosis as it has shown to increase diagnostic accuracy while maintaining a good specificity 28. Meanwhile, with the advent of a new generation of handheld confocal microscopes, acquisition of videos instead of only still images is becoming increasingly useful31 to consistently and rapidly display tumor morphology and surrounding normal tissue over much larger areas of skin (from 2 mm × 2 mm up to 10 mm × 10 mm).Several clinical studies have consistently reported RCM imaging to be capable of diagnosing melanocytic and non-melanocytic cancers with high sensitivity and specificityμm) in living tissue, resulting in varying resolution, exaggerated motion and distortion between images in any video. Moreover, the regions to be imaged differ greatly from subject to subject, skin site to skin site, and lesion to lesion, so operator flexibility in acquisition is necessary. Although “playback” of videos can be useful, the ability to spatially “map” the imaged region and display the entire field of view at once, by constructing a mosaic, allows much more “natural” analysis by trained clinicians or (semi-)automated algorithms29. Reading and interpreting a mosaic more closely mimics the standard procedure for examining pathology slides, which usually starts by looking at 10 mm × 10 mm areas of a tissue section with 2× magnification. Thus, there is a need in the clinic for video-mosaicking of RCM images, toward wider acceptance and adoption for routine use.In such settings, the special challenges are that the microscope is manually controlled and focused at varying depths  during registration. The properties of the resulting affine transformations are also used to detect abrupt motion artifacts between frames. Finally, we developed an approach, based on a graph-cuts based model33, to stitch overlapping frames together along arbitrary (non-rectilinear) boundaries, driven by the image data itself, without the loss of resolution that, otherwise, would occur from blurring or other blending techniques, and without imposing arbitrary pre-defined stitching boundaries. Herein, we describe our method in detail, and then present illustrative video-mosaicking results on human skin lesions in vivo. The advantages and limitations of our algorithm are discussed, as well as the possible future directions for both research and clinical applications.We address all these challenges by (i) directly modeling deformations, (ii) automatically detecting abrupt motion artifacts (discontinuities) in the image sequence, and (iii) robustly stitching the registered frames with an approach that combines overlapped images flexibly, in a data-driven fashion, without blurring or loss of resolution. Our algorithm is based in part on adaptation of methods from computational photographyVideo-mosaicking is of particular interest for rapid assessment of skin lesions that can be arbitrarily large and/or located at curved, irregular anatomical sites. We illustrate the capabilities of our approach by presenting results for three distinct clinical cases, each of which presents a particular set of imaging requirements. The flexibility and robustness of our approach is demonstrated for producing useful video-mosaics in all three settings.The first case is a lentigo maligna melanoma (LMM), where the need for accurate planning of surgical margins at non-flat corrugated and irregular anatomical sites is met by video-mosaicking over arbitrarily large predefined areas of tissue. In the second case, we show how video-mosaicing can help in adaptively detecting small and focal sites of suspicion within widely spread areas for diagnosing and monitoring of extramammary Paget disease. In the third case, we illustrate how video-mosaicking can help in rapid intraoperative detection of residual basal cell carcinoma in surgical wounds. All the videos acquired for this study were obtained after Institutional Review Board approval.In Fig. 34. Several days later, if all biopsy reports result in negative findings at the margin, the lesion is excised along the surgical margins. In case any of the biopsies turn out to be positive, the surgical margin is extended further along that direction. After the surgery, if residual tumor is found at the edges of the excised tissue, then further surgery is performed on a third visit.Following an initial diagnostic biopsy (at the location of the red asterisk at the center of panels (A), (B), and (C)), the clinical margin (yellow dotted circle) of the lesion is determined with dermoscopy and Wood’s lamp examination. In the standard surgical procedure, removal of the disease is the primary concern and tissue conservation the secondary one. Therefore, as shown in Fig. In specialized cancer care centers, in order to conserve more of the healthy tissue, alternative multi-stage excision guided by histology is sometimes performed Fig. . After tIn contrast, our video-mosaicking method offers a streamlined diagnostic and therapeutic procedure, as illustrated in Fig. Histopathology from this particular case confirmed the margin determined in the video-mosaic. Note that he white arrows in panel D show two locations at which our tailored video-mosaicking algorithm automatically compensates for ‘‘jumps’’ (discontinuities) in the video, allowing correct mosaicking of the three distinct arcs. The individual sub-mosaics are put together manually in an image editor. This is a simple procedure as the first and last frame of consecutive sub-mosaics are spatially located next to (or very close to each other), and their relative location to each other can typically be estimated using the RCM video.35. The excision is tissue-conserving, similar to that of the staged excision approach used in specialized clinics, but all accomplished in a single visit without the need for repeated visits and repeated biopsies.The resulting video-mosaicking method allows sufficiently precise determination of the appropriate surgical marginIn Fig. 36.In order to cover such large areas with curved surface topography, clinicians move the confocal microscope over the region of interest in a free-hand fashion. The approach is to acquire RCM videos in the suspicious area, starting from the center of the lesion and navigating towards the outer borders in a spiral path, aiming to cover the entire area in a rapid fashion without missing any regions. This approach allows identification of areas with higher tumor cell density, which can then be selectively biopsied to confirm diagnosisBeing able to image in a user-determined irregular trajectory is important in EMPD as tumor cells are sparsely distributed in a patchy fashion. To illustrate, in Fig. Fig. 30. This enhances the ease of correlating microscopic cellular detail with macroscopic location in the wound, which would be much more difficult with standard RCM imaging or by simply observing the original videos. Since the video-mosaic has the same resolution as in the original images, the surgeon can zoom in on suspicious areas and examine them in detail, with higher magnification, without loss of any information . Video-mosaicking thus solves this problem by registering, concatenating, and stitching together microscopic images to produce a large field of view.In this paper, we present a new way of imaging large areas of tissue at microscopic resolution, even when the anatomical sites of interest are hard to access and/or present uneven, corrugated and irregular topology. To image such large and challenging regions of interest, clinicians must often rely on video-microscopy. Typically, a video sequence is captured over the area of interest by moving a handheld microscope manually over the tissue. The need to maintain cellular resolution means that each frame of these videos has a small field of view, and thus the video presents a rapid succession of small correlated areas of tissue but viewing them as a video does not reveal their spatial relationship. As an example of possible consequences, in the case of RCM imaging of skin, a recent studyin vivo microscopic video-mosaicking is challenging for both image capture and mosaic composition. The quality of the final mosaic is sensitive to the motion of the microscope over the imaged area. The operator must to avoid rapid motion, while maintaining smooth translation across the skin and constant depth of the focal plane, with micron-level accuracy. Rapid motion can result in blurring and warping deformations in the final mosaic, while rapid change in depth can result in jumps in the imaging plane between frames. Although such artifacts can not be totally avoided during clinical imaging, one of the novelties of our approach, the scene cut detection step, can detect and handle both types of rapid motion and thus minimize these artifacts in the mosaic.However Another novelty that distinguishes other video-mosaicking methods in the literature is our graph-cuts based seamless stitching approach. This method has previously been used in computational photography; however, as far as we know, this is the first study that utilizes it in the microscopy setting. The challenge it solves is that traditional blending methods will lead to blur, and thus loss of the cellular resolution that is critical to diagnosis, while image-to-image variability and edge artifacts in the images make an a priori fixed cut-and-stitch approach suboptimal. Using graph-cuts based stitching, we allow the data itself to drive the merging of adjacent overlapping images, thus preserving the resolution of the original images without altering pixel values, while providing maximally smooth transitions, across stitching seams.39. In addition, all the registration steps, including the SIFT keypoint extraction, matching, and RANSAC-based keypoint elimination steps, are parallelizable and could also be implemented on GPUs for further acceleration. Therefore, we believe that substantial increase in computational speed will be achieved, enabling stitching that essentially keeps up with the image acquisition.However, one current challenge of video-mosaicking is processing delay. Our current implementation stitches a 500 frame video  into a video-mosaic in less than 30 minutes, using a MATLAB based implementation with a few C MEX libraries. The timing varies depending on the specifics of the scene geometry, which affects the number of reliable keypoint matches that are used in the registration step. This is adequate for some clinical settings and indeed is being actively validated by our clinicians, as in the examples shown in the Results section. However this delay is clearly an impediment to wider adoption; being able to present video-mosaics on-the-fly to the clinician would provide valuable live visual feedback, which would not only make it more clinically useful but would also help decrease motion artifacts and ensure appropriate coverage of the region of interest. Currently, the registration step is the bottleneck to increasing the speed of video-mosaic composition. We note that our MATLAB code is not optimized for such processing, and thus we are confident that significant increase in speed is feasible without significant loss of performance. Highly efficient codes have already been developed in C and C++ that provide real time feature extraction and tracking capabilities, such as in the OpenCV computer vision libraryAnother approach to lessen motion artifacts is the potential availability of smaller and faster microscopes with frame rates higher than the current 7 frames per second. Besides, with increase in processing speed, smaller microscopes would allow enhanced operator control of free-hand movement and, along with higher frame rates, further facilitate real-time stitching, thus improving both the quality of the mosaics as well as the ability to rapidly and reliably cover an entire arbitrarily shaped region of interest. Availability of real-time video-mosaicking has the potential to enable the clinicians to be more time/cost efficient and precise in their diagnostic assessments and surgical practice.Finally, as shown by the clinical examples in the results section, video-mosaicking has the potential to open new alleys in imaging based diagnosis and surgery, that in the past the clinicians were not able to do due to technical limitations. Moreover, the initial clinical success in imaging skin, is being translated to other setting and applications. One example is video-microscopy in the oral cavity. Head and neck surgery group in our institute is currently testing handheld microscope with a new telescopic probe for intra-oral imaging during head-neck surgery. Testing has thus far been performed on then patients, with video-mosaic being assessed for correlation to pathology for delineation of margins. Again real-time mosaicing has the potential to provide live feedback to the surgeon and make surgical planning and procedure more efficient.Need for video-mosaicking has already raised in other fields such as video-microscopy of oral cavity, where confocal microscopy has also begun to show its usability. Head and neck group in our institute is currently testing a new telescopic attachment on the handheld microscope, which was designed by our group, to acquire video-microscopy videos. Their investigate the potential use of video-microscopy in image guided surgery of the oral cancer patients. The videos that they acquire during in the OR are converted into video-mosaics for diagnostic correlation studies. Again, real-time video-mosaicking has the provide critical feedback to the surgeon throughout the surgery and make surgical planing and procedure more efficient.Prior to enrollment to the study and RCM imaging, all patients signed an informed consent to an institutional review board (IRB) approved protocol at Memorial Sloan Kettering Cancer Center. The study and the experiments were conducted in accordance with the MSKCC IRB regulations and the Declaration of Helsinki principles.32 and added methods to identify discontinuities  between frames, requiring separation into individual “subvideos” and to stitch with maximal preservation of image fidelity, both specifically tailored for application to video-microscopy.The algorithm is composed of a series of steps Fig.  designed40 and keypoint matching32. During keypoint extraction, uniquely identifiable pixel locations in each frame are determined. Then, through keypoint matching, keypoints are matched between consecutive frames and their inter-frame displacement is tracked. These displacement parameters are then used to estimate registration parameters to map the pixels of each successive frame to the reference frame of the mosaic. These registration parameters also provide quantitative estimates of inter-frame motion. Therefore, the algorithm uses them to identify rapid and/or tilted motion in the videos where inter-frame registration is either not feasible or unreliable; at those frames the algorithm automatically imposes scene cuts 40 keypoints and descriptors, among several available descriptors in the literature32. SIFT is invariant to commonly encountered variation in images such as uniform scaling and changes in orientation, and also robust against affine distortion and changes in intensity. The first step in the SIFT algorithm is keypoint identification. Keypoints are typically high contrast pixels within their neighbors. SIFT uses a difference-of-Gaussian (DoG) representation  during video capture, the scale, orientation, and intensity value of keypoints may change slightly between consecutive frames. To improve robustness, we use “Scale-Invariant Feature Transform”  in the local neighborhood of the keypoint; moreover we normalize orientation using the direction of the maximum gradientWe used the two step keypoint matching procedure described in ref. ℓ2 (Euclidean) distances between their SIFT descriptors, which we use as our keypoint similarity metric. A keypoint is then considered non-uniquely matched, and therefore the match is designated as unreliable, if the two most similar keypoints in the previous frame are nearly equally similar to the given keypoint, as measured by their similarity metric ratio being below a threshold . For example, a keypoint located in a region with similar or repeating textures is typically not unique. We prune our set of keypoint matches by discarding matches deemed unreliable by this procedure.Since the keypoint estimator itself is noisy with many outliers, we systematically eliminate unreliable matches that do not comply with this restricted type of motion. All keypoints in a subsequent frame are provisionally matched with the two “most similar” keypoints in the previous frame as determined by the 42. RANSAC iteratively estimates models from randomly chosen subsets of the set of observations and then chooses the model that generalizes best to the whole set. In each iteration, an affine transformation between consecutive frames is estimated using a randomly chosen subset of three matches and an error measure is calculated by generalizing this transformation to the rest of the matches. We then chose the affine transformation that minimizes the generalization error over the random subsets. In our experiments we observed that 3000 trials gave a good balance between computational complexity and generalizability. The list of matches is then pruned again by discarding keypoints whose displacement cannot be estimated with at least 50 pixel  accuracy by the chosen transformation matrix.Next, the pruned matches are used to model the transformation that best registers consecutive frames. In principle, only 3 keypoint matches are needed to determine the three registration parameters, and those parameters should be consistent across the choice of any three keypoints. However, in practice there will still be outlier correspondences, even after the previous refinement step, since even after refinement we typically have more than 1000 pairs of keypoint matches between frames. Thus, we find a consistent single transformation using the well-known “RANdom SAmple Consensus (RANSAC)” algorithmℓ2 distances between the estimated location of the keypoint matches in the latter frame and their real locations. Since we are matching consecutive frames that were collected through smooth motion of the microscope, the scale and warping introduced through the final registration transformation should be relatively small. However, when the set of matches remaining after RANSAC pruning happen to be spatially concentrated in a relatively small spatial portion of the frames, the transformation matrix is susceptible to over-fitting to that region, leading to distortions in the rest of the frame. In order to avoid such distortions, we added an additional penalty term to the cost that penalizes scale change and shear in the final registration, and thus avoids distortions due to over-fitting. The effect of this new penalty parameter is minimal as long as the matches are relatively homogeneously spread across the frames.We then run a final optimization over all matches that remain in this refined set and find the best transformation that describes their motion. The cost that we minimize is the sum of the To avoid distortion of the mosaic due to artifacts such as warping/distortion in frames or abrupt motion between them Fig. , we cut After registration, given the large overlap between consecutive frames, we need to “merge” frames to construct the mosaic. As noted above, typical blending methods such as averaging or multi-resolution pyramid blending will lead to blurring, while the common alternative of choosing the maximum (or minimum) pixel value across the overlapped frames is not appropriate here since there is no reason to prefer dark versus bright intensities. In addition, the highly textured nature of RCM images will cause a loss of coherent structure and important cellular detail if extremal methods are used. Thus, we needed a method that keeps the actual pixel values while maintaining image fidelity. Simply choosing acquisition frame boundaries will lead to artifacts as well, due to imaging aberrations at frame edges as well as illumination changes between frames. Thus we devised an approach that meets this criteria by objectively choosing a single continuous boundary through the overlap area, driven by the pixel values themselves.33, an approach which has been applied to many computer vision applications including photomontage41, texture synthesis43 and segmentation44, resulting in a data-driven optimized boundary. .For simplicity of exposition, we concentrate here on the case where only two frames overlap; the extension to multi-frame overlap is straightforward. Since we want to retain the original pixel intensities, we have two candidate pixels to choose between, one from the previous and one from the subsequent frame. Our stitching algorithm determines a contiguous boundary through the overlap region, with the mosaic on one side coming from the previous frame and on the other from the subsequent frame Fig. . From a Supplementary MaterialsLaTeX Supplementary FileLaTeX Supplementary FileLaTeX Supplementary FileLaTeX Supplementary File


TRPM6 ion channel and development of meningomyelocele (MMC). Therefore, we examined a total of 150 children with MMC, along with age- and gender-matched controls. DNA collected from whole blood was analyzed for the presence of two polymorphisms, rs2274924  and rs3750425 , in TRPM6. Serum Mg2+ and calcium levels were also examined.To evaluate whether there is an association between single nucleotide polymorphisms in magnesium-permeable 2+ levels were lower in the GG genotype of rs3750425 compared with the GA and AA genotypes (p = 0.003).A statistically significant difference in the distribution of rs2274924 genotypes (p = 0.049) was observed between the groups. Decreases in the AA genotype, and increases in the AG heterozygous genotype were also detected in the study group. The distribution of polymorphisms in the rs3750425 genotype and alleles was not statistically different between groups. Serum Mg2+ concentrations in these patients. Taken together, these results suggest that genetic variations in the Mg2+-permeable TRPM6 ion channel may play a role in the etiopathogenesis of MMC during embryonic development.A statistically significant difference in rs3750425 genotypes was observed between the patients with MMC and the controls, which corresponded to lower serum Mg Neural tube defects (NTDs) occur during embryogenic period when the neural tube lefts uncovered to close normally and neural tissue exposed to the intrauterine environment. It affects about 0.1–0.4 % in worldwide, 0.3 % in Turkey  is the second most abundant cation in cell and is essential for all stages of life, from the early embryo to adult. Regulation of cellular as well as whole body Mg2+ homeostasis is critical for human health. Although the concentration of Mg2+ in the extracellular environment can vary significantly, the total intracellular Mg2+ concentration is actively maintained within a relatively narrow range (14–20 mM) via tight , located on chromosome 9q21.13, plays a central role in the regulation of Mg2+ homeostasis via absorption and reabsorption of Mg2+ from the small intestines and kidneys, respectively . Oral and written consent were obtained from all families prior to inclusion in this study. A total of 150 patients diagnosed with MMC admitted to the outpatient clinics of neurosurgery, pediatric surgery, and pediatrics were enrolled in this study. An additional 150 patients admitted to the pediatric surgery outpatient clinic with no evidence of abnormal sacral spine examination findings were used as a control group. The demographic characteristics and clinical history of each patient were investigated in detail. Evidence of consanguineous marriage between parents or earlier generations was also noted. Due to the possibility of teratogenic effects, maternal use of any drug during the periconceptional period was recorded. Serum calcium and Mg® Genomic DNA Isolation Kit according to the manufacturer’s instructions. The DNA concentration was measured using a MaestroNano® , and diluted to 1–10 ng.DNA isolation was performed using the Promega WizardTRPM6 were analyzed using TaqMan® probes in combination with an ABI 7500 Fast Real-Time PCR System . The genomic sequence of rs2274924 was assessed using the probe AGTCCTTGAGTATTCTTCTTTTTCT[C/T]TGACAGTCTCCTGTCTTTGGTTAGC (Assay ID: C_16183233_20). Polymorphism at this nucleotide confers an AAG to GAG substitution, resulting in a leucine to glutamic acid substitution at residue 1579. For genotyping, the kit used FAM and VIC florescent dyes for the A and G alleles, respectively, as forward and reverse primers. The genomic sequence of rs3750425 was assessed using the probe TCCACTGATGCCCAGTCAGAGACAA[C/T]TGGGGTCTGCCCAGTCAGATGAACA (Assay ID: C_25943493_20). Polymorphism at this nucleotide confers an ATT to GAG substitution, resulting in a valine to isoleucine substitution at residue 1393. For genotyping, the kit used FAM and VIC florescent dyes for the A and G alleles, respectively, as forward and reverse primers. All polymerase chain reaction (PCR) reaction mixtures were prepared on ice.SNPs of rs2274924  and rs3750425  in From each sample, we combined 2.5 µL DNA with 7.5 µL PCR reaction mixture for a final volume of 10 µL. Plates were then covered with optical film, centrifuged, and analyzed using a 7500 Fast Real-Time PCR System for 40 cycles. The device software was used to identify homozygous mutant, heterozygous, and homozygous normal genotypes for each allele.U test was used to evaluate differences between groups. Data were expressed as the mean ± standard deviation. A p value <0.05 was considered statistically significant.Data were analyzed using the IBM Statistical Package for the Social Sciences v. 21 . Relationships between parameters were assessed using Pearson’s correlation analysis. Distributions of genotype and allele frequency were analyzed using a Chi square test. The Mann–Whitney The study group n = 150) consisted of 60 female (40 %) and 90 (60 %) male patients. The mean age of the study group was 3.1 ± 3.22 years (range 1 month–18 years). The mean age of the mothers was 28.15 ± 4.71 years (range 19–44 years). A detailed list of demographic, clinical, and laboratory findings are presented in Table 50 consisTRPM6 rs3750425 polymorphisms were evident between the groups  and 0.15–0.36 for the rs2274924 G allele (1584Glu) in four ethnic groups  babies in humans (Venu et al. 2+ supplementation given before the 25th week of gestation is also associated with a lower frequency of preterm births, a lower frequency of low birth weight, and fewer SGA infants compared with placebo (Makrides and Crowther 2+ deficient diet demonstrated an increased incidence of resorptions, gross malformation, growth retardation, abnormal fat metabolism, insulin resistance and diabetes (Hurley et al. 2+ levels and some embryonic and fetal anomalies, no correlation was found between maternal or neonatal serum Mg2+ levels and MMC (Golalipour and Mansourian 2+ or Ca+ levels evident between the groups, although lower serum Mg2+ levels were detected in patients with GG genotypes in rs3750425 compared with either the GA or AA variants. As Mg2+ homeostasis in the embryo is critically dependent on the maternal blood supply, a lack of genetic and biochemical analysis of maternal serum samples to assess whether Mg2+ homeostasis was altered during prenatal development was the main limitation of our study.Mg2+ transporters and ion channels involved in Mg2+ homeostasis during embryogenesis. The expression pattern of TRPM6 during embryogenesis demonstrated a significant increase at E10, suggesting TRPM6 is also temporally required for early development. Heterozygous TRPM6 knockout mice exhibited mild hypomagnesemia (Walder et al. 2+ diet slightly suppressed the embryonic lethality caused by knockout of TRPM6, suggesting that embryonic lethality may in part be due to Mg2+ deficiency. However, in the second study by Woudenberg-Vreken et al. (2+ diet failed to rescue the lethality caused by homozygous deletion of TRPM6. Rare mutations or polymorphic variants in TRPM6, including amino acid substitutions that prevent its heterooligomerization with TRPM7 or changing channel activity, may cause the imbalance in intracellular magnesium level and likely to contribute the abnormal bone development during embryogenesis.Recent studies have continued to add to the growing number of Mgn et al.  a high MTRPM6, as well as its activity (Cao et al. TRPM6 synthesis during pregnancy, this gene is likely to play an important role in the regulation of Mg2+ homeostasis in both the mother and fetus. Genetic variations in TRPM6 may therefore affect intracellular Mg2+ levels during embryonic development, with significant implications for fetal development.Recent studies have shown that estrogen directly increases the transcription of Several limitations in present study must be completed with further consideration. First, our study had relatively small sample size, however, consistency with laboratory findings makes our results acceptable. Second, population’s etnic diversity may be a concern but imposible to clarify our findings, because our populations are racially homogeneous, all participant lived in Elazig region. Third, althought serum magnesium may be a good indicator for heavy magnesium deficiency, we didn’t performed the measures of extracellular or intracellular magnesium status and the patch clamp experiment to display the effect on channel activity of studying polymorphic variant in TRPM6 gene.TRPM6 gene (Altıncık et al. TRPM6 in active intestinal magnesium absorption and renal reabsorption happen very important. Based on these observations, we strongly recommend checking serum Mg2+ values of babies born with MMC. Further studies performed on a large number of patients will be necessary to confirm these findings, as well as clarify the etiology of MMC, particularly in the context of Mg2+ homeostasis.Clinical data show that sufficient magnesium intake can partially compensate for the severe magnesium deficiency caused by genetic defect in the


In Phase 2, national and regional coaches (n = 7) in 2001 who attended training camps identified the most talented players. In Phase 3, current novice and advanced handball players (n = 12 in each group) selected the most talented from short videos of matches played during the talent camp. Analyses compared predictions among all groups with a best model-fit derived from the motor tests. Results revealed little difference between regional and national coaches in the prediction of future performance and little difference in forecasting performance between novices and players. The best model-fit regression by the motor-tests outperformed all predictions. While several limitations are discussed, this study is a useful starting point for future investigations considering athlete selection decisions in talent identification in sport.In most sports, the development of elite athletes is a long-term process of talent identification and support. Typically, talent selection systems administer a multi-faceted strategy including national coach observations and varying physical and psychological tests when deciding who is chosen for talent development. The aim of this exploratory study was to evaluate the prognostic validity of talent selections by varying groups 10 years after they had been conducted. This study used a unique, multi-phased approach. Phase 1 involved players ( One of the most important and difficult tasks in our society is forecasting a person’s potential. In music, dance, and theater, choices regarding who is cast are made based on a few minutes interaction with potential candidates. Similarly, top-rated reality TV-shows like American Idol or Australia’s Next Top Model attempt to forecast potential based on sometimes very brief performance exposures. In each of these situations decisions are made on the basis of available information and assumptions about how this information relates to future performance. In sport, the entire field of talent identification is grounded in the notion that performance during early stages of development gives some indicator of an athlete’s future potential .raison d’être is usually to provide coaches and trainers with information to improve judgments about talent selection and development. Despite its potential importance to sports systems worldwide, there has been very little research considering whether performance parameters measured earlier in development  are valid predictors of expertise later in development .One of the cornerstones of sport science is understanding the factors explaining exceptional performance . AlthougOne of the factors limiting research in this area is the difficulty of obtaining high quality youth samples and managing to track them over extended periods of time in order to determine the level of performance they ultimately attain. Moreover, because most talent development systems operate via a ‘de-selection’ process  the number of athletes who start in the system and the number exiting the system at its highest level are considerably different, which increases the administrative, logistical and financial demands on researchers aiming to obtain samples of sufficient size for inferential and comparative analyses. Although demanding, longitudinal studies of talent development have begun to emerge – most notably from Elferink-Gemser, Visscher and their colleagues as well as Available evidence of the accuracy of talent decisions by coaches and scouts is not compelling. In the sections below we describe the results of an analysis that evolved over an extended timespan. One of the authors was involved in the initial assessment of a group of youth handball players over 10 years ago and we are now able to determine how these athletes advanced across their athletic careers. The primary aim of this exploratory study was to evaluate the prognostic validity of talent selections by varying groups 10 years after they had been conducted. In a first step of this study, motor test results acquired during a youth talent camp were used to determine a possible model fit. Following In part two of this study, the original subjective predictions of national and regional coaches who scouted during the tournament of this youth talent camp were evaluated. Determining the long-term accuracy of their predictions is the main focus of our study. In part three of this study, these coach predictions were compared to those made by amateur handball players and novices  who viewed short videos from the tournament matches. This final element was included to compare the expert decisions of the national and regional coaches with advanced performers  and novices  to deterBecause much of the research design was determined by serendipity as much as it was by conscious planning, we are cautious in how we present these results; however, given the lack of data on the efficacy of coach/scout decision-making in talent settings, we believe the comparisons  provide us with the opportunity to explore the prognostic validity of talent selections over an extended period cf. .This study was conducted in three parts. These parts evolved over time and are presented in temporal order below.Sixty-eight female handball players participated in a talent camp, 56 born in the older year (age 13–14) and 12 born in the younger year (age 12–13). All participants were chosen by regional coaches to participate in the talent camp as a preparation for the national talent selection. Twelve of the 68 players in this camp were invited to the national talent selection of the German Handball Federation. Ten years later, 16 of the original 68 players were playing handball as professionals, four in the first German Handball league and twelve in the second league. Two were later National team members. These 16 were considered as ‘high achievers.’ The remaining 50 players either were not playing handball any longer or were playing in lower leagues. These were considered as ‘low achievers.’The 2001 talent selection camp used a range of motor tests to identify the best players of the 68 participants. These tests collected anthropometric data such as body height and body weight, performance data from handball specific games  as well as data from 12 basic fitness and coordinative tasks. These 12 tasks are described below.In this task, participants had to stand with one foot on a wooden block (2 cm wide and 10 cm high) and balance for as long as possible. Hands are kept on the hip and the “free foot” can be used to assist balance. Time was measured until either one foot touched the floor, the hands moved away from the hip or 1 min elapsed. The best of two trials was recorded.In this task, participants walked on 4.5 cm wide by 2 m long beam fixed to the floor. Participants’ score was the distance traveled in 45 s or when they fall off the beam. The best of two trials was counted.Participants began skipping for 15 s forward bipedally. At a signal, they had to change to skipping backward. A recorder noted the number of achieved jumps from the best of two trials.Participants stood with one foot on a balance board using their “free foot” for balance. In one hand, they held a handball with their palm facing upward. On top of the handball in their hand, participants had to bounce another handball for 15s. The “free hand” could be used for balancing movements. One trial was performed with the right foot/hand and the second performed with the left foot/hand. The total number of bounces from both trials was recorded as the participant’s score on this task.In this task, participants laid on a gym mat on their back holding a handball with both hands at their chest. The task required the participants to throw the ball upward, roll 360° to the side and catch the falling ball. After this the same procedure was performed moving to the other side. The number of caught balls in 20 s in the best of two trials was recorded.Participants jumped alternately with the right and left foot on a 10 cm small beam placed between their legs 30 cm above the floor. Simultaneously, they had to bounce a handball on the beam, with the hand from the same side as the foot that is on the beam. If the participant lost control of the ball, a second one was immediately provided. The score was the highest number of bounces in 20 s across two trials.A normal handball goal was covered so that only the upper corners of the goal (50 cm × 70 cm) were open. Standing at 9 m distance from the goal, participants had 30 s to make as much goals as they could with the number of trails and the number of achieved goals recorded as the score. The best of two trails was counted.Participants had one trial each to sprint 30 and 100 m.Participants had three trials to achieve the greatest distance in a standing long jump.The distance between reachable height in normal standing position and reachable height jumping with both legs was measured. The best of three trials was counted.This task required participants to throw the handball as far as possible. They were allowed to take a short run-up and the longest of three trials was counted.For this part of the study, participants included the two national coaches who observed the try-outs and the five regional coaches from the teams participating. All had the highest German handball coaching license and several years of experience coaching and selecting girls of this age group. Therefore, the information they could use were the observations during these try-outs as well as the data from the motor tests.All coaches were asked to nominate the best 14 players for an all-star game at the end of the try-outs. The national coaches selected together while the regional coaches discussed their selections with their trainer team . Therefore, we received five separate nominations from the regional coaches, while the two national coaches decided jointly.SD = 3.4. They had no handball playing experience and only occasional experience watching handball games. The advanced group consisted of six male and six female handball players with a mean age of 31.3 years, SD = 12.7. Their average handball experience was 20.3 years of playing, SD = 10.7, and 1.9 years of coaching, SD = 2.9.Participants in the final phase of the experiment included 12 novice and 12 advanced handball players. The novice group consisted of six male and six female participants with a mean age of 22.8 years, n = 5) played against each other in normal handball formation (six on six) and from these games video footage of offensive play from one half game was selected. This selection resulted in the creation of five different video pools, with each pool containing the matches of one team against the other four teams.Given the differences in knowledge between the coaching and other groups, we decided to limit the information given to the lesser skilled groups. This was done since it was not possible to replicate the level of information national and regional coaches would be able to access in making their decisions . To this end, the stimulus materials were videos from the handball talent section camp from 2001. During the camp, different teams  and selected who they felt were the most talented players. When participants perceived a talented player, they said “stop” at which time shirt number and playing position of the selected person as well as the time of participants’ verbalization were written down by the experiment supervisor. The five different video pools were presented in counterbalanced order. The duration of the offensive scenes per match  varied between 6–8 min, but showed exactly 5 min of playing time per match . The experiment took approximately 160 min to complete and there was a 10-min break after the completion of three video parts.Table 1). Because of the exploratory nature of this study and because we had no clear hypotheses regarding comparisons between the different models, we restrained from presenting inferential results and compare instead the correct classifications as well as both types of error.Of the possible 68 female athletes, complete data sets for 58 players were obtained. These data were used to compare varying prediction models and the best model-fit from the motor tests. In contrast to other prediction studies in sports, we were less interested in the correlations between those predictions , and morTo determine the best model-fit for the motor tests, a logistic regression analysis was calculated. The logistic regression analyses allowed for prediction of group membership on the basis of a set of variables. For the logistic regression analyses, these group memberships can be dichotomous and the variable mix can consist of continuous, discrete and dichotomous variables, which makes it more robust for predictions. The probability criterion was set from 0.15 to 0.20 to ensure that all important variables were included in the model.For the a-priori-probability, which was used as the baseline comparison for other models, all players were forecasted as not-talented. Therefore, 75.9% were correctly forecasted as low-achievers, while 24.1% were predicted incorrectly since they were high-achievers (type II error).post hoc model fit was calculated on the basis of the motor test data . As with previous studies, motor tests were considered a measure of general motor abilities and can therefore predict the motor potential for future performances  and should be kept in mind when interpreting these results. While this is an often-administered approach, testing the prognostic validity requires a two-step process. The first step requires generating the prediction formula of data from a first talent selection camp and then using this formula in a second step to determine its efficacy for predicting talent. However, in this study we only had data for the first step and therefore consider these results cautiously.Subsequently, a ormances . Importa2(1) = 10.12, p < 0.01, R2 = 0.26. In the second “step roll with ball catching” was identified as a significant predictor and increased the correct classification to 79.6%, χ2(1) = 6.34, p = 0.01, R2 = 0.39. A third significant predictor, number of goals in the “precision throw task,” decreased the correct classification to 75.9%, χ2(1) = 2.49, p = 0.11, R2 = 0.44. Finally, number of throws in the precision throw task was identified in Step 4 and improved the correct classification to 85.2%, χ2(1) = 4.73, p = 0.03, R2 = 0.53. The number of correct classifications was higher than the a-priori-probability and the type-I-error (9.2%) was higher than the type-II-error (5.6%).A forward stepwise logistic regression revealed “bench hopping” as a significant predictor in the first step with a correct classification of 74.1%, χAnalysis of the national coaches indicated they correctly classified 79.3% of participants in the training camp, which is 3.4% more than the a-priori-probability. However, they had a type-I-error of 8.6% and a type-II-error of 12.1%. The regional coaches had a total of 75.8% correct classifications with 12.1% for both type-I- and type-II-errors.Table 2). For the players this was reached with five nominations and for the novices with six. The group of players were able to predict 75.8% correctly. Their type-II-error was 13.8% and the type-I-error was 10.3%. For the group of novices, the percentage of correct classifications went down to 72.4% and their type-II-error was 15.5% with type-I-error as 12.1%.Next, the groups of handball players and novices were analyzed. In a first step, the optimal model fit was determined by testing varying numbers of nominations, similar to the method used with the regional coaches . The best forecast came from the national coaches, but they were only slightly better than the a-priori-probability. The advanced players performed with the same accuracy as the regional coaches and the forecast by novices was also quite similar. Comparing the type-II-error, the logistic regression on the motor tests outperformed all other models. Here, the regional and national coaches performed similarly but were only slightly better than the forecasts by the advanced players and laypersons.Comparison of the different methods revealed that the best classification was by the post hoc logistic regression using the motor tests outperformed all other forecast models, presumably because this approach used all available data to create the best model-fit. It needs to be restated here that it can only be considered cautiously, because it is no real prediction, but it provides us with an idea how much variance might be possible to explain. This caution increases even, when you see that the prognostic validity of the predictors seems questionable. From a practitioner’s viewpoint, these would likely not be the tests chosen as the most predictive of later performance because they do not measure handball-specific skills . Future studies need to confirm the prognostic validity of these tests ; moreover, and perhaps more importantly, they may have had a role in influencing future performance by training the selected players in the national team and also identifying them as highly talented players for the professional clubs, which may have resulted in greater quantity and quality of training. The same holds for the regional coaches who are usually ‘active agents’ in the training of some of the athletes. In the light of these confounds, the issue is perhaps not that the well-educated and trained coaches were superior to the performance of the less educated/knowledgeable groups but that the differences between these groups were so small. Future studies are necessary to replicate these results, ideally with greater test power, and determine the influence confounding variables may have had.In these future studies, some limitations should be overcome if possible. Most significantly, our design limited the information to which the advanced and novice groups had access. While this may have prevented lesser skilled decision-makers from being overwhelmed by the amount of data see , it resupost hoc model, there may be some inherent value to the use of objective tests that was not explored in the present study. For instance, objective tests have a pedagogical aspect to them. By controlling for specific skills with these tests, every players gets feedback on his or her performance, something that can be very useful in the learning process , these types of errors are not affordable. Prediction models that fail to identify an athlete with high potential result in lost talent, which can be particularly damaging if the numbers participating in the sport are low. While the effectiveness of talent development systems has been questioned , the oveThird, given the amount of error that is found in all predictions, it seems reasonable to create a system in which athletes are not only considered as potential high-performers at a single time point. Therefore, multiple testing or observations should be included in the talent system. For the German Handball federation, for example, this was implemented by having two national try-outs that are a year apart, as well as having the national coaches scout at elite level youth matches . A similThe use of longitudinal data for our analyses was a strength of this study. Most studies in sport consider how specific tests  predict selection at one point in time. Little research has considered how early performance predicts achievement at later points of time , with stIt is noteworthy that two athletes within the sample participating at the talent camp later became National team players. One of them, a goalie, was recognized by all of the coaches. The other, a field player, was not recognized by either of the national coaches and only two of the regional coaches. At the age under investigation here, she seemed to have nothing outstanding one could see. Anecdotally, she reported that she started training quite hard after the first selection camps and subsequently became a national team player. These examples illustrate how difficult the forecasting of future national players might be, because the process of talent development is often non-linear, highlighting again the problematic nature of type-II-errors .In summary, talent selection is a very difficult task; ideally it is a decision that should be re-considered often, in order to consider performer-specific changes over development. Based on the results of the current study, understanding the foundation on which these decisions are, and should be, made seems an open field for future research in all kinds of talent domains.The study was conducted over 10 years ago. At this time the institute of the first author did not have an ethic committee. But the study was conducted in accordance with the ethical declaration of Helsinki. We received written informed consent from all participants that made decisions.JS conceptualized and designed the study. He acquired, analyzed and interpreted the data. He also drafted substantial parts of the manuscript. RR and LF wrote and revised parts of the manuscript. JB conceptualized and designed the study. He also drafted substantial parts of the manuscript. All authors read and approved the final manuscript.The 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. The reviewer AV and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


This study explored mid-adolescents’ views and experiences of socio-ecological influences on their drinking practices in order to help inform the development of interventions to reduce alcohol-related risk. We conducted 31 in-depth interviews with young people aged 13–17 in North East England. Verbatim interview transcripts and field notes were coded systematically and analysed thematically, following the principles of constant comparison. We adopted Bourdieu’s idea of social game-playing and elements of his conceptual toolkit  during analysis. Analysis yielded three intersecting themes: (1) ‘drinking etiquette’: conveying taste and disgust; (2) ‘playing the drinking game’: demonstrating cultural competency; (3) ‘hidden habitus’—the role of alcohol marketing. Our work demonstrates that there is a nexus of influential factors which come together to help shape and reinforce mid-adolescents’ behaviour, norms and values in relation to alcohol consumption. Drinking practices are not just formed by friendships and family traditions, these are also subject to wider cultural shaping including by the alcohol industry which can encourage brand identification, and gear specific products to add ‘distinction’. However young people are not inactive players and they use aspects of capital and social games to help cement their identity and present themselves in particular ways which in turn are influenced by age, gender and social status. Guided by promising work in the tobacco field, interventions which focus on critical awareness of the framing of alcohol products by key stakeholders, such as policymakers, commercial industry and public health professionals, and by wider society may facilitate behaviour change among young people. Alcohol use is the leading cause of death and disability adjusted life years in both 15–19 year-olds and 20–24 year-olds globally ,4,5,6, iMarketing is embedded in a rich milieu of other social and cultural influences on behaviour and it is increasingly recognised that behavioural drivers work together to collectively influence young people’s drinking practices. Whilst there are a number of conceptual lenses that could be drawn upon to make sense of why people act the way they do, one way in which to interpret young people’s drinking practices is by adopting Bourdieu’s theory of practice ,20, a thBourdieu also presents the inter-related idea of ‘game-playing’ as a functional metaphorical tool ,20. To sAspects of Bourdieu’s theoretical framework have previously been applied to the study of young people’s alcohol use ,34. MostPrevious work in this area only partially links together the role of alcohol marketing and social game-playing with other influential factors within underage drinking practices. Data from Lunnay et al. was collected in Australia and relates to females only , whilst Our approach to data collection, coding and analysis was guided by COREQ  . Our fulA semi-structured approach was undertaken, with a topic guide used to inform but not direct the interviews. This topic guide was developed iteratively throughout data collection and covered a range of issues including experiences and reasons for drinking alcohol; social networks, peer groups and drinking contexts; tastes and preferences; awareness of and/or engagement with alcohol products and marketing techniques; access to alcohol and the importance of price; and risk and vulnerabilities. All interviews were conducted by the first author (Stephanie Scott), who at the time of the study was a PhD student (educated to Masters Level) and classed as a young female (aged 24 years). As part of her doctorate, the interviewer received detailed training in qualitative interviewing and analysis. All participants were approached face-to-face, no young people approached for interview refused to take part and there were no drop-outs during the interview process. Interviews took place at the recruiting site or a nearby public coffee shop. All young people consented to their interview being audio recorded. Interviews were transcribed verbatim, either by the researcher who conducted the interview (Stephanie Scott) or by a research administrator at the university, with observational field notes maintained in a research diary. Ethical approval for the study was provided by Newcastle University . No relationship between the interviewer and participants was established prior to study. All participants received a study information leaflet, which included details about the interviewer’s credentials and reasons for conducting the study. Participants provided written informed consent before taking part and anonymity was assured. Data collection and analysis took place concurrently, in order to continually re-evaluate emergent findings. Verbatim interview transcripts and field notes were analysed thematically , followi‘drinking to get drunk… that’s not really what I want to do… not your entire life focused on getting drunk or whatever, it’s sort of something that happens rather than something that you set out to do…’—Female, aged 16). These young people demonstrated ‘distinction in restraint’, taking pride in being able to recognise their limits and control the effects of intoxication . As such, they deliberately chose drinks which were not overly strong and did not result in immediate and obvious drunkenness, a practice referred to in the wider literature as the ‘intoxication tightrope’  money, we go out, sometimes...not all the time, sometimes have a little drink and we just dance about, talk, tell jokes, take pictures and then go in...we don’t talk to anyone, we don’t give grief to anyone, we don’t cause trouble, we just stay in one drinking place. Like if we don’t drink we just go down the park and we just play in the park’—Female, aged 13). Thus, the value derived from a night out was extended both retrospectively and prospectively, through sharing stories, high levels of planning, getting ready together and use of social networking sites to validate and re-live experiences, a finding which accords well with other literature  on the next morning and people’s put ‘oh I had a good night last night but hangover now’…Loads put photos on there’—Male, aged 15). For young people in our study, drinking alcohol required them to learn and understand distinct ‘rules of play’ and involved the acquisition, maintenance, development and mobilisation of resources or capital . Further, friendships and social networks offered a ‘micro economy’ to allow young people to effectively pool limited resource. Young people took turns to purchase alcohol or ‘subbed’ each other, pooled money together and ‘pre-loaded’ before going out for the night. Whilst interviewees gave a number of reasons for pooling resources—suggesting that this represented a cheaper and fairer system and thus stopped arguments, that alcohol lasted longer that way, and that it enhanced drinking as a social experience—predominantly there seemed to be a reciprocal understanding i.e., ‘if I pay this time, they’ll pay when they have money next time’, which also helped to cement relationships . In addition to peer networks, young people drew on their parents when developing new drinking practices . Young people regularly discussed drinking with their parents, suggesting that their parents encouraged them to drink sensibly, and to try alcohol at an early age, in order to eradicate the mystique around drinking alcohol, and as a ‘least worst’ strategy in comparison to smoking and drug taking, findings which accord well with other studies of parental influence on adolescent alcohol use  down cheaper’. When asked if price was critical to his choice, he commented: ‘Aye sometimes. ‘Cause if I haven’t got enough I’ve got to get something else’; whilst some interviewees also articulated the influence of visual promotions in pubs and clubs, often related to price . As well as criticising their own younger selves, interview participants felt that the drinking behaviour of ‘younger’ young people was more likely to be influenced by external factors such as marketing (Different forms of alcohol consumption were used to help construct identity, cement young people’s place within their peer group and mark out others as being ‘different’ to them. Whilst some young people reported that they drank to demonstrate maturity, others felt that they drank to lose control. These findings are supported by other studies conducted with young people, especially with regard to: established and embedded drinking norms or dispositions , the symWhat this and other studies indicate is that youth drinking can only be fully understood within a wider social and societal context. Whilst industry-driven marketing is an important aspect of the external environment for young people, it seems to be firmly embedded alongside other key influences such as cultural and geographical traditions, peer groups and family-based drinking practices. In our study, we saw an interplay between aspects of the internal and external world which helped to build early social identity, drive learning and shape what individuals and others around them do. Further, others have suggested that young people inhabit an ‘intoxigenic’ environment where social, physical, cultural and commercial influences shape youth drinking but are not always consciously recognised as doing so . Such inAn engagement model of marketing extends to nightlife activity, often referred to as the ‘P’ for ‘Place’ in the marketing lexicon. Historically, differences in the two core ‘traditions’ of youth research—youth transitions and youth cultural analyses—have resulted in a fragmented, dichotomous youth studies field. However, recently, Hollands has reflected on points of convergence, one of which is around studies which have begun looking at the structuring of nightlife activity and what this can tell us about where and how social inequalities and distinctions are displayed and acted out by young people . In partThis study has some limitations which warrant discussion. Our research was conducted with one sample of young people aged 13–17 within one geographical area of the UK, which is a part of England with a strong industrial past and a traditionally heavy drinking culture, reflected in some traditional gender views which may not be found in other cosmopolitan areas. Whilst interviews were conducted in 2010, our analysis and subsequent findings resonate with other work in the field of adolescent drinking. All participants in this study were current drinkers, since we wished to understand the influence of contextual factors on drinking behaviour rather than the decision to drink or not in the first place. Nevertheless, the absence of views from individuals who chose not to drink alcohol is a limitation of our work on shaping influences, i.e., what makes the difference between youth living ostensibly in the same external environment who do and do not drink? Bourdieu suggests that, in order to fit in, one must be able to keep up with changes in the field. Further, that falling behind may induce ‘hysteresis’, defined as a mismatch between habitus and field . Such miThis study provides useful insights for future alcohol prevention policy and practice. All interviewees were below the UK legal age of being able to buy alcohol. Nevertheless, they often described themselves as ‘well practised’ in the use of alcohol and they often described ‘learning’ how to be so. In some instances parents were clearly initiating or ‘instructing’ them, perhaps in an attempt to mitigate the various physical and psycho-social risks to these young people whilst growing up, particularly in public drinking environments. The finding that risky or heavy drinking or loss of control due to alcohol can have a ‘cultural logic’ to specific groups of young people is important. This has been recognised by other authors, particularly in relation to young adults, gender and alcohol use . Consequ


As countries scale up adult voluntary medical male circumcision (VMMC) for HIV prevention, they are looking ahead to long term sustainable strategies, including introduction of early infant male circumcision (EIMC). Although a number of devices for EIMC are prequalified by the World Health Organization, evaluation of additional devices can provide policy-makers and clinicians the information required to make informed decisions. We undertook a field evaluation of the safety and acceptability of the AccuCirc device in Kisumu County, Kenya.Procedures were performed by four trained clinicians in two public facilities. Participants were recruited from surrounding public health facilities through informational talks at antenatal clinics, maternity wards, and maternal neonatal child health clinics. Healthy infants ages 0–60 days, with no penile abnormality, without a family history of bleeding disorder, with current weight-for-age within –2 Z-scores of WHO growth standards, and whose mother was at least 16 years of age were eligible for EIMC. The procedure was performed after administration of a penile dorsal nerve block using 2% lidocaine and administration of Vitamin K. The mother was given post-operative instructions on wound care and asked to remain in the clinic with the baby for an observational period of one hour, during which a face-to-face questionnaire was administered.Of 1259 babies screened, 704 were enrolled and circumcised. Median age of the infants was 16 days (IQR: 7–32.5) and of the mothers was 26 years (IQR: 22–30). Median time for the procedure was 19 minutes (IQR: 15–23). There were no serious adverse events (AE), and 20 (2.8%) moderate AEs, all of which were due to bleeding that required application of one to three sutures. There were 22 (3.8%) procedures in which the device did not fully incise the entire circumference of the foreskin and had to be completed using sterile scissors. 89.9% of mothers had knowledge of EIMC, but few (8.1%) had any knowledge of devices used for EIMC. Protection against HIV/AIDS was the most cited reason to circumcise a baby (65.3%), while the baby being ill (38.1%) and pain (34.4%) were the most cited barriers to uptake. 99% of mothers were “very satisfied” or “completely satisfied” with the procedure.This evaluation of the AccuCirc device is the largest to date and indicates that the device is safe and acceptable, achieving high levels of parental satisfaction. The AccuCirc device should be considered for WHO prequalification to increase options for safe and sustainable provision of EIMC. Medical male circumcision (MMC) is a proven HIV prevention intervention, reducing the risk of heterosexual acquisition of HIV in men by 57–67% in three randomized controlled trials and in long-term follow-up studies –6. The WIn 2010, the WHO published the “Manual for Early Infant Male Circumcision under Local Anaesthesia ,” which A crucial consideration in assessing the effectiveness and acceptability of any EIMC device is safety. Although several studies have shown low rates of serious adverse events (AE) associated with EIMC ,16–18, AEvaluations of specialized devices for EIMC are important to provide programs with the information required to make informed decisions about which devices could meet specific local needs. The WHO male circumcision device prequalification process is designed to assist program managers and sponsors supporting expansion of circumcision programs in making decisions about which devices are safest and most appropriate for integration with comprehensive HIV prevention programs . The AccThe study was carried out at the University of Nairobi, Illinois, and Manitoba (UNIM) Research and Training Centre (URTC) in Kisumu and the Ahero Sub-County Hospital (ACH) in Ahero by the Nyanza Reproductive Health Society (NRHS) in collaboration with the University of Illinois at Chicago (UIC) and the Brigham and Women’s Hospital (BWH).Prior to the onset of the study, training was provided to study clinicians  all of whom had previous experience with EIMC using the Mogen clamp. The training curriculum was developed based on the WHO “Manual for Early Infant Male Circumcision under Local Anaesthesia ,” as welMembers of the study team identified health facilities in the Kisumu and Ahero catchment areas and gave informational presentations to all staff at each facility. The presentations included information about the risks and benefits of both adult and early infant male circumcision, different methods and devices used in EIMC, and the design and objectives of the AccuCirc study. Staff at each facility were requested to disseminate information about EIMC to pregnant mothers, as well as new mothers and fathers of a male. Study staff as well as some staff at the facilities actively identified and recruited eligible mothers at the antenatal clinics (ANC) and visited maternity wards and post-natal clinics to speak directly with mothers about the risks and benefits of EIMC and the possibility of participating in the study. Mothers who requested time to consult other relatives or their spouse were given time to do so and, when requested, the study staff also met with the relatives/spouse and explained EIMC. Those who wished to have their infants circumcised were referred to URTC or ACH depending on location. Once at the study clinic, they were provided with additional information on EIMC. Parents were given the option of participating in the study, in which the AccuCirc was used, or having EIMC performed as part of the national VMMC program using the Mogen clamp. Those opting to join the study provided written informed consent and their infants were screened for eligibility.Subjects were recruited between February 2, 2015 and July 21, 2016. Eligibility criteria were: 1) male infant born within the study catchment area; 2) ages 24 hours to 60 days; 3) ability to attend scheduled study follow-ups; 4) provision of written informed consent by at least one parent or guardian; 5) no evidence of neonatal infection/sepsis or other current illness; 6) no penile abnormality that might require reconstructive surgery in the future; 7) no family history of bleeding disorder; 8) estimated infant gestational age ≥37 weeks; and 9) current weight-for-age for boys of at least -2 Z-scores according to WHO growth standards . This laFollowing eligibility determination, the infants underwent a complete head-to-toe physical examination to rule out conditions that could preclude them from EIMC. To minimize risk of bleeding, all infants received vitamin K (1 mg) approximately one hour before the procedure. The area was prepped and draped in accordance with sterile procedures. Local anaesthesia was achieved by the use of 2% lidocaine dorsal penile nerve block with a maximum dose of 0.15 mg/kg. Anaesthesia was assessed by pinching the foreskin using an artery forceps 15 to 30 minutes following administration. Once no signs of pain were detected, AccuCirc circumcision was performed as previously described , 30. FolDuring the hour that the parents and baby remained in the clinic for observation, a research assistant administered a questionnaire in either English, Kiswahili or DhoLuo to the mother to assess demographics, birth history, prenatal care and delivery, knowledge about EIMC, source(s) and timing of information about EIMC, beliefs and attitudes about MMC and EIMC, reasons for accepting EIMC, father’s role in decision-making, knowledge of EIMC devices and the role of devices in decision-making about EIMC . Only moFollowing the interview, the baby was re-assessed and the mother was again provided with instructions on wound dressing, detection and management of bleeding, infection or any abnormalities, and was given emergency contact information, which included a 24-hour hotline. The mother was encouraged to call the emergency numbers or to come to the clinic if she had any concerns or detected any unanticipated events between scheduled visits.The first 50 participants were followed up at 24 hours, three days, one week, and four weeks following the procedure. The subsequent 654 participants were scheduled for follow up at three days post-procedure only. All mothers were asked to bring their infants to the clinic at any time between scheduled visits or any time after the last scheduled visit if they had any concerns. During follow-up visits the clinician conducted a physical examination of the infant, including inspection of the circumcision site, and asked the parent if she had any concerns about the procedure or progress of the wound. In this paper we do not report the first 50 participants separately. We report the results of only the Day 3 post-procedure visit or of any unscheduled visits for the full sample of 704 infants and 700 mothers.Safety was measured by the number of moderate and severe adverse events (AEs). The primary EIMC-related AEs assessed were categorized as bleeding, infection, hematoma, inadequate or excessive skin removal, or penile injury . We assessed EIMC acceptability by the proportion of mothers who reported being satisfied with the procedure, and who expressed willingness to adopt EIMC for a future son. Other measures of interest included: median surgical time and parental satisfaction with AccuCirc EIMC services.All data were entered by the clinician or the research assistant on paper forms and entered into a REDCap database by the data manager. All statistical analyses were conducted using Stata SE version 13 . We summarized characteristics of parents and infants, including socio-demographic characteristics, father’s reported circumcision status, and knowledge of circumcision and HIV. To evaluate the safety of the procedure, we calculated the proportion of procedures associated with AEs with 95% confidence intervals (CI). We estimated that the 95% CI around an AE rate of 0.02 would be 0.011 to 0.033 given a sample size of 700 circumcisions. 2% was chosen based on the historical proportion of males who experience an AE with surgical MC , 25 of 919 (2.7%) have resulted in incomplete cuts. In the great majority of cases, these cause no harm, since they are easily managed by completing the cut using sterile scissors or a scalpel. However, in a few cases (n = 6), completing the cut can cause bleeding that may not be managed by applying pressure alone and may require one or more sutures. Midway through our study, we discovered that applying steady continuous pressure when activating the lever arm of the device reduced the risk of an incomplete cut, and this lesson should be incorporated into any training programs for use of the AccuCirc. In addition, the manufacturer should explore revising the device to minimize instances of incomplete cuts. An alternative would be to restrict EIMCs to infants younger than 30 days, since we found a trend toward a higher proportion of babies who were older than 30 days of age experiencing an incomplete cut (5.1%) compared to those 30 days or younger (2.4%). However, the 60-day window for achieving large numbers of EIMCs is already short; reducing it by half would adversely impact the efficacy of EIMC programs substantially. For example, 30% of our participants were older than 30 days.The potential for incomplete cuts that may result in the need for sutures would restrict the use of the device to facilities with standard minor surgical instruments and personnel capable of applying sutures. However, this is the case for EIMC programs using any device .Limitations of this study include our inability to assess the proportion of parents exposed to demand creation messages who ultimately sought EIMC services. This limitation is inherent to our recruitment activities that included general talks at ANC, at maternal neonatal child health clinics and at maternity wards, as well as training of personnel at numerous health facilities to educate women about EIMC and to assist with recruitment and referral to our study. These conditions are likely to be similar when EIMC programs are scaled up. Because some data were collected via face-to-face interviews, information bias, such as social desirability, is possible. This may be especially true regarding mothers’ levels of satisfaction, although the interviewers were not themselves clinicians and were not involved in performing procedures. Our study was not designed as a head-to-head comparison of EIMC using the Mogen clamp versus the AccuCirc device. One such trial has been published  and therWith a sample size of 704 infants ages 0–60 days, this is the largest study to date of EIMC using the AccuCirc device. The device appears to be safe and acceptable, achieving very high levels of satisfaction among parents of babies circumcised with the device. The AccuCirc should be considered for WHO pre-qualification to increase the options for safe and sustainable provision of EIMC.S1 Questionnaire(DOCX)Click here for additional data file.


The abbreviated Alcohol Use Disorder Identification Test for Consumption (AUDIT-C) is rapidly becoming the alcohol screening tool of choice for busy practitioners in clinical settings and by researchers keen to limit assessment burden and reactivity. Cut-off scores for detecting drinking above recommended limits vary by population, setting, country and potentially format. This validation study aimed to determine AUDIT-C thresholds that indicated risky drinking among a population of people seeking help over the Internet.The data in this study were collected in the pilot phase of the Down Your Drink trial, which recruited people seeking help over the Internet and randomised them to a web-based intervention or an information-only website. Sensitivity, specificity, and positive and negative likelihood ratios were calculated for AUDIT-C scores, relative to weekly consumption that indicated drinking above limits and higher risk drinking. Receiver-operating characteristic (ROC) curves were created to assess the performance of different cut-off scores on the AUDIT-C for men and women. Past week alcohol consumption was used as the reference-standard and was collected via the TOT-AL, a validated online measure of past week drinking.AUDIT-C scores were obtained from 3720 adults  searching the internet for help with drinking, mostly from the UK. The area under the ROC curve for risky drinking was 0.84   and 0.80  . AUDIT-C cut-off scores for detecting risky drinking that maximise the sum of sensitivity and specificity were ≥8 for women and ≥8 for men; whereas those identifying the highest proportion of correctly classified individuals were ≥4 for women and ≥5 for men. AUDIT-C cut-off scores for detecting higher risk drinking were also calculated.AUDIT-C cut-off scores for identifying alcohol consumption above weekly limits in this largely UK based study population were substantially higher than those reported in other validation studies. Researchers and practitioners should select AUDIT-C cut-off scores according to the purpose of identifying risky drinkers and hence the relative importance of sensitivity and/or specificity. Early identification of people drinking at risky levels followed by brief intervention is the key individual-level intervention approach for reducing alcohol intake to safer levels , 2, withSince the development of the AUDIT there have been a number of abbreviated versions that allow screening to take place in busy environments where time is limited . The AUDIn 2007, a review of abbreviated versions of the AUDIT recommended an AUDIT-C cut-off score of ≥3 (women) and ≥4 (men) for detecting hazardous or harmful drinking . This reThere may be many reasons for the heterogeneity in findings in previous studies including differences in populations, settings and cultures, where both prevalence and recommended drinking limits vary. Validation studies use different reference standards and forms of measurement for determining hazardous or harmful drinking, e.g. time-line follow-back, 10-item AUDIT, International Classification of Diseases (ICD-10 criteria) and the Diagnostic and Statistical Manual of Mental Disorders  , 14. TheScreening and brief intervention delivered over the Internet has grown in popularity over the past decade and is now a substantial field of research . ElectroThe purpose of this study was to determine a suitable cut-off score for the AUDIT-C for identifying risky drinkers in a general population sample of people seeking online help with their drinking. Objectives were to determine the sensitivity, specificity, likelihood ratios and area under the Receiver-operating characteristic (ROC) curves of different cut-off scores for the AUDIT-C, with a goal of identifying people drinking above the recommended UK weekly consumption limits. To the best of our knowledge, this is the first study that seeks to validate the AUDIT-C in a population of people seeking help with their drinking over the Internet.The data for this study were collected during the eight month pilot phase (February to October 2007) of an online randomised controlled trial investigating the effectiveness of an internet-based intervention , Monthly or less (score 1), 2–4 times per month (score 2), 2–3 times per week (score 3), 4+ times per week (score 4);How many units of alcohol do you drink on a typical day when you are drinking? Answer: 1–2 (score 0), 3–4 (score 1), 5–6 (score 2), 7–9 (score 3), 10+ (score 4);How often have you had 6 or more units if female, or 8 or more if male, on a single occasion in the last year? Answer: Never (score 0), Less than monthly (score 1), Monthly (score 2), Weekly (score 3), Daily or almost daily (score 4).The AUDIT-C constitutes the following three questions:Our reference standard is the TOT-AL measure of past week drinking, which is used here to identify two conditions: (1) risky drinking, and (2) higher risk drinking.14 units of alcohol per week for women;21 units of alcohol per week for men, where (1 UK unit is 8 grams of ethanol). At the time of writing there was a consultation on reducing this to 14 units .Weekly drinking limits recommended by the Royal Colleges of General Practitioners and Psychiatrists  and prev35 units of alcohol per week for women;50 units of alcohol per week for men.We were also interested in evaluating performance against the accepted UK threshold for a level of heavy drinking at which problems are likely to be occurring. These “higher risk” thresholds were:The TOT-AL is a reliable and valid online measure that presents drop-down menus on the type, brand, size and quantity of alcohol consumed on each of the past seven days and calculates total units of alcohol consumed (measured in UK units) . There iThe sensitivity and specificity of cut-off scores between two and ten on the AUDIT-C were examined separately for males and females using the recommended weekly drinking limits (measured by the TOT-AL) as the reference-standard. Positive and negative likelihood ratios were calculated to estimate how different cut-off scores change the odds of being a risky drinker and a higher risk drinker, where positive likelihood ratios = sensitivity/(1 − specificity), and negative likelihood ratios = (1 − sensitivity)/specificity. Receiver-operating characteristic (ROC) curves were created to assess the performance of different cut-off scores on the AUDIT-C for men and women. ROC curves plot the sensitivities of different cut-off scores against 1-specificities . The area under the ROC curve quantifies the ability of the AUDIT-C to discriminate between those people drinking above and within weekly drinking limits. A perfect test is indicated by an area under the ROC curve of 1.0, whereas a worthless test is indicated by 0.5. Analyses were conducted in Stata V13 .Validation studies of the AUDIT-C in general population settings tend to report a cut-off score that maximises the sum of sensitivity and specificity. We refer to this as an ‘optimal’ cut-off score . This cuWhen the same data are used both to select a cut-off score and to evaluate performance  at the cut-off, performance tends to be over-estimated—a phenomenon known as “optimism” or “overfitting” . To avoiA total of 3720 participants completed baseline measures in the pilot trial. All participants included in this study had data for both TOT-AL and AUDIT-C measures, there was no drop out or withdrawals. Participants were mostly female (55%), with an average age of 37 years SD 11), mostly ‘White British’ (84%) and living in the UK (89%). Participants living outside the UK were most commonly from other Anglophone countries  with small numbers from other countries (highest France n = 28). Half of all participants were educated to university degree level or above (51%). Average (geometric mean − given the skewed distribution of the data) alcohol intake at baseline was 38 UK units in the past week (SD 4) and the mean AUDIT-C score was 8 (SD 2), for distribution of AUDIT-C scores in men and women see Fig. 1, mostlyThe area under the ROC curve was 0.84  for females and 0.80  for males Fig. .Fig. 2ReThe ‘optimal’ AUDIT-C cut-off scores for identifying people drinking above weekly limits were found to be ≥8  and ≥8 . Performance at optimal cut-offs are reported in Table The most accurate AUDIT-C cut-off scores for identifying people drinking above weekly limits were ≥4  and ≥5 , with corresponding sensitivity of 99%  and specificity of 25%  for females, and sensitivity of 99%  and specificity of 25%  for males. These cut-off scores led to a high proportion of participants correctly identified as drinking above recommended limits for both females (92%) and males (91%). The positive likelihood ratios corresponding to a cut-off score of ≥4 was 1.32  for women and ≥5 was 1.33  for men; the negative likelihood ratios were 0.05  for women and 0.03  for men Tables .The area under the ROC curve was 0.79  for females and 0.78  for males Fig. .Fig. 3ReThe ‘optimal’ AUDIT-C cut-off scores for identifying higher risk drinkers, i.e. more than 35 units/week for women and more than 50 units/week for men) was found to be ≥8 for women and ≥9 for men, with corresponding sensitivity of 70% 95% CI 67, 73) and specificity of 71%  for women, and sensitivity of 77%  and specificity of 65%  for men. The positive likelihood ratios corresponding to a cut-off score of ≥8 was 2.39  for women and ≥9 was 2.19  for men; the negative likelihood ratios were 0.42  for women and 0.36  for men  population, with almost the entire sample drinking above recommended limits . DYD participants also differ from the general population as a whole by reporting fewer problems with mobility, self-care, usual activities and pain because they were younger. However, they were more likely to report experiencing anxiety and/or depression  . The natWhen selecting a suitable threshold for the identification of risky drinkers there are various factors that need to be considered, such as prevalence, the regularity of screening, and any physical, psychological and economic costs related to the identification of false positives or false negatives . For exaOne of the key strengths of this study is the use of past week drinking data as the reference-standard with which the AUDIT-C scores were compared. In using a very detailed online measure of past week drinking (the TOT-AL), we were able to determine the AUDIT-C cut-off score at which participants were drinking above the recommended UK weekly limits of 14 units per week for women, and 21 units per week for men. Many of the studies investigating different cut-off scores for abbreviated versions of the AUDIT have used the full AUDIT as their reference-standard which “violates the independence of data assumption underlying the use of these statistical tests” p. 22 . FurtherThe TOT-AL measures past week consumption, known as actual or exact recall, which leads to easier and more accurate recall due to the recency of consumption, and avoids difficulties in attempts to estimate average consumption , 47. ThiThe use of weekly limits as a reference-standard is also arguably a weakness. Despite the focus of the AUDIT-C on consumption, it was principally developed as a screening tool for problematic drinking . As suchThis study did not investigate whether different AUDIT-C scores were necessary for identifying risky drinking in different age groups as participants were aged 18 years or above, and due to the online nature of the DYD trial, older people may have been under-represented. Previous research has found that lower cut-off scores may be necessary in younger age groups , and theThe ‘optimal’ AUDIT-C scores for identifying people drinking above recommended weekly drinking limits were substantially higher in this study than in any previous study undertaken in any form of general population sample. This is one of the few studies that has validated the AUDIT-C in an adult UK population, and the web browsing nature of this sample is emphasised in interpreting these data. Researchers should consider carefully the basis for selecting AUDIT-C cut-off scores according to the purposes of identifying risky drinkers, the relative importance of sensitivity and/or specificity, and the setting in which screening is undertaken.