Combining the algorithm with an all terrain vehicle (ATV)-mounted image acquisition system, we demonstrate a feasible method for robust coverage and species distribution mapping of 225 ha of mixed crops at a median capacity of 17 ha per hour at 173 images per hectare.Crop mixtures are often beneficial in crop rotations to enhance resource utilization and yield stability. While targeted management, dependent on the local species composition, has the potential to increase the crop value, it comes at a higher expense in terms of field surveys. As fine-grained species distribution mapping of within-field variation is typically unfeasible, the potential of targeted management remains an open research area. In this work, we propose a new method for determining the biomass species composition from high resolution color images using a DeepLabv3+ based convolutional neural network. Data collection has been performed at four separate experimental plot trial sites over three growing seasons. The method is thoroughly evaluated by predicting the biomass composition of different grass clover mixtures using only an image of the canopy. With a relative biomass clover content prediction of R Crop mixtures of grass and clovers have many advantages such as reduced use of industrial fertilizer, lower production costs, increased protein self-sufficiency, increased yield stability, and improved feed quality . To optiThe problem of automatically assessing the mixed crop composition using computer vision and top-down images has been investigated during the previous 15 years. Before the introduction of deep learning , all appSkovsen et al.  introducSkovsen et al.  proposedBateman et al.  proposedDue to the inherent entanglement of the deep learning model architecture, training data, hyper parameters, and image quality, a direct comparison between proposed methods and their results is increasingly inaccurate. As a consequence, an increase or decrease in either of the components greatly influence the final result.Mapping the spatial species distribution of mixed crops is a difficult task, but necessary, to fully benefit from targeted fertilization. Bakken et al.  systematIn this work, we have three contributions: (1) Extension of biomass-labeled image data for grass clover segmentation evaluation. Compatible with the publicly available GrassClover dataset , we exteThis study uses images from three sources: (1) images from a high-resolution mirrorless system or digital single-lens reflex (DSLR) camera from plot trials, (2) images from a high-velocity camera mounted on an ATV from large fields, and (3) synthetic images. Each of these three image types has a purpose. The images from the high-resolution system camera were taken in plot trials, and are used to find the correlation between dry weight and image information. The images from the high-velocity camera on the ATV are used to find clover grass variations at field level, and the synthetic images are used to create large amounts of realistic training data. Each of these image types will be covered in the following sections.The sample pairs of biomass samples and corresponding canopy images originate from four grass clover plot trials located 60–250 km apart. Each plot trial was designed to answer research questions unrelated to this paper but introduced a large variation with regards to soil conditions, fertilization strategies, seed compositions, and herbicide usage. Due to the variation in growth conditions and composition of crops, we consider that the images are representative of most clover-grass fields. A comparison of the four sites is shown in Plant samples were cut with electrical cutters in plots of 0.5 × 0.5 m, in the same plots as the images were taken. The harvested material was hand sorted into four categories: red clover, white clover, grass, and weeds. Each sample category was weighed, dried at 60 °C for 48 h and weighed again to determine dry matter yield and botanical dry matter species composition. The dry matter yield of sample categories with less than 5 g of fresh weight was estimated assuming a 25% dry matter content. The sample weight distribution across plot trial sites and seasonal cuts is shown in Image acquisitions in plot trial C and D were carried out with a Sony  A7 Mirrorless interchangeable-lens camera (MILC) with a Sony Sonnar T* FE 35 mm F2.8 ZA lens. With the camera mounted on a horizontal rod connecting two tripods, images were captured facing down. Without moving the camera, two images were collected before each biomass sample as shown in All images were manually developed from raw formats, with guidance from a cardboard color calibration target. Image crops, corresponding to each biomass sample of 0.5 × 0.5 m, were then generated as demonstrated in The primary desire of predicting the biomass composition from images is the possibility of estimating the species compositions at scales where manual plant fractioning is unfeasible. To demonstrate the robustness of the proposed method in this context, large scale image acquisitions were carried out at the three dairy farms, located in Jutland, Denmark, with 25–68 km spacing in between. All fields were seeded with perennial ryegrass and white clover following the growers’ conventional procedure. One farmer did not use pesticides for weed control in his fields. Image acquisition was carried out in May, a few days before the high-yielding first cut of the season, and, in October, with sparser vegetation. A summary of the large scale acquisitions is shown in −1. The camera is mounted in the center of the ring flash, using a 3D printed adapter. The adapter is made with a series of spring elements to center the camera inside the ring flash. The ring flash itself is mounted on three oil-spring dampeners to protect camera and flash from mechanical shocks. The camera assembly is mounted on the front of the ATV  using aluminum extrusions tied to the front and rear luggage racks. The camera is mounted approximately 0.9 m above the ground. The camera system is controlled by a computer , TX2) running the Robot Operating System [The image acquisition system, shown in g System . Images −1. The image sampling was triggered every 5 m in the driving direction with a typical track-to-track distance of 6–12 m. In October, each field was additionally sampled along the field border prior to the interior sampling.Each field was systematically traversed with the ATV-mounted camera at approximately 18 km hThe images were digitally developed from Bayer pattern images using the approach of Malvar et al. . To miniHierarchically labeled synthetic images provide a multitude of benefits compared to a flat real dataset of similar manual labor. Namely: (1) 100% pixel accurate labels, (2) compute-limited sample size as opposed to labor-limited, (3) controlled data distributions and biases, (4) emphasis on under-sampled parts  using intra-class labels.For training the convolutional neural networks for semantic segmentation, the publicly available GrassClover dataset was used . This daThe existing data-set of 15 labeled image crops by Skovsen et al.  is used Consistent with previous research in the field, the biomass composition of the mixed crop is estimated based on the visible canopy in a top-down camera view ,8,10,19.From demonstrated success in other domains, and the inherent use of multi-scale feature processing, the DeepLabv3+  with a bThe convolutional neural network (CNN) architecture was trained in Tensorflow version 1.15.0 based on the official deeplab model repository . ChangesFollowing the principle of Skovsen et al. , two sepAlthough this structure doubles the total inference time, it permits a direct comparison with the existing literature in the field when ignoring the second stage.The models were initialized with weights from a pre-trained Xception65 trained on ImageNet , followeBoth models were trained with the same set of hyper parameters: Adam optimizer, learning rate of The aim of this work was to create an image recognition model that handles images from ordinary grown fields. However, training entirely on synthetic data introduces the risk of overfitting on a domain, different from the target domain. To reduce the decoupling between the artificial and the real world, three strategies were employed, namely sub-class weighting, aggressive image augmentation, and style-transfer augmentation. While sub-class weighting and image augmentation were employed to improve the performance in generic real world conditions, style transfer was introduced as a semi- or unsupervised approach to deal with unfamiliar conditions, not present in the training dataset. In principle, this could reduce the need to manually extend the training data when new sampling conditions arise.c, c, Based on the hierarchical data distributions of the training data , a defauIn the case of red clover leaves and white clover leaves, which combined makes up half of the image data, an extension was made to regularize the weights based on the overall presence of clovers in the dataset. Weeds species were trained with a common weight, based on the combined weed coverage.The final class weights used for the training of the CNNs were 3.97, 1.46, 2.86, 6.07, 1.46, 6.68, 6.07, 6.07, 6.07, 8.58, 2.41, 13.28, 0.88, and 1.46 corresponding to soil, clover, grass, weeds, white clover, red clover, dandelion, shepherds purse, thistle, white clover flower, white clover leaf, red clover flower, red clover leaf, and unknown clover leaf, respectively.In order to make the GrassClover dataset consistent with the real images, the resolution was initially set to fit a ground-sampling distance of 6 pixels per mm. However, to increase the robustness of the semantic segmentation outside of the evaluated scope, an aggressive augmentation strategy was introduced. The image scale of the training images was uniformly scaled by 60% as illustrated in Changing weather conditions during the acquisition of the 915 biomass sample pairs introduced diverse visual effects. This means that some plants have strongly reflective leaves due to rain or morning dew, as illustrated in The publicly available implementation of GLStyleNet  was usedThirty-two images with severe reflections from morning dew were selected to provide image styles. Using GLStyleNet, 493 synthetic image crops of 1600 × 1600 pixels were then augmented to express wet weather conditions in an unsupervised approach.Model 1, which handles classification of soil, clover, grass, and weeds, was subsequently fine-tuned for 6000 iterations at a learning rate of The 29,848 images, distributed across 16 fields, as shown in 2, the biomass composition at field scale cannot be performed manually through fractioning into plant species. Sparsely distributed biomass samples, providing the exact biomass composition in sub-m2, might not be representative to a larger region due to local variations at multiple scales in the mixed crop.Evaluation of the large scale biomass composition mapping in crop mixtures introduces new challenges for the research community. With mapped areas five orders of magnitude larger than the manually fractioned biomass samples of 0.25 mNo images from the ATV-based image acquisition platform have corresponding biomass samples. As a consequence, the predictive performance from canopy image to biomass composition, specifically for that camera system, cannot be evaluated directly. In order to validate a consistent segmentation accuracy of Model 1 on the ATV-based images, three other approaches were used: (1) Test on five pixelwise labeled image crops of 1000 × 1000 pixels of varied clover content. These images are included as part of the GrassClover semantic segmentation test set. (2) Qualitative semantic segmentations of the five image crops along with ground truth labels and previous work. (3) Qualitative results of semantic segmentation for each of the four days of data acquisition with a variation in both biomass quantity and composition.The result section is ordered into sections regarding semantic segmentation of images, biomass composition prediction using images, and large scale mapping using images.The two cascaded models were evaluated for pixelwise classification on the Grass-Clover dataset  evaluatiThe baseline FCN-8s based model  reached Biomass composition predictions was made for all 915 biomass samples from the cascaded model predictions, similar to those shown in 2) between the predicted canopy segmentations and the corresponding biomass fractions using a first order linear model on four different model cascade combinations. Focusing on the 1st stage image segmentation into clover, grass, and weeds, there was a clear improvement moving from the FCN-8s model to the DeepLabv3+ of 4.5 percentage points for clover fraction predictions. Since the grass fraction prediction was not improved, the improvement presumably came from better separation of clovers, weeds, and soil in the biomass images.2 of 2.7, 3.2, and 10.8 percentage points were observed for clover, grass, and weeds prediction, respectively. Similar to the semantic segmentation results of 2 originates. The linear correlations from canopy image to the secondary biomass fractions of weeds and specific clover species were not too reliable, in part due to the varying vertical distributions of the fractions in the sward. However, the correlation of the primary fractions of clovers and grasses demonstrated a convincing robustness across the four locations, three seasons, three camera setups, multiple seed mixtures, and changing weather conditions.The introduction of style-transfer augmentation of synthetic data to improve weather condition invariance, as described in overfitted to the evaluated conditions in a degree that penalizes generalizability. This is especially true in areas of expensive sample acquisition, such as biomass sampling and cumbersome fractioning of mixed crops. As a consequence, the evaluated samples are often limited in quantity or variation. To evaluate the generalizability of recent methods, the 915 biomass samples were organized according to (1) acquisition date, (2) experimental site, and (3) seasonal cut no. The correlations between the detected canopy clover fraction and the biomass clover fraction for each set of samples were then determined, as shown in Computer vision techniques can often be 2 ≈ 88%). When red clover was added in the mixture  the average correlation was significantly reduced (R2 ≈ 65%). The inherent sensitivity to scale in morphological filtering was likely insufficient to equally detect the different sized leaves of the two clover species. When used for multiple acquisition dates, the average correlation was reduced to R2 = 48.2%, R2 = 51.4% and R2 = 36.9%, across cuts, sites and all samples, respectively.Morphological filtering from Mortensen et al. , similar2 = 84.1%, the data driven approach, albeit cumbersome to train, provided a single model, suitable for multiple locations and species compositions for the duration of the productive season.The deep learning based FCN-8s model from Skovsen et al. , similar2 < 80%), but all acquisitions demonstrated a higher correlation. Five out of the eighteen acquisition dates demonstrated an R2 < 90%, but four of those were sampled in rainy  or dewy  weather conditions. Across all 915 biomass samples, the DeepLabv3+ST demonstrated an R2 = 91.3%, and, within the common mixture of ryegrass and white clover (site C), the correlation was increased to R2 = 94.6%. The presence of red clover in the mixtures had a small impact on the correlation, but this was more likely the result of a harder task, than unbalanced detection of the two clover species.The DeepLabV3+ST further improved the predictive performance from the analyzed canopy image. Compared to FCN-8s, the biggest improvements for individual sites were those of lower correlations  better spatial accuracy of image segmentations using an encoder–decoder architecture, and (2) incorporating local context features in form of a local context pyramid pooling (LCPP) block [The LC-Net implementation in  improved2 = 94.6% on the extensive dataset of 240 biomass samples across five seasonal cuts. Although the DeepLabv3+ model architecture was not directly compared with the LC-Net model, the combination of dataset and model improved the correlation by 12.1 percentage points.The DeepLabv3+ST model implementation in this work surpassed all previously published results in ryegrass and white clover mixtures with an RTo test the predictive clover content accuracy of the proposed DeepLabv3+ST model on unrelated test locations, the biomass samples were split into four folds, corresponding to the four plot trial sites. Based on a first order linear model, fitted to the data from three sites, the clover fraction for each biomass sample at the fourth site was predicted. The results for each plot trial site are shown in −1. Locally, at each plot trial site, the detected canopy coverage explained a gradual increase in yield, with the most clear trends at sites C and D. The difference in trends between the sites is thought to originate from a combination of species composition, growing conditions , establishing method, and cutting height. By leaving the interpretation up to the agricultural advisor or crop consultant, we demonstrate field maps of canopy coverage in Comparable to the biomass fraction estimates of clover, weeds, red clover, and white clover in Using the 1st stage DeepLabv3+ST model, each large scale acquired image was semantically segmented as demonstrated in Twelve of the clover content mapped fields are shown in The same twelve fields were similarly mapped for canopy coverage in With the popularity of deep learning and data driven methods, improvements in state-of-the-art model architectures for general computer vision can often be transferred to agronomy. However, as the complexity in the experimental design increases, so does the difficulty in obtaining, reproducing, and comparing results. In the chain of events from biomass sampling, to image acquisition and computer vision, inaccuracies at a single point reduce the observed result in the end. With the introduction of data driven models, additional potential sources of error have been introduced in the form of insufficient training data, inaccurate image labels, and suboptimal model hyper parameters. While deep learning can provide great improvements for computer vision in agronomy, as demonstrated in this work, care must be taken to: (1) acquire a representative dataset, adequately spanning the problem at hand, and (2) recognize the impact of camera systems and optimal lighting for computer vision tasks. Although we have presented state-of-the-art results in image based prediction of the biomass clover fraction; this is also the result of a high quality dataset not being the limiting factor in the analysis. With the recent release of the GrassClover dataset , we hopeThe proposed transition from a VGG16  based FCWith the increased reliability of accurate clover content predictions from images, camera based prediction systems are in rising demand. In contrast to manually fractioned biomass samples or inconsistent visual inspections, computer vision opens up for large scale research experiments in the form of (1) hundreds or thousands of plots, or (2) plot sizes large enough to be used for satellite imagery. Albeit the prediction error is not zero in the proposed method, an increased number of repetitions will not only reduce the average image prediction error, but also reduce the consequence of random events occurring in the physical research plots.−1 and without day to day adjustments, was not possible nine years ago. Although future investigations are still needed to evaluate the need for high quality image data, this work has demonstrated the feasibility of automated species distribution mapping of mixed crops.The detailed evaluation of model generalization in 2 of predicting the relative biomass composition from the color images. Evaluated on 915 canopy images with corresponding biomass samples, collected at four different experimental sites across three seasons, the grass and clover fractions were predicted with an R2 of 90.5% and 91.3%, respectively, between the two domains. In mixtures of only ryegrass and white clover, the correlation for clover fraction prediction was increased to a new state of the art of R2 = 94.6%.It has been shown that the Xception-65 based DeepLabv3+ model architecture can significantly outperform the earlier VGG16 based FCN-8s model for semantic segmentation of grass clover images into grass, clover, weeds, and soil. With an extended data augmentation scheme, including style transfer for environmental effects, the DeepLabv3+ model increased the semantic segmentation Intersection over Union (IoU) by 13.4 percentage points. The increased image segmentation IoU greatly improved the RBased on the evaluation of 29,848 spatially distributed high quality images across 225 ha, the local clover fraction of 16 grass clover fields was mapped, as well as the projected leaf area index. With the demonstrated large scale capabilities of highly accurate biomass composition predictions in mixed crops, it has been shown that computer vision sensors can provide reliable, valuable metrics into both research and management of mixed crops in real world conditions, without the need for ongoing hyper-parameter adjustments.


Individuals with depressive symptoms were approximately five times more likely to be at risk of malnutrition or malnourished  than individuals without depression. Residents taking three or more prescription drugs daily  were more likely to be at risk of malnutrition or malnourished. In summary, poor nutritional status was associated with depression, while the intake of numerous prescription drugs was associated with being at risk of malnutrition or malnourished.This study evaluated the association between nutritional status, depressive symptoms, and the number of prescription drugs taken by older adults living in nursing homes in Mexico City. In a cross-sectional study, 262 participants were subjected to anthropometric and nutritional ) evaluations; additionally, their depression ) and functional status were assessed. Multiple logistic regression was used for identifying factors associated with the risk of malnutrition/malnourishment. The mean age of participants was 83.1 ± 8.6 years. A total of 59.9% and 21.1% were at risk of malnutrition and malnourished, respectively. With respect to depression, 27.9% of the participants had mild depression, while 11.4% showed severe depression. An inverse correlation between MNA evaluations and depression scores was found (Spearman’s  Nutrition is one of the most important determinants for the overall health of nursing home residents, where malnutrition is a common finding. Malnutrition during aging has a multifactorial etiology and may severely influence the health status of older adults . MalnutrA recent meta-analysis study including cross-sectional, prospective, retrospective, and case-control studies showed that the prevalence of malnutrition is quite different in older adults living in the community 4.0%) compared to those who are hospitalized 22.3%), those who are living in nursing homes 15.2%), or those in long-term care institutions (30.0%) [%, those .0% compa.2%, or tOlder adults suffer multiple chronic conditions and their emotional health is frequently affected by feelings of depression, such as sadness, misfortune, and unhappiness, leading to broken spirits. Depression is a potentially disabling psychiatric disorder that is commonly associated with an increase in mortality rate . Older pFurthermore, the multiple chronic conditions affecting older people quite often require the simultaneous use of several prescription drugs for treatment. The groups of drugs more frequently used by older patients are directed to the cardiovascular and nervous systems, digestive tract, and metabolism ; thus, mA few studies have investigated the association between nutritional status and polypharmacy; however, their findings are conflicting ,17. For A cross-sectional study was conducted in three private nursing homes located in the south-eastern area of Mexico City. A total of 337 residents were distributed in these three residences as follows: 100, 116, and 121 older adults, respectively. Middle-income older adults paying around one hundred and twenty US dollars a month reside in these nursing homes, which were managed by a private assistance non-profit organization. The study was carried out from May to October 2018. This study was in accordance with the ethical standards of the Declaration of Helsinki. The protocol was registered by the Council of the Division of Biological Sciences and Health of the Metropolitan Autonomous University and approved by the Institutional Ethics Committee .The objectives of the study and procedures involved were explained to each participant, and a signed written consent form was obtained from each of them. The inclusion criteria were: men or women, 65 years and older, staying for more than one year in the nursing home, capable of answering the questionnaire by him/herself, and providing written consent. Only five older adults were unwilling to participate in the study, resulting in a participation response rate of 98.5%.The Charlson Index, Cumulative Illness Rating Scale (CIRS) was used to establish the inclusion and exclusion criteria for the participants, where the criteria are based on the information obtained from the medical records in the file of each patient .Older adults with cognitive impairment or dementia, bedridden, functionally dependent, severe dysphagia, chronic obstructive pulmonary disorder (COPD), and end-stage illnesses were not included since these patients usually have a pre-existing history of weight loss, decreased body mass index, or deteriorated nutritional status and a higher risk of infections ,24,25,26Based on the residents’ medical history, 70 older adults were not included because 47 of them had cognitive impairment or dementia in advanced stages, while the remaining 23 had one or more of the medical conditions mentioned above. Thus, data from only 262 participants were analyzed.The following sociodemographic information was used from the medical records of the participants: date of birth and admission to the nursing home, educational attainment, pre-existing medical conditions, use of prescription drugs, and current antidepressant use (within the past 30 days).Two certified dieticians performed the anthropometry. The standardization procedures were conducted by a certified dietitian ,28. DuriThe anthropometric evaluation considered the following measurements: weight, height, circumferences , mid-upper-arm (MUAC), arm muscle (AMC)), and tricipital skinfold (TSF) . The cutTo evaluate the nutritional status, the Mini Nutritional Assessment (MNA) was used. Older adults with a score <17 were classified as malnourished, those with an MNA score of 17–23.5 were considered at risk of malnutrition, while older people with an MNA > 23.5 were classified as well-nourished .The Barthel Index (BI) was used to evaluate the activities of daily living (ADL). This index, ranging from 0 to 100, is a scale for estimating disability or dependence in ADL in geriatric patients , toilet use, transfers (bed to chair and back), walking with the help of a person, going up/down the stairs (needs help)). A score of ≤60 indicated the presence of physical disability .The presence of depressive symptoms was assessed using the Geriatric Depression Scale (GDS). This scale ranges from 0 to 15, with a higher score indicating depression. The cut-off points used for the classification of depression were as follows: 0–5 normal, 6–10 mild depression, and ≥10 established depression .The results were disclosed to the participants and the personnel responsible for the nursing home medical services.ρ correlation coefficient, owing to the non-normal distribution of the variables. Pearson’s χ2 test was used for comparison of the categorical variables.The data are presented as means ± standard deviations for continuous variables and percentages for categorical variables. Correlation analysis was performed using Spearman’s p < 0.05. The statistical package STATA V15  was used for the data analysis.The MNA score was used to classify older persons into two groups: (1) at risk of malnutrition, and (2) malnourished, both of which were assigned to a single group, and the well-nourished residents were assigned to another group. Logistic regression models were constructed to determine nutritional status. Crude, adjusted odds ratios (ORs), and 95% confidence intervals (95% CIs) were obtained; considering that the participants were living in three different nursing homes, the analysis was performed using the cluster option and robust standard estimators were obtained. The goodness of fit of the model was evaluated using Hosmer–Lemeshow’s test. Interactions between sex, depressive symptoms, and the number of prescription drugs were explored in the multiple logistic regression model. Statistical significance was set at n = 153), type 2 diabetes mellitus 22.5% (n = 59), and cardiovascular diseases 17.6% (n = 46), while 20.2% (n = 53) were diagnosed with depression. A total of 37 (14.0%) patients were taking antidepressants, while the most frequently used medications were sertraline, fluoxetine, and paroxetine.A total of 262 older adults were included in the study: 88 men and 174 women. The mean age was 83.1 ± 8.6 years. No significant differences in age, sex, anthropometry, and MNA were found between the three nursing homes included. n = 193), while those taking five or more drugs (polypharmacy) accounted for 46.2% (n = 121) of the total.Regarding the number of prescription drugs taken by the participants, an average of 5.1 ± 3.9 drugs/24 h was observed, with no significant difference observed when stratified by sex. The percentage of participants taking three or more drugs was 73.7% (2) showed that 10.7% of the participants were underweight. Instead, when the Lipschitz criterion (BMI < 22 kg/m2) was applied, more than one-third (34.4%) of the participants were classified as underweight. Significant differences in the anthropometric values by sex were observed only in height and TSF (BMI determinations using the WHO criterion (BMI < 18.5 kg/m and TSF .p = 0.0275). Estimations of the depression level of the study subjects showed that 39.3% (n = 103) of them had mild (27.9%) or severe (11.4%) depression (p < 0.001), as shown in The MNA results indicated that only a low percentage (17.9%) of participants were well nourished. No significant differences between males and females were observed. The GDS data showed statistical differences between men and women . No significant differences were found in the dependency levels by sex. When the results were grouped by nutritional status, statistical differences were found for the BI and some anthropometric measurements . Furthermore, the BI and MNA were significantly associated with depression .For depression, the results were dichotomized into non-depression and mild-and-severe depression groups. It was found that some anthropometric measurements, such as calf circumference, MUAC, and TSF were significantly different between these depression categories . The results of the multiple logistic regression model (p > 0.05).on model  indicateThis study confirmed and extended previous reports indicating that malnutrition is tightly associated with depression and that taking three or more prescription drugs daily is associated with a worsening nutritional status in older residents living in nursing homes ,14,35,36In general, malnourished patients developed weight loss in association with anorexia, loss of skeletal muscle, low anthropometric indexes, energy intake deficiency, and altered biochemical markers. Thus, it is important to identify malnutrition in older people in the face of its relationship with adverse outcomes, such as functional impairment, poor quality of life, and increased healthcare costs and mortality rates .However, poor nutritional status is not only associated with depression or polypharmacy in older people. Previously published studies showed a strong association between physical dependency and poor nutritional status in Mexican nursing home residents . In a crOur results revealed that 39.3% of the nursing home participants had depression (27.9% mild and 11.4% severe depression). Furthermore, recent widowhood was found to be a risk factor for the appearance of depressive symptoms in the participants . In thisA comparison with other studies showed a higher prevalence of depression in nursing home residents in Germany (34.7% mild and 21.3% severe depression)  than in In the present study, depression was higher in women than in men; these results are similar to other studied populations, such as in Chinese older people, where a higher percentage of depression was found in women . AdditioAlthough depression is a common health problem in older persons living in nursing homes, this condition goes frequently undiagnosed. In fact, in the current study, more than half of the patients with depressive symptoms were previously undiagnosed. The delay in the diagnosis of depression consequently produces a delay in the medical treatment, which may affect the health of the patients, reducing their social and physical activities and their quality of life . In thisIn the current study, the association between malnutrition and depression was tight (OR = 5.82); likewise, in a study of German older people, the GDS score was the only factor that had a significant influence on nutritional status . FurtherAlthough it is not clear whether depression is a cause or an effect of malnutrition, depression is clearly a major factor affecting appetite, nutritional behavior, and dietary intake. Alterations in several neurotransmitters and hormonal changes are associated with depression. These alterations may reduce appetite . AdditioOn average, approximately five medications were taken daily by the participants included in this study, which is similar to a study of Japanese home-care patients, where 5.9 medications were taken daily . In IsraIn an article based on longitudinal studies of the risk factors for malnutrition, age and frailty in institutionalized subjects, poor general health, decreased physical function, and excessive polypharmacy, among other factors, were associated with an increased risk . The relThe impact of polypharmacy on malnutrition is complex and a two-way relationship may actually be at play. Drugs could alter the taste buds and cause hyposalivation and loss of appetite, thus decreasing food intake. Conversely, poor nutritional status is associated with the development of comorbidities that will, in turn, require the use of drugs for treatment . BecauseThe complex relationship between malnutrition, depression, and polypharmacy during aging has been explored and there is evidence of reverse causation ,57,58,59Some strengths of our study are the high participation rate of the nursing home residents and the use of validated instruments in the assessment of nutritional status and depression with the participation of qualified professionals. However, one limitation of our study is its cross-sectional design, which did not allow for a causal relationship to be established. Unfortunately, a follow-up study in older home residents poses important ethical restrictions when no therapeutic interventions are provided. Another limitation is that the studied sample was selected from private nursing homes and therefore our findings may not be directly extrapolated to public nursing homes in Mexico.The majority of the older adults studied were at risk of malnutrition or malnourished. Moreover, depression was highly prevalent among nursing home residents. A strong association was found between poor nutritional status and depression. Additionally, an association was observed between poor nutritional status and taking more than three drugs daily. Systematic assessment of nutritional status and depression is of utmost importance in nursing home residents.


PREX1, UBE3B, PCDHGA2, and ZSWIM6 genes, which occurred only in the BJA-affected calf and were absent in the global control cohort of more than 4500 cattle. Assuming a germline mutation as etiology, one of these variants might be responsible for the vascular malformations identified in this calf.Vascular anomalies represent a heterogeneous group of rare disorders encompassing both vascular malformations and tumors, which can be congenital or arise shortly after birth. They often pose a diagnostic challenge in human and veterinary medicine, and the referring nomenclature is equivocal. Bovine juvenile angiomatosis (BJA), a clinical condition belonging to this group of disorders, encompasses vascular malformations and tumors arising in calves. Usually, such vascular anomalies are not further investigated on a molecular genetic level, mainly because of a lack of resources and diagnostic tools, as well as the low value and short lifespan of the affected animals. Here we report the clinical, pathological, immunohistochemical, and genetic features of a Simmental calf that displayed multiple cutaneous, subcutaneous, and visceral vascular hamartomas compatible with a generalized form of BJA. Whole-genome sequencing identified six coding variants, including four heterozygous variants in the PREX1, UBE3B, PCDHGA2, and ZSWIM6 genes may represent a possible candidate pathogenic variant for this rare form of vascular malformation.Bovine juvenile angiomatosis (BJA) comprises a group of single or multiple proliferative vascular anomalies in the skin and viscera of affected calves. The purpose of this study was to characterize the clinicopathological phenotype of a 1.5-month-old Simmental calf with multiple cutaneous, subcutaneous, and visceral vascular hamartomas, which were compatible with a generalized form of BJA, and to identify genetic cause for this phenotype by whole-genome sequencing (WGS). The calf was referred to the clinics as a result of its failure to thrive and the presence of multiple cutaneous and subcutaneous nodules, some of which bled abundantly following spontaneous rupture. Gross pathology revealed similar lesions at the inner thoracic wall, diaphragm, mediastinum, pericardium, inner abdominal wall, and mesentery. Histologically, variably sized cavities lined by a single layer of plump cells and supported by a loose stroma with occasional acute hemorrhage were observed. Determined by immunochemistry, the plump cells lining the cavities displayed a strong cytoplasmic signal for PECAM-1, von Willebrand factor, and vimentin. WGS revealed six private protein-changing variants affecting different genes present in the calf and absent in more than 4500 control genomes. Assuming a spontaneous de novo mutation event, one of the identified variants found in the  Congenital vascular tumors and malformations are rare anomalies that develop during pregnancy or within the first three months of life . The IntHerein, we aimed to characterize the clinical and pathological phenotype of multiple cutaneous, subcutaneous, and visceral vascular hamartomas compatible with a generalized form of BJA in a Simmental calf. In addition, whole-genome sequencing (WGS) was carried out to identify putatively pathogenic variants.2O2 for 10 min, counterstained with hematoxylin, and mounted. Tissue sections from normal haired skin from a cow were used as positive control for the PECAM-1, von Willenbrand, vimentin, and MNF116 IHCs, while a tissue section from the small intestine from a cow was used as positive control for the SMA IHC. Tissue sections from the nodules from the affected calf without primary antibody incubation were used as negative controls.A 1.5-month-old female Simmental calf with a body weight of 64 kg was referred to the Clinic for Ruminants at the Vetsuisse Faculty, University of Bern, as a result of poor weight gain and skin lesions. Previous treatment by the referring veterinarian included anthelmintics  and antibiotics . The calf was euthanized with an intravenous injection of pentobarbital  and was subsequently submitted to the Institute of Animal Pathology at the Vetsuisse Faculty, University of Bern, for necropsy and histologic examination. Tissue samples from the subcutaneous and internal nodules, as well as from several inner organs, were immediately collected, fixed in 4% buffered formalin, embedded in paraffin, cut at 4 µm, and stained with haematoxylin and eosin (H&E) for further histologic evaluation. Immunohistochemical (IHC) analysis for platelet endothelial cell adhesion molecule (PECAM-1), von Willebrand factor, smooth muscle actin (SMA), vimentin, and a broad-spectrum cytokeratin marker (MNF116) were performed from one subcutaneous and one mediastinal nodule. For the PECAM-1 IHC, antigen retrieval using pressure cooking  in basic EDTA buffer (pH 9.0) was performed, and the primary antibody  was incubated for 1 h at room temperature (RT) (1:1000 dilution). For the von Willebrand factor IHC, pressure cooking in citate buffer  was performed for antigen retrieval, and the primary antibody  was incubated for 40 min (1:100 dilution) at RT. Peroxidase blocking  was performed prior to primary antibody incubation for 10 min in both cases, followed by incubation with Envision+system HRP rabbit  for 30 min at RT, labeling with 3,3′-diaminobenzidine (DAB)  for 10 min, counterstaining with hematoxylin, and mounting. For the SMA IHC, no antigen retrieval was performed, primary antibody incubation  took place for 1 h at RT (1:400 dilution), and the Mach 4 Universal HRP Polymer kit  was used as secondary antibody at RT. Peroxidase blocking and DAB labeling were performed as previously described. For the vimentin IHC, antigen retrieval was performed with Bond Epitope Retrieval Buffer Type 2  for 10 min at 95 °C, and was followed by primary antibody incubation  for 15 min at RT (1:1000 dilution). For the MNF116 IHC, antigen retrieval was performed with Bond Enzyme 1 (AR9551 Leica Biosystems) for 5 min at 37 °C, followed by primary antibody incubation  for 15 min at RT (1:400 dilution). In both cases, all further steps were performed using reagents of the Bond Polymer Refine Detection Kit , namely peroxidase blocking for 5 min, incubation with rabbit anti-mouse secondary antibody for 8 min at RT, and peroxidase-labelled polymer incubation for 8 min. Finally, slides were developed in DAB/Hwww.1000bullgenomes.com) [http://www.ebi.ac.uk/en) (www.1000bullgenomes.com accessed on 15 November 2020) [Genomic DNA was isolated from the liver of the calf using the Promega Maxwell RSC DNA system . WGS using the Illumina NovaSeq6000 was performed on the genomic DNA of the calf. The sequenced reads were mapped to the ARS-UCD1.2 reference genome, resulting in an average read depth of approximately 17.5× , and sinmes.com) , except mes.com) . Furthermes.com) . In ordec.uk/en) . The filer 2020) . Integraer 2020)  softwareIn order to evaluate possible chromosomal abnormalities, the read depth along all chromosomes was calculated. A sliding window approach was used where 3 different window sizes were executed . Using the function bedcov of the program Samtools , the outPROVEAN , MutPredAt clinical examination, the calf was alert but moderately reduced in its general body condition. The rectal body temperature was 39.0 °C, the heart rate was 80 beats per minute, and the respiratory rate was 40 beats per minute. Examination of the cardiovascular, respiratory, digestive, and urinary systems did not reveal any abnormalities.The integumentary system of the calf revealed multiple, cutaneous and subcutaneous, soft, movable nodules, which measured up to 5 cm in diameter. These were predominantly present in the caudal aspect of the back, the pelvis, and the hind limbs a. The cuAt necropsy, the cutaneous and subcutaneous nodules displayed either a white or reddish cut surface. Additionally, mostly pedunculated but similar looking nodules were occasionally present at the inner thoracic wall and diaphragm c, the meHistologically, all analyzed nodules consisted of encapsulated (with the exception of the nodule present in the mesentery adjacent to the duodenum), well demarcated, expansively growing, moderately cellular masses. These consisted of abundant, variably sized, mostly empty cavities, which were lined by a single layer of plump cells and supported by a loose, partially edematous fibrovascular stroma a,b, withTo determine the cellular origin of these nodules, IHC was performed. In both nodules, the plump cells lining the cavities displayed a strong cytoplasmic signal for PECAM-1 a, von WiThe above described histologic and immunohistochemical features resemble previously described vascular anomalies in calves ,8. In spAssuming a spontaneous mutation as etiology for this most likely congenital condition, the sequencing of the whole genome of the affected calf was carried out. Filtering of the obtained variant catalogue for private variants exclusively present in the BJA-affected calf and absent in 496 available control genomes identified 31 private protein-changing variants, and subsequent visual inspection using IGV software confirmed 29 as real variants. Analyzing the occurrence of these variants in the global control cohort of 4110 genomes of a variety of breeds , six hetPREX1, UBE3B, ZSWIM6, and PCDHGA2 were considered the most likely candidate pathogenic variants for the observed phenotype. Unfortunately, no biological samples of the sire and dam were available to evaluate if one of these variants has occurred de novo in the calf.Based on the function of the identified genes and in the predicted impact on the protein, the variants identified in No evidence for chromosomal abnormalities were detected by analyzing the obtained read depth or coverage along all chromosomes.We performed a comprehensive clinical, pathological, and genetic investigation in a Simmental calf displaying multifocal subcutaneous and visceral vascular hamartomas compatible with a generalized form of BJA.PREX1, UBE3B, PCDHGA2, ZSWIM6, NR1H3, and C23H6orf132 genes that were exclusively present in the genome of the affected calf and absent in a global control cohort of more than 4500 cattle genomes of a variety of breeds. Therefore, we considered these apparently rare coding variants as possible candidates for the observed BJA phenotype. In the following, literature and in silico effect predictions are used to discuss a conceivable causal role. The missense variants identified in the NR1H3 and C23H6orf132 genes were not considered as putative causes for these vascular malformations because they were predicted to have a neutral or benign effect.We evaluated the hypothesis of a spontaneous mutation as the possible cause for this most likely congenital phenotype. Analysis of the genome sequence revealed six heterozygous protein-changing variants in the PREX1 was predicted to be deleterious by different tools such as PROVEAN and PredictSNP1 [PI3K) and the β-gamma subunits of the heterotrimeric-G proteins (Gβγ) [PREX1 has an important role in the control of many fundamental cellular functions, including cell migration, actin cytoskeletal rearrangement, adhesion, and the production of reactive oxygen species (ROS) [PREX1 and the occurrence of vascular tumors has been reported to date.However, the missense variant found in dictSNP1 ,36. Thisns (Gβγ) . Furtheres (ROS) . Additioes (ROS) . Evidences (ROS) , breast es (ROS) , prostates (ROS) , and oraes (ROS) . HoweverUBE3B was predicted to be deleterious by PredictSNP1 [UBE3B is associated with an autosomal-recessive inherited disorder called PIRM syndrome in Finnish Ayrshire cattle, which causes intellectual disability, retarded growth, and mortality (OMIA 001934-9913) [TRIB3 with MYC is associated with high proliferation and self-renewal of lymphoma cells [UBE3B was heterozygous, and the calf did not show a phenotype compatible with these disorders.The missense variant in dictSNP1 . An inde34-9913) , and whi34-9913) . In addima cells . HoweverPCDHGA2 was predicted to have a deleterious impact in the protein using PROVEAN [PCDHGA2 most likely does not belong to the group of LoF haploinsufficient genes. In addition, PCDHGA2 protein is a potential calcium-dependent cell-adhesion protein that might be involved in the establishment and maintenance of specific neuronal connections in the brain [PCDHGA2 have been associated with cell and biological adhesion in aggressive papillary thyroid microcarcinomas [PREX1, no association between this gene and the occurrence of vascular tumors has been reported to date.Moreover, a disruptive in-frame deletion in  PROVEAN . This ge PROVEAN . Conside PROVEAN , PCDHGA2he brain ,50. In hrcinomas . HoweverZSWIM6 have been reported in domestic animals. Interestingly, recent data obtained from human genome sequencing studies presented in the gnomAD [ZSWIM6 falls into the class of LoF haploinsufficient genes. The encoded protein, so-called Zinc finger SWIM domain-containing protein 6, is a protein of unknown function that is involved in the nervous system development and regulation [ZSWIM6 enables the zinc ion binding [ZSWIM6 gene (OMIM 615951) are associated to acromelic frontonasal dysostosis [GLMN gene (OMIM 138000) are associated with the development of glomuvenous malformations (GVMs) [To date, no pathogenic variants of e gnomAD  showed tgulation . Moreove binding  and is p binding . In humasostosis  and neursostosis ,55. Addisostosis , GLMN [5sostosis , HECW2 [sostosis , HNRNPH1e OMIM 6151 are assostosis . Similarsostosis . Particus (GVMs)  and Blues (GVMs)  in humanBased on these findings, we speculate whether the above-described vascular lesions in this calf could be due to an impaired interaction between ZSWIM6 and GLMN. However, it cannot be excluded that the phenotype displayed by this calf is due to one of the other remaining variants found exclusively in the sequenced case, or even to a combination of these. Either way, we hypothesize that one of these variants either occurred post-zygotically during the fetal development of the affected calf or represent a germline mutation that occurred in one of the parents. To prove that the private variants occurred was indeed de novo, genotyping of the sire and dam would be needed. Unfortunately, no samples from these animals were available at the time the genetic analysis was performed, therefore this hypothesis cannot be confirmed.We hope that these findings may contribute to a better knowledge and characterization of BJA. However, it is highly unlikely that the candidate causal variants identified in the genome of the studied calf are responsible for other BJA cases since this condition encompasses several kinds of vascular anomalies, including both vascular malformations and tumors .This report highlights the utility of WGS-based precision diagnostics for understanding the underlying genetics of rare disorders in animals with an available reference genome sequence and the value of surveillance for harmful genetic disorders in cattle breeding populations.


Dry-cured ham has a characteristic flavor that originates from biochemical reactions during processing and seasoning of hams. In the case of Toscano dry-cured ham, the Protected Designation of Origin (PDO) states the minimum seasoning length in 12 months, but seasoning can be extended achieving favorable outcomes on sensory characteristics, and above all on aroma. The present study focused on subcutaneous fat of ham. Color of seasoned ham and fat composition of green and seasoned hams were studied. Special attention was paid on the study of volatile compounds, the main substances perceived by smell, present in fat. These compounds are present in large numbers, and they can be used as markers of a specific seasoning stage. For this purpose, they were analyzed by different statistical techniques to select the ones which are the most characteristic of each specific processing  and seasoning  classes.During ham processing the action of endogenous proteolytic and lipolytic enzymes leads to the development of volatile compounds (VOCs) responsible of typical aromas. Protected Designation of Origin (PDO) of Toscano ham requires at least 12 months of ripening but extended seasoning might improve flavor and economic value. This study aimed at assessing the evolution of color, fatty acids, and VOCs profile in subcutaneous fat, and, among VOCs, at identifying possible markers characterizing different seasoning length. For this purpose, a reduced pool of VOCs was selected by 3 multivariate statistical techniques  to classify hams according to ripening (<12 months) or seasoning (≥12 months) periods and also to seasoning length . The main VOCs chemical families steadily increased along ripening. Aldehydes and hydrocarbons reached their peaks at S16, acids and ketones remained constant from R6 to S16, whereas esters started decreasing after 12 months of seasoning. Stepwise analysis selected 5 compounds able to discriminate between ripening and seasoning periods, with 1,1-diethoxyhexane and dodecanoic acid being the most powerful descriptors for ripening and seasoning period, respectively. Instead, 12 compounds were needed to correctly classify hams within seasoning. Among them, undecanoic acid methyl ester, formic acid ethyl ester, 2,4,4-trimethylhexane, and 6-methoxy-2-hexanone had a central role in differentiating the seasoning length. Fat content is one of the main factors characterizing the quality of the meat. Its quantitative and qualitative characteristics affect many aspects linked both to the general quality of products and to consumer acceptability ,2,3. In The aim of this work was to study the evolution of color, fatty acids, and volatile compounds (VOCs) profile in subcutaneous fat, and, among VOCs, to identify possible markers characterizing different seasoning length.In an industrial plant, thirty hams weighing 15.60 ± 1.06 kg were randomly selected and underwent the same manufacturing “Toscano” PDO Consortium manufacturing protocol, consisting of the following stages: salting (15–18 days), pre-resting (15 days), resting (60–70 days), drying (10 days), and ripening (~240 days). At the end of the ripening, hams were randomly allotted into 3 groups of 10 hams each. The first group (S14) was seasoned until 14 months, the second group (S16) until 16 months, and the third group (S18) up to 18 months. At the end of each established seasoning time, hams were dissected, and the external fat was trimmed and analyzed. Hams reached the average final weight of 10.73 ± 0.88 kg, in accordance with the PDO protocol.Moreover, the external fat of hams belonging to group S18 was sampled along the whole processing and ripening periods to assess VOCs profile. Specifically, samples were taken at 0, 1, 3, 6, 12, 14, 16, and 18 months as described below, in paragraph 2.3 “Volatile compounds analysis”.Results on chemical composition of sliced hams, comprehensive of lean and fat, were reported in a previous research . At the n = 10). Sampling took place at time 0  and after 1 (R1), 3 (R3), 6 (R6) months of ripening and at 12 (S12), 14 (S14), 16 (S16), and 18 (S18) months of seasoning. Fat was sampled using a 5-mm punch corer positioned approximately in the same location every sampling time. After each sampling, the hole was filled with a mixture of lard, salt, and pepper to prevent oxidation reactions and microbial contaminations. Subsequently, 1g of homogenized fat was grounded by liquid nitrogen and then transferred to 10 mL screw cap headspace vials adding for each sample 1 mL of distilled water and approximately 1 g of NaCl. The vials were supplemented with 40 μL of internal standard mix , either isotopologues, i.e., deuterated analogues of compounds present in the samples, added to the samples immediately before the analyses [Analysis on VOCs profile were carried out by repeated sampling on each ham of group S18  applying 3 multivariate statistical techniques: stepwise discriminant analysis (SDA), CDA, and DA. The aim of multivariate analysis was to assess if there were groups of VOCs able to characterize the different ripening and seasoning times studied. Furthermore, multivariate approach was also used to outline the contribution of the identified VOCs in properly classifying hams according to their seasoning time only.Groups separation was tested by Hotelling’s T-square test . Howevernc are the canonical coefficients (CCs) and Xi are the scores of original variables. CCs indicate the partial contribution of each variable in composing the CAN. The greater the CC, the more the variable contributes to compose the CAN.The CDA derives a set of new variables, called canonical functions (CAN), that are linear combination of the original compounds. For k-groups involved in the CDA, k-1 CANs are extracted. The structure of a CAN is:The distance between groups was evaluated by using the Mahalanobis’ distance, whereas the effective groups’ separation was tested with the corresponding Hotelling’s T-square test . Finally, the DA was performed to classify ham samples into seasoning groups.The above-mentioned statistical approaches were successively applied to volatile compounds data according to the following two scenarios. In the first scenario, VOCs data were arranged in two major seasoning classes: the low maturing class (LMC) with samples belonging to 0, 1, 3, and 6 months; the high maturing class (HMC) with samples belonging to 12, 14, 16, and 18 months of seasoning. In the second scenario, only samples belonging to HMC were considered.The discriminant procedures were applied to detect the most discriminant compounds able to correctly separate groups involved in the two scenarios. To validate the results, considering the reduced number of involved hams, the leave-one-out cross-validation technique was adopted. In practice, in each scenario, SDA, CDA, and DA were applied 10 times (being 10 the hams involved in the study) by using, at each run, one ham as validation sample. At the end, ten datasets of variables were obtained. Since compounds selected at each round could be different, the ten groups of variables were joined. The resulting compounds were used to develop the final run of CDA and DA.Subcutaneous fat is an important component of the final product, indeed, even when ham is sold in slices, fat is commonly left on. However, few studies have investigated its characteristics separately from the other section of the slice. Instrumental color parameters are shown in Total lipids  significVolatile compounds develop over the ripening process, leading to the typical aroma of dry-cured products. Among the several compounds normally generated by the enzymatic and oxidative processes taking place in the tissues, aldehydes play a prominent role in characterizing dry-cured products’ aroma. Aldehydes are considered important contributors to overall aroma both for being strongly present in finished products and for their low thresholds making them easily detectable by assessors and consumers . Twenty-Three branched aldehydes were observed. Two-methylbutanal and 3-methylbutanal were observed in low concentrations, but they are both considered important contributors to dry-cured ham’s aroma. Two-methyl butanal is associated with nutty, cheesy, and salty notes, while 3-methyl butanal is characterized by fruity, acorn-like, cheesy notes ,32. TheyBiceps femoris or Semimembranosus muscles, in which the fat content was very low compared to our samples. So, being the esters produced by the interaction of two lipid oxidation products, the free fatty acids and the alcohols, the greatest number of esters identified in the present work could be well explained by the matrix used. Additionally, a microbial contribution cannot be excluded being the sampling carried out on subcutaneous fat not covered by skin, where moulds and yeasts develop during the ripening and tissues are easily accessible for microbial esterase enzymes. Eventually, subcutaneous fat is also in close contact with salt used for the manufacturing, in which a considerable number of microbial communities belonging to Micrococcaceae was found [Another important chemical family in dry-cured products is the esters. These compounds were generally associated to the microorganism esterase activity, but, due to the low bacterial count in ham, an alternative pathway was hypothesized. Flores et al.  proposedas found . These mas found . Esters as found . AccordiThirteen hydrocarbons were identified from R0 to S18. Hydrocarbons generally followed the trend showed by aldehydes, with an overall gradual increase until S16 and a final drop at S18. Four n-alkanes  were detected; they are likely products derived from lipid oxidation, as reported by several authors ,50. The Twelve ketones were found in subcutaneous fat. Three of them were aliphatic ketones . Aliphatic ketones are characteristics of dry-cured ham, being already reported by several authors both in lean and fat tissues ,33,44,54Alcohols detected in subcutaneous fat consisted of 12 compounds. Most of them were also observed in fat , whole sCarboxylic acids showed an increasing trend from green to seasoned hams. Most of the identified compounds reached the highest concentration between 12 and 16 months of seasoning and they generally decreased at S18. This is in accordance with their origin being products from hydrolysis of triglycerides and phospholipids, or from the oxidation of unsaturated fatty acids . Twelve Three last compounds were identified, one nitrogenous compound, one furanone, and one furan. Both furan and furanone reached their peak during the early stage of seasoning. Two-pentylfurane was detected in subcutaneous fat of Teruel white hams, Iberian hams, Spanish white hams, and French white hams ,39. Its p < 0.001) split hams belonging to LMC  from hams belonging to HMC  (Semimembranosus muscle (CC = 4.20) [Several authors proposed a multivariate approach to classify dry-cured ham relying on VOCs profile. The main approach used was PCA ,33, but 16, S18) . In deta = 4.20) . In subc = 4.20) , so despSemimembranosus muscle [In the second scenario, 12 VOCs were identified by SDA to discriminate samples into seasoning classes  and thens muscle , a lowerIn conclusion, instrumental color of subcutaneous fat was affected by time, especially L* score was higher for longer seasoning time, partially in agreement with the greater degree of saturation observed in the higher maturing classes. Oleic acid, the main contributor to MUFA amount, showed no difference among seasoning groups. According to VOCs profile, almost every identified compound was affected by ripening and seasoning times. Regardless of single compounds, the main chemical families steadily increased until R6, then different trends were observed. Aldehydes and hydrocarbons reached their peaks at S16, ketones and acids instead showed the highest total content at R6 and R12, respectively. Lastly, esters started to decrease after 12 months of seasoning. Moreover, at S18 most of the main compounds involved in dry-cured ham overall aroma already declined to value similar to ripening phases. In the future, it would be interesting to thoroughly investigated also the enzymatic activity taking place during the different processing stages. The multivariate approach adopted highlighted the importance of 5 compounds present in subcutaneous fat to discriminate between ripening and seasoning stages . Instead, 12 compounds were selected to classify hams according to seasoning length. Among them, 4 VOCs with CCs > 1  had a central role in differentiating the clusters.


Introduction: If a woman suffers from congenital fibrinogen deficiency (CFD), she might undergo hypermenorrhea monthly and possibly to suffer from hemoperitoneum due to a ruptured follicle or corpus luteal cyst every month, which seriously threaten her health and quality of life. Here, we creatively used a combination of the levonorgestrel-releasing intrauterine system (LNG-IUS) and the combined oral contraceptives (COC) for a girl with CFD.Case presentation: A 14-year-old girl presented with no obvious cause of persistent and severe lower abdominal pain that began 8 h prior. After examination, she was diagnosed as hemoperitoneum. CFD was diagnosed when she was 2 years old and she had two hospitalizations due to hemorrhagic anemia caused by menorrhagia. Therefore, after successful conservative treatment of hemoperitoneum, a combination of LNG-IUS and COC was used for the long-term conservative management of hypermenorrhea and hemoperitoneum. During the half-year of follow-up, she had hypomenorrhea without hemoperitoneum.Conclusions: To the best of our knowledge, this was the first patient treated with such a procedure in the literature, and we recommend every woman with CFD at puberty or reproductive age receives this two-pronged treatment. Fibrinogen and fibrin play important and overlapping roles in blood clotting and fibrinolysis . CongeniIn CFD patients, hypermenorrhea is the most common gynecological complication. The incidence exceeds 70% , seriousA 14-year-old girl presented to the Department of Gynecology of the First Affiliated Hospital of Xi'an Jiaotong University with no obvious cause of persistent and severe lower abdominal pain that began 8 h prior. After receiving 450 mL of plasma at another hospital, she was referred to our facility since her test results suggested that her fibrinogen was only 0.10 g/L. She developed spontaneous epistaxis bleeding 7 days after she was born. At 2 years of age, CFD was diagnosed. She had a history of spontaneous auditory canal bleeding at 7 years of age. Her menarche took place at the age of 13, every menstrual period she needed to use more than 40 sanitary pads, accompanied by fatigue, rapid heartbeat, and unable to attend physical education classes or daily physical exercise, showing that she may have hypermenorrhea. She even had two hospitalizations due to hemorrhagic anemia caused by menorrhagia 9 and 3 months prior. But no medical treatment was performed afterwards. Upon presentation at our hospital, she was in the middle of her menstrual cycle. Her abdominal pain was accompanied by nausea, vomiting, dizziness, and palpitation. Besides, she denied the history of smoking.After admission to the Department of Gynecology, we had a detailed physical examination. She was 155 cm and weighed 50 kg. Her vital signs exhibited blood pressure of 107/74 mmHg, a heart rate of 105 beats/min, a respiration rate of 23 breaths/min, and the body temperature of 36.5°C. General examination revealed marked pallor, but the mind was clear and no bleeding spots on skin. Abdominal examination revealed a soft abdomen without tenderness or rebound pain, but the mobile voiceless was positive. No obvious abnormalities were found in other physical examinations. Since the girl and her parents firmly asserted a history of asexual life, we didn't perform a gynecological examination.Then the patient underwent transabdominal ultrasound demonstrating that a visible 3.1 cm × 2.4 cm cystic mass next to her right ovary. The mass boundary was clear and the morphology was regular. No obvious blood flow signal was observed. The fluid dark area in front of her uterus was approximately 8.3 cm × 8.0 cm, and the fluid dark area of her uterus rectal lacuna was 5.5 cm × 2.8 cm, where sparse spot reflection was visible. The right iliac fossa level was 3.9 cm, and that of the left iliac fossa was 2.0 cm. There was no abnormality in the appendix.The patient's coagulation function examination revealed signs of difficult coagulation. Her prothrombin time was 23.30 s , prothrombin activity was 37% , international standard ratio was 2.07 , activated partial thromboplastin time was 43.8 s , thrombin time was 33.3 s , fibrinogen was 0.30 g/L . Her hemoglobin was 71 g/L.When the girl was admitted to our hospital due to abdominal pain, we considered it may be ruptured corpus luteum, ruptured follicles, ectopic pregnancy, torsion of the ovarian cyst or appendicitis. Since the girl was too young, and deny the history of sex life resolutely, we ruled out the possibility of ectopic pregnancy. Her abdominal pain was not metastatic, and there were no signs of peritoneal irritation. Ultrasound showed the appendix was normal, so appendicitis can also be ruled out. Ultrasound images confirmed the presence of hemoperitoneum, so the possibility of torsion of ovarian cyst pedicles was also low, thus the initial judgment was ruptured corpus luteum or ruptured follicles. The complete diagnosis of the girl included: hemoperitoneum (ruptured corpus luteum? ruptured follicles?), CFD, abnormal uterine bleeding-coagulation (AUB-C), and hemorrhagic anemia (moderate).Considering the patient's vital signs were stable, based to the advice of a Department of Hematology consultation, we utilized conservative treatment including rehydration support, aminocaproic acid hemostatic treatment, 2 units of red blood cell suspension, 200 mL of frozen plasma, and 6 g of fibrinogen. After 3 days of hospitalization, her fibrinogen reached 0.50 g/L. We then placed a LNG-IUS (52 mg/piece) under intravenous anesthesia. Diagnostic curettage was performed simultaneously. The pathological results suggested that it was the proliferative phase of the uterus , it alsoIn the seven CFD cases reported with hemoperitoneum  5–11), –11, 5–11Since hemoperitoneum caused by ovulation may recur every month, most doctors –11 preveA network follow-up was conducted when the first withdrawal bleeding occurred 2 months after the patient was discharged from the hospital. She had no abdominal pain and the number of sanitary pads used had dropped to 23 pieces, using the pictorial blood loss assessment chart (PBAC) (Both COC and LNG-IUS are common methods for ordinary being to treat AUB, COC monotherapy was used for the long-term management of CFD patients, in the currently reported literature. Since CFD patients usually experience hypermenorrhea shortly after menarche, at that time, almost all of them are minors who have no sex life; doctors will be more conservative than reproductive women when choosing treatments. However, the high failure rate of COC to treat of hypermenorrhea continues to put patients at risk. In our case, after full communication with the girl and her parents, we implant a LNG-IUS for the girl besides taking COC. A long follow up still necessary to evaluate the impact and tolerance of the LNG-IUS, but for the moment, our case had proved that COC combined with LNG-IUS can be chose when needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Written informed consent was obtained from the girl's parents for the publication of any potentially identifiable images or data included in this article.QilL conceptualized the case report. YW, LZ, XF, and QinL collected the history. YW wrote the first draft of the manuscript. SM, DL, ML, PY, and QirL revised the manuscript. All authors contributed to the revision of the manuscript and approved its final version.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.


Those affected may have intellectual disabilities, dysmorphic facial features, and ocular alterations such as ptosis, hypertelorism, nystagmus, and chorioretinal coloboma. The prevalence of this syndrome is unknown, there are only approximately 100 cases reported. However Dup22q11.2 should have a similar prevalence of DiGeorge syndrome (1 in each 4000 new-borns), in which the same chromosomal region that is duplicated in Dup22q11.2 is deleted.We report a patient with intellectual disability, psychomotor development delay, hearing loss with disyllable pronunciation only, hyperactivity, self-harm, hetero-aggressive behaviour, facial dysmorphism, left facial paralysis, post-axial polydactyly, and for the first time in patients with Dup22q11.2, optic nerve coloboma and dysplasia in optic nerve. Array comparative genomic hybridization showed a 22q11.23 duplication of 1.306 million base pairs.New ocular findings in Dup22q11.2 syndrome, such as coloboma and dysplasia in the optic nerve, are reported here, contributing to the phenotypic characterization of a rarely diagnosed genetic syndrome. A complete characterization of the phenotype is necessary to increase the rate of clinical suspicion and then the genetic diagnostic. In addition, through bioinformatics analysis of the genes mapped to the 22q11.2 region, it is proposed that deregulation of the SPECC1L gene could be implicated in the development of ocular coloboma. The same chromosomal region involved in this syndrome is deleted in DiGeorge syndrome. Although both deletion and duplication are expected to occur in equal proportions as reciprocal events, very few duplications have been identified. While the calculated prevalence of DiGeorge syndrome is 1 of each 4000 new born, only around of 100 cases of Dup22q11.2 had been reported. This disparity in the prevalence of both syndromes could be explained at least in part because the phenotype of the duplication is not well understood.In the majority of cases, the size of the duplication varies between 1.5 and 3.0 million base pairs (Mb). The phenotype is variable and has been reported to include intellectual disabilities, severe psychomotor development delays, language disorders, dysmorphic facial features and hypotonia; however, others with duplication do not present particular phenotypic findings [In patients with Dup22q11.2, ocular findings such as ptosis, down-slanting palpebral fissures, epicanthal fold, hypertelorism, astigmatism, strabismus, hyperopia, myopia, nystagmus, chorioretinal coloboma, retinal vascular tortuosity and primary congenital glaucoma have been described [Array comparative genomic hybridization (aCGH) is a molecular test that evaluates the entire genome and detects numerical and structural chromosome alterations throughout all chromosomes; this diagnostic technique surpasses karyotyping by light microscopy by finding deletions and duplications up to 10,000 times smaller. The aCGH is a test used in the differential diagnosis of patients with intellectual disabilities, psychomotor development delay, autism, and multiple congenital anomalies –5.We report a patient with Dup22q11.2 with optic nerve coloboma and dysplasia in the contralateral optic nerve, findings not described in the reviewed literature, contributing to the phenotypic characterization of the syndrome and the epidemiology of this rare genetic disorder. In addition, through bioinformatics analysis of the genes mapped to the 22q11.2 region, deregulation of the SPECC1L gene could be implicated in the development of coloboma.A female patient 6 years and 3 months of age at the time of consultation, the product of a non-consanguineous relationship, was accompanied by her paternal grandmother, who had legal custody. According to the information provided by the caregiver, the patient was the product of a first pregnancy. At the time of conception, the father was 20 years old, and the mother, who was 18 years old, did not attend prenatal care appointments during pregnancy. She had a vaginal delivery without complications, but the new born required hospitalization for 3 days for phototherapy management of Rh incompatibility.Regarding psychomotor development, the patient crawled at 16 months, walked at 3 years, and has spoken disyllables since 4 years of age. Currently, she does not have a comprehensible verbal language. In addition, she has hyperactivity disorder, has difficulty following instructions, displays self-harm and hetero-aggressive behaviour, for which she is treated pharmacologically with risperidone, and is enrolled in first grade, without meeting the learning requirements.philtrum, right palpebral ptosis, right ear with poor differentiation of the antihelix, left ear with prominent antihelix and hypoplastic lobe; short and wide neck; winging of the scapula occurs with myopathy of the muscles around the shoulder; high exit crease on palm; café-au-lait macule, approximately 6 cm in the hypogastrium; and hypotonia. A post-axial polydactyly surgical scar was found on the left hand. She had hypoacusis and only pronounced disyllables. During the consultation, the patient wandered spontaneously and showed signs of hyperactivity and a delay in psychomotor development, and intellectual disability became evident.Among the relevant findings in the physical examination are weight, 16.3 kg (− 1.9 SD); height, 107 cm (− 1.9 SD); head circumference, 50 cm (− 1.64 SD); distance between outer canthi, 8 cm (50th percentile); distance between inner canthi, 2.6 cm (3rd to 25th percentile); interpupillary distance, 4.5 cm (25th percentile); wide The following relevant findings were observed on ophthalmological physical examination: left facial paralysis, with secondary lagophthalmos of approximately 4 mm, with Bell’s phenomenon; and right palpebral ptosis with a palpebral fissure of 9 mm, unlike the left palpebral fissure of 12 mm. In the exploration of visual acuity, she fixed, followed and maintained with both eyes. The pupils had a diameter of 3 mm and were reactive to light, and no afferent pupillary defect was found. Extraocular movements were preserved, and the Hirschberg corneal reflex was centred. No significant findings were obtained in the exploration of the eye anterior segment, the cornea was clear, the anterior chamber was wide, the iris had a normal appearance, and crystalline lenses were transparent. Intraocular pressure measured by digital palpation was considered normal. Upon examination of the right fundus, a macrodisc was observed, with marked pallor and only a remnant of the superior part of the neuroretinal rim, marked peripapillary atrophy and the presence of an inferotemporal coloboma  was larger than the right kidney (5.7 × 2.2 × 2.3 cm), both without alterations in sonographic morphology. Four months later, videofluoroscopy showed a decrease in the oesophageal lumen at the T1 level.Electroencephalography showed moderate diffuse cortical dysfunction without epileptogenic characteristics. Using electromyography, severe axonal injury of the left facial nerve was observed.At 5 years, the patient was diagnosed with hypothyroidism, and treatment with levothyroxine was started, with an adequate therapeutic response.The patient has been examined using multiple diagnostic aids, with alterations found in the following:Based on the history of the patient, the findings in the physical examination and the observed congenital anomalies, it was concluded that the origin was most likely genetic, and aCGH was requested. The result was a 22q11.23 duplication of 1.306 Mb .The grandmother, who was the legal guardian of the patient, signed an informed consent form allowing the child to be photographed and the use of medical history data.The first case of Dup22q11.2 syndrome was reported in 1999 by Edelmann et al., who described a patient younger than 4 years old with a duplication of 3.0 Mb. The main phenotypic characteristics described were delayed motor development, marked hypotonia, significant delay in language skills and mild facial dysmorphic features . In 2003Dup22q11.2 syndrome is infrequent, and its prevalence has not been established. However, in recent years, with the increasing use of comparative genomic hybridization in patients with congenital anomalies, intellectual disabilities and autism, the diagnosis of this syndrome has increased; thus far, approximately 100 cases have been reported .The chromosome region implicated in Dup22q11.2 syndrome is the same as that involved in DiGeorge syndrome or 22q11.2 deletion syndrome. The prevalence of the latter is 4:10,000 live births, and given that the genetic mechanism that causes the syndrome is also responsible for the duplication, it would be expected that this would occur in a similar proportion; however, that is not observed. This is because the deletion, whose penetrance is 100%, leads to the expression of a characteristic phenotype widely known that, although variable from mild to severe, leads to clinical suspicion and diagnostic testing, while in duplication, in addition to variable expression with a not yet clearly defined phenotype, incomplete penetrance is presented, which explains its under diagnosis .Dup22q11.2 syndrome is a product of chromosomal rearrangement that can also cause the deletion of that same locus because this region contains low copy repeats (LCRs) that are susceptible to non-allelic homologous recombination. Specifically, this locus has 8 LCRs, A through H, and depending on which of these have been deleted or duplicated, can be either a proximal -LCRs close to the centromere: A to D or distal -LCRs close to the telomere: E to H- deletion or duplication. The most common 22q11.2 duplications are those of 3.0 Mb, which involve the LCRs from A to D, and those of 1.5 Mb, which encompass LCRs from A to B . For theIn Dup22q11.2 syndrome, a variable phenotype has been described; even in members of a family with the same duplication, different phenotypes have been reported. The main non-ocular phenotypic characteristics of Dup22q11.2 syndrome include cognitive deficit, psychomotor development delay, decreased hearing, mild micro-retrognathia, cleft palate, elongated face, urogenital tract malformations, and congenital heart defects. Other less common phenotypic characteristics are hyperactivity, macro-microcephaly, seizures and skeletal defects , 11. TheIn addition to the characteristics mentioned above, ocular involvement in Dup22q11.2 has been reported; the most frequently described findings are hypertelorism, down-slanting palpebral fissures, and sparse and thin eyebrows. Less frequently, palpebral ptosis, epicanthal fold, astigmatism, strabismus, hyperopia, myopia, nystagmus, chorioretinal coloboma, retinal vascular tortuosity, and primary congenital glaucoma have been described [Cat eye syndrome (CES) is found in the differential diagnosis of Dup22q11.2 syndrome. Coloboma of the iris, retina and choroids, preauricular pits and appendages and anal atresia have been reported in those affected by CES; however, these characteristics are not consistently found in patients with CES. Common findings include intellectual disability, down-slanting palpebral fissures, cleft palate, congenital heart defects, renal and urinary tract abnormalities, and bone defects. The patient described here presented several of the phenotypic features of CES .CES is caused in most cases by the presence of a bisatellite supernumerary marker chromosome, formed by two copies of the proximal part of chromosome 22, which includes satellites, the entire short arm, the centromere and part of the long arm corresponding to the 22q11 region. This additional chromosome, which occurs in mosaics in one-third of cases, usually has two centromeres and is the product of an inverted duplication 22q11, resulting in partial tetrasomy of this segment, whose distal limit is superimposed on the common distal deletion breakpoint of the 3 Mb deletion seen in 22q11.2 deletion syndrome , 14. TheTo understand the molecular pathways involved in optic nerve coloboma is necessary to review the embryologic development of the eye. The formation of the eye begins early in embryonic development, day 22, by interactions between the neuroectoderm, the superficial ectoderm and the mesenchyme, and the ocular fields are formed . These aAlthough some environmental factors that could be associated with the development of colobomas have been described, it is believed that most have genetic causes . At leasThe noteworthy phenotypic characteristics of the patient with Dup22q11.23 are developmental delay, facial dimorphism and coloboma and optic nerve dysplasia. To determine which genes in the duplication could potentially explain these alterations, a bioinformatic analysis of the altered chromosomal region was performed.In the 22q11.23 region, 66 genes are mapped between nucleotides 23.654.163–24.959.827, and 22 of these genes are annotated in the Gene Ontology gene function database  with divAccording to disease phenotypes registered in the OMIM database, two genes with high potential of being related to the Dup22q11.23 phenotype were prioritized: SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1) and sperm antigen with calponin homology and coiled-coil domains 1-like (SPECC1L).The SMARCB1 protein is part of one of several subunits of the SWI/SNF complex that functions as a chromatin remodelling factor . MutatioOptic nerve coloboma, another of the prominent characteristics of the phenotype secondary to 22q11.23 duplication in the patient reported here, could be related to an increase in the gene dose of SPECC1L. This gene codes for the protein cytospin-A, which stabilizes microtubules. This protein is necessary for the migration of neural crest cells to form the forehead, the nasal bridge and the lower jaw. Mutations in the SPECC1L gene produce oblique facial clefting , and OpiFor the development of the optic nerve and cup, concentration gradients of transcription factors (TFs), such as PAX2, PAX6, VAX1, VAX2, TBX2, TBX3, TBX5, VSX2 and MITF , are necAlthough the exact causes of the patient findings cannot be determined by our analyses, they do allow us to propose that the increase in the number of copies of some genes from the 22q11.23 region could be directly related to the development of the abnormal phenotype. Specifically, we postulate alterations in SPECC1L with optic nerve coloboma as a potential transcriptional target of PAX2 and PAX6. Additional research is necessary to identify the exact mechanisms related to the abnormal phenotype caused by the duplication.In conclusion, based in this case report, the ophthalmologic phenotype of 22q11.23 duplication syndrome should be expanded to additional ocular alterations as coloboma and dysplasia in the optic nerve. By the analysis of the genes in the duplicated region in patients with the syndrome, it is plausible that the triple genetic doses of SPECC1L gene could be involved in the development of ocular alterations by the deregulation of the PAX2 and PAX6 signalling pathways. Further research is necessary to confirm the previous hypothesis.