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3840500	pes2o/s2orc	v3-fos-license	Beyond Sharing Weights for Deep Domain Adaptation Deep Neural Networks have demonstrated outstanding performance in many Computer Vision tasks but typically require large amounts of labeled training data to achieve it. This is a serious limitation when such data is difficult to obtain. In traditional Machine Learning, Domain Adaptation is an approach to overcoming this problem by leveraging annotated data from a source domain, in which it is abundant, to train a classifier to operate in a target domain, in which labeled data is either sparse or even lacking altogether. In the Deep Learning case, most existing methods use the same architecture with the same weights for both source and target data, which essentially amounts to learning domain invariant features. Here, we show that it is more effective to explicitly model the shift from one domain to the other. To this end, we introduce a two-stream architecture, one of which operates in the source domain and the other in the target domain. In contrast to other approaches, the weights in corresponding layers are related but not shared to account for differences between the two domains. We demonstrate that this both yields higher accuracy than state-of-the-art methods on several object recognition and detection tasks and consistently outperforms networks with shared weights in both supervised and unsupervised settings. Introduction Deep Neural Networks [1,2] have emerged as powerful tools that outperform traditional Computer Vision algorithms in a wide variety of tasks, but only when sufficiently large amounts of training data are available. This is a severe limitation in fields in which obtaining such data is either difficult or expensive. For example, this work was initially motivated by our need to detect drones against complicated backgrounds with a view to developing anti-collision systems. Because the set of possible backgrounds is nearly infinite, creating an extensive enough training database of representative real images proved to be very challenging. Domain Adaptation [3] and Transfer Learning [4] have long been used to overcome this difficulty by making it possible to exploit what has been learned in one specific domain, for which enough training data is available, to effectively train classifiers in a related but different domain, where only very small amounts of additional annotations, or even none, can be acquired. Following the terminology of Domain Adaptation, we will refer to the domain in which enough annotated data is available as the source domain and the one with only limited amounts of such data, or none at all, as the target arXiv:1603.06432v1 [cs.CV] 21 Mar 2016 domain. In the drone case discussed above, the source domain can comprise synthetic images of drones, which can be created in arbitrary quantities, and the target domain a relatively small number of annotated real images. Again as in the domain adaptation literature, we will refer to this scenario as the supervised one. An even more difficult situation arises when there is absolutely no annotated data in the target domain. We will refer to this as the unsupervised scenario. In this paper, we will investigate both of the aforementioned scenarios. Recently, Domain Adaptation has been investigated in the context of Deep Learning with promising results. The simplest approach involves fine-tuning a Convolutional Neural Network (CNN) pre-trained on the source data using labeled target samples [5,6]. This, however, results in overfitting when too little target data is available. Furthermore, it is not applicable in the unsupervised case. To overcome these limitations, other works have focused on using both source and target samples to learn a network where the features for both domains, or their distributions, are related via an additional loss term [7][8][9][10]. To the best of our knowledge, all such methods use the same deep architecture with the same weights for both source and target domains. As such, they can be understood as attempts to make the features invariant to the domain shift, that is, the changes from one domain to the other. In this paper, we postulate that imposing feature invariance might be detrimental to discriminative power. To verify this hypothesis, we introduce a different approach that explicitly models the domain shift by allowing the CNN weights to compensate for it. To this end, we rely on the two-stream architecture depicted by Fig. 1. One stream operates on the source domain and the other on the target one. Their weights are not shared. Instead, we introduce a loss function that is lowest when they are linear transformations of each other. In short, our approach models the domain shift by learning features adapted to each domain, but not fully independent, to account for the fact that both domains depict the same object categories. Motivated by our drone detection application, we demonstrate the benefits of our approach on a Unmanned Aerial Vehicles (UAV) image dataset, consisting of a large amount of synthetic data and a small amount of real images. We show that our approach lets us successfully leverage the synthetic data. In particular, we can reach the same level of accuracy as a model trained on a large set of real data by using only 5-10% of real data in conjunction with synthetic examples. This could have a huge impact in many research areas, such as human pose estimation, where labeling new data is timeconsuming and costly, but generating synthetic examples is relatively easy. Furthermore, to demonstrate the generality of our approach, we also apply it to two standard benchmark Domain Adaptation datasets, covering both the supervised and unsupervised scenarios and different network architectures. Our non-shared weight approach consis-tently outperforms those that share all the weights and improves on the state-of-the-art in all cases. Related work In many practical applications, classifiers and regressors may have to operate on many kinds of related but visually different image data. The differences are often large enough for an algorithm that has been trained on one kind of images to perform badly on another. Therefore, new training data has to be acquired and annotated to retrain it. Since this is typically expensive and time-consuming, there has long been a push to develop Domain Adaptation techniques that allow re-training with minimal amounts of new data or even none. We review here briefly some recent trends, with a focus on Deep Learning based methods, which are most related to our work. A natural approach to Domain Adaptation is to modify a classifier trained on the source data using the available labeled target data. This was done, for example, using SVM [11,12], Boosted Decision Trees [13] and other classifiers [14]. In the context of Deep Learning, fine-tuning [5,6] essentially follows this pattern. In practice, however, when little labeled target data is available, this often results in overfitting. Another approach is to learn a metric between the source and target data, which can also be interpreted as a linear cross-domain transformation [15] or a non-linear one [16]. Instead of working on the samples directly, several methods involve representing each domain as one separate subspace [17][18][19]. A transformation can then be learned to align them [19]. Alternatively, one can interpolate between the source and target subspaces [17,18]. In [20], this interpolation idea was extended to Deep Learning by training multiple unsupervised networks with increasing amounts of target data. The final representation of a sample was obtained by concatenating all intermediate ones. It is unclear, however, why this concatenation should be meaningful to classify a target sample. Another way to handle the domain shift is to explicitly try making the source and target data distribution similar. While many metrics have been proposed to quantify the similarity between two distributions, the most widely used in the Domain Adaptation context is the Maximum Mean Discrepancy (MMD) [21]. The MMD has been used to re-weight [22,23] or select [24] source samples such that the resulting distribution becomes as similar as possible to the target one. An alternative is to learn a transformation of the data, typically both source and target, such that the resulting distributions are as similar as possible in MMD terms [25][26][27]. In [28], MMD was used within a shallow neural network architecture. However, this method relied on SURF features [29] as initial image representation and thus only achieved limited accuracy. Recently, using Deep Networks to learn features has proven effective at increasing the accuracy of Domain Adaptation methods. In [30], it was shown that using DeCAF features instead of hand-crafted ones mitigates the Domain Shift effects even without performing any kind of adaptation. However, performing adaptation within a Deep Learning framework was shown to boost accuracy further [31,[7][8][9][10]. For example, in [31], a Siamese architecture was introduced to minimize the distance between pairs of source and target samples, which requires training labels available in the target domain thus making the method unsuitable for unsupervised Domain Adaptation. The MMD has also been used to relate the source and target data representations [7,8] thus making it possible to avoid working on individual samples. In [9,10], a loss term that encodes an additional classifier predicting from which domain each sample comes was introduced. This was motivated by the fact that, if the learned features are domain-invariant, such a classifier should exhibit very poor performance. All these Deep Learning approaches rely on the same architecture with the same weights for both the source and target domains. In essence, they attempt to reduce the impact of the domain shift by learning domain-invariant features. In practice, however, domain invariance might very well be detrimental to discriminative power. As discussed in the introduction, this is the hypothesis we set out to test in this work by introducing an approach that explicitly models the domain shift instead of attempting to enforce invariance to it. We will see in the results section that this yields a significant accuracy boost over networks with shared weights. Our Approach In this section, we introduce our Deep Learning approach to Domain Adaptation. At its heart lies the idea that, for the model to adapt to different domains, the network weights should be related, yet different for each of the two domains. This constitutes a major difference between our approach and the competing ones discussed in Section 2. To implement this idea, we therefore introduce a two-stream architecture, such as the one depicted by Fig. 1. The first stream operates on the source data, the second on the target one, and they are trained jointly. While we allow the weights of the corresponding layers to differ between the two streams, we prevent them from being too far from of each other by introducing appropriately designed loss functions. Additionally we use the MMD between the learned source and target representations. This combination lets us encode the fact that, while different, the two domains are related. More formally, let be the sets of training images from the source and target domains, respectively, with Y s = {y s i } and Y t = {y t i } being the corresponding labels. To handle unsupervised target data as well, we assume, without loss of generality, that the target samples are ordered, such that only the first N t l ones have valid labels, where N t l = 0 in the unsupervised scenario. Furthermore, let θ s j and θ t j denote the parameters, that is, the weights and biases, of the j th layer of the source and target streams, respectively. We train the network by minimizing a loss function of the form where c(θ · \|x · i , y · i ) is a standard classification loss, such as the logistic loss or the hinge loss. r w (·) and r u (·) are the weight and unsupervised regularizers discussed below. The first one represents the loss between corresponding layers of the two streams. The second encodes the MMD measure and favors similar distributions of representations of source and target data. These regularizers are weighted by coefficients λ w and λ u , respectively. In practice, we found our approach to be robust to the specific values of these coefficients and we set them to 1 in all our experiments. Ω denotes the set of indices of the layers whose parameters are not shared. This set is problem-dependent and, in practice, can be obtained from validation data, as demonstrated in our experiments. Weight regularizer. While our goal is to go beyond sharing the layer weights, we still believe that corresponding weights in the two streams should be related. This models the fact that the source and target domains are related, and prevents overfitting in the target stream, when only very few labeled samples are available. Our weight regularizer r w (·) therefore represents the distance between the source and target weights in a particular layer. The simplest choice would be the L 2 norm θ s j − θ t j 2 2 . This, however, would penalize even small differences between the weights and makes the whole system too rigid. To add more flexibility, we therefore use the exponential loss function and write While the exponential loss of Eq. 6 gives more flexibility than the L 2 loss, it still tends to keep the weights of both streams very close to each other, which might be too restrictive when the domains differ significantly. We therefore propose to further relax this prior by allowing the weights in one stream to undergo a linear transformation. We express this as where a j and b j are scalar parameters that encode the linear transformation. These parameters are different for each layer j ∈ Ω and are learned at training time together with all other network parameters. While simple, this approach lets us model parameter transformations such as those depicted by Fig. 2 and has proven to be effective in practice. For example, this parametrization can account for global illumination changes in the first layer of the network. We have tried replacing the simple linear transformation of Eq. 7 by more sophisticated ones, such as quadratic or more complicated linear transformations. However, this has not resulted in any performance improvement. Unsupervised regularizer. In addition to regularizing the weights of corresponding layers in the two streams, we also aim at learning a final representation, that is, the features before the classifier layer, that is domain invariant. To this end, we introduce a regularizer r u (·) designed to minimize the distance between the distributions of representations of the source and target data. Following the popular trend in Domain Adaptation [32,7], we rely on the Maximum Mean Discrepancy (MMD) [21] to encode this distance. As the name suggests, given two sets of data, the MMD measures the distance between the mean of the two sets after mapping each sample to a Reproducing Kernel Hilbert Space (RKHS). In our context, let f s i = f s i (θ s , x s i ) be the feature representation at the last layer of the source stream, and similarly for the target stream. The squared MMD between the source and target domains can be expressed as where φ(·) denotes the mapping to RKHS. In practice, this mapping is typically unknown. However, expanding Eq. 8 yields By using the kernel trick, the inner products can be replaced by kernel values. This lets us rewrite our regularizer as where the dependency on the network parameters comes via the f · i s, and where k(·, ·) is a kernel function. In practice, we make use of the standard RBF kernel k(u, v) = exp (− u − v 2 /σ 2 ), with σ 2 being the bandwidth of the kernel. In all our experiments, we found our approach to be insensitive to the choice of σ 2 and we therefore set it to 1. Training. To learn the model parameters, we first pre-train the source stream using the source data only. We then simultaneously optimize the weights of both streams according to the loss of Eqs. 2-5 using both source and target data, with the target stream weights initialized from the pre-trained source weights. Note that this also requires initializing the linear transformation parameters of each layer, a j and b j for all j ∈ Ω. We initialize these values to a j = 1 and b j = 0, thus encoding the identity transformation. All parameters are then learned jointly using backpropagation with the AdaDelta algorithm [33]. Note that we rely on mini-batches, and thus in practice use all the terms of our loss over these mini-batches rather than over the entire source and target datasets. Depending on the task, we use different network architectures, to provide a fair comparison with the baselines. For example, for digit classification we rely on the standard network structure of [34] for each stream, and, for the Office benchmark, we adopt the AlexNet [35] architecture, as was done in [7]. Experimental Results In this section, we show the benefits of our approach in both the supervised and unsupervised scenarios using different network architectures. Since our motivating application was drone detection, we first thoroughly evaluate our method for this task. We then demonstrate that it generalizes well to other problems by testing it on standard benchmark datasets. Leveraging Synthetic Data for Drone Detection As drones and UAVs become ever more numerous in our skies, it will become increasingly important for them to see and avoid colliding with each other. Unfortunately, training videos are scarce and do not cover a wide enough range of possible shapes, poses, lighting conditions, and backgrounds against which they can be seen. However, it is relatively easy to generate large amounts of synthetic examples, which can be used to supplement a small number of real images and increase the detection accuracy [36]. We show here that our approach allows us to exploit these synthetic images more effectively than other state-of-the-art Domain Adaptation techniques. Dataset and Evaluation Setup. We used the approach of [36] to create a large set of synthetic examples. We also collected three sets of real images, such as those depicted by Fig. 3, which we used for training, validation, and testing, respectively. In our experiments, we treat the synthetic images as samples from the source domain and the real ones as samples from the target domain. We report results using two versions of this dataset, which we refer to as UAV-200 (small) and UAV-200 (full). Their sizes are given in Table 1. They only differ in the number of synthetic and negative samples used at training and testing time. The ratio of positive to negative samples in the first dataset is more balanced than in the second one. For UAV-200 (small), we therefore express our results in terms of accuracy, which is commonly used in Domain Adaptation and can be computed as Using this standard metric facilitates the comparison against the baseline methods whose publicly available implementations only output classification accuracy. In real detection tasks, however, the training datasets are typically quite unbalanced, since one usually encounters many negative windows for each positive one. UAV-200 (full) reflects this more realistic scenario, in which the accuracy metric is poorlysuited. For this dataset, we therefore compare various approaches in terms of precisionrecall. Precision corresponds to the number of true positives detected by the algorithm For both datasets, we follow the supervised Domain Adaptation scenario. In other words, training data is available with labels for both source and target domains. Testing is then done on the images from the test data, as described in Table 1. Network Design. Since drone detection is a relatively new problem, there is no generally accepted network architecture, and we had to design our own. As illustrated by Fig. 1, our network consists of two streams, one for the synthetic data and one for the real data. Each stream is a CNN that comprises three convolutional and max-pooling layers, followed by two fully-connected ones. The classification layer encodes a hinge loss, which was shown to outperform the logistic loss in practice [37,38]. Some pairs of layers in the two streams share their weights while others do not. To identify what the optimal arrangement should be, as well as which loss function should be employed between the non-shared layers, we trained different models corresponding to all possible combinations and evaluated them on the validation data. Fig. 4 depicts the results. In this figure, the + and − signs indicate whether the weights are stream-specific or shared. We can see that using the exponential loss to connect the layers that do not share their weights typically yields better accuracy then using the L2 norm. The best performance overall is obtained when the first two convolutional layers are connected by an exponential loss function and the others share their weights. Our understanding is that, even though the synthetic and real images feature the same objects, they differ in appearance, which is mostly encoded by the first network layers. Thus, allowing the weights of the streams to differ in these layers lets each network better adapt to its domain. In any event, we used this architecture in the remainder of our drone detection experiments. Evaluation. We first compare our approach to other Domain Adaptation methods on UAV-200 (small). As can be seen in Table 2, it significantly outperforms many stateof-the-art baselines in terms of accuracy. In particular, we believe that outperforming DDC [7] goes a long way towards validating our hypothesis that modeling the domain shift in more effective than trying to be invariant to it. This is because, as discussed in Section 2, DDC relies on minimizing the MMD loss between the learned source and target representations much as we do, but uses a single stream for both source and target data. In other words, except for the non-shared weights, it is the method closest to ours. Note, however, that the original DDC paper used a slightly different network architecture than ours. To avoid any bias, we therefore modified this architecture so it matches ours in our own DDC implementation. We then turn to the complete dataset UAV-200 (full). In this case, the baselines whose implementations only output accuracy values become less relevant because it is not a good metric for unbalanced data. We therefore compare our approach against DDC [7], which we found to be our strongest competitor in the previous experiment, and against Accuracy ITML [15] 0.60 ARC-t assymetric [16] 0.55 ARC-t symmetric [16] 0.60 HFA [39] 0.75 DDC [7] 0.89 Ours 0.92 Table 2 Loss: Ls + Lt [36] 0.569 DDC [7] (pre-trained on S and fine-tuned on R and S) 0.664 Our approach (pre-trained on S and fine-tuned on R and S) Loss: Ls + Lt + Lw 0.673 Loss: Ls + Lt + LMMD 0.711 Loss: Ls + Lt + Lw + LMMD 0.757 Table 3. Comparing our method against baselines approaches on the UAV-200 (full) dataset. As discussed in Section 3, the terms Ls, Lt, Lw, and LMMD correspond to the elements of the loss function, defined in Eqs. 2, 3, 4, 5, respectively. the Deep Learning approach of [36], which also tackled the drone detection problem. We also turn on and off some of our loss terms to quantify their influence on final performance. We give the results in Table 3. In short, all the loss terms contribute to improving the AveP of our approach, which itself outperforms all the baselines by large margins. More specifically, we get a 10% boost over DDC and a 20% boost over using real data only. By contrast, simply using real and synthetic examples together, as was done in [36], does not really yield much improvement. Interestingly, allowing the weights of both streams to be independent while the outputs of their last fullyconnected layers are regularized with an MMD loss (Our approach: Loss: Ls + Lt + LMMD) achieves lower accuracy than our approach, which regularizes the weights of both streams. We attribute this to overfitting of the target stream to the target data. Influence of the Number of Real and Synthetic Samples. Using synthetic data in the UAV detection scenario is motivated by the fact that it is hard and time consuming to collect large amounts of real data. We therefore evaluate the influence of the ratio of synthetic to real data. To this end, we first fix the number of synthetic samples to 32800, as in UAV-200 (full) and vary the amount of real positive samples from 200 to 5000. The results of this experiments are reported in Fig. 5(a), where we again compare our approach to DDC [7] and to the same CNN model trained on the real samples only. Our model always outperforms the one trained on real data only. This suggests that it remains capable of leveraging the synthetic data, even though more real data is available, which is not the case of DDC. More importantly, looking at the leftmost point on our curve shows that, with only 200 real samples, our approach performs similarly to, and even slightly better than, a model trained using 2500 real samples. In other words, one only needs to collect 5-10% of labeled training data to obtain good results with our approach, which, we believe, can have a significant impact in practical applications. Fig. 5(b) depicts the results of a second experiment in which we fixed the number of real samples to 200 and increased the number of synthetic ones from 0 to 32800. Note that the AveP of our approach steadily increases as more synthetic data is used. DDC also improves but we systematically outperform it except when we use no synthetic samples, in which case both approaches reduce to a CNN trained on real data only. Evaluation on Standard Classification Benchmarks To demonstrate the generality of our approach, we further evaluate it on two standard domain adaptation benchmarks for image classification, one to test the supervised scenario and the other the unsupervised one. Following standard practice, we express our results in terms of accuracy, as defined in Eq. 11. Supervised domain adaptation using the Office dataset. The Office dataset [15] comprises three different sets of images (Amazon, DSLR, Webcam) featuring 31 classes of objects. Fig. 6 depicts some images from the three different domains of the Office dataset. For our experiments, we used the evaluation protocol proposed in [15], which corresponds to the supervised scenario. Specifically, following [15], we used the labels of 20 randomly sampled images for each class for the Amazon domain and 8 labeled images per class for the DLSR and Webcam domains, when used as source datasets. For the target domain, we only used 3 randomly selected labeled images per class. The rest of the dataset, however, was used as unlabeled data for the calculation of the MMD loss of Eq. 5. Fig. 7(a) illustrates the network architecture we used for this experiment. Each stream corresponds to the standard AlexNet CNN [35] with the additional adaptation On the x-axis we briefly describe the configuration that we use. For each layer '+' denotes that the weights of the network are not shared, while '-' means that corresponding layers share weights. layers proposed in the DDC model of [7]. As in [7], we start with the pre-trained model and fine tune it. However, instead of forcing the weights of both streams to be shared, we allow them to deviate from each other and regularize this deviation using the loss function introduced in Section 3. To identify which layers should not share their weights and which ones should, we used a validation set consisting of 5 examples per object category in the Amazon → Webcam domain adaptation task. Fig. 7(b) depicts some of the results of this validation procedure. As we can see, not sharing the last two fully-connected layers achieves the highest accuracy on this subset. Note that we only performed this validation on the Amazon → Webcam task and used the same architecture for the other tasks reported below. In Table 4, we compare our approach against other Domain Adaptation techniques on the three commonly-reported source/target pairs. It outperforms them on almost all pairs. More importantly, the comparison against DDC confirms that allowing the weights not to be shared increases accuracy. Unsupervised domain adaptation using MNIST-USPS. The MNIST [34] and USPS [41] datasets for digit classification both feature 10 different classes of images corresponding to the 10 digits. They have recently been employed for the task of Domain Adaptation [42]. (a) (b) Fig. 8. Architecture for the MNIST-USPS dataset. (a) Best CNN architecture based on our validation procedure. (b) Results of some of our validation experiments to determine, which layers should not share weights and instead be connected by a regularization loss. The x-axis denotes the network configuration, where '+' denotes that the weights of a particular layer are not shared, while '-' means that they are. vised setting, and thus ignore the target domain labels at training time. Following [32], as the image patches in the USPS dataset are only 16 × 16 pixels, we rescaled the images from MNIST to the same size and applied L 2 normalization of the gray-scale pixel intensities. For our tests, we rely on the standard CNN architecture of [34] and followed a similar validation procedure to that used for the previous two datasets to determine which layers should not share their weights. Specifically, as for the Office dataset, since there is no explicit validation set, we randomly sampled 5 images for each class from the target domain and used them to select the best network architecture. We found that allowing all layers of the network not to share their weights yielded the best performance. Fig. 8(a) depicts the final structure of the CNN. In Fig. 8(b), we provide the results of some of the validation experiments. In Table 5, we compare our approach against other Domain Adaptation techniques. Our approach outperforms the baselines by a large margin. Overall we have compared our approach with various methods [30,28,7], which involve using deep architectures to extract domain invariant representations for both source and target domains. We further evaluated our approach with respect to methods [43,18,19,42] that do not use deep networks. In all cases we believe that our method shows superior performance due to its ability to adapt the feature representation to each domain, while still keeping these representations close to each other. Discussion In all the experiments reported above, allowing the weights not to be shared in at least some of the layers of our two-stream architecture boosts performance. This validates our initial hypothesis that explicitly modeling the domain shift is generally beneficial. However the optimal choice of which layers should or should not share their weights is application dependent. In the UAV case, allowing the weights in the first two layers to be different yields top performance, which we understand to mean that the domain shift is caused by low-level changes that are best handled in the early layers. By contrast, for the Office dataset, it is best to only allow the weights in the last two layers to differ. This network configuration was determined using Amazon and Webcam images, such as those shown in Fig. 6. Close examination of these images reveals that the differences between them are not simply due to low-level phenomena, such as illumination changes, but to more complex variations. It therefore seems reasonable that the higher layers of the network should be domain-specific, since they typically encode this type of highlevel information. Finally, for MNIST+USPS, it is optimal not to share any weights, which we take to be a consequence of the networks being relatively small and therefore requiring adaptation of all the parameters to provide sufficient flexibility. Fortunately, we have shown that it is possible to use validation data to decide which configuration is best, which makes our two-steam approach a practical one. Conclusion In this paper, we have postulated that Deep Learning approaches to Domain Adaptation should not focus on learning features that are invariant to the domain shift, which makes them less discriminative. Instead we should explicitly model the domain shift. To prove this, we have introduced a two-stream CNN architecture, where the weights of the streams are not shared. To nonetheless encode the fact that both streams should be related, we encourage these weights to remain close to being linear transformations of each other by introducing an additional term in the loss function. Our experiments have clearly validated our hypothesis. Our approach consistently yields higher accuracy than networks that share all weights for the source and target data. Furthermore, we have outperformed the state-of-the-art on three challenging object detection and recognition datasets involving both supervised and unsupervised scenarios. In the future, we intend to study if more complex weight transformations could help us improve our results, with a particular focus on designing effective constraints for the parameters of these transformations.	2016-03-21T14:20:41.000Z	2016-03-21T00:00:00.000	{ "year": 2016, "sha1": "4080edb464e5373c5fe271015dcd716e6544650e", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1603.06432", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "3da60c0209d05d6bbd3a77b38b61ed5daef9208b", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Medicine" ] }
254130490	pes2o/s2orc	v3-fos-license	A critical assessment of the association between postnatal toxoplasmosis and epilepsy in immune-competent patients While postnatal toxoplasmosis in immune-competent patients is generally considered a self-limiting and mild illness, it has been associated with a variety of more severe clinical manifestations. The causal relation with some manifestations, e.g. myocarditis, has been microbiologically proven, but this is not unequivocally so for other reported associations, such as with epilepsy. We aimed to systematically assess causality between postnatal toxoplasmosis and epilepsy in immune-competent patients. A literature search was performed. The Bradford Hill criteria for causality were used to score selected articles for each component of causality. Using an arbitrary but defined scoring system, the maximal score was 15 points (13 for case reports). Of 704 articles, five case reports or series and five case–control studies were selected. The strongest evidence for a causal relation was provided by two case reports and one case–control study, with a maximal causality score of, respectively, 9/13, 10/13 and 10/15. The remaining studies had a median causality score of 7 (range 5–9). No selection bias was identified, but 6/10 studies contained potential confounders (it was unsure whether the infection was pre- or postnatal acquired, or immunodeficiency was not specifically excluded). Based on the evaluation of the available literature, although scanty and of limited quality, a causal relationship between postnatal toxoplasmosis and epilepsy seems possible. More definite proof requires further research, e.g. by performing Toxoplasma serology in all de novo epilepsy cases. Introduction Toxoplasmosis is caused by the intracellular parasite Toxoplasma gondii, which has felines as the definitive host and a variety of vertebrates as intermediate hosts. The clinical manifestations in humans depend on whether it is transmitted pre-or postnatally, on gestational age in case of prenatal transmission and on host immune status. A recent publication based on registered diagnoses from private insurance records reported significant morbidity and mortality related to toxoplasmosis, underscoring the relevance of the subject [1]. The seroprevalence of toxoplasmosis, as reflected by the presence of IgG antibodies against T. gondii, differs worldwide. In the USA, the seroprevalence was approximately 14% by the age of 40 years old [2], while in the Netherlands, the seroprevalence was 47% in the age category 40-44 years of age [3]. In postnatal infected immunecompetent individuals, the clinical course is asymptomatic in 90% of the cases, while in the remaining 10%, it usually presents as lymphadenopathy, in which especially the cervical nodes are involved [4]. Lymphadenopathy can be accompanied by fever, rash, sore throat, hepatosplenomegaly and atypical lymphocytosis. Myocarditis, hepatitis, pneumonitis, polymyositis and encephalitis have been reported but are rare manifestations [4]. During the past several years, seropositivity for T. gondii has been associated with various diseases, including e.g. malignancies (e.g. breast cancer, melanoma or non-Hodgkin lymphoma) and auto-inflammatory diseases (systemic lupus erythematosus, rheumatoid arthritis or granulomatosis and Electronic supplementary material The online version of this article (doi:10.1007/s10096-016-2897-0) contains supplementary material, which is available to authorized users. polyangiitis) [5]. However, the association was neither supported by a solid hypothesis on the pathogenesis nor by evidence of causality and the actual role of T. gondii in these diseases therefore remains speculative. An association with neurological manifestations such as Alzheimer's disease, Bell's palsy, migraine and epilepsy has also been reported [5], which would be more plausible given the neurotropic nature of T. gondii. In a recently published meta-analysis by Ngoungou and colleagues, which included studies of immune-competent patients with toxoplasmosis as well as of patients with congenital toxoplasmosis or with human immunodeficiency virus (HIV) infection, it was concluded that toxoplasmosis should be regarded as a risk factor for epilepsy [6]. While the risk of epilepsy in congenital toxoplasmosis or in a setting of impaired immunity is unequivocal, this is not the case for postnatal toxoplasmosis in immune-competent individuals. In the present study we, therefore, specifically aimed to evaluate the possible causal role of postnatal toxoplasmosis in immune-competent patients for the development of epilepsy. Literature search A literature search was performed on February 5th 2015 of the PubMed, Medline, Embase, Web of Science and Cochrane databases. The keywords were 'toxoplasmosis' and 'epilepsy'; for the full search, see the supplementary material. An additional search was conducted on July 14th 2016 using Medline and the keywords were 'toxoplasm* epilepsy 2015 2016'. Selection of studies Articles were selected based on the title and, when potentially relevant, the abstract. Included were studies on toxoplasmosis and epilepsy published between 1966 and July 2016. Exclusion criteria were toxoplasmosis in patients with HIV infection or any other primary or secondary immune deficiency, congenital toxoplasmosis, publications in any language other than English, French or Dutch and animal studies. Scoring of causality (Bradford Hill) In order to construct a semi-quantitative measure for causality in each article, the nine criteria for causality were used as published by Sir Austin Bradford Hill in 1965 [7]. These criteria consisted of strength, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment and analogy, as shown in Table 1, which shows the arbitrary scoring system that was specifically developed for this study. Scores for each article were given by both authors and in case of discrepancy, consensus was obtained. The maximal achievable score was 15 (for case reports, this was 13, as the criterion of strength was not applicable). Scoring for bias and confounding Articles were qualitatively assessed on potential bias/confounding (comprised of information bias, selection bias and confounding). Citation index and citations Articles were assessed with regard to scientific impact, using the impact factor at the time of publication and citation score at the time of the present study, as reported on the Web of Science. Results The search performed in February 2015 resulted in 701 articles, of which nine fulfilled the selection criteria. 215 articles were excluded due an immune deficiency in the patients, 248 articles because it was primarily about a different subject, 63 were about congenital toxoplasmosis, 61 because of the language, 32 were animal studies, 72 were not accessible, while one was a meta-analysis of earlier studies [8]. The nine remaining articles consisted of three case reports, one case series and five case-control studies, with a publication year between 1966 and 2014 [9][10][11][12][13][14][15][16][17]. The additional search in July 2016 resulted in seven articles, of which three were selected based on their title and abstract. One of these three were excluded due to the presence of HIV infection and one was excluded due to low quality; only the remaining case report was included in this study [18], resulting in a total of ten included articles. The causality scores of the ten included studies are shown in Table 2. The total score per article ranged from 5 to 10, with two articles being allocated ten points [9,17]. Four criteria were not applicable to individual articles but were scored simultaneously for all articles. For plausibility and analogy, all ten publications were allocated one point, as T. gondii is known to infect the brain and the development of epilepsy is, therefore, biologically plausible, while other parasitic infections (e.g. neurocysticercosis caused by Taenia solium) can cause epilepsy as well [19]. Along the same line, each article was allocated one point for coherence because the data didn't seriously conflict with current knowledge. Regarding consistency, the studies comprised of various settings and different patient groups. Therefore, each study was allocated two points. Thus, all ten studies received the maximal score for these four characteristics; therefore, the minimal score was five points and the maximal score 15 (13 for case reports). The strength of association was not applicable in the case reports or series. Of the five case-control studies, two reported a significant association between seropositivity for toxoplasmosis and epilepsy [15,17], while it was not significant in the other three [12,14,16]. Yazar et al. found a significantly higher seropositivity rate in patients with cryptogenic epilepsy compared to patients with epilepsy due to a known cause or to healthy volunteers (p < 0.01) [15]. Kamuyu [16]. Critchley et al. did not report statistical data but concluded that the prevalence of T. gondii antibodies in 204 patients with epilepsy was not in excess of that found in a non-epileptic population from another study [12]. Stommel et al. showed, after adjustment for age and gender, no difference in seropositivity between 22 patients with cryptogenic epilepsy and 23 healthy controls, but did report a higher antibody titre for T. gondii in patients with epilepsy compared with nonepileptic controls (Wilcoxon rank-sum test, p = 0.013) [14]. (0) a These four criteria were not applicable to individual articles but were scored simultaneously for all articles in the present study (see the Results section) For case reports, the maximal score was 13, as the criterion of strength was not applicable c After excluding HIV-infected patients A gradient, here defined as higher T. gondii antibody titres among a group of patients with epilepsy, was found in one case-control study, in which high antibody titres were more strongly associated with epilepsy (OR = 1.36; p < 0.015) than lower antibody titres (OR = 1.25; p < 0.069) [17]. The case-control studies were more or less representative for a general conclusion (specificity), while single case reports were allocated one point. Regarding temporality, only three case reports and the case series demonstrated unequivocally that the epileptic seizures took place after infection with T. gondii [9,11,13,18]. Regarding the criterion of experiment, four individual patients had received anti-toxoplasmosis therapy. In four out of six patients, the epileptic insults resolved [9,13], while two others did not recover [10,18]. Due to lack of controls, the effect of treatment could not be interpreted with certainty. Only the case series and case report were allocated one point for this criterion. In all, the strongest evidence for a causal relation was provided by one case report, one case series and one case-control study, each with a causality score of 9 or 10 points [9,13,17]. The remaining studies had a median causality score of 7 (range 5-9). We found no selection bias, but six out of ten studies contained potential confounders, as indicated in Table 3 (in five, it was unsure whether the infection was pre-or postnatally acquired; in one study, immunodeficiency was not specifically excluded). The scientific impact of the selected articles was limited. The impact factor of the journals in which the selected studies were published varied from 1.1 to 4.5 and the number of citations per article varied from 0 to 31. Discussion In this study, we performed a systematic assessment of causality of postnatal toxoplasmosis for the development of epilepsy in immune-competent patients. Worldwide, approximately 50 million individuals suffer from epilepsy. Among known causes are, e.g. genetic syndromes, prenatal or perinatal brain damage, infections of the brain or brain tumours, but in 60% of the patients the cause remains unknown [20]. It would, therefore, be relevant to know whether a common infection such as postnatal toxoplasmosis can cause epilepsy, and, if it can, how frequent this occurs. Previous studies of epilepsy and toxoplasmosis included cases with congenital as well as with postnatal toxoplasmosis and, in addition, often included immunocompromised individuals. The recent meta-analysis by Ngoungou and colleagues of the relation between toxoplasmosis and epilepsy, which included studies of congenital toxoplasmosis and immunocompromised patients, found an estimated OR of 2.25 [95% confidence interval (CI) 1.15 to 3.93] [6]. In a large data analysis based on reported diagnoses from insurance records, a significant association between toxoplasmosis and epilepsy was reported (OR 3.51, 95% CI 3.00-4.12), thus in the same range as was found in the meta-analysis, but as a result of the study design, it could not be ascertained which proportion concerned patients with congenital toxoplasmosis or with an immune deficiency [1]. It was acknowledged that directionality and causality of observed relationships between toxoplasmosis and associated comorbidities were not clear. The specific focus of our study was to assess the causality of postnatal toxoplasmosis for the development of epilepsy specifically in immune-competent individuals. Based on a literature search, ten articles were selected, five of which were case reports or case series and the remainder were case-control studies. In general, case reports are not used in formal meta-analyses, but for the particular purpose of this study, i.e. assessment of causality, the case reports actually provided the most convincing evidence by fulfilling the criterion for temporality, i.e. certainty that the exposure had occurred before and not after the patient had developed epilepsy. In all five case-control studies, it could not be excluded that infection with T. gondii and resulting positive serology had occurred after the patients had developed epilepsy because those studies included patients with previously diagnosed epilepsy and not de novo epilepsy as in the case reports. Even the presence of IgM antibodies, as was reported in some studies, does not prove recent infection because IgM may remain detectable for years [21]. Along the same line, because the time since infection was not known, a potential confounder in the case-control studies was that some or all of the patients could have had congenital toxoplasmosis which first manifested as epilepsy later in life. These limitations of the case-control studies justify the inclusion of case reports in the present assessment. Acknowledging temporality as an essential causality factor, this was most convincingly illustrated in the case report by Beltrame and colleagues [18]. In summary, this report describes an immune-competent 15-year-old boy who developed epileptic insults after having eaten raw meat and vegetables while on a vacation in Ethiopia. Abnormalities were seen on the electroencephalogram (EEG) and serology for T. gondii was positive for both IgM and IgG with low avidity, together convincingly indicating recent infection. The patient received anti-toxoplasmosis therapy, after which the EEG pattern was restored to normal. However, 5 months later, the patient again had seizures with EEG abnormalities, and anti-epileptic drug treatment had to be restarted. In our opinion, this case history convincingly supports a causal relation between toxoplasmosis and epilepsy in an immune-competent person. The presumed pathogenesis of how T. gondii would cause epilepsy is not yet fully understood, but if a causal relation actually exists, then the process is most likely multifactorial, with a contribution by both the immune response of the host and parasite-induced altered neurotransmission. The potential mechanisms contributing to the pathogenesis have recently been reviewed in detail by Ngoungou and colleagues [6]. In short, in the intermediate host, which includes humans, T. gondii forms cysts in several tissues, including the brain, infecting both neurons and glial cells [6]. A rupture of the cysts leads to an expulsion of bradyzoites, followed by a T cell immune response of the host, resulting in inflammation and scar tissue, which has been suggested as one of the main mechanisms leading to epilepsy [8,18]. A recent study in mice showed that the presence of T. gondii tissue cysts led to an alteration in the gamma-aminobutyric acid (GABA) pathway, GABA being an inhibitory neurotransmitter [22]. This led to seizures in the infected mice and, while still highly speculative, this mechanism might contribute to the development of epilepsy in humans as well. In human patients with ocular toxoplasmosis, certain genotypes were overrepresented in immune-competent patients and, although speculative at present, strain-specific virulence, parasite stage and size or type of inoculum may contribute to the development of severe manifestations of toxoplasmosis in immune-competent individuals [23,24]. Based on the present study, it is not possible to either finally prove or disprove a causal relation between postnatal toxoplasmosis and epilepsy. Because the seroprevalence of T. gondii is high and the proportion of patients with cryptogenic epilepsy is considerable, a chance co-occurrence could not be excluded. Not all of Hill's criteria of causation have to be fulfilled to transform an association into belief of causation and Hill himself stated that our decision to take action is not a matter of causation but a matter of its merits [7]. Because epilepsy is a chronic disease that affects many aspects of a patient's life and often requires prolonged use of antiepileptic drugs, we think further study into a potentially treatable cause of epilepsy is justified. Obviously, experimental studies in humans are not possible for ethical reasons. A feasible study design could be to perform T. gondii serology, including IgM and IgG antibodies plus avidity in order to differentiate between recent and remote infection, in all incident cases of epilepsy. If available, a biobank of sera of de novo cases of epilepsy would provide suitable samples for such a study in a retrospective fashion. Patients with positive serology indicating recent toxoplasmosis could be included in a randomised controlled trial, comparing treatment with antitoxoplasmosis therapy with placebo (no such study was found at http://www.clinicaltrials.gov). However, if a causal relation actually exists, the interval between toxoplasmosis and the first manifestation of epilepsy may vary and serology indicating past infection could still be relevant. Additional case-control studies including incident epilepsy cases and adequate controls would be useful. In this regard, it could be interesting to also study cell-mediated immune responses to Toxoplasma, which, after all, is mainly an intracellular pathogen, analogous to e.g. the interferon-gamma release assays as are presently used for the diagnosis of tuberculosis infection [25]. The balance between humoral and cellular responses might reveal a relevant association with neurological manifestations such as epilepsy. A limitation of our study is the fact that the scoring system for the assessment of causality was arbitrary, but we think it, nevertheless, provides an objective, albeit possibly imprecise, measure of causality. The main limitation was the lack of high-quality data in the case-control studies because the temporal relation between infection with T. gondii and epilepsy could not be assessed. Finally, in some of the case reports, an underlying immune deficiency was not explicitly excluded, but because most of these reports were published long before the start of the HIV epidemic, it was unlikely that these patients were immunocompromised. In conclusion, based on the available data, we think that postnatal toxoplasmosis in immune-competent individuals may cause encephalitis featuring as epilepsy, which can persist beyond the acute infection. More definitive proof of causality and an assessment of the frequency of this association require further study. Compliance with ethical standards Funding No funding. Conflict of interest None for both authors. Ethical approval This was not required because it was a literature study using only data from previously published articles and did not involve any experiments with or the use of clinical information of human subjects. Informed consent Not applicable. Financial support None.	2022-12-02T14:53:18.426Z	2017-01-12T00:00:00.000	{ "year": 2017, "sha1": "daf1882b8cc47bfaf3cf634f0866222959e8f9a8", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10096-016-2897-0.pdf", "oa_status": "HYBRID", "pdf_src": "SpringerNature", "pdf_hash": "daf1882b8cc47bfaf3cf634f0866222959e8f9a8", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [] }
500069	pes2o/s2orc	v3-fos-license	The longest diameter of tumor as a parameter of endoscopic resection in early gastric cancer: In comparison with tumor area Background and aim Tumor burden is important to predict clinical behaviors of cancer such as lymph node metastasis (LNM). Tumor size has been used as a parameter of tumor burden such as indication of endoscopic resection in early gastric cancer (EGC) to predict LNM. Thus, we aimed to investigate whether tumor area can be more helpful to predict clinical behaviors than longest diameter of tumor in EGC. Patients and methods 3,059 patients who underwent gastrectomy for EGC were reviewed retrospectively. Tumor area was calculated by multiplying long and short diameter of the tumor in surgical specimen. Longest diameter means maximal longitudinal diameter of tumor in specimen. Clinicopathologic features were compared between longest diameter and area using area under receiver operating characteristic (AUROC) curves. Results Longest diameter and area of tumor showed a strong correlation (correlation coefficient 0.859, p<0.01). The cutoff value for prediction of LNM was 20 mm of longest diameter of tumor and 270 mm2 of tumor area. There was no significant difference between longest diameter and area for prediction of LNM (AUC 0.850 vs. 0.848, respectively). In differentiated-type EGC and undifferentiated-type EGC, there was no significant difference between longest diameter and area for prediction of LNM. Among mucosal or submucosal cancer prediction value of LNM between longest diameter and area was not significantly different. Conclusion Tumor area may not be more helpful to predict LNM than longest diameter in EGC. Therefore, the longest diameter of tumor may be sufficient as an indicator of tumor burden in EGC. Introduction Early gastric cancer (EGC) is defined as confined to gastric mucosa or submucosa with low incidence of nodal metastasis [1], endoscopic resection (ER) is the curative treatment option for EGC. Curative ER means en bloc resection, negative horizontal and vertical margins, no lymphovascular invasion (LVI), and that meet the absolute or expanded indication [2]. However, following pathological evaluations, any ER does not satisfy these criteria is considered a noncurative resection. The prognosis of EGC tends to be good, but some patients still require additional surgery after ER. Although nodal status does not affect the designation of EGC, lymph node metastasis (LNM) is one of the most meaningful prognostic factors. The incidence of LNM in EGC ranges from 0% to 3% for intramucosal cancer and from 11% to 20% for submucosal cancer [3,4]. In some large-scale studies from Japan and Korea, the overall survival rate of lymph node-positive EGC fell to 70%-80% [5], and the local recurrence rate after ER of EGC ranges from 0.4% to 3.7% [6] and the metachronous recurrence rate from 2.7% to 14.0% [7]. Thus, the LNM prediction before ER for EGC plays an important role in the prognosis of EGC. Jung et al. [8] reported that the elevated type was significantly associated with LNM in differentiated-type EGC. They represented that clinical behaviors vary by the endoscopic gross appearance of EGC and tumor burden is predicted by endoscopic gross appearance. They suggested tumor burden is important to predict clinical behaviors of cancer such as LNM. In order to represent tumor burden of EGC, usually the longest diameter of tumor; maximal longitudinal diameter of tumor was measured [9,10]. The generally accepted indications of ER for EGC, based Gotoda's data, are used the longest diameter of tumor [2]. However, several studies reported that the clinical significance of tumor area or volume measured by endoscopic ultrasound in esophageal cancer and EGC [11,12]. Therefore, we aimed to identify whether tumor area can be more helpful to predict clinical behaviors than the longest diameter of tumor in EGC. If tumor area is more helpful to predict clinical behaviors, we thought it would be able to use to predict LNM in ER. Patients and definition We retrospectively reviewed clinicopathologic findings of 3,059 patients underwent gastrectomy for EGC from January 2005 to December 2012 at Severance and Gangnam Severance Hospital. These data included the longest diameter of tumor, the presence of LNM, depth of invasion, and histologic grade using pathologic specimen after surgical resection. All surgical specimens were routinely fixed in 10% formalin and were then serially sectioned at 5-mm intervals, embedded in paraffin blocks, and stained with hematoxylinand eosin. The depth of tumor invasion was then evaluated with lymphovascular involvement and degree of differentiation.LNM was identified using hematoxylinand eosin staining. We defined the longest diameter of tumor means maximal longitudinal diameter of the tumor in pathologic specimen and the tumor area was calculated by multiplying the longestdiameter and its perpendicular short diameter of the tumor in specimen. The study was conducted according tothe provision of the Declaration of Helsinki. This study was approved by the ethics committee and written informed consent was obtained from all subjects. Comparison of LNM prediction value by the longest diameter and area of tumor First, we analyzed the relationships between the longest diameter and area of tumor in EGC and measured cut-off values of the longest diameter and area of tumor in EGC. And then, we compared the LNM prediction value of the longest diameter and area of tumor in total EGC. In addition, we performed subgroup analysis for the LNM prediction value of the longest diameter and area of tumor according to the histologic classification and the tumor depth. Statistical analysis Continuous variables are reported as means ± standard deviation (SD). The relationship between longest diameter and area of tumor analyzed by pearson correlation coefficient. It is a measure of the linear correlation between two variables that is defined as the covariance of the variables divided by the product of their standard deviations. The predictive performance of the longest diameter and area of tumor for lymph node metastasis was evaluated using receiver operating characteristic (ROC) curves. The Youden index is the difference between the true positive rate (sensitivity, %)and the false positive rate (1-specificity, %) [13]. Finding the point on the ROC curve that maximizes the Youden index provides an optimal cutoff value that is independent of the prevalence rate. Using this index, we get cut-off values of longest diameter and area of tumor in EGC. The cutoff value was defined the highest sum of the sensitivity and specificity. Comparisons of AUCs were performed using the method described by DeLong et al [14] for correlated data. Clinicopathologic features were compared between tumor area and the longest diameter of tumor using area under receiver operating characteristic (AUROC) curves. Analyses were undertaken using SPSS ver. 20.0 for Windows (SPSS, Chicago, IL, USA), SAS ver. 9.2 (SAS Institute, Cary, NC, USA) and MedCalc ver. 12.7.0 (MedCalc Software, Ostend, Belgium). Two tailed p-value <0.05 indicated statistical significance. Results Baseline characteristics of study group Table 1 shows the baseline characteristics of total enrolled patients. The median age of total patients was 57 years (range, 23-86 years), and the male to female ratio was 1.88:1. The median the longest diameter was 26mm (range,1-220mm) and the median tumor area was 634mm 2 (range, 1-16900mm 2 ). Among 3,059 patients LNM was observed in 321 patients (10.4%). In addition, we analyzed the associated factors for the LNM in total EGC patients by multivariate analysis at Table 2. Analysis of LNM prediction value between the longest diameter and area of tumor in total EGC The longest diameter and area of tumor showed a strong correlation (Pearson correlation coefficient 0.859, p<0.01,S1 Fig). When analyzed among the total EGC patients, the cutoff value for prediction of LNM was 20 mm of the longest diameter and 270 mm 2 of tumor area. If the value of the longest diameter or tumor area was lower than the cutoff value, we considered that it showed the low probability of LNM.ROC curve was made using the cut off value, the AUROC value of the longest diameter was 0.850 and tumor area was 0.848 (Fig 1). There was no significant difference between the longest diameter and area of tumor for prediction of LNM. Subgroup analysis by histologic type and depth of invasion First, we performed subgroup analysis according to histologic differentiation by Japanese classification. In differentiated type-EGC, the cutoff value was 16mm of the longest diameter and 216mm 2 of tumor area. AUROC value of the longest diameter and tumor area was 0.863 and 0.866, respectively (Fig 2). There was no significant difference between the longest diameter and area of tumor for prediction of LNM. In undifferentiated type-EGC, the cutoff value of the longest diameter of tumor was 23mm and tumor area was 390mm 2 . AUROC value of the longest diameter of tumor was 0.841 and area was 0.836 (Fig 3). There was no significant difference between the longest diameter and area of tumor. Secondly, we analyzed LNM prediction value between the longest diameter and area of tumor according to depth of invasion in EGC specimen. In mucosal cancer, the cutoff value was 15mm of the longest diameter of tumor and 495mm 2 of tumor area. The AUROC value of the longest diameter of tumor was 0.780 and tumor area was 0.730 (Fig 4). There was no significant difference between the longest diameter and area of tumor for prediction of LNM. In submucosal cancer, the cutoff value was 20mm of the longest diameter of tumor and 400mm 2 of tumor area. AUROC value of the longest diameter of tumor was 0.568 and tumor area was 0.564 (Fig 5). There was no significant difference between the longest diameter and area of tumor for prediction of LNM. Discussion Even though 5-year survival rate of EGC exceeds 90%, LNM status is still the most important prognostic factor. Overall survival is worst in node positive compared to node negative EGC with 10 year survival rate of 72% and 92%, respectively [4]. ER is local treatment as standard curative treatment option for EGC. It is now widely performed in standard and expanded indications. So it is important the prediction of the clinical behavior of the tumor before performing ER for EGC. To predict clinical behaviors and prognosis of EGC, endoscopic gross appearance may be useful. Endoscopic gross appearance associated with Is tumor area more helpful to predict lymph node metastasis than longest diameter in early gastric cancer? histological differentiation and clinical behavior of EGC. LNM were significantly different according to the gross appearance of EGC [8]. Some previous studies reported that tumor size is known to be independent predictor of LNM [15,16]. Katsube et al. [17] reported that in addition to submucosal invasion !0.5 mm, a diameter ! 30mm was a risk factor for LNM. Generally, maximal diameter of the tumor was used to evaluating the longest diameter of tumor. In the ER indication and other guidelines of EGC, one-dimensional diameter is commonly used [10]. However, the tumor shape of EGC is usually oval or round, some studies represented measuring tumor volume of EGC is associated with LNM [11,18]. Jeon et al. [19] suggested calculating tumor area as 3.14 x 0.25 x maximal diameter x short diameter. In our study, we used multiplying the longest diameter and its perpendicular short diameter of the tumor in a surgical specimen to express the tumor area roughly. We added short diameter to conventional maximal diameter of tumor, represent tumor area as maximal diameter x short diameter. When we analyzed of LNM prediction value between the longest diameter and area of tumor in total EGC, there is no significant difference between the longest diameter and area of tumor for prediction of LNM. We performed subgroup analysis in histologic type (Japanese classification), but there is no significant difference between the longest diameter and area of tumor for prediction of LNM. We also performed subgroup analysis according to the depth of tumor invasion, there is no significant difference between the longest diameter and area of Is tumor area more helpful to predict lymph node metastasis than longest diameter in early gastric cancer? tumor. Generally, the bidirectional growth of the tumors was more common tumor biologically, it could be related that the longest diameter of tumor, used in current ER indication, is sufficient to reflect tumor burden. Recently, Kim et al. [20]suggested that tumor size using 2-dimensional method was significantly useful to predict for LNM in differentiated minute submucosal cancer. However, when we also analyze LNM prediction value between the longest diameter and area among differentiated minute submucosal cancers, there was no statistically significant difference Although our study concluded no statistical significance between the longest diameter and area of tumor, our study is the meaningful study to represent the tumor burden of EGC using tumor area calculated by multiplying the longest and short diameter through large numbers of the patients with EGC. Furthermore, our results suggested that the maximal diameter used in current ESD criteria of EGC could be a useful indicator of tumor burden to predict LNM. However, further prospective studies with a large sample size may be necessary to determine the role of tumor area for LNM prediction precisely. In addition, our study has some limitations. First, there could be a selection bias because our study evaluated patients only underwent surgery and not included those underwent ESD. However, although ESD was performed based on endoscopically determined longest size of tumor, curative resection or non-curative resection was determined by pathological review after ESD. Therefore, to predict the possibility of LNM, we considered being sufficient using surgical specimen after gastrectomy for EGC. Second, by the limitation of retrospective analysis, a prospective analysis of undergoing ESD for EGC is necessary to confirm of the role of tumor area. In conclusion, tumor area may not be more helpful to predict LNM than one-dimensional longest diameter of tumor in EGC. Therefore, maximal longitudinal tumor size may be sufficient as an indicator of tumor burden in EGC.	2018-04-03T03:34:54.280Z	2017-12-20T00:00:00.000	{ "year": 2017, "sha1": "4635d176afcd6fa9b9b1cfe4425523bfe00a54bd", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0189649&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4635d176afcd6fa9b9b1cfe4425523bfe00a54bd", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
252780276	pes2o/s2orc	v3-fos-license	Variational quantum non-orthogonal optimization Current universal quantum computers have a limited number of noisy qubits. Because of this, it is difficult to use them to solve large-scale complex optimization problems. In this paper we tackle this issue by proposing a quantum optimization scheme where discrete classical variables are encoded in non-orthogonal states of the quantum system. We develop the case of non-orthogonal qubit states, with individual qubits on the quantum computer handling more than one bit classical variable. Combining this idea with Variational Quantum Eigensolvers (VQE) and quantum state tomography, we show that it is possible to significantly reduce the number of qubits required by quantum hardware to solve complex optimization problems. We benchmark our algorithm by successfully optimizing a polynomial of degree 8 and 15 variables using only 15 qubits. Our proposal opens the path towards solving real-life useful optimization problems in today’s limited quantum hardware. Introduction.-Quantumcomputing is evolving rapidly thanks to significant advances in hardware.Current Noisy Intermediate-Scale Quantum (NISQ) processors [1] are starting to show promise in specific tasks, including claims on quantum advantage [2,3], fault-tolerant gates [4], and industrial applications for specific problems [5].Nonetheless, NISQ devices are still quite limited at the time of solving certain problems of relevance, such as complex optimization problems.This is true with the current implementation of quantum optimization algorithms such as Variational Quantum Eigensolvers (VQE) [6] and Quantum Approximate Optimization Algorithms (QAOA) [7]. In this paper we attack this limitation by developing new quantum optimization schemes where classical variables are not encoded in orthogonal qubit states, but rather in other degrees of freedom of the quantum computer.An example is the case in which individual qubits are allowed to represent more than one classical bit variable each, by considering the degrees of freedom of the Bloch sphere [8,9].As we shall see, this allows to significantly reduce the number of qubits needed to solve complex discrete optimization problems in universal gatebased quantum computers, thus boosting the practical utility of NISQ devices for real-life problems. The problem.-A key relevant problem for which NISQ devices are very limited is optimization, which amounts to the minimization of a cost function, typically under some constraints which can also be included via, e.g., Lagrange multipliers.At the end of the day, an arbitrary cost function H of a discrete optimization problem takes the form with q α = 0, 1, • • • , p − 1 a discrete variable that can take up to p different values, with α = 0, 1, • • • , N − 1.For example, if p = 2 for all α, then we have the case of an optimization problem with usual bit variables, q α = 0, 1 for all α.Finding the minimum of such a cost function, for N bit variables, and for a discrete polynomial of degree 2, is known as a Quadratic Unconstrained Binary Optimization (QUBO) problem and is well-known to be NP-Hard.Higher-order polynomials, and higherdimensional variables, make the problem even harder.In many optimization schemes for NISQ devices, cost functions such as the one described above are typically considered by first boiling it down to bit variables.In a binary encoding scheme, this implies that the original variables q α are expressed in terms of m = log 2 (p) bits each, i.e., As an example, if p = 4, then we have that m = 2 and the correspondence x 0 x 1 → q between the individual bits and the original variable (for a given α) is [00 → 0; 01 → 1; 10 → 2; 11 → 3].With this decomposition in mind, the cost function H can be written as in terms of m × N classical bit variables. As we can see, the number of bit variables quickly explodes in classical optimization problems.It is not surprising, therefore, that when solving such problems on quantum computers, their capabilities are quite limited if we consider one qubit in the quantum register for each bit variable of the cost function.Say, for instance, that you run a VQE algorithm on a universal gate-based quantum computer.The largest such machine built as of today is the IBM System One with 127 supercoducting qubits [10].This implies that, with such an algorithmic < l a t e x i t s h a 1 _ b a s e 6 4 = " L b 4 z s e 8 d c U p Z g 7 h D 5 z P H / S g k 0 s = < / l a t e x i t > classical < l a t e x i t s h a 1 _ b a s e 6 4 = " / Q 3 r p Y E a s r p f 3 5 r 1 r n w p 6 c B p P i w approach, one can optimize cost functions up to 127 bits only, so that m × N = 127.This is very far from real-life useful problems.As an example, a static portfolio optimization over the N = 500 assets of the SP500 index, with p = 8 investment positions per asset would require of m × N = 1500 qubits [11]. Method.-Our proposal to overcome the above problem is to modify the assignment between the classical variables in the optimization problem and the quantum states in the quantum computer.As such, a quantum state of N qubits has many more degrees of freedom than those in N classical bit variables.If we were to assign one qubit per classical bit, then the correspondance of degrees of freedom would clearly be and this is a waste of quantum resources.However, this can be dramatically improved by assigning nonorthogonal quantum states to classical configurations [8]. The simplest example is the case of a single qubit: we can represent the different configurations of the classical discrete variable q α using different non-orthogonal states of the qubit, i.e., by breaking the Bloch sphere into "chunks".In particular, we can use p maximallyorthogonal states of one qubit to represent the values of the classical variable q α = 0, 1, • • • , p − 1 (such states are not mutually orthogonal, but minimize globally the mutual scalar products).Such states are maximally distinguishable by single-qubit measurements.These states also correspond to the vertices of convex polyhedra inscribed inside the qubit's Bloch sphere of the qubit [8,9], see Fig. 1 for an example.In addition, this scheme can also be generalized to multi-qubit non-orthogonal quantum states.The readout of the classical configurations is thus done via quantum state tomography, which can be implemented in several ways.A simple approach is, again, to read out the quantum states of the individual qubits at each iteration of a variational quantum optimization algorithm, say VQE or QAOA.This would approximate the probability distribution P (q 0 , q 1 , • • • , q N −1 ), needed to estimate the expectation value of the cost function and its gradient at each iteration (epoch) of the variational algorithm, by P (q 0 , q 1 , • • • , q N −1 ) ≈ P (q 0 )P (q 1 ) • • • P (q N −1 ). ( This approach, while missing correlations, is very efficient to implement and turns out to work quite well in many practical situations, at the expense of sometimes lowering the performance in the convergence of the variational quantum optimization algorithm.An alternative, more precise approach, is to implement quantum state tomography of the whole quantum state, which can be done using, e.g., compressed sensing [12].The single-qubit measurements chosen here work well when the final state is not an entangled state, but rather a separable state.Forcing this type of measurements together with the optimization of the variational parameters in the quantum circuit, implies that the highest degree of entanglement typically happens towards the middle of the circuit evolution, with the qubits tending to be disentangling towards the end of the circuit.Notice also that, while multiqubit tomography could also be implemented, it is not efficient, in the sense that it cannot be scaled up in general for an arbitrary number of qubits. Using this simple idea, it turns out that we can fit much larger optimization problems in NISQ devices.As an example, take again the processor of 127 qubits from IBM, which is currently available.For this processor, and using 6 states per qubit, we could optimize cost functions of up to ≈ 328 bit variables, being this already sufficient for a real-life static portfolio optimization problem of all the companies in the NASDAQ-100 index with 8 positions per asset.To be more specific, we used the relation Number of bit variables = Number of qubits × log 2 (p), (6) rounded to the closest integer from below.In this scheme the technological problem is to be able to distinguish 6 different quantum states in the Bloch sphere of a single qubit.While this may sound harsh at first, notice that it is a single-qubit technical problem, and not a multiqubit one.As a matter of fact, distinguishing 6 states per qubit is also well within the capabilities of current quantum technology.And what is more: this can only improve as larger quantum processors are fabricated.For instance, for a 433 qubit quantum processor, such as the one planned for 2022 in the IBM quantum roadmap [10], we could optimize the whole SP500 with 8 positions per asset with just 12 non-orthogonal states per qubit.To understand this graphically, we show in Fig. 2 the number of qubits required to represent a given number of classical binary variables, for different numbers p of nonorthogonal quantum states per qubit.p = 4 < l a t e x i t s h a 1 _ b a s e 6 4 = " j M 3 H g z 4 9 a H B d 3 W j 2 Z g p / L i e G a V A = " > A A A B 6 n i c b V D L S g N B E O y N r x h f U Y 9 e B o P g K e z 6 v g h B L x 4 j m g c k S 5 i d d J I h s 7 P L z K w Q l n y C F w + K e P W L v P k 3 T p I 9 a G J B Q 1 H V T X d X E A u u j e t + O 7 m l 5 Z X V t f x 6 Y W N z a 3 u n u L t X 1 1 G i G N Z Y J C L V D K h G w S X W D D c C m 7 F C G g Y C G 8 H w d u I 3 n l B p H s l H M 4 r R D 2 l f 8 h 5 n 1 F j p I b 4 + 7 x R L b t m d g i w S L y M l y F D t F L / a 3 Y g l I U r D B N W 6 5 b m x 8 V O q D G c C x 4 V 2 o j G m b E j 7 2 L J U 0 h C 1 n 0 5 P H Z M j q 3 R J L 1 K 2 p C F T 9 f d E S k O t R 2 F g O 0 N q B n r e m 4 j / e a 3 E 9 K 7 8 l M s 4 M S j Z b F E v E c R E Z P I 3 6 X K F z I i R J Z Q p b m 8 l b E A V Z c a m U 7 A h e P M v L 5 L 6 S d m 7 K J / e n 5 U q N 1 k c e T i A Q z g G D y 6 h A n d Q h R o w 6 M M z v M K b I 5 w X 5 9 3 5 m L X m n G x m H / 7 A + f w B 1 H O N g g = = < / l a t e x i t > p = 5 case in a two-dimensional space with coordinates x 1 , x 2 .Again, the number of epochs was 15-20, and here in all cases the accuracy obtained was always 100%.We see also that the higher the density of points, the better the algorithmic convergence and stability. Our results for several qubits are shown in Fig. (2), for gaussian blobs and a similar scenario to that in the previous figure in terms of coordinates, epochs and accuracies, with increasing accuracy for increasing point density.Interestingly enough, in all the shown cases the Hilbert space allowed for more clusters to be considered.We see that in this case, an iterative strategy, increasing the number of clusters until we reach good accuracy, perfectly provides the number of clusters also without having to tell the algorithm a priori.Datasets with a larger number of clusters could also be considered. For the sake of explanation, in Fig. (3) we include the idea of the variational quantum circuits that have been used for the di↵erent benchmarks, for 1, 2 and 3 qubits, in the case of one single epoch.For one qubit only the circuit involves only a single qubit rotation.In the case of two qubits, the most generic circuit involves two onequbit rotations and one CNOT gate.For three qubits, we implement three one-qubit rotations and two sequential CNOTs.This strategy is also used for more qubits, namely for 8 and 10 qubits: one single-qubit rotation per qubit, and sequential entangling gates such as CNOTs and/or To↵olies.These circuits o↵er a good performance for the required data.What is more, they actually correspond to variational quantum states that are complex and intricate datasets, we can always use more complex quantum circuits involving a large degree of entanglement between the qubits, so that they cannot be e ciently simulated with tensor networks (TN) [5,6].However, and as a matter fact, even a complex quantum circuit could be simulated using TNs at the expense of some controllable error, hence producing by construction a generic quantum-inspired clustering algorithm that can run on current classical hardware.In fact, with TNs one could target directly the optimization of the cost function without any quantum circuit behind, using e.g., variational, imaginary-time, or tangent space methods [5,6]. Conclusions.-In this paper we have proposed a new quantum clustering algorithm that can run on NISQ devices.The algorithm is based on an optimization problem, that is subsequently solved via VQE using, on top, non-orthogonal states in the Hilbert space.The combination of all these approaches allows to cluster large datasets into a large number of clusters, accurately, and even with few qubits.The algorithm is benchmark by performing clusterings of the Iris dataset and gaussian blobs, using from one to ten qubits.Additionally, the algorithm can also be simulated and refined with tensor networks, producing a quantum-inspired clustering. We believe that the results in this paper are a significant step forward in the enormous challenge of finding useful applications of NISQ devices.As such, the proposed method can label real data, in an unsupervised way, even with few noisy qubits.The applications of such an algorithm are transversal in many fields of science, engineering and industry.Our algorithm shows that current prototypes of quantum computers can be applied in real-life settings, beyond toy academic models. FIG. 3: Example of variational quantum circuit used for optimization of the 8-degree polynomial in Eq.( 7), where the structure shown for 3 qubits should be generalized to 15 (we show only 3 for space reasons).This variational quantum circuit has a sequential, one-dimensional structure of entanglement, but more generic circuits can also be implemented.Measurements at the end of the circuit are meant to implement single-qubit quantum state tomography.The initial unitary operators set the initial quantum state, and the variational parameters {θi} correspond to single-qubit rotations for each qubit. Benchmark.-Wehave benchmarked the idea by running a VQE optimization, on a classical simulator, to find the minimum of the following degree-8 polynomial: H = (q 3 0 − q 3 1 q 0 − q 2 q 3 + q 3 4 − q 5 q 6 − q 2 7 + q 3 8 + q 8 q 10 /5 + q 11 q 1 + q 2 12 + q 3 13 + q 14 q 0 q 3 ) 2 .(7) In the above equation, we consider the discrete values so that each classical variable in the polynomial can take 20 different values.A standard VQE algorithm, codifying one bit per qubit, would need at least 65 qubits using a binary encoding of the variables, and all the bits in the cost function would be fully-connected, therefore implying deep and complex variational quantum circuits.Additionally, a quantum annealer would need thousands of qubits due to embedding for such a fully-connected graph.In our implementation, however, the simulation solves the minimization problem using 15 qubits and 20 quantum states per qubit with single-qubit tomography, implying a reduction of 50 qubits with respect to VQE and thousands of qubits with respect to quantum annealing.Our simulation was able to find both the trivial and non-trivial minima of the cost function, converging with good accuracy after some epochs, and using a simple variational quantum circuit such as the one in Fig. 3 but for 15 qubits.The evolution of the cost as a function of the number of iterations is shown in Fig. 4. The algorithm finds the optimal value in roughly O(10 3 ) steps, which is to be compared to the O(10 20 ) steps that it would take for a bare classical sampling of 65 bits, and the O(10 10 ) that it would take for an unstructured quantum search (so, even this simple implementation is seven orders of magnitude faster than a quantum search by using Grover's algortithm).The obtained minimum is ≈ 3 × 10 −3 , with 6 circuit layers, and a learning rate of 0.008 using Adam optimizer.In parallel to this example.we have also tested the performance of the algorithm for other problems, including non-polynomial cost functions and even with discontinuities, and the performance of the method has also been remarkably good. At this point, it is worth comparing the evolution of the cost in Fig. 4 to that of a standard VQE algorithm with p = 2, and even with that of the continuum limit p = ∞, analyzed in detail in Ref. [13].While for those two limiting cases one may observe a quite smooth behavior of the cost function, we do not find it for the intermadiate cases explored in this paper.We believe this is due to the fact that, in our current encoding, the p maximallyorthogonal states of a qubit are distributed across the surface of Bloch's sphere, and are not naturally ordered.It should be possible to refine the embeddings and labelings of the states in the sphere, so as to improve the smoothness of the evolution.This will be explored in detail in further extensions of this work. Conclusions and outlook.-Inthis paper we have presented a scheme to significantly reduce the number of qubits needed in variational quantum optimization algorithms.This is based on the fact that configurations of the classical variables can be mapped to non-orthogonal quantum states of the quantum register, and these can be recovered via quantum state tomography.In the simplest case, we map several classical configurations to nonorthogonal states of each qubit, and perform single-qubit tomography.We have benchmarked this idea by successfully optimizing via VQE a polynomial function of 15 variables and 20 configurations per variable, using only 15 qubits. We stress that, similarly to other variational quantum algorithms, our method is heuristic.This means that there are only heuristic arguments about how this algorithm processes information, and the ultimate justification for using it, is that it works in practice.And this is not a sloppy justification, since it is the same one being applied to all heuristic algorithms, even to neural networks and deep learning.Our algorithm here is no exception: it introduces an extra degree of freedom in the optimization (the number of states per qubit), that is indeed another parameter to play with in the heuristic algorithm.the benchmarks presented here, show that this heuristics works in practice, and may be useful in dealing with certain types of optimization problems. The scheme presented here is remarkably simple and powerful.As we have discussed throughout the paper, it dramatically reduces the number of qubits required to solve optimization problems in universal quantum computers.Traditionally, optimization problems had been better solved by means of quantum anneal-ing approaches, with gate-based approaches being disregarded for optimization due to the low number of qubits.The fact that universal NISQ devices are getting more and more powerful (with 4000+ qubits being in the IBM roadmap for 2025) together with algorithmic improvements such as the one discussed here, is certainly an indication that universal quantum computers are closer to industrial applications than commonly expected.4000 qubits with 40 non-orthogonal states per qubit could solve a classical optimization problem of 200.000 bit variables, and these numbers are within reach in the near future for universal quantum computers.This is to the detriment of quantum annealers, in which this scheme cannot be applied as such and are therefore more limited in this respect.Additionally, optimization is the basis of many other algorithms, and the ideas discussed here are already being used as the basis of further variational quantum algorithms solving a wide range of problems.This will be investigated in upcoming works. FIG. 1: [Color online]The 20 configurations of classical variable qα (left) are codified in 20 maximally-othogonal quantum states of a single qubit, which correspond to the 20 vertices of a dodecahedron inscribed in the qubit's Bloch sphere (right). < l a t e x i t s h a 1 _ b a s e 6 4 = " I 0 Q U 7 r e H z y M o g V I 4 2 1 s a I / C h M 5 o = " > A A A C A 3 i c b V C 7 S g N B F J 2 N r x h f q 3 b a D A b B K u y q q G X Q x j K C e U A S w u x k N h k y j 3 U e Q l g i N v 6 K j Y U i t v 6 E n X / j J N l C E w / c y + G c e 5 m 5 J 0 o Y 1 S Y I v r 3 c w u L S 8 k p + t b C 2 v r G 5 5 W / v 1 L S 0 4 j 7 S C F s X G w F F 0 I 4 e / I 8 q R 2 X w r P S y c 1 p s X y Z x Z E H + + A A H I E Q n I M y u A Y V U A U Y P I J n 8 A r e v C f v x X v 3 P q a j O S / b 2 Q V / 4 H 3 + A J E A l 3 w = < / l a t e x i t > number of qubits < l a t e x i t s h a 1 _ b a s e 6 4 = " p 0 8 A h R o Z z m c K O t D l O C B v e F z A y w R A d p t t I E H l m l B y O p 7 B E G Z u r v j h R x r c c 8 t J U c m Y G e 9 6 b i f 1 4 7 M d F V k F I R J 4 Y I P H s o S h g 0 E k 7 z g T 2 q C D Z s b A n C i t q / Q j x A C m F j U y z Z E P z 5 l R d J 4 7 T i X 1 T O 7 s 7 L 1 e s 8 j i I 4 A I f g G P j g E l T B L a i B O s D g E T y D V / D m P D k v z r v z M S s t O H n P P v g D 5 / M H 4 I m d o A = = < / l a t e x i t > number of classical variables < l a t e x i t s h a 1 _ b a s e 6 4 = " W m m 8 j k N M u m b C Q j A 8 M A L e n z T h 6 o g w L w p n 9 + e 5 c i W N I w O O w D H I A x e U Q B n c g C q o A Q z G 4 B m 8 g j f r y X q x 3 q 2 P W e u S l c 4 c g D + w P n 8 A b 3 W U 8 w = = < / l a t e x i t > IBM Eagle (127) < l a t e x i t s h a 1 _ b a s e 6 4 = " u r B N O n b 6 Y / G N S m x 3 m e b C p V o 5 S B c = " > A A A C A n i c b V D L S s N A F J 3 U V 6 2 v q C t x M 1 i E u i m J L e q y 1 I 0 u x A r 2 A W 0 o k + m k H T q Z h J m J E E L r x l 9 x 4 0 I R t 3 6 F O / / G a Z u F t h 6 4 c D j n X u 6 9 x w 0 Z l c q y v o 3 M 0 v L K 6 l p 2 P b e x u b W 9 8 g 1 f w Z j w Z L 8 a 7 8 T F r z R j p z D 7 4 A + P z B 5 K j l Z 0 = < / l a t e x i t > IBM Ospray (433) < l a t e x i t s h a 1 _ b a s e 6 4 = " 9 x 7 B y Q B d R + 8 L l J 7 l Y m T n o Y Q M r I s = " > A A A B 6 n i c b V D L S g N B E O y N r x h f U Y 9 e B o P g K e x G U S 9 C 0 I v H i O Y B y R J m J 5 1 k y O z s M j M r h C W f 4 M W D I l 7 9 I m / + j Z N k D 5 p Y 0 F B U d d P d F c S C a + O 6 3 0 5 u Z X V t f S O / W d j a 3 t n d K + 4 f N H S U K I Z 1 F o l I t Q K q U X C J d c O N w F a s k I a B w G Y w u p 3 6 z S d U m k f y 0 Y x j 9 E M 6 k L z P G T V W e o i v K 9 1 i y S 2 7 M 5 B l 4 m W k B B l q 3 e J X p x e x J E R p m K B a t z 0 3 N n 5 K l e F M 4 K T Q S T T G l I 3 o A N u W S h q i 9 t P Z q R N y Y p U e 6 U f K l j R k p v 6 e S G m o 9 T g M b G d I z V A v e l P x P 6 + d m P 6 V n 3 I Z J w Y l m y / q J 4 K Y i E z / J j 2 u k B k x t o Q y x e 2 t h A 2 p o s z Y d A o 2 B G / x 5 W X S q J S 9 i / L Z / X m p e p P F k Y c j O I Z T 8 O A S q n A H N a g D g w E 8 w y u 8 O c J 5 c d 6 d j 3 l r z s l m D u E P n M 8 f z + e N f w = = < / l a t e x i t > p = 2 < l a t e x i t s h a 1 _ b a s e 6 4 = " B r L w Q H 3 f 9 B t b Z r j X C E C T Y 1 i L t 7 g = " > A A A B 6 n i c b V D L S g N B E O y N r x h f U Y 9 e B o P g K e w a U S 9 C 0 I v H i O Y B y R J m J 5 1 k y O z s M j M r h C W f 4 M W D I l 7 9 I m / + j Z N k D 5 p Y 0 F B U d d P d F c S C a + O 6 3 0 5 u Z X V t f S O / W d j a 3 t n d K + 4 f N H S U K I Z 1 F o l I t Q K q U X C J d c O N w F a s k I a B w G Y w u p 3 6 z S d U m k f y 0 Y x j 9 E M 6 k L z P G T V W e o i v K 9 1 i y S 2 7 M 5 B l 4 m W k B B l q 3 e J X p x e x J E R p m K B a t z 0 3 N n 5 K l e F M 4 K T Q S T T G l I 3 o A N u W S h q i 9 t P Z q R N y Y p U e 6 U f K l j R k p v 6 e S G m o 9 T g M b G d I z V A v e l P x P 6 + d m P 6 V n 3 I Z J w Y l m y / q J 4 K Y i E z / J j 2 u k B k x t o Q y x e 2 t h A 2 p o s z Y d A o 2 B G / x 5 W X S O C t 7 F + X K / X m p e p P F k Y c j O I Z T 8 O A S q n A H N a g D g w E 8 w y u 8 O c J 5 c d 6 d j 3 l r z s l m D u E P n M 8 f 0 W u N g A = = < / l a t e x i t > p = 3 < l a t e x i t s h a 1 _ b a s e 6 4 = " T z t F e 9 + k T 7 7 w Y T U t G + s 1 2 R v P l Y E = " > A A A B 6 n i c b V D L S g N B E O y N r x h f U Y 9 e B o P g K e x q U C 9 C 0 I v H i O Y B y R J m J 5 N k y O z s M t M r h C W f 4 M W D I l 7 9 I m / + j Z N k D 5 p Y 0 F B U d d P d F c R S G H T d b y e 3 s r q 2 v p H f L G x t 7 + z u F f c P G i Z K N O N 1 F s l I t w J q u B S K 1 1 G g 5 K 1 Y c x o G k j e D 0 e 3 U b z 5 x b U S k H n E c c z + k A y X 6 g l G 0 0 k N 8 X e k W S 2 7 Z n Y E s E y 8 j J c h Q 6 x a / O r 2 I J S F X y C Q 1 p u 2 5 M f o p 1 S i Y 5 J N C J z E 8 p m x E B 7 x t q a I h N 3 4 6 O 3 V C T q z S I / 1 I 2 1 J I Z u r v i Z S G x o z D w H a G F I d m 0 Z u K / 3 n t B P t X f i p U n C B X b L 6 o n 0 i C E Z n + T X p C c 4 Z y b A l l W t h b C R t S T R n a d A o 2 B G / x 5 W X S O C t 7 F + X z + 0 q p e p P F k Y c j O I Z T 8 O A S q n A H N a g D g w E 8 w y u 8 O d J 5 c d 6 d j 3 l r z s l m D u E P n M 8 f 0 u + N g Q = = < / l a t e x i t > < l a t e x i t s h a 1 _ b a s e 6 4 = "U u O 1 X 8 a o s z n O r p K 4 d 6 N V X u 2 9 B A Q = " > A A A B 6 3 i c b V B N S 8 N A E J 3 U r 1 q / o h 6 9 L B b B U 0 l U 1 I t Q 9 O K x g v 2 A N p T N d t M u 3 d 2 E 3 Y 1 Q Q v + C Fw + K e P U P e f P f u G l z 0 N Y H A 4 / 3 Z p i Z F y a c a e N 5 3 0 5 p Z X V t f a O 8 W d n a 3 t n d c / c P W j p O F a F N E v N Y d U K s K W e S N g 0 z n H Y S R b E I O W 2 H 4 7 v c b z 9 R p V k s H 8 0 k o Y H A Q 8 k i R r D J p e T G 9 / p u 1 a t 5 M 6 B l 4 h e k C g U a f f e r N 4 h J K q g 0 h G O t u 7 6 X m C D D y j D C 6 b T S S z V N M B n j I e 1 a K r G g O s h m t 0 z B R z N 6 K y A g r T I y N p 2 J D 8 B d f X i a t s 5 p / W T t / u K j W b 4 s 4 y n A E x 3 A K P l x B H e 6 h A U 0 g M I J n e I U 3 R z g v z r v z M W 8 t O c X M I f y B 8 / k D P P i N u A = = < / l a t e x i t > p = 10 < l a t e x i t s h a 1 _ b a s e 6 4 = " L Z 0 t 8 D w d w 6 0 S r y c V C B H c 1 D + 6 g 8 j k g g q D e F Y 6 5 7 n x s Z P s T K M c D o r 9 R N N Y 0 w m e E R 7 l k o s q P b T + a 0 z d G a V I Q o j Z U s a N F d / T 6 R Y a D 0 V g e 0 U 2 I z 1 s p e J / 3 m 9 x I T X f s p k n B g q y W J R m H B k I p Q 9 j o Z M U W L 4 1 B J M F L O 3 I j L G C h N j 4 y n Z E L z l l 1 d J u 1 b 1 L q s X D / V K 4 z a P o w g n c A r n 4 M E V N O A e m t A C A m N 4 h l d 4 c 4 T z 4 r w 7 H 4 v W g p P P H M M f O J 8 / P n 2 N u Q = = < / l a t e x i t > p = 20 < l a t e x i t s h a 1 _ b a s e 6 4 = " Q w d w 6 0 S r y c V C B H c 1 D + 6 g 8 j k g g q D e F Y 6 5 7 n x s Z P s T K M c D o r 9 R N N Y 0 w m e E R 7 l k o s q P b T + a 0 z d G a V I Q o j Z U s a N F d / T 6 R Y a D 0 V g e 0 U 2 I z 1 s p e J / 3 m 9 x I T X f s p k n B g q y W J R m H B k I p Q 9 j o Z M U W L 4 1 B J M F L O 3 I j L G C h N j 4 y n Z E L z l l 1 d J + 6 L q X V Z r D / V K 4 z a P o w g n c A r n 4 M E V N O A e m t A C A m N 4 h l d 4 c 4 T z 4 r w 7 H 4 v W g p P P H M M f O J 8 / Q A K N u g = = < / l a t e x i t > p = 30 < l a t e x i t s h a 1 _ b a s e 6 4 = " E C O s W T v e z v p U W H FIG. 2: [Color online] Number of qubits required to represent a given number of classical binary variables, for different numbers p of non-orthogonal quantum states per qubit.The dashed lines correspond to two IBM quantum processors: one with 127 qubits, which is already available, and one with 433 qubits, expected in principle before the end of 2022. FIG. 3 . FIG. 3. Three variational quantum circuits for one epoch: (a) one qubit, (b) two qubits, and (3) three qubits.For qubit i, the set of variational angles is {✓i}, and the set of angles for the initial rotations is {⇢ init i }. FIG. 4 : FIG. 4: [Color online] Cost as a function of the number of iterations in the optimization of a degree-8 polynomial of 15 variables, with 20 possible values per variable, as explained in the text.	2022-10-11T01:16:25.236Z	2022-10-06T00:00:00.000	{ "year": 2023, "sha1": "aee65578c5429ea21d8a0c4df1160db3c36e6fec", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-023-37068-2.pdf", "oa_status": "GOLD", "pdf_src": "ArXiv", "pdf_hash": "aee65578c5429ea21d8a0c4df1160db3c36e6fec", "s2fieldsofstudy": [ "Physics", "Computer Science" ], "extfieldsofstudy": [ "Medicine", "Physics" ] }
262125947	pes2o/s2orc	v3-fos-license	Possible role of selenium in ameliorating lead-induced neurotoxicity in the cerebrum of adult male rats: an experimental study Chronic lead (Pb) poisoning is one of the greatest public health risks. The nervous system is the primary and most vulnerable target of Pb poisoning. Selenium (Se) has been shown to be a potential protection against heavy metal toxicity through anti-inflammatory and antioxidant properties. Therefore, the present study aimed to elucidate the possible protective role of Se in ameliorating the effects of Pb on rat cerebral structure by examining oxidative stress and markers of apoptosis. The rats were divided into 6 groups: control group, Se group, low Pb group, high Pb group, low Pb + Se group, high Pb + Se group. After the 4-week experiment period, cerebral samples were examined using biochemical and histological techniques. Pb ingestion especially when administered in high doses resulted in cerebral injury manifested by a significant increase in glial fibrillary acidic protein, malondialdehyde (MDA) marker of brain oxidation and DNA fragmentation. Moreover, Pb produced alteration of the normal cerebral structure and cellular degeneration with a significant reduction in the total number of neurons and thickness of the frontal cortex with separation of meninges from the cerebral surface. There was also a decrease in total antioxidant capacity. All these changes are greatly improved by adding Se especially in the low Pb + Se group. The cerebral structure showed a relatively normal histological appearance with normally attached pia and an improvement in neuronal structure. There was also a decrease in MDA and DNA fragmentation and an increase TAC. Selenium is suggested to reduce Pb-induced neurotoxicity due to its modulation of oxidative stress and apoptosis. Experiment protocol.The rats were randomly divided into 6 groups of 10 animals each: control group, Se group, low Pb group, high Pb group, low Pb + Se group and high Pb + Se group.The post-acclimation period for the experiment was 4 weeks.Control group received balanced diet only without any treatment.Se group was given 0.25 mg/kg/day of Se by oral gavage 14 .Low Pb group animals were given 20 ml/kg/day of Pb acetate dissolved in 1ml distilled water by oral gavage 8 .High Pb group was given 50 ml/kg/day of Pb acetate dissolved in 1ml distilled water by oral gavage 21 , low Pb + Se group was given 20 mg/kg/day of Pb acetate along with 0.25 mg/ kg/day of Se by oral gavage 8,14 .High Pb + Se group was given with 50 mg/kg/day of Pb acetate along with 0.25 mg/kg/day of Se by oral gavage 14,21 . Animal weight.For measuring body weight, each animal was put in a closed plastic container and weighed day before the experiment (bwt) and at the last day (Lwt).The results were written in a record for each labeled rat.Moreover, the whole brains were harvested after animal slaughtering and weighted before dissection.The relative brain weight was obtained as a percentage of brain weight (br) to Lwt (br/Lwt %) 22 . Samples preparation. All animals were anaesthetized by intra-peritoneal injection of thiopental 30 mg kg. Then, the brain was carefully excised and weighted; and the two cerebral hemispheres were split.One-half was wrapped in aluminum foil and frozen at − 80 °C until it was needed for biochemical research.For histopathological examinations, the other was fixed in 10% formol saline for 48 h, or buffered glutaraldehyde solution at pH 7.4 for 2-24 h in a refrigerator at 4 °C19 . Comet assay for DNA fragmentation.By measuring the length of DNA migration and the percentage of migrated DNA, the quantitative and qualitative amount of DNA damage in preprocessed cells was determined.CCD camera was used in conjunction with Comet 5 image analysis software created by kinetic imaging, Ltd. (Liverpool, UK).The research was carried out at the Cairo-based Animal Reproduction Research Institute (ARRI).It was performed to evaluate the apoptotic effect of Pb.Its steps were followed according to guidelines of Tice et al. 23 . Homogenate tissue analysis.Tissues were homogenized in ice-cold phosphate buffers (50 mM, pH 7.4) 5 times their tissue weight, and centrifuged at 5000 rpm for 30 min.Then, supernatants were preserved in a deep freeze until being used for the following assays 24 . Lipid peroxidation assay.Thiobarbituric acid (TBA) (0.2%) in H 2 SO 4 (0.05 M) was combined with cerebral homogenate samples and heated in a boiling water bath for 30 min.N-butanol was used to extract TBA-reactive compounds; and absorbance was measured at 532 nm.The standard was MDA; and the results were given in nmol/mg protein 25 . www.nature.com/scientificreports/Total antioxidant capacity (TAC) assay.Samples were assessed by investigating their ability to decrease Fe 3+ to Fe 2+ .The results were expressed in nanomoles per milligram (nmol/mg) of protein 25 .The aim of the lipid peroxidation and TAC assays was to assess the oxidative damage of Pb. Histological examination. Light microscopy (LM) examination. The aim was to assess the structural changes of the cerebrum.The cerebral samples were fixed in a 10% neutral buffered formalin solution; then tissues were processed for LM examination, and stained with hematoxylin and eosin (H&E) 26 .Five different nonoverlapped sections from each animal, were examined and evaluated under LM and photographed.Ten fields from 3 different non-overlapped horizontal sections from each rat were coded enabling blind examination and evaluation.Assessment images were analyzed for the following: (1) thickness of the frontal cortex; from each section, 20 perpendicular lines between the white and pia mater were quantified at 100× magnification, (2) total number of vesicular neurons (pyramidal or granular) per field; at 400× magnification 27 . Electron microscopy (EM) examination.The aim was to assess the ultrastructural changes of the cerebrum.Cerebral sections from each group were immediately placed in 3% glutaraldehyde in 0.1 M phosphate buffer for a few hours and post fixed in 1% osmium tetroxide for 1 h.Ultrathin sections of 50 nm were cut by an ultramicrotome from selected areas, were contrasted with uranyl acetate and Pb citrate 9 .Sections were examined under JEM-2100, EM unit; and images were captured using AMT CCD camera (software version AMTV600). Immunohistochemical analysis. GFAP immunohistochemical aimed to evaluate Pb effect on astrocytes. The streptavidin-biotin immunoperoxidase method was used for immunohistochemistry.The slides were then incubated overnight with primary the antibodies: polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP) delivered from Sigma Laboratories was used.Universal kit used avidine biotin peroxidase system produced by Novacastra Laboratories Ltd. Incubation with a secondary antibody and product visualization were performed with diaminobenzidine chromogen.Counterstaining with Mayer's hematoxylin and slides washing with distilled water and phosphate-buffered saline (PBS) were done.PBS was used instead of primary antibody as negative controls 28 .Ten fields from 3 different non-overlapped horizontal sections from each rat were coded enabling blind examination and evaluation under LM and photographed. Statistical analysis. The sample size was calculated to be 60 (10 in each group) according to Galal et al 9 . The exposed/unexposed ratio was 5/1, using open-EPI at confidence level 95% and power 80%.The SPSS 18.0 program was used to statistically analyze all the data.The Shapiro-Wilk test was used to examine if the data had a normal distribution.The mean values of various groups were compared using a one-way ANOVA, and multiple comparisons were evaluated using the Tukey HSD Post-hoc Test.Normal distributed data were reported as mean and standard deviation (SD) (Statistical Package for Social Science).Kruskal-Wallis H tests were used to compare the median values of the groups in non-normally distributed data, multiple comparisons were evaluated using the Mann-Whitney test, and non-normally distributed data were displayed as median (range).A value of P < 0.05 was accepted as statistically significant; and value of P < 0.001 was considered highly statistically significant 29 . Ethical approval and consent to participate.All studies and procedures involving rats were approved by Institutional Animal Care and Use Committee at Zagazig University in Egypt (ZU-IACUC/3/F/93/2020) and by the National Institute of Health (NIH) guidelines.The publication of this manuscript has been approved by all authors.We attest that we have studied the Journal's stance on matters pertaining to ethical publication and declare that our report complies with those standards. Results Regarding to body weight day before study, there was no significant difference (P: 0.97) between all groups.In contrast, rats treated with Pb only "low-Pb and high-Pb groups" and high Pb + Se group showed a significant decrease (P < 0.001) in Lwt, br, and br/Lwt% in comparison to control group.Combination of Pb with Se in low Pb + Se and high Pb + Se groups showed significant increase in Lwt, br and br/Lwt% in comparison with low Pb and high Pb groups.Comparing with control group and low Pb + Se group showed no significant difference, while high Pb + Se group showed a significant decrease in Lwt, br, and br/Lwt% in comparison with other two groups (Table 1). Low Pb, high Pb, low Pb + Se, and high Pb + Se groups showed a significant increase in cerebral DNA fragmentation (tailed, tail length, tail DNA and tail moment) compared to control group.Also, low Pb + Se and high Pb + Se groups showed significant decrease in cerebrum DNA fragmentation in comparison with low Pb and high Pb groups respectively (Table 2). Regarding to biochemical analysis for MDA, low Pb, high Pb, low Pb + Se and high Pb + Se groups showed significant increase in cerebral MDA in comparison with control group.Combination of Pb with Se in low Pb + Se and high Pb + Se groups showed a significant decrease in cerebral MDA in comparison with low Pb and high Pb groups respectively.On the other hand, low Pb, high Pb, low Pb + Se, and high Pb + Se groups showed significant decrease in cerebral TAC in comparison to control group.Combination of Pb with Se in low Pb + Se and high Pb + Se groups showed a significant increase in cerebral TAC compared to the low Pb and high Pb groups, respectively (Table 3). Examination of H&E-stained cerebral sections of rats of control and Se groups showed the well-known normal histological structure of the cerebral cortex, which was arranged in six successive layers: (1) the molecular layer underneath the meninges and consisted of homogenous neuropil and few cell bodies of neurons, (2) the external granular layer, (3) the external pyramidal layer, (4) the internal granular layer, (5) the internal pyramidal layer, and (6) the multiform layer was dominated by elongated spindle shaped cells.External cortical layers both granular and pyramidal were crowded with small cortical vesicular neurons while internal cortical layers have fewer but larger neurons.Neurons had rounded pale nuclei and little basophilic cytoplasm.Glial cells appeared small with small well-demarcated nuclei.The acidophilic background (neuropil) was composed of neuronal and glial cell processes and blood vessels with a narrow perivascular space (Fig. 1A-F). Table 1.Effect of Pb ± Se on body and brain weights in different study groups.One-way ANOVA, and Tukey HSD Post-hoc Test, P > 0.05: no significant differences, P < 0.05: significant differences, P < 0.001: highly significant differences.# % of difference = mean of (each) group-mean of control group)/mean of control group %, bwt body weight at the beginning of the experiment, Lwt last day body weight, br brain weight, br/ Lwt% brain weight/body wt. at last day% a Significant vs control group, b highly significant vs control group.c Significant vs low Pb group, d highly significant vs low Pb group.e Significant vs high Pb group, f highly significant vs high Pb group.Table 2. DNA fragmentation and biochemical analysis in different study groups.Photos of CCD camera show effects of Pb, Se and their combinations on cerebrum comet assay in male albino rats after 4 weeks of exposure.Blue arrows = intact cells; yellow arrows = cells with a shadow, indicating damaged DNA.# nmol/ mg, One-way ANOVA, and Tukey HSD Post-hoc Test, P > 0.05: no significant differences, P < 0.05: significant differences, P < 0.001: highly significant differences.a Significant vs control group, b highly significant vs control group.c Significant vs low Pb group, d highly significant vs low Pb group.e Significant vs high Pb group, f highly significant vs high Pb group.www.nature.com/scientificreports/Examination of H&E-stained cerebral sections of rats of low Pb group showed slight separation in pia matter with cortical layers still can be distinguished.Molecular layer was vacuolated.Most of neurons were with rounded pale nuclei and thin rim of basophilic cytoplasm while, other neurons were irregular in shape with darkly stained nuclei and surrounded by pericellular halos.Acidophilic neuropil contained blood vessels with slight wide perivascular spaces and numerous glial cells which appeared small with small well-demarcated nuclei.Moreover, some neuroglia showed sign of mitotic activity.High Pb group's H&E-stained cerebral sections revealed marked separation of pia matter with disorganization of the six less cellular layers of the cerebral cortex and large congested blood vessels.Most of neurons were irregular in shape, darkly stained nuclei, strong acidophilic cytoplasm and surrounded by pericellular halos while, few presented large rounded pale nuclei.Acidophilic neuropil showed blood vessels with wide perivascular spaces, vacuolation, and numerous glial cells with rounded nuclei and pale cytoplasm (Fig. 2A-F). Low Pb + Se and high Pb + Se groups' H&E-stained cerebral sections exhibited a relatively normal appearance with regular normally attached pia matter.Neurons had rounded pale nuclei and basophilic cytoplasm while, few neurons had darkly stained irregular nuclei surrounded by pericellular halos.Acidophilic neuropil showed few vacuolation and blood vessels with wide perivascular spaces.Sign of mitotic activity in some neuroglia was observed (Fig. 2G-L). Table 3. Morphometric analysis for H&E-stained cerebral sections in different study groups.One-way ANOVA, and Tukey HSD Post-hoc Test, P > 0.05: no significant differences, P < 0.05: significant differences, P < 0.001: highly significant differences.At 100 magnification, & at 400 magnification, # % of difference = mean of (each) group-mean of control group)/mean of control group %. a Significant vs control group, highly significant vs control group.c Significant vs low Pb group, d highly significant vs low Pb group.e Significant vs high Pb group, f highly significant vs high Pb group.Regarding to morphometric analysis for H&E-stained cerebral sections, low Pb, high Pb and high Pb + Se groups showed a significant decrease in frontal cortex thickness and vesicular neurons number/field in comparison to control group; while low Pb + Se group showed no significant difference with control group.Moreover, low Pb + Se and high Pb + Se groups showed significant increase in frontal cortex thickness and vesicular neurons number/field in comparison with low Pb and high Pb groups (Table 3; Fig. 2M,N). EM examination of cerebral sections of rats of control and Se groups revealed cortical neurons with vesicular euchromatic nucleus surrounded by regular nuclear envelope with a dark prominent nucleolus.Their cytoplasm contained well developed strands of rough endoplasmic reticulum, numerous mitochondria and free ribosomes.The astrocyte cells appeared with sharply demarcated nucleus and electron lucent cytoplasm containing mitochondria.The neuropil contained regular myelinated nerve fibers (Fig. 3A-F).Cerebral sections of rats of low Pb group revealed neurons with small heterochromatic nucleus surrounded by regular nuclear envelope with a dark prominent nucleolus.Cytoplasm was dark and contained lysosomes, free ribosomes, normal mitochondria, normal rough endoplasmic retinaculum (RER), and other dilated ones.The astrocytes contained sharp demarcated nucleus with chromatin and vacuolated cytoplasm containing swollen mitochondria losing its cisterna.In high Pb group, cortical neurons were shrunken with more lysosomes and swollen mitochondria losing its cisterna (Fig. 4A-F).In low Pb + Se group, the cerebral sections showed normal cortical neurons with lysosomes and swollen mitochondria, the neuropil contained normal mitochondria and myelinated nerve fibers some of them has irregularity in myeline sheath.On the other hand, high Pb + Se group's cerebral sections displayed cortical neurons with heterochromatic nucleus surrounded by irregular nuclear envelope and indentations (Fig. 4G-L). Examination of GFAP-stained cerebral sections of rats of control and Se groups showed positive GFAPstaining in the cytoplasm of astrocytes and their processes.The cells appeared small and few with short thin few processes.On the other hand, low Pb group showed cytoplasm and processes of astrocytes appeared variable size with thick, few, and short processes.In the low Pb + Se group, the cytoplasm and processes of astrocytes appeared variable in size with thin and long branching processes.Moreover, in high Pb and high Pb + Se groups, there was much positive GFAP staining in the cytoplasm and processes of astrocytes which appeared multiple with thick, long and branched processes (Fig. 5A-F). Discussion In recent years, environmental pollution with heavy metals has increased radically with the rapid development of modern industry.Excess amounts of heavy metals including Pb in animal feed and forage are often the result of human actions resulting from either agricultural or industrial production or accidental or intentional misuse 30 .The average human diet contains about 3 μg of Pb per day; 1-10% are really absorbed.In the presence of a nutritional deficiency, the degree of Pb absorption may even increase 31 .Pb poisoning is often caused by the consumption of high concentrations in drinking water 32 .Our study investigated the potential neuroprotective effects of Se against the neurotoxic effect of Pb in rats.We used male animals to avoid any potential effects of hormonal changes during estrous cycles that occur in females 33,34 . The Pb-treated groups (low Pb group and high Pb group) displayed significant decreases in body weight, brain weight and relative brain weight compared to control and Se groups; these results are consistent with those of Singh et al. 7 .However, concomitant use of Se with Pb improved the feeding status resulting in improvement of these anthropometric measures especially in low Pb + Se group.These results contradict those of Nehru et al. 35 who reported that Se administration has no effect on brain and body weight gain. The brain is the organ most affected by oxidative stress due to its high rate of oxygen consumption 36 .The level of oxidative stress was measured by TAC and MDA which are end oxidation product of polyunsaturated fatty acid peroxidation.An elevated MDA level is an important indicator of lipid peroxidation and thus an indicator of oxidative stress.Oxidative stress damages the cell membrane and alters its permeability.The biochemical results of the current study showed a significant decrease in TAC and a significant increase in the level of MDA in the Pb-treated rats in both the low Pb and high Pb groups.These results are in general agreement with other studies 7,10,37 .Our biochemical data reinforced that Se is a potent antioxidant as there was significant decrease in MDA level and significant increase in TAC level in rats of Pb + Se treated groups (low Pb + Se and high Pb + Se groups) compared to the Pb-only groups.These results are generally consistent with other studies investigating the role of Se in the alleviation of oxidative stress induced by chromium (VI), cypermethrin, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 13,38,39 .This antioxidant effect of Se is proved by Rahbardar et al. 40 , who confirmed increased glutathione content, and reduced MDA level.Moreover, these results agree with Zakeri et al. 6 who stated that Se has antioxidant, anti-inflammatory, and antiviral properties, and with Shahidi et al. 41 who showed that administration of Se to rats with Alzheimer disease led to an increase in the hippocampal TAC, a decrease in MDA level and an increase in recovered memory with improved cognition.These results are not consistent with those of Gholamigeravand et al. 42 who reported that Se administration did not significantly increase TAC, but reduced MDA. Separation of pia matter was detected in rats in low Pb and high Pb groups.This was demonstrated by AL-Mzaien et al. 30 who reported that this segregation may contribute to edema as a result of oxidative stress www.nature.com/scientificreports/resulting to a change in permeability leading to abnormalities in hemodynamics and fluid leakage into neural tissue.Some authors suggested that BBB disturbance is a causative mechanism in Pb neurotoxicity [43][44][45] .In case of co-administration of Se with Pb, the pia matter was found to be regular and normally attached in low Pb + Se group but still slightly detached in high Pb + Se group. Oxidative stress regulates apoptosis associated with the activation of the intrinsic pathway of apoptosis within the mitochondria.Polyunsaturated fatty acids in plasma and organelles are the target of oxidative agents.Oxidative DNA damage induced by ROS leads to cell injury 46 .In our study, Pb has a toxic effect on the cerebral cortex of rats by inducing apoptosis which has been demonstrated histologically and biochemically.Histological examination of brain sections stained with H&E showed a change in normal cerebral architecture in the low Pb and high Pb groups.Most of the cortical neurons were shrunken with dark stained nuclei and surrounded by a peripheral halo.The vacuolar halo may be attributed to cell shrinkage and regression of cell processes as a result of the strain of the cytoskeleton creating pericellular spaces.Moreover, reduction in the total number of cortical www.nature.com/scientificreports/neurons as well as thickness of frontal cortex morphometrically confirmed apoptosis.EM examination of rats' cerebral sections of Pb treated groups revealed small shrunken cortical neurons with heterochromatic nucleus.This finding is in harmony with Galal et al. 9 .The degenerative changes detected in the neurons as well as nuclear changes in the form of shrinking and darkening (pyknosis) seen in our study reflect a certain stage of apoptosis; this result is in accordance with Afifi and Embaby 47 .In addition, ultrastructural examination of cerebral sections of low and high Pb groups revealed that the cytoplasm of cortical neurons contained many lysosomes, swollen mitochondria losing their cisterna, normal RER and another dilated RER.The astrocytes contained sharp demarcated nucleus with chromatin and vacuolated cytoplasm containing swollen mitochondria losing cisterna.Wakabayashi 48 documented that swollen mitochondria with partial or complete loss of cristae might be correlated to oxidative stress.The detected mitochondrial changes may be measured as an early manifestation of apoptosis and an adaptive progression to undesirable surroundings due to extra exposure of the cell to free radicals at the level of intracellular organelles 49 .Biochemically, our study showed a significant increase in apoptosis index "DNA fragmentation"; this result agrees with Khalaf et al. 50and Shaban et al. 51 .This Pb-apoptotic effect was approved by Zhou et al. 52 who reported that Pb increases Bcl-2 associated X protein (Bax) expression and the ratio of Bax to B-cell lymphoma-2 (Bcl-2) "Bax/Bcl-2". The co-administration of the Se with the Pb produced different degrees of improvement in the histopathological changes of H&E-stained sections of rat's cerebral cortex, and protected cerebrum from Pb-apoptotic effect.This was signified by improvement in neuronal status in several areas with moderate restoration of the normal cerebral cortex organization layers.These results are in harmony with others 14,53,54 .This was confirmed by increased number of vesicular neurons number neurons and thickness of frontal cortex morphometrically.Also, EM examination of cerebral sections of rats of Pb and Se treated groups (low Pb + Se and high Pb + Se groups) revealed improvement in cortical neurons status.Cortical neurons with heterochromatic nucleus and a dark prominent nucleolus.The nuclear envelope was regular in (low Pb + Se group) and irregular showing indentations in (high Pb + Se group).Their cytoplasm was dark containing lysosomes, normal mitochondria, free ribosomes, normal RER, and little dilated RER.The Se-protective effect is confirmed by a significant decrease in DNA fragmentation percentage in groups treated with Pb and Se (low Pb + Se and high Pb + Se groups).Our results in DNA fragmentation are in synchronization with those of Khalaf et al. 55 and Sadek et al. 56 .Other authors have said that Elsie is a double-edged sword with both beneficial and toxic effects 57,58 .Despite its importance in cell functions and as an antioxidant, excess intake can be fatal, leading to a serious disease called selenosis.Moreover, excessive exposure to Se even at low levels may lead to adverse effects on human health 58 . Astrocytes are a target of Pb toxicity 59 .GFAP is an intermediate filament protein which is expressed by several cell types of CNS including astrocyte cells.GFAP is involved in some significant CNS functions, like cell communication and the effectiveness of the BBB.Also, GFAP has been shown to play an important role in mitosis by regulating the filament network present inside the cells 60 .Sections stained with GFAP immunohistochemistry of the Pb-treated groups showed rich positive GFAP staining of cytoplasm and astrocyte processes.The cells were increased in number and appeared larger with multiple thick processes in comparison to control and Se groups.GFAP is an indicator protein for astrogliosis.Astrocytes make up the majority of the neuroglial cells, which respond rapidly to many neurodegenerative changes, resulting in marked astrogliosis 61 .This result agrees with other studies reporting that the increase in GFAP-immunopositive astrocytes is a compensatory mechanism against neuronal cell damage 62,63 .In contrast to our study, Cai et al. 58 reported that the GFAP-immuno-positive astrocyte numbers were significantly decreased after Pb acetate exposure.The cells appeared small with few short thin processes in control and Se groups compared to Pb-treated groups.This agrees with the study of Ibrahim et al. 64 who stated that the GFAP reaction was significantly increased in the cyclophosphamide group and significantly decreased when combined with nano-Se. Study limitations include using only a single dose of Se administered as 0.25 mg/kg/day to examine its protective role against cerebral Pb poisoning.However, our main goal was to investigate its potential efficacy against neurotoxicity of both low and high doses of Pb exposure through various methods including biochemical and immunohistochemical and histological screening.Although Se is essential to life, its safety is limited with regard to its dosage levels.In other words, toxic dose levels are close to those normally required for the body's needs or appropriate treatment 11 .Therefore, determining its dosage is crucial.Future studies to investigate different doses of Se in this regard are recommended. In conclusion, the oxidative stress and apoptosis pathways have important roles in Pb-neurotoxicity.The Se is suggested to significantly improve Pb-induced histopathological changes by modulating oxidative stress and apoptosis.However, it is recommended for use in humans after more prospective experimental studies are conducted to evaluate the most effective doses that could be taken as prophylaxis or treatment for lead neurotoxicity. Figure 1 . Figure 1.H&E photomicrographs of the cerebral tissues.Control (A-C) and Se (D-F) groups.(A,D) Layers of the cerebral cortex: (I) the molecular layer, (II) the external granular layer, (III) the external pyramidal layer, (IV) the internal granular layer, (V) the internal pyramidal layer, (VI) the multiform layer, in addition to white matter (WM), blood vessels (BV), thin and normally attached pia matter (arrow) are noticed.External layers (B,E), and internal layers (C,F) are presenting cortical neurons (N) with rounded pale nuclei and basophilic cytoplasm.Acidophilic neuropil () shows blood vessels (BV) with narrow perivascular spaces (arrowheads) and neuroglia cells (G) that are smaller with small well-demarcated nuclei (bar A,D: 200 μm ×100-B,C,E,F: 50 μm ×400). Figure 2 . Figure 2. H&E photomicrographs of the cerebral tissues.Low Pb group (A-C), high Pb group (D-F), low Pb + Se group (G-I), high Pb Se group (J-L).(A,G,J) Micrographs show layers of the cerebral cortex I-VI.(D) Micrograph show severe disruption nearly in all layers (CL) with vacuolated (v) molecular layer (I).Pia matter (arrow) was irregular and separated in (A,B,D) and regular attached in (G,H,J,K).WM white matter, Bv blood vessels, CBV congested blood vessels, arrowheads wide perivascular spaces, HG hemorrhage, N cortical neurons with rounded pale nuclei and thin rim of basophilic cytoplasm, n neurons are irregular in shape with darkly stained nuclei and surrounded by pericellular halo, * acidophilic neuropil, G glial cells with rounded nuclei and pale areas of cytoplasm, most probably astrocytes, mitotic activity in some neuroglia (circle) (bar A,D,G,J: 200 μm ×100-B,C,E,F,H,I,K,L: 50 μm ×400).(M,N) Charts show quantitative assessment of the cortical thickness and total no. of neuron/filed.Data were expressed as mean ± SD. Figure 5 . Figure 5. Immunohistochemical staining for anti-GFAP (A control group; B Se group; C low-Pb group; D high-Pb group; E low Pb + Se group; F high Pb + Se group).The stained cerebral sections show a positive GFAPstaining in the cytoplasm and processes of astrocytes (arrow) which appear small with thin, few short in groups (A) and (B), variable size with thick, long, branched processes in group (C), multiple, thick, long, branched processes in group (D), variable size with thin, long branched processes in group (E), appear with multiple, thick branched processes in group (F) (bar A-F 50 μm ×400).	2023-09-23T06:17:40.442Z	2023-09-21T00:00:00.000	{ "year": 2023, "sha1": "9344bdefdd037aab616c574edc3ae42ad5dd70a0", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-023-42319-3.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "94663be003575363a4527e3134ed2bd5e66ebd10", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
208916979	pes2o/s2orc	v3-fos-license	(E)-N′-Benzylidene-5-methylisoxazole-4-carbohydrazide The molecule of the title compound, C12H11N3O2, is approximately planar with an r.m.s. deviation of 0.0814 Å from the plane through all the non-H atoms. The dihedral angle formed by the benzene and isoxazole rings is 6.88 (16)°. The molecular conformation is stabilized by an intramolecular C—H⋯N hydrogen bond, forming an S(6) ring, and the molecule displays an E configuration with respect to the C=N double bond. In the crystal structure, intermolecular N—H⋯O hydrogen bonds form centrosymmetric dimers which are further linked by weak C—H⋯N interactions augmented by very weak C—H⋯π contacts, forming layers parallel to (120). The molecule of the title compound, C 12 H 11 N 3 O 2 , is approximately planar with an r.m.s. deviation of 0.0814 Å from the plane through all the non-H atoms. The dihedral angle formed by the benzene and isoxazole rings is 6.88 (16) . The molecular conformation is stabilized by an intramolecular C-HÁ Á ÁN hydrogen bond, forming an S(6) ring, and the molecule displays an E configuration with respect to the C N double bond. In the crystal structure, intermolecular N-HÁ Á ÁO hydrogen bonds form centrosymmetric dimers which are further linked by weak C-HÁ Á ÁN interactions augmented by very weak C-HÁ Á Á contacts, forming layers parallel to (120). Experimental Cg1 is the centroid of the C7-C12 ring. Data collection: APEX2 (Bruker, 2004); cell refinement: SAINT (Bruker, 2004); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97. (Fun et al. 2008;Wei et al. 2009;Khaledi et al. 2008) and their biological activity (Molina et al. 1994;Khattab 2005) and coordination ability (Reiter et al.1985) have also been noted. Isoxazole compounds have also attracted much interest as they exhibit some fungicidal, plant-growth regulating and antibacterial activity (Stevens et al.1984). In order to study the properties of a new compound containing both the hydrazine and isoxazole groups, we synthesized the title compound and report its crystal structure here, Fig 1. The title compound C 12 H 11 N 3 O 2 , is approximately planar (rms deviation 0.0814 from the plane through all non-hydrogen atoms)with a dihedral angle of 6.88 (16)°. between the C7···C12 benzene and C2···C4,N1,O1 isoxazole rings. The molecule displays an E configuration with respect to the C6═N3 double bond, with a C7-C6-N3-N2 torsion angle of -179.3 (3)°. The dihedral angle formed by the benzene and isoxazole rings is 2.457 (114)°. An intramolecular C-H···N hydrogen bond generates an S6 ring motif (Bernstein et al. 1997) and locks the molecule into a planar configuration. Bond lengths (Allen et al.,1987) and angles are unexceptional and similar to those found in related stuctures (Fun et al., 2008;Wei et al., 2009;Khaledi et al., 2008). Experimental Benzaldehyde (4.6 g,0.02 mol) and 5-methylisoxazole-4-carbonyl hydrazine (2.8 g, 0.02 mol) was mixed with glacial acetic acid (50 ml). The mixture was heated at 65° C for 3 h, the precipitate collected by filtration and washed with water, chloroform and ethanol. The product was recrystallized from ethanol, then dried under reduced pressure to give the title compound in 85% yield. Colourless, block-shaped crystals were obtained by slow evaporation of a dimethylformamide solution. Refinement The H atom bound to N2 was located in a difference Fourier map and refined freely with the N-H distance restrained to 0.90 Å. All other H atoms were positioned geometrically and allowed to ride on their parent atoms, with C-H = 0.93-0.96 Å, and with U iso = 1.2 U iso (C) or 1.5 U iso (C) for methyl groups.	2014-10-01T00:00:00.000Z	2009-12-12T00:00:00.000	{ "year": 2009, "sha1": "b45724693fba653351c2a2c1c30cd52e21200600", "oa_license": "CCBY", "oa_url": "http://journals.iucr.org/e/issues/2010/01/00/sj2706/sj2706.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "e135cb369e0f179329b5a2ffc4ef2031ee68c694", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Medicine", "Computer Science", "Chemistry" ] }
236419641	pes2o/s2orc	v3-fos-license	Effects of physical and chemical properties on the dissolution of sea salt Use your smartphone to scan this QR code and download this article ABSTRACT Salt plays a crucial role in human health. However, excess use of NaCl in food products can be harmful to health. One suggestion for this problem is optimization salt dissolution to increase the content of salt ions in themouth. For this purpose, it is important to understand the solubility properties of salt crystals in saliva. The dissolving process is not only affected by the physical properties but also by the chemical composition of the salt. This study compared the solubility of four commercial grain salts in four regions in Vietnam (Bac Lieu, Thanh Hoa, Sa Huynh, Vung Tau), one flower salt in Sa Huynh and a control sample with two particle sizes 1 2mm and 2 3mm in a Saliva Artificial Gal – Fovet solution (SAGF). Dissolutionwas determinedby analyzingmicroscopic images taken by the time and analysis by Bayesian and Partial Least Squared methods. The research evaluated the influence of physical properties (area, Feret's diameter, circularity, aspect ratio and solidity) and chemical compositions (sodium, potassium, magnesium, calcium and moisture content) on the dissolving process. Salt samples showed significant differences in physical and chemical properties by region. Morphological parameters are affected by conditions of salt crystallization that indicated through region of origin. Dissolution is evaluated through solubility coefficient, Sa Huynh flower salt and control salt have the highest solubility coefficient, simultaneously, it is also the smallest value of roundness and surface index. The projected area, magnesium and sodium content are the factors which strongly affecting on dissolution of salt samples. These results demonstrated the possibility to exploit these factors to adjust the solubility of salt as well as the perceived salinity over time. INTRODUCTION Sodium is an essential nutrient for maintaining blood plasma, acid-base balance, transmit nerve impulses and participate in necessary functions for human cells 1,2 . Sodium is found naturally in many foods, such as milk, meat, and seafood. Besides, in some processed foods, snacks, spices (sodium glutamate) are also sources of sodium for the body 3 . Nowadays, most people are consuming too much salt, averaging 9 to 12 g per day, which is approximately twice the maximum recommended intake (World Health Organization, 2014). High sodium in the diet can lead to high blood pressure, cardiovascular disease, and stroke 4,5 . It can also cause calcium losses, some of which may be pulled from bone 6 . Therefore, it is necessary to develop strategies that reduce the level of sodium intake while maintaining the salt taste perception in products. There are many different suggestions for increasing salt salinity, such as using salt alternatives or altering the chemical composition by adding a salt additive, thereby reducing sodium intake body 7 . Previous studies showed that from 70 to 95% sodium (or NaCl salt) can remain in food matrix after a consumer has swallowed it 8,9 . For dry foods that are salted directly to the surface, a significant amount of sodium can be swallowed completely without the consumer perceiving the salty taste. The difference in salty taste is actually caused by the dissolution of the salt in the mouth by saliva. In it, saliva acts as a solvent to distribute ions in salt to the taste receptors on the tongue 10 . Therefore, the dissolving process of salt will directly affect the taste of salt in consumers. Several methods have been developed to study salt dissolution in the mouth e.g. installing ionic electrodes in the mouth. However, this technique has limitations as the levels recorded may not fully reflect the signal received by the taste buds. Furthermore, having a string in the mouth can make chewing difficult 11 . For this reason, in vitro methods have been developed with easier implementation while still providing data for screening 12 . These methods developed systems that simulate conditions in the mouth and the use of natural or artificial saliva in experiments in which salt solubility is measured using a conductivity probe or observation system. By this method, Vella, D. et al (2012) found a significant difference between the size of salt particles and its close correlation with the dissolution rate 12 . Beside that, Quilaqueo, M. et al (2015) analyzed the dissolution rate of the salt by video microscopy images taken at different times. The results revealed that the solubility rate of salt crystals in water is higher than in artificial saliva and at higher temperatures. The increased surface area after fragmentation results in pyramid-shaped crystals having the highest dissolution rates 8 . Hence, significant changes in salt dissolution can be achieved depending on its crystal structure and soluble form. In order to optimize the salty taste felt in the mouth, it is essential to understand the solubility of salt in saliva. The differences in salt origin, raw seawater sources and production specifications lead to sea salt having different particle sizes and shapes that result in the difference of salty taste 13 . Besides, the difference in mineral composition such as potassium, calcium, and magnesium also affects the salty taste of salt 14 . It can be explained by the difference in dissolution kinetics. This research focused on correlation and influence of physico-chemical and particle shape on the dissolution of sea salt in some region in Vietnam. From there, it can shown the characteristics of sea salt in different geographical regions in Vietnam. Normally, conventional linear regression is used to estimate the parameters of equations and their confidence amounts. However, this paper used Bayesian statistics as an alternative method with many advantages over conventional regression method 15,16 . The Bayesian method can be basically applied to approach the linear model for the nonlinear first-order model, or any other model that is deemed suitable 15 . Materials Five commercial salt samples and one control were used to evaluate physico-chemical, morphological properties and solubility. Commercial salt samples were collected from other production areas (Table 1): four samples of grain salt from Thanh Hoa (Hoa Loc, Hau Loc Province), Sa Huynh (Duc Pho Province, Quang Ngai), Vung Tau (An Ngai, Long Dien Province), Bac Lieu (Long Dien, Dong Hai Province) and sample of Sa Huynh flower salt (Duc Pho Province, Quang Ngai). Samples are stored at room temperature under conditions of low humidity. Each salt (500 g) is sieved for 20 min and the material between sieves net of size 3.00 mm, 2.00 mm and 1.00 mm is used for analysis dynamics of the dissolution process. This size is present in all samples and is sufficient to be observed with a microscope. The classification of salt samples into two particle sizes helps to minimize errors when analyzing the physical properties and the effect of particle size factors on the dissolution process. The salt samples after sieving are vacuum packed and stored. Prepare a control sample Take 371g of 99.5% pure NaCl dissolved in distilled water at 60 • C. Then filter the solution through the filter paper and then pour it into the stainless steel trays (600 x 400 x 35 mm) so that the water level reaches 3 cm. Crystallize this solution in an oven at 70 • C for two days. After crystallizing, the residual brine is removed and the salt is dried to constant weight within the next 12 hours before being packed for storage. Prepare a solution of artificial saliva Artificial saliva (SAGF) was described by Gal & Fovet (1998) 12 . The artificial saliva mixture was stirred for 1 hour at 37 • C on the magnetic stirrer. The SAGF solution was tested to ensure a pH of 6.8 and a conductivity of 530 -560 µS/cm. Analysis of chemical composition The moisture content of salt was determined by MB90 type infrared moisture analyzer (Ohaus, USA), drying at 130 • C until constant weight. The mineral composition including sodium, potassium, magnesium, calcium and magnesium in salt was determined by flame atomic absorption spectroscopy (VISTA-pro, Varian Canada, Mississauga, ON, Canada). Samples were analyzed in 3 replicates. Analysis of morphological parameters The morphology of the salt was assessed by analyzing the images obtained with an optical microscope. The salt particles were placed on a concave slide and observed under the microscope (OPTIKAM-B9, Optika, Italy) and the images obtained with the Optikam digital camera (OPTIKAM-B3, Optika, Italy) were combined with a microscope and using OptikaView software (Optika, Italy). The captured images were processed and analyzed using ImageJ 1.8.0 software (National Institutes of Health, USA). Parameters describing 2D shape and dimension are determined based on the image analysis. These parameters include: projected area (A); roundness (C = 4π.(A/P 2 )) where P is projected perimeter; Maximum ferrite diameter (Fmax is the maximum distance between two parallel tangent lines of a particle projected), aspect ratio (AR = Spindle / Minor axis of the circumcircle), surface index (S = A / Ac) where Ac is the area of the smallest circumcircle 17 . Dissolution process analysis in artificial saliva solution Dissolution analysis method includes image analysis of microscopic images obtained from Optikam digital cameras (OPTIKAM-B3, Optika, Italy) combined with microscopes (OPTIKAM-B9, Optika, Italy). Image sequences were collected using OptikaView software (Optika, Italy) and recording was started when a grain of salt was placed on a concave slide. Immediately 500 µL of a previously preheated SAGF solution at 37 • C was added. The chosen temperature is close to human body temperature (37 • C) because many salty foods are consumed in this range. The captured images were analyzed using ImageJ 1.8.0 software (National Institutes of Health, USA) and converted into 8-bit images, sharpened and adjusted to the color threshold. The dissolution rate is calculated based on the reduction of the projected area of the salt particle over time and the dissolution kinetics are described by the following three models 8,18 : Zero-order model, has the following equation: First-order model, has the following equation: Hixon Crowell model, has the following equation: Where A t is the projected area of the insoluble salt particles at the time t(s), K 0 , K 1 K S (s −1 ) is the solubility coefficient and A 0 is the initial projected area. Statistical methods Differences between samples were determined using ANOVA analysis and LSD test with R software version 4.0.2 (CRAN) 19 . Correlation between morphological parameters and chemical composition was analyzed by Principle Components Analysis -PCA. The dissolution results were calculated by constructing the Bayesian regression equation and the relationship between physical and chemical properties and dissolution was determined by Partial Least Squared -PLS. Table 2 shows the chemical composition of salt samples. Depending on the production area, other mineral components such as K, Mg and Ca were also found in the samples apart from Na. Those non-Na elements have been found to contribute to the salty taste [20][21][22] . The sodium content varies between samples from different production regions. Vung Tau grain salt has the highest sodium content (337 to 339 mg/g), while Sa Huynh flower salt has the lowest content (287 to 289 mg/g). Control salt contains few minerals than other samples, while flower salt and Sa Huynh seed salt have the highest total content of other minerals. Humidity showed significant differences between the types of salt (Table 2). Flower salt and Sa Huynh grain salt with the highest humidity (from 10.3% to 13.7%) differ significantly from other salts (from 3.30 to 8.51%). In general, most commercial grain salts have a low moisture content, ranging from 3.30% to 8.51%, which is consistent with the current moisture content requirement of no more than 10%. Table 3 compares roundness and surface index between salt samples. Statistically, Sa Huynh flower salt had the smallest area value. The roundness of the salt is affected by the salt crystallization. If the salt samples are crystallized in stable conditions, the salt particles grow evenly between the edges, creating a cube shape and a flat surface, making the roundness and surface index small. The grain salt samples have more angular shapes like convex polygons due to crystallization under unstable conditions with higher roundness and surface index (Figure 1). In terms of frame rate, Sa Huynh flower salt (Figure 1c, Figure 1d) and Sa Huynh grain salt (Figure 1g, Figure 1h) have the highest value, and samples of grain salt in other production areas such as Bac Lieu (Figure 1a, Figure 1b), Thanh Hoa (Figure 1i, 1j), Vung Tau (Figure 1k, Figure 1l) have smaller aspect ratio. Dissolution analysis results The kinetics of the solubility of salt granules in artificial saliva solution shows that there are differences between the different salts. Figure 2 shows the reduction of the projected area with time dissolving in the artificial saliva solution of each salt sample. Two salt samples of Vung Tau (VT1 and VT2) and Bac Lieu (BL1 and BL2) exhibited very slow reduction in particle projected area with dissolution time and the linear graph is similar to that of the zero-order model. This result is similar to observed in previous study for salt samples with block shape having slower dissolution rate compared to pyramid-shaped samples 8 . On the other hand, Sa Huynh flower salt (FS1 and FS2) and reference salt (M1 and M2) tended to dissolve faster, the salt particle projected area of these samples decreased very quickly, with sugar dissolution graph. Curved is suitable for the first order model. When dissolved in artificial saliva solution, these salt samples have fragmentation phenomenon, increasing the surface area in contact with saliva and dissolving rapidly. Fragmentation of the salt grain may be related to the crystal's internal structure. Samples of grain salts without fragmentation during dissolution indicated that these samples were structurally tight. In contrast, Sa Huynh flower salt has a porous, lightgrain structure with many pores so fragmentation is observed during the dissolution. In addition, samples of Thanh Hoa grain salt (TH1 and TH2), Sa Huynh (SH1 and SH2) have curve-shaped solubility graph but do not show the nonlinear model clearly, it is possible to apply Hixon Crowell model to show the best solubility of all salt samples 16 . The solubility coefficient results of salt samples calculated from the regression equation of Hixson Crowell model by Bayesian method are presented in Table 5. Samples of Sa Huynh flower salt and control salt have the highest solubility coefficient. While the salt samples in Bac Lieu and Vung Tau have a lower solubility coefficient than other salt samples. This is shown when the greater the solubility coefficient, the faster the salt dissolves or the shorter the total dissolution time, and vice versa. This result also shows the effect of particle size on the dissolution rate when larger salt samples (BL2, FS2, M2, SH2, TH2 and VT2) have lower solubility coefficients. Discussion on characteristics Correlation between physical and chemical properties. The analysis results from the PCA correlation circle showed a very large correlation between the roundness of the grain and the chemical composition of the salt (Figure 3). This result clearly shows the specifics and differences in physical and chemical properties in the two samples of flower salt and Sa Huynh grain salt compared to the remaining salt samples, which are AR and moisture, magnesium, potassium content. At the same time, morphological parameters also showed that projected area parameters and maximum ferrite diameter also have a very clear positive correlation. Most physical properties exhibit an inverse correlation with the potassium, magnesium and moisture content, while positively correlating with the sodium and calcium content. This shows that during natural crystallization, the purer the salt samples or the higher their sodium content, the shape and size of the salt also differ from those with lower sodium content. Salt samples also showed significant differences in physical and chemical properties by region of production. Sa Huynh flower salts and grain salt have high levels of magnesium, potassium, and moisture, which are very round. Salt from Vung Tau, Bac Lieu has high sodium content, high roundness and surface index. Thanh Hoa salt showed a distinct difference in the high calcium content. Evaluate the influence of chemical and physical properties on the solubility of salt To evaluate the effect of physical and chemical properties on the salt solubility, we conduct PLSR analysis with solubility coefficient calculated from the regression equation according to Bayesian method as dependent variable and 5 physical parameters (Area, Feret's Diameter, Circularity, Aspect Ratio and Solidity) as well as 5 chemical parameters (sodium, potassium, magnesium, calcium and moisture content) as independent variables. The result of RMSEP shows that when evaluating combinations of factors, the combination of 6 factors shows the best explanation due to the lowest RMSEP value of the 10 combinations is considered (Figure 4). The analysis results of Variable Importance in Projected -VIP coefficients in the combination of 6 factors showed a clear influence of physical and chemical properties which is presented in Table 6. The results showed that the chemical composition has a great influence on the dissolution process when the sodium, magnesium, potassium and calcium contents have the highest VIP coefficients of the factors assessed. In there, magnesium showed the highest influcence on dissolution process. This is possible because magnesium binds hydration water more tightly than calcium, potassium, and sodium when dissolved, as a result, the hydrated magnesium cation is difficult to dehydrate. Among physical properties, Area parameter has the highest coefficient, proving that this is an important physical factor to consider when evaluating the solubility of salt. The results also show that the moisture content also has a slight influence on the dissolution process as the moisture's VIP coefficient is clearly different from the remaining physical parameters. CONCLUSIONS The results of the solubility of salt samples from 1 to 2 mm and 2 to 3 mm in size in artificial saliva solution have shown that salt type, morphology and chemical composition are influential factors affecting dissolving speed. Flower salts dissolve much faster than grain salt. Projected particle area parameters and sodium, potassium, calcium, and magnesium content are the main physical and chemical factors that have great correlation with the solubility process. The mineral composition influences the taste perception of salt (potassium is acidic and magnesium is bitter). Therefore, the adjustment of salt crystallization in production and the chemical composition change the sensory properties of salt. This problem required further, study on the effect of the physical and chemical factor on the salinity sensory properties of salt samples of different origin.	2021-07-27T00:05:15.652Z	2021-05-29T00:00:00.000	{ "year": 2021, "sha1": "249d0d725a27bbfd499c580c86fcb3c984f131ca", "oa_license": "CCBY", "oa_url": "http://stdjet.scienceandtechnology.com.vn/index.php/stdjet/article/download/822/1134", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "80ac1c045e54ed4aeb66453a9194b80bbeb7b422", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Chemistry" ] }
4953927	pes2o/s2orc	v3-fos-license	An internet-based intervention for people with psychosis (EviBaS): study protocol for a randomized controlled trial Background Evidence shows that internet-based self-help interventions are effective in reducing symptoms for a wide range of mental disorders. To date, online interventions treating psychotic disorders have been scarce, even though psychosis is among the most burdensome disorders worldwide. Furthermore, the implementation of cognitive-behavioral therapy (CBT) for psychosis in routine health care is challenging. Internet-based interventions could narrow this treatment gap. Thus, a comprehensive CBT-based online self-help intervention for people with psychosis has been developed. The aim of this study is the evaluation of the feasibility and efficacy of the intervention compared with a waiting list control group. Methods The intervention includes modules on delusion, voice hearing, social competence, mindfulness, and seven other domains. Participants are guided through the program by a personal moderator. Usage can be amended by an optional smartphone app. In this randomized controlled trial, participants are allocated to a waiting list or an intervention of eight weeks. Change in positive psychotic symptoms of both groups will be compared (primary outcome) and predictors of treatment effects will be assessed. Discussion To our knowledge, this project is one of the first large-scale investigations of an internet-based intervention for people with psychosis. It may thus be a further step to broaden treatment options for people suffering from this disorder. Trial registration NCT02974400 (clinicaltrials.gov), date of registration: November 28th 2016. Electronic supplementary material The online version of this article (10.1186/s12888-018-1644-8) contains supplementary material, which is available to authorized users. Background Schizophrenia and other psychotic disorders are severe mental disorders with heterogeneous symptom profiles encompassing positive symptoms such as persecutory delusions and auditory verbal hallucinations as well as negative symptoms such as social isolation and avolition [1]. In addition, they are accompanied by neuropsychological impairments in attention, memory, and executive functioning [2][3][4][5][6]. Sleep is impaired in the majority of people experiencing persecutory delusions [7] and levels of worrying are high [8,9]. Besides symptoms, stigmatization is a major source of distress in people diagnosed with schizophrenia [10], even in the context of mental health care [11]. Lifetime prevalence of schizophrenia is about 1% and stable across different regions of the world and cultures [12]. Schizophrenia is accompanied by an enormous individual and societal burden [13,14] and lies on position eight of the leading causes of disability-adjusted life years in 15-to 44-year-olds [15]. About 65% of individuals with a first episode relapse during the subsequent three years [16], resulting in inpatient costs about two to five times higher compared to non-relapsed patients [17]. As a complementary or alternative treatment option to antipsychotic medication [18], cognitive behavioral therapy for psychosis (CBTp) has emerged as an evidencebased treatment option for patients with schizophrenia and related disorders [19][20][21][22][23][24]. CBTp targets psychological mechanisms of symptom formation and maintenance that were primarily identified or corroborated using experimental psychopathology research [25][26][27]. The therapeutic framework and techniques of CBTp are to a large extent similar to those of cognitive behavioral therapy (CBT) for depressive or anxiety disorders (cognitive restructuring, reality testing, etc.). For example, the distress (consequence) related to hearing voices (situation) is assumed to be determined not by hearing voices per se, but predominantly by automatic thoughts and the according belief system. Consequently, alternative helpful beliefs about voices established with the help of cognitive techniques are supposed to result in less distress [28]. CBTp is likely to be effective for patients who choose not to take antipsychotic medication, too [29]. In regular mental health care, the effectiveness of CBTp has also been asserted [30], and neurocognitive deficits, comorbidity and poorer functioning pose no barrier to improvement during CBTp [28]. Consequently, national regulations such as the United Kingdom National Institute for Health and Care Excellence (NICE) guideline recommend that CBTp should be offered to every person with psychotic symptoms [31]. Acceptance and Commitment Therapy (ACT) focuses on noticing rather than changing thoughts and feelings [32]. ACT seems to be effective in treating mental health problems [33]. In schizophrenia, ACT helps people to cope with psychotic experiences using strategies such as cognitive distancing, which is characterized by learning to see one's belief as a hypothetical statement rather than a fact. Instead of trying to change, modify, or control odd cognitions or disturbing sensory states, patients are encouraged to instead simply be aware of these experiences [34]. A meta-analysis showed a mediumsized effect of ACT on symptoms of psychosis [35]. The third type of treatment is the Metacognitive Training for psychosis (MCT), developed specifically for people with schizophrenia [36,37]. MCT invites participants to critically evaluate cognitive biases such as jumping to conclusions and overconfidence in their thinking (metacognition). These biases might increase the likelihood of psychotic symptoms [38]. Studies show that MCT is efficacious in reducing psychotic symptoms [39,40]. Despite the availability of evidence-based treatments for schizophrenia, 69% of patients remain untreated in countries with low and middle income [41]. In particular, the need for psychosocial treatments including CBTp remains unmet [42]. Even in highly developed countries such as the United Kingdom or Germany, the treatment gap for schizophrenia is large. In theory, the NICE guidelines proclaim that CBTp is mandatory for the treatment of psychosis [31]. In practice, more than 50% do not receive even a single session of CBTp [43]. In Germany, CBTp is virtually not represented in the mental health service [44]. To sum up, CBTp is effective, recommended, and has great potential to alleviate psychological distress, but only a small fraction of patients with psychosis receives CBTp. Internet-based cognitive behavioral therapy (iCBT) can help to overcome treatment gaps in many mental disorders [45]. In several psychological disorders, including anxiety and depression, internet-based treatments have proven to be efficacious and effective in randomized controlled trials (RCTs; for a comprehensive review, see [46]). Most of the growing body of evidence comes from studies evaluating guided internet-based self-help treatments. While patients work their way through a structured selfhelp program that is typically based on CBT manuals, therapists or coaches assist and support them via a secured e-mail system. Meta-analyses on internet-based treatments show a superiority of guided interventions in comparison to unguided, automated programs in terms of efficacy, adherence to treatment, and drop-out rates [47][48][49]. Main advantages of guided internet-based treatments include: (1) low-threshold accessibility, (2) flexible usage independent of time and place at a selfdetermined pace, (3) high levels of anonymity and privacy (which is an attractive feature for many persons with a mental disorder due to their fear of stigmatization) and (4) low costs of delivery to large populations [50]. People diagnosed with schizophrenia use the internet [51] and are able and willing to use mental health services on the internet, such as peer-to-peer support [52]. The feasibility of internet-based treatments for people with psychosis (iCBTp) is well documented for webbased interventions [53,54] and also reported for smartphone interventions [55]. However, current internetbased programs differ in their comprehensiveness and focus. For instance, mixed results have been reported regarding the efficacy of internet-based psychoeducation programs [56], and the efficiency of internet-based programs targeting medication management [53,57]. There is a pilot study on a more comprehensive web-based, CBTp-oriented program for auditory verbal hallucinations, but this program was delivered via computers in mental health care centres (and not online). The study provided promising results using an uncontrolled pre-postdesign (Cohen's d = 0.58) [58]. None of the 21 participants with schizophrenia reported that the program was unhelpful and the authors report no adverse events, highlighting the feasibility of iCBTp in a computerized self-help format. A recent investigation of aforementioned program in an RCT design showed a comparable effect of the web-based intervention and usual care on levels of auditory hallucinations [59]. The study was able to show that patients with schizophrenia who used the web-based program, however, had increased significantly in social functioning and their knowledge about CBTp was larger than of those who did not use the program. In another study that investigated iCBT for people with schizophrenia, the web-based program specifically targeted comorbid depressive symptoms. The intervention lead to a significant decline in depression severity [60]. In summary, there is preliminary evidence that iCBTp for people diagnosed with schizophrenia could be beneficial. However, to the best of our knowledge, no larger trials on comprehensive treatments have been conducted. The overarching goal of this RCT is to evaluate a guided internet-based self-help intervention for people with psychosis. We developed a web-based program that is comprehensive in many respects: The program is not only based on CBT but also includes elements from its third wave, specifically ACT and MCT [34,36]. Schizophrenia patients often have comorbidities, such as depression, which should be addressed in an appropriate treatment [61]. This program offers additional interventions for such comorbidities. Disrupted sleep and worrying, among other secondary symptoms, are crucial in the formation and maintenance of psychotic disorders [25]. These factors are considered in the intervention as well. According to a review, the effects of smartphoneenhanced self-help are promising [62]. The intervention therefore includes an accompanying smartphone app for access in symptom-relevant situations in daily life. The app is expected to facilitate a transfer of skills to real world settings. Finally, a specific goal of the intervention was not to overstate negative consequences of the disorder [63] and solely focus on deficits, but to specifically target resources of the participants [64]. Treatment adherence in schizophrenia has been a well discussed topic predominantly in medication treatment [65]. But also in psychological treatments, rather high dropout rates are reported (e.g. prematurely terminated treatments by 45% of patients) [66]. This led us to look for factors that might influence treatment adherence. Among others, suggested mediators are treatment motivation [67] and working alliance with the therapist [68]. Overall, the study tests whether a comprehensive internet-based self-help program with an accompanying smartphone app reduces symptomatology in people with schizophrenia. Study design The study is an RCT of parallel design comparing the efficacy of guided internet-based self-help treatment for patients with schizophrenia to a waiting list control group ( Fig. 1). Participants in the control group receive access to treatment after the intervention period of eight weeks. The long-term effect of the intervention is measured by a follow-up assessment six months after the intervention period has ended and is not part of the RCT design. Sample size A power analysis with the software G*Power [69] resulted in a target sample size of 128 to detect a medium-sized effect (f = 0.25) with α = 0.05 and a power of 0.80 for an ANCOVA. Including an assumed attrition rate of about 10%, the final number of participants should reach 140. Recruitment Participants will be recruited in three different ways: First, the study staff will contact former patients with diagnoses of schizophrenia who consented to get contacted and inquire whether they are interested in participating in this study via e-mail. Second, study information will be sent out to psychiatric institutions in Switzerland and Germany. The health staff at those institutions can then distribute the information to interested and suitable patients directly, for example those patients who leave the institution and look for a continuation treatment. Lastly, online bulletin boards and informative websites on psychoeducation specifically created for people affected by schizophrenia will be targeted for publishing descriptions and links to the study. Eligibility criteria To be included, participants must fulfill all inclusion criteria and not show any exclusion criteria described below. Suffering from other psychiatric disorders such as depression or anxiety disorders (part of the former 'axis I' disorders) does not lead to exclusion as long as the schizophrenia spectrum disorder is the primary diagnosis. Inclusion criteria are: interview. These diagnoses are allowed to be partly remitted. 6 A score of three or higher on the items assessing delusions (P1), hallucinations (P3) or suspiciousness/ persecution (P6) on the Positive and Negative Syndrome Scale (PANSS) [71] showing that some positive symptoms remain. 7 Simultaneous treatment with antipsychotic medication (or regular psychological or psychiatric care in Germany). 2 Representing an acute danger for others. 3 No agreement on compiling an emergency plan. 4 Diagnosis of an acute neurological disease of the central nervous system that needs to be treated. Randomization Participants eligible for inclusion will be randomly allocated to one of the two groups (intervention or waiting list control group). Randomization and allocation will be prepared in advance by an independent researcher. This researcher will remain blinded to all processes within the intervention. An automated, web-based randomization service (www.random.org) [72] will be used to generate the randomization list. The allocation ratio will be 1:1. Intervention The intervention called EviBaS (for Evidence-Based Selfhelp intervention) consists of an online program based on CBT principles, while also including components of ACT and MCT treatments. A smartphone app provides the possibility of exercising the modules in everyday life. Reflect on thoughts, feelings and behavior to understand and decrease the symptom burden. Feelings of Threat Review the effects of paranoia on individual goals and needs. Voice Hearing Learn strategies to reduce distress caused by hearing voices (better coping, influence the evaluation of voices). Self-Worth Find forgotten strengths and train a balanced sense of self. Overcoming Depression Set up activities and scrutinize depressing thoughts. Worrying Minimize upholding factors of worry and tackle worries with problem solving skills. Sleep Discuss maintaining factors of the sleep disorder, such as sleep hindering thoughts or disadvantageous surrounding factors. Mindfulness Exercise to direct your attention on one thing without judgement. Metacognition Learn in interactive exercises to avoid jumping to conclusions and overconfidence in errors. Social Competence Plan and train three different types of social situations: enforcing interests, shaping relationships, and winning sympathies. Relapse Prevention Collect individual warning signs and plan ahead. There are 11 text-based modules in the online program, addressing a variety of topics (see Table 1). Each module includes texts and a worksheet. The worksheets can also be accessed via the optional app. The only mandatory module is the introductory one. After completion, the participants can choose from the remaining ten modules freely. Relapse prevention is recommended as the last module. Study participants are asked to work on approximately one to two modules per week. The time required to finish one module may vary, but will usually not exceed 60 min. The intervention is self-paced, so that participants are able to work on topics they prioritize, such as emotional issues rather than positive symptomatology [73]. While working with the program, participants will be in contact with a personal moderator if they want to. The moderator will guide the participants through the program with at least one message per week. The main goal of this steady contact is to help participants structure their usage of the program and to encourage regular participation [47]. If necessary (in case of a participant not using the intervention for seven days), the moderator reminds the participant to interact with the program. Participants' questions are answered within three workdays by moderators. There is a biweekly supervision of all the moderators in the study team led by a licenced psychotherapist with extensive experience in CBTp. Measures The primary outcome is the reduction of psychotic symptoms (positive symptoms such as voice hearing and paranoid delusions) at post assessment (directly after the completion of the intervention). Secondary outcomes include the level of symptomatology at follow-up, the number of dropouts and the results of all secondary questionnaires which evaluate quality of life, depression severity, treatment satisfaction, the influence of treatment expectancy, and process measures, among others (see Table 2). Assessments will be completed at baseline, eight weeks and 32 weeks. For an overview of all primary and secondary outcome measures, predictors and moderators, as well as process measures, see Table 2. Primary outcome measures Positive and Negative Syndrome Scale (PANSS) The PANSS [71] was the first standardized evaluation tool for symptoms of schizophrenia [74]. It assesses 30 symptoms, which can be grouped into five factors: positive symptoms, negative symptoms, disorganization, excitement, and emotional distress [1]. The positive symptom factor serves as the primary outcome of this study. It includes nine items (delusions, hallucinations, unusual thought content, suspiciousness, grandiosity, somatic concern, active social avoidance, lack of judgment and insight, and (less) difficulty in abstraction). In this study, clinicians administer the PANSS via telephone. MINI International Neuropsychiatric Interview (MINI) The MINI is a diagnostic structured interview for the assessment of psychiatric diagnoses [70], with a corresponding German version [75]. The specificity of the MINI was reported as good for all diagnoses (ranging from 0.72 to 0.97) [70]. In this study, a part of the MINI (depressive episode, suicidality, manic episode, and psychosis) was also administered via telephone. Paranoia Checklist The Paranoia Checklist was developed by Freeman et al. in 2005 [76] and assesses the frequency, degree of conviction, and associated distress of a wide range of paranoid thoughts. The three subscales each include the same 18 items, which are rated on a five-point Likert scale: The first subscale measures the frequency of paranoid thoughts (ranging from 'does not apply at all' to 'applies very well'; adapted), the second subscale measures the degree of conviction (from 'not at all convinced' to 'absolutely convinced') and the third subscale surveys the level of distress (from 'not distressing' to 'very distressing'). Cronbach's alpha, as an estimate of reliability, is reported as .90 or above, which stands for an excellent internal consistency of the Paranoia Checklist [76]. Launay-Slade Hallucination Scale (LSHS-R) The LSHS-R is the revised version of the LSHS, developed by Launay and Slade in 1981 [77]. It includes 12 items and measures the predisposition to hallucinations on a wide spectrum [78]. There is a German version of the LSHS-R, which shows a Cronbach's alpha of .87 in a patient sample and therefore is comparable to the original [79]. The five-point Likert scale ranges from 0 ('certainly does not apply to me') to 4 ('certainly applies to me') and there is a sum score that will be compared between both groups of participants. Secondary outcome measures Delusion and Voices Self-Assessment (DV-SA) The DV-SA has two subscales, a Delusions Scale (DS) and a Voices Scale (VS). The former contains five items to assess patients' opinions about the dominant delusional idea and the latter includes ten items about the subjective dimensions of auditory hallucinations [80]. All responses of the participants are rated on two fourpoint Likert scales, from 0 (absence of problems) to 3 (the severest of problems), with the highest achievable total score of 15 for the DS and 33 for the VS [80]. For this study, we changed the time period referenced in the DV-SA from 1 month to 1 week. Incongruence questionnaire (K-INK) The K-INK [81] is a short version of a questionnaire (INK) that measures the degree of realization of motivational goals in the participant's life. Those goals can be classified into two groups: approach goals and avoidance goals. On a five-point Likert scale, the K-INK measures approach incongruence, avoidance incongruence, and total incongruence. Internal consistency (as measured by Cronbach's alpha) for the K-INK is reported as ranging between .52 and .87 [81]. World Health Organization Quality of Life Assessment (WHO-QoL-BREF) The WHO-QoL-BREF consists of 26 items and is a standard questionnaire to measure the quality of life [82]. Cronbach's alpha values range from .66 to .84, indicating an acceptable internal consistency [82]. The WHO-QoL-BREF demonstrates good discriminant validity [82]. In this study, it is administered both in baseline and post assessments and the participants have to indicate their level of agreement on a five-point rating scale with changing answer formats. Rosenberg Self-Esteem Scale (RSES) The RSES measures self-esteem on a four-point Likert scale. The RSES shows a high reliability and validity for global self-worth [83]. It demonstrates an excellent internal consistency (Guttman scale coefficient of reproducibility of .92) [84]. Moreover, the two week test-retest reliability revealed high correlations of .85 and .88, indicating excellent stability [84]. Higher scores on the RSES indicate higher self-esteem. Insomnia Severity Index (ISI) The ISI is a brief measure for insomnia and is composed of seven items. Each of these items is rated on a fivepoint Likert scale, ranging from 0 ('not at all') to 4 ('extremely'). Studies reported adequate psychometric properties for ISI versions in English and French [85,86]. Penn State Worry Questionnaire -Abbreviated (PSWQ-A) The PSWQ [87] is a questionnaire designed to assess the tendency to worry. In this study, an abbreviated eight-item version (PSWQ-A) was used [88]. The measure is scored on a five-point Likert scale from 1 ('not at all typical') to 5 ('very typical'). The PSWQ-A items have good internal consistency highlighted by reported Cronbach's alpha ranging from .87 to .89 and .94 [88,89]. Scores for the PSWQ-A range from 8 to 40 [90]. Patient Health Questionnaire (PHQ-9) The PHQ-9 measures depression severity [91]. It scores all nine DSM-IV criteria for depression on a rating scale from 0 ('not at all') to 3 ('nearly every day'). A score of 20 represents severe depression. Internal consistency of the PHQ-9 is excellent with a Cronbach's alpha of between 0.86 and 0.89 [91]. In this study, the short German version (PHQ-D) was used [92] and the item on suicidality also serves as an indication of the necessity of exclusion from the study. Box Task In the Box Task [93], participants are confronted with grey boxes on the computer screen, concealing two distinct colours. Participants have to gather information about which of the two colours is more frequent by clicking on said boxes. When they decide that they have gathered sufficient information, they can choose the more frequent colour. This experimental paradigm has been administered in a previous study [94]. If the amount of information an individual gathers before making a decision is low this indicates a tendency to jump to conclusions [27]. Jumping to conclusions has been found to be associated with schizophrenia and delusions [95]. Mindful Attention Awareness Scale (MAAS) The MAAS is a questionnaire measuring mindfulness on a six-point Likert scale. Cronbach's alpha of the MAAS has been reported as .81 [96]. Interpersonal Competence Questionnaire (ICQ) The ICQ is originally a 40-item questionnaire for the assessment of five domains of interpersonal competence [97]. In this study, only two of the five domains are surveyed: initiation of relationships and negative assertion. Moreover, a recently published short version of the ICQ (called ICQ-15) [98] was used to pick out the six items of the two subscales (three for each domain). The internal consistency of the total scale was high (Cronbach's alpha = .87) and the reliability coefficients of the subscales were the highest two coefficients of the five subscales: .73 (for initiation of relationships) and .75 (for negative assertion) [98]. The German version of this questionnaire has been validated as well [99]. Internalized Stigma of Mental Illness (ISMI) -Short version The ISMI is a questionnaire measuring the internalized stigma of participants [100]. In this study, the short version of the ISMI was used, which includes 10 items with four-point Likert scales [101]. To evaluate the results, one calculates the mean of those items. A mean score between 1.00 and 2.50 points stands for no internalized stigma, while a mean score between 2.51 and 4.00 stands for high internalized stigma. The German version of the ISMI showed a high internal consistency (Cronbach's alpha = .92), which was calculated in a study with 139 participants [102]. Predictors and moderators Medication Adherence Rating Scale (MARS-D) The MARS [103] consists of five items rated on a five-point Likert scale. These items measure a participant's non-adherent behavior from 1 ('always') to 5 ('never'). A higher score indicates higher adherence to the prescribed medication. MARS-D is the German adaption of this questionnaire developed by Mahler et al. [104]. Internal consistency of the MARS-D (Cronbach's alpha ranging from .60 to .69) was reported as satisfactory and comparable to the original [104]. Attitudes towards Psychological Online Interventions (APOI) The APOI measures patients' attitudes towards an online intervention [105]. It reveals certain prejudgments of a study participant, which might influence the outcome parameters and the motivation. Sixteen items are displayed and the level of agreement with each item can be indicated on a five-point rating scale (ranging from 'no agreement' to 'total agreement'). A factor analysis of the APOI showed four dimensions: (a) scepticism and perception of risks (b) confidence in effectiveness (c) technologization threat and (d) anonymity benefits. The APOI shows acceptable to good internal consistency and a good content validity is assumed, because the construction of items was done deductively as well as inductively [105]. Credibility/Expectancy Questionnaire (CEQ) The CEQ is an economic scale to measure treatment expectancy and rationale credibility [106]. The dimensions measured by the CEQ can possibly moderate the outcome. In this study, participants rate items according to two dimensions -one dimension is related to thinking and one is related to feeling. On four of six items, the rating scale ranges from 'no agreement' to the treatment rationale to 'total agreement' on a nine-point rating scale. For the remaining two items, participants can indicate the subjective symptom improvement from 0% to 100% in steps of 10%. The CEQ demonstrated a high internal consistency of between .84 and .85 [106]. University of Rhode Island Change Assessment (URICA) The URICA [107] is a measure of readiness to change. The 32-item URICA consists of four subscales (eight items each) that correspond to four stages of change (precontemplation, contemplation, action and maintenance) [108]. Internal consistency for the total URICA was reported as excellent (Cronbach's alpha = .83) [109]. This study used a short version of the German URICA (URICA-S) [110], where items are rated on a five-point Likert scale ranging from 1 ('not applicable at all') to 5 ('very applicable'). Client Satisfaction Questionnaire (CSQ) The CSQ asks participants to what extent they were satisfied with the intervention [111]. The internal consistency of the CSQ was .93, which stands for an excellent score. There is evidence for a strong construct validity of the CSQ as well [111]. Because it measures treatment satisfaction, it can only be administered at post assessment. Due to its shortness and comprehensiveness, the CSQ is very suitable for mailed surveys [112] and can therefore also be administrated in an online format. The German version (called ZUF-8) [113] is being used in this current study. The eight items can be answered on a four-point rating scale [113]. Questionnaire about Side Effects Psychosis and Internet (QueSPI) This questionnaire assesses the negative effects of internetbased interventions for psychotic patients. It was developed within the research group as part of a pilot study leading up to the current project [114]. Detailed information can be found in the Additional file 1. Process measures Working Alliance Inventory -Short Revised (WAI-SR) As a measure of the weekly variation of the therapeutic alliance, the WAI-SR [114] was included in the study in its German version [115]. It goes back to the Working Alliance Inventory (WAI) [116] and differentiates between goal, task and bond alliance dimensions. Each of those dimensions is represented by four items. The WAI-SR uses a five-point rating scale representing the frequency of positive alliance experiences (ranging from 'rarely' to 'always'). Cronbach's alpha of the subscale scores ranges from .85 to .90, and of the total scores from .91 to .92, indicating excellent internal consistency [114]. Intermediate Assessments Questionnaire The study group designed a questionnaire to briefly measure symptoms and mental states that were expected to change over the course of the online intervention. Each therapeutic topic covered in the online intervention is represented by a single item in the intermediate assessments questionnaire, such as auditory hallucinations, quality of sleep, self-worth, worry, or depression. Additionally, potentially psychosis-related thinking styles (e.g. jumping to conclusions) are assessed via single items. Five out of 14 items were newly created by the authors, the remaining 9 items were adopted from German versions of established questionnaires (e.g. PHQ-9) [117], or taken and translated from experience sampling studies on schizophrenia (e.g. 'I feel suspicious') [118]. The intermediate assessments questionnaire uses a five-point rating scale ranging from 'not at all true' to 'absolutely true' and can be read in the Additional file 2. Data collection and management At baseline, participants complete an online assessment including several questionnaires described above. An electronic informed consent and demographic questions will be displayed at the beginning of this online survey. Participants must also indicate their e-mail address and telephone number, which are processed independently from other data. The process of baseline assessment lasts approximately 35 to 40 min. After completion and if no inclusion criteria is not met, a telephone interview with the participant will be arranged. This telephone interview includes two diagnostic interviews (MINI and PANSS, also described above) and the development of an individual emergency plan in case of acute suicidality or psychotic relapse with each participant. The interview lasts approximately 45 to 60 min. If the participant scores below the cutoff on all of the PANSS items (delusions, hallucinations or suspiciousness/persecution), or if the participant reports neither current nor past psychotic symptoms, he or she is excluded from participation and receives a short self-help manual as a compensation for the assessment participation. When inclusion criteria are met, the participants are randomized to one of two groups. The participant either gets access to the online intervention program immediately or after a waiting period of eight weeks. After completion of the intervention period, post assessment and a second telephone interview are administered. The same procedure takes place during the follow-up assessment six months after the intervention period. Statistical analyses Based on the intention to treat sample, a linear mixedmodel repeated measures ANOVA with time (T1-T2) as a within-group factor and study condition as a betweengroup factor will be used for the main research question. Mixed-model repeated measures ANOVA uses all available data of each subject and does not require the substitution of missing values. Sensitivity analysis will be conducted to analyze the impact of dropout on the results. The significance level is set at 5%. We are also interested in possible mediators and/or moderators of the relation between the internet-based self-help intervention and positive psychotic symptoms. We will therefore test whether predictors identified in the literature, such as treatment motivation, working alliance or usage of the intervention could mediate and/or moderate the main effect [119]. To evaluate these possible predictors of treatment outcome, we use change scores of outcome measures as the dependent variables. Ethical aspects and data safety The Cantonal Ethics Committee Bern (ID: 03/14) as well as the German Society for Psychology (ID: SM052015_CH) have approved of this study. Data safety is ensured by several means: The program and app usage are independent of any personal data. Conversely, the communication via the secured e-mail system contains no information that would allow the identification of a participant in EviBaS. Sensitive data (where personal information such as the email address can be linked to login data for the online program) is stored exclusively non-electronically in a locked closet at one study site. Diagnostic staff will not know the identification of the participants in the program and will be blinded for the allocation of participants in the two groups, whereas moderating staff will not know the contact information of the participants. Breach of blinding will be reported. Network security is achieved through SSL encryption. All staff members who are in contact with study participants, are required to fill out a non-disclosure agreement. Discussion EviBaS has been developed as one of the first fully encompassing iCBT programs for people with psychosis. People with schizophrenia spectrum disorders suffer from a heavy burden of symptoms and stigmatization [13]. Psychological evidence-based treatments for schizophrenia exist, but only a small portion of affected people receive them [120]. Bridging the treatment gap in the psychological care for people with severe mental disorders is therefore of utmost importance. This project wants to reach people who do not receive psychological treatment but are looking for support. Internet-based interventions might even be able to reach a group of patients that discontinued a previous face-to-face therapy [121]. Given the efficacy of CBTp on hallucinations and delusions [122], as well as of iCBT in other mental disorders [46], the EviBaS self-help program is expected to reduce symptomatology. Negative effects and long-lasting effects of the treatment will be assessed. This study will also add to our understanding of how people with schizophrenia use internet-based interventions. Jelinek, Helena Meyer, Johanna Schröder and Ruth Veckenstedt for their contribution in writing the self-help modules. Funding This study was funded by the Swiss National Science Foundation (project number 159384) and the German Research Foundation (project number DFG Mo 969/17-1). The funding body played no role in the design of the study, the collection of data or in writing the manuscript. Availability of data and materials Not applicable. Trial status Trial start date: December 6th 2016. Currently recruiting (N current = 66 as of 30th October 2017) Authors' contributions NR wrote the first draft of the manuscript. SW, SM, TB and NR designed the study. All authors contributed to and approved the final manuscript. Ethics approval and consent to participate The Cantonal Ethics Committee Bern (ID: 03/14) as well as the German Society for Psychology (ID: SM052015_CH) have approved of this study. Informed consent to participate in the study is being obtained from every participant. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests.	2018-04-18T06:34:50.589Z	2018-04-13T00:00:00.000	{ "year": 2018, "sha1": "b4638df01f44d8483c4aa7004c03394581a93f15", "oa_license": "CCBY", "oa_url": "https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/s12888-018-1644-8", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b4638df01f44d8483c4aa7004c03394581a93f15", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
233893954	pes2o/s2orc	v3-fos-license	White Matter Integrity Correlates of the Reading Span Although working memory (WM) is crucial for intellectual abilities, not much is known about its brain underpinnings, especially the structural connectivity. We used diffusion tensor imaging (DTI) to look across the whole brain for the white matter integrity correlates of the individual differences in the reading span (verbal WM capacity during reading) in healthy adults. Right-handed healthy native Russian speakers (N = 67) underwent DTI on a 3T Philips Ingenia scanner. Verbal WM was assessed with the Daneman-Carpenter reading span test (Russian version). Fractional anisotropy maps from each participant were entered into the group tract-based spatial statistics analysis with the reading span as a covariate; the results were TFCE-corrected. After taking into account effects of age, sex, education and handedness, reading span positively correlated with the white matter integrity in multiple sites: the body, the genu and the splenium of corpus callosum; bilateral corona radiata (anterior, posterior, and superior); bilateral superior longitudinal fasciculus; several tracts in the right hemisphere only, including the internal and external capsule; bilateral superior parietal and frontal white matter. Although the left hemisphere is central for verbal processing, we revealed the important role of the right hemisphere white matter for the verbal WM capacity. Our finding indicates that larger verbal working memory span may originate from additional processing resources of the right hemisphere. Introduction Working memory is a limited capacity system for short-term information storage and manipulation which lies in the core of human cognitive and executive functions. It is crucial for learning and intellectual abilities (Cowan, 2014;Süß et al., 2002;Oberauer et al., 2005), and many neurological conditions and developmental disorders are characterized by working memory deficits (Goldman-Rakic, 1994;Sandry, 2015;Alloway et al., 2009). Working memory models stem from the multiple memory systems approach which suggests that memory is composed of at least two subsystems, a short-term and a long-term memory. Extending the idea of short-term memory, working memory concept implies that information is not only maintained within a temporary storage system, but is also actively manipulated to complete the cognitive task at hand (Miller et al., 1960;Baddeley and Hitch, 1974;Cowan, 2008). Multicomponent model of the working memory, proposed and elaborated by Baddeley and Hitch (Baddeley & Hitch, 1974;Baddeley, 2001Baddeley, , 2003Baddeley, , 2012 includes specific storages for phonological and visuospatial formats along with the central executive component operating on representations in different formats. This approach received support from individual difference studies which revealed a corresponding threefactor structure of the working memory abilities across different age groups (Alloway et al., 2004;Gathercole et al., 2004;Hornung, Brunner, Reuter, & Martin, 2011). In assessment of individual capacities of storages for phonological and visuospatial formats a distinction between verbal and non-verbal working memory is widely used (Baddeley, 2003). Being the most influential, Baddeley's multicomponent model is not the only. Questions of whether working memory is a separate memory subsystem or an activated part of the long-term memory (Cowan, 1988(Cowan, , 1999Oberauer, 2002) and on specific cognitive and executive mechanisms within the working memory such as information maintenance, updating or protection from interference have been highly debated for several decades (Miyake, Friedman, Emerson, Witzki, Howerter, & Wager, 2000;Engle, 2002;Ecker, Lewandowsky, Oberauer, & Chee, 2010;Schmiedek, Lövdén, & Lindenberger, 2014). In accordance with these different theoretical perspectives a variety of working memory 3 capacity measures were developed, tapping verbal and non-verbal information storage and processing and including simple span tasks (immediate recall of a list of words, digits, letters or object spatial locations), complex span tasks (dual-task requiring that items are maintained in memory during a concurrent cognitive activity such as reading), updating tasks, such as the n-back task (comparing the present item in a continuously updating sequence with an item presented n steps back), and some other (see Wilhelm et al., 2013 for a review). While simple spans are widely adopted in cognitive ability test batteries (Wechsler scales, AWMA, CANTAB), complex spans have shown to better predict real-life cognitive tasks performance. The reading span test (Daneman & Carpenter, 1980) which was originally developed as a proof of concept for the complex span tasks, was found to predict reading comprehension much better than word span or digit span tasks addressing mostly short-term storage of the verbal material (Daneman & Merikle, 1996) and is nowadays widely used in psycholinguistics. Neural underpinnings of the working memory are not yet described in such detail as its behavioral aspects. Neurocognitive models have shown importance of both cortical and subcortical structures for different working memory processes and components, including language areas of the left hemisphere; visual cortices; fronto-parietal network, which recruits the dorsolateral prefrontal cortex, parietal cortex, and anterior cingulate; and even basal ganglia (see Chai et al., 2018 for review). As these models are built mostly on the functional neuroimaging data, they describe predominantly the grey matter impact, while underlying structural connectivity may also be of great importance. Therefore white matter tract-based correlates of the complex span working memory performance remain unknown, although they may much better describe the neural substrates of working memory functioning in the real-life cognitive behavior such as reading and writing, and better predict academic and professional success in language-related spheres. To fill this gap, we conducted a tract-based spatial statistics (TBSS) study in the healthy adult population with a focus on reading span which characterizes the verbal working memory capacity during reading (Daneman, Carpenter, 1980). We used diffusion tensor imaging (DTI) to obtain the fractional anisotropy (FA) measures for each voxel across the whole brain which were further correlated with the individual differences in the reading span measured outside of the scanner. Participants Ninety two healthy volunteers from Moscow, Russia (35 males, 57 females, aged 24.4±5.6 y.o., mean years of education 14. 9±2.6) took part in the study. The following inclusion criteria were used: native speaker of Russian; right-handed (handedness was assessed with laterality quotient 10 [LQ10] from Edinburgh Handedness Inventory; Oldfield, 1971); no contraindications for the MRI procedures (assessed with a screening questionnaire); no reported history of neurological or mental conditions. The project was approved by the Interuniversity Ethics Committee of Moscow. Participants gave written informed consent to the MRI and psychological assessment and received financial compensation for their time and effort. Data from 2 participants were excluded from the analysis due to technical issues (wrong imaging protocol or signal loss induced by metallic dental implant), from 2 more participants due to distraction from neuropsychological tasks, and from 21 due to substantial checkerboard artifact (Oguz et al., 2014) revealed by radiologists during visual inspection of their DWI images. Therefore data analysis was performed on a subset of 67 participants (23 males, 44 females, mean age 24.0±5.3 y.o, mean years of education 15.1±2.5, handedness: LQ10 range 60-100, median 100). Materials Reading span (verbal working memory) was assessed with the Russian version (Fedorova, 2010) of the Daneman-Carpenter test (Daneman, Carpenter, 1980). Participants were tested individually in a quiet room. They were shown sentences via PowerPoint presentation on a laptop, one sentence per slide. The task was to read each sentence aloud and immediately proceed to the next slide. Time per slide was not limited in a way other than participant's reading speed. After a set of a few sentences, a blank slide appeared, and participants were asked to recall the last word of each sentence from the set they just have read in the correct order and grammatical form (gender, number, case, and tense). All responses were audio recorded. Overall the test contained 20 sets of sentences, from 2 up to 5 per set, 5 sets of each size (i.e., 5 sets of 2 sentences, 5 sets of 3 sentences etc.). Performance was measured as a percentage of correctly recalled groups (without any omission or grammatical and ordinal error), 5% per set. Procedure The DWI was performed in either the beginning or end of the one hour and a half neuroimaging session which included data collection for a speech perception study. The working memory assessment (reading span test) was administered either before or after the scanning session along with other behavioral tests. Data Analysis As a first step of the DTI data processing, two radiologists (L.M. and D.B.) performed a visual screening of the T1-weighted images for anatomy anomalies potentially critical for the present study and DWI data for image artifacts while being blind of the behavioral test results of the participants. Then an automated quality check procedure was applied with the DTIPrep software (Oguz et al., 2014) to detect volumes corrupted by excessive head motion or intensity abnormalities. Volumes affected by any data quality issue were removed from further processing; there were no subjects for whom the portion of the remaining DWI data was less than 75%. Further processing was performed with FSL software (Jenkinson et al., 2012; http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) and included correction of the eddy currents (eddy_correct), correction of the metric distortions with a fieldmap calculated from non-DW images with the opposite phase encoding direction (topup and applytopup), skull stripping of all data and spatial coregistration of the DTI and structural images. Diffusion tensor model was fitted voxelwise on individual participant's preprocessed data, and fractional anisotropy (FA) maps were computed. Using the FSL Tract-Based Spatial Statistics (TBSS) workflow (Smith et al., 2006), FA individual maps were then registered to the common FA image 1 mm template in MNI space and a group white matter skeleton was created from FA maps; individual diffusion tensor imaging (DTI) metrics were projected onto the skeleton. One-sample t-tests with the mean-centered reading span score as a covariate of interest were performed on skeletonized FA images using nonparametric permutation inference (Winkler et al., 2014); mean-centered age, sex, years of education and handedness (laterality quotient) were included into the model as covariates of no interest to account for extra FA variability. The number of permutations was set to 5000 and the results were corrected for multiple comparisons using the thresholdfree cluster enhancement method (TFCE) with p<0. 05. The anatomical localization of significant clusters was defined with ICBM-DTI-81 white-matter labels probabilistic atlas (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Atlases). For better visualization of the results on the skeleton (Figure 1), we thickened the thresholded p-value image using tbss_fill function. Results Reading span scores ranged from 20 to 95% (mean 45±15%). After taking into account effects of sex, age, education and handedness, reading span positively correlated with the white matter integrity in multiple sites: the body, the genu and the splenium of corpus callosum; bilateral anterior, posterior, and superior corona radiata; bilateral superior longitudinal fasciculus; cerebral peduncle, anterior and posterior limb of the internal capsule, retrolenticular part of internal capsule, posterior thalamic radiation, sagittal stratum, external capsule, and fornix in the right hemisphere; bilateral parietal and frontal white matter outside the scope of the atlas (see Figure 1). Discussion The present study has revealed that the verbal working memory capacity in real-life cognitive activity, such as reading, is associated with the integrity of extensive portions of the white matter tracts in both hemispheres. Implication of the corpus callosum, corona radiata, and superior longitudinal fasciculi in working memory capacity is consistent with the earlier DTI studies on different populations and other working memory measures, both verbal and non-verbal (Bathelt et al., 2017;Chung et al., 2018;Darki & Klingberg, 2015). At the same time, unlike most of the previously reported data, our results indicate bilateral nature of the verbal working memory capacity neural substrate, even with a right-sided lateralization of the white matter integrity correlates in several structures. Noteworthy, Takeuchi et al. (2011) have previously reported right-sided parietal white matter integrity correlates of an index reflecting information processing component in the verbal working memory. Our finding indicates that larger verbal working memory span may originate from additional processing resources of the right hemisphere. Looking for potential white-matter right-hemisphere correlates of the verbal working memory deficits seems to be a promising direction of future research. Figure 1. Parts of the white matter skeleton that were significantly associated with working memory capacity (reading span) after taking into account effects of age, sex, education and handedness, threshold-free cluster enhancement method (TFCE) with p<0. 05. Skeleton is shown in green. Results are overlaid on the target FA image. Slice indices represent MNI coordinates (z).	2021-05-08T00:03:50.062Z	2021-02-17T00:00:00.000	{ "year": 2021, "sha1": "c2b394f5e2a13138d1a1dfdd447d7e8d47d38c3c", "oa_license": "CCBY", "oa_url": "https://www.preprints.org/manuscript/202101.0254/v2/download", "oa_status": "GREEN", "pdf_src": "Adhoc", "pdf_hash": "a18ddab502987c096ed4409397991df6175b34be", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [ "Psychology" ] }
228093788	pes2o/s2orc	v3-fos-license	Secrecy Enhancement of Cooperative NOMA Networks With Two-Way Untrusted Relaying The two-way relaying (TWR) technique has been confirmed to achieve higher spectral efficiency and average sum rate compared with the one-way relaying (OWR) technique in ultra-dense next generation networks with limited spectal resources. In this paper, an enhanced secrecy cooperative TWR scheme for cooperative non-orthogonal multiple access (NOMA) networks against untrusted relaying is highlighted. Specifically, with the application of NOMA principle, a base station (Bs) communicates with two trusted users, i.e., namely a near and a far users, where the communication with the far user takes place only via an untrusted relay (UR) employing both TWR and analog network coding (ANC) mechanisms. To minimize information leakage at the UR, the far user transmits its uplink signal simultaneously with Bs’s downlink signal to confuse the eavesdropping capability of the UR by increasing inter-user interference (IUI). To investigate the benefits of the proposed scheme, asymptotic lower bound expressions of the ergodic secrecy sum rate (ESSR) of uplink/downlink rates and their scaling law are derived to characterize the secrecy performance. The system parameters have been carefully studied to maitain the desired ESSR performance where we obtain an optimal value for the uplink power sharing coefficient with an arbitrary known value of the downlink one. Analytical and simulation results show that the proposed scheme can achieve scaling gain of $\boldsymbol {\frac {1}{8}\ln {\rho }}$ of positive ESSR over the untrusted OWR scheme with adaptive uplink/downlink jamming and a significant gain over the other conventional NOMA uplink/downlink schemes in two time slots of communication. I. INTRODUCTION The ever-extending massive connectivity, low latency, high spectral efficiency demands makes non-orthogonal multiple access (NOMA) techniques having the urgent necessity to be performed to break the orthogonality requirement in time/frequency/code resources by partially overlapping wireless signals with a tolerable level of interference [1]. In the context of increasing the multiplexing gain by exploiting different domains, NOMA have been recently classified into power-domain and code-domain NOMA. The key concept of the power-domain NOMA is to enable the The associate editor coordinating the review of this manuscript and approving it for publication was Xingwang Li . signals of multiple users to occupy the same resource block (time/frequency/code) but with different power levels. In particular, NOMA exploits superposition coding (SC) at the transmitters to transmit superimposed signals within the same resource while the receivers of better channel conditions carry out successive interference cancellation (SIC) technique to separate superimposed signals of the poorer ones before decoding their own signals [2]. Thus, NOMA can support massive connectivity [3], [4], achieve higher spectral efficiency [5]- [8] and improve the user fairness [9]- [11]. Cooperative NOMA is an effective approach to enhance the network coverage extension and the reception reliability of the users of poor channel conditions by exploiting the spatial diversity. Particularly, cooperative NOMA can VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ be classified in terms of the elements of the network into: user-aided and relay-aided cooperative NOMA. In user-aided cooperative NOMA, the users of better channel conditions act as a relay to enhance the reception capability of the users of poorer channel conditions as they previously have to decode the signals intended to the poorer users. There exist several studies concerning this topic [12]- [16]. In [12] the outage performance of user-aided cooperative NOMA was investigated where it was shown to be better than the noncooperative case. To improve the performance of cell-edge users in a cellular NOMA network, the cell-center users can act as relays in both half-duplex (HD) and full-duplex (FD) modes of operation to achieve a diversity gain [13]. Cooperative FD NOMA based user-aided relaying was proposed in [5], [14] to further save the resources and improve the spectral efficiency. Employing a user in cooperative relaying may consume its dedicated power and degrade its life time. Thus, energy harvesting (EH) techniques were exploited in cooperative NOMA which termed as simultaneous wireless information and power transfer (SWIPT) to compensate the user for the required power of relaying [15]. A joint power allocation and splitting factor design was proposed in [16] to enhance the system throughput and user fairness. On the other hand, relay-aided cooperative NOMA schemes have gained also a considerable attention in practical design of NOMA networks [17]- [19]. This can be implemented by deploying dedicated relays to increase the diversity. Coordinated direct and relayed transmission based cooperative NOMA was proposed in for both downlink and uplink communications [17], [18]. In [19] a multi-relay multi-antenna NOMA system was proposed to further increase the spectral efficiency. The above contributions considered only one-way relaying (OWR) or unidirectional communication protocol which does not fulfill the dense mode of NOMA network operation. Twoway relaying (TWR) permits a simultaneous bidirectional communication to further improve the spectral efficiency. HD and FD TWR NOMA based networks were proposed in [20], [21] where the outage probability, diversity analysis and network throughput were derived and compared with OWR networks. A joint rate splitting and user grouping protocol for multi-pair TWR NOMA network was proposed in [22] for better utilization of resources and enhanced throughput. In [23] a joint antenna and relay selection scheme was proposed to enhance the transmission reliability for multiple access and time division broadcast relaying strategies in a TWR NOMA based networks where the optimal transmit power allocation was obtained. However, due to the broadcast nature of wireless signals in a cooperative NOMA network, confidential information in this network is vulnerable to two main types of attack: 1) external eavesdropping: in which an illegitimate attacker that is not belonged to the network elements tries to overhear confidential signals and decode them for its own sake, 2) internal (untrusted) eavesdropping: in which a legitimate element in the network tries to decode confidential information of the other elements. There exist recent works that considered the external eavesdropping in NOMA networks. The secrecy performance and power allocation analysis was investigated in [24]- [28]. The beamforming design in multiple input single output (MISO) and multiple input multiple output (MIMO) NOMA networks to minimize information leakage was investigated in [29]- [35]. Exploiting relay selection and FD relaying techniques to confront external eavesdropping in cooperative NOMA networks was proposed in [5], [36]- [40]. Secure TWR NOMA based networks were also of a considerable interest. In [41], [42] the security issue of NOMA based TWR networks with artificial noise jamming (AN-jamming) were analyzed. However, the above works considered the external eavesdropping in NOMA networks while the impact of internal eavesdropping on the secrecy performance may be more dangerous as the network elements that share the same resource blocks may distrust each other. In the context of untrusted users, the authors of [43] investigated the case of untrusted far user that can decode confidential information of the near user via SIC where the secrecy outage probability of the near user and the outage probability of the far user were derived. The authors of [44] considered the case of multiple relay selection schemes to enhance the secrecy performance of a cooperative NOMA network of untrusted far user. The secrecy performance of a MISO NOMA system with untrusted far user was proposed in [30]. Unlike [30], [43], [44], two joint beamforming and power allocation schemes were proposed in [45] for a cooperative NOMA network with both untrusted near user and external eavesdroppers. To enhance the secrecy performance in a HD untrusted OWR cooperative NOMA network, a novel NOMA-inspired jamming and relaying approach was proposed in [8] where the base station sends a superimposed mixture of destination and jamming signals and a positive secrecy sum rate can be always guaranteed. In [46], [47] the secrecy performance of cooperative NOMA networks with untrusted relay (UR) was studied in which a base station communicates with two users and one random user or the two users or the base station emit an AN-jamming signal to combat the UR. As the emitting of AN-jamming signal may degrade the eavesdropping capability of the UR, it may cause a simultaneous degradation in the reception quality of the other elements of the network. To overcome this issue, a novel secrecy-enhancing design for cooperative uplink/downlink NOMA with an UR was proposed in [48] where the near and far users send an adaptive jamming signal of a similar symbol structure. Thus, enabling legitimate users to cancel out the jamming signal while preventing the UR from eavesdropping. However, in cooperative NOMA networks with multiple superimposed signals, the use of internal network interference can be a sufficient mean to degrade the eavesdropping capability and enhance the secrecy rate [49]. Thus, imposing an intended jamming signal may increase interference sources rather than combat eavesdropping which leads to inefficient information decoding at receivers, and this motivates our work as follows. A. MOTIVATIONS AND CONTRIBUTIONS The recent work of cooperative NOMA network with untrusted relaying focused on transmitting jamming signals simultaneously along with information signals to confuse eavesdropping against untrusted relaying. However, this may confuse the legitimate channels themselves and we have the following observations: • Exploiting inter-user interference (IUI) inherent in cooperative NOMA networks can deteriorate the eavesdropping capability more than confusing legitimate decoding of information signal. Therefore, IUI can play the role of cooperative jamming by performing a tradeoff design between the minimization of IUI at legitimate receivers and the maximization of IUI at the eavesdroppers to enhance the secrecy performance. This can be achieved by hindering SIC at the eavesdropper, particularly, by mixing a superimposed version of adaptive jamming and information signals, i.e., making a composite signal, at the UR. Consequently, the UR cannot be able to determine the decoding order of the mixed signal which can significantly degrade its ability to retrieve the confidential information signals coherently. • Employing TWR in cooperative NOMA networks enables a bidirectional communication which leads to better utilization of the resources and recover the spectral efficiency loss inherent to OWR. In addition, combining TWR with NOMA forces the UR to exchange a dense information between nodes against the facility of its decoding process. Motivated by the previous observations, in this paper, we propose an enhanced secrecy performance of uplink/ downlink cooperative TWR NOMA network against untrusted relaying. In particular, the base station (Bs) communicates with the near (NU) and the far users (FU) where the communication with FU takes place by untrusted TWR. The main contributions can be summarized as follows: • An uplink/downlink cooperative TWR NOMA scheme against untrusted relaying is proposed in which Bs transmits a superimposed downlink signal for NU and FU where the communication to FU takes place via an UR. Meanwhile, FU transmits its uplink signal to the UR. The received signals at the UR incurs an IUI with unknown decoding order which impair the ability of the UR to separate and decode the composite signal and confuses its eavesdropping capability. Then, amplify and forward (AF) relaying is employed at the UR to forward the received signals to the destinations. To this end, an unavoidable level of IUI can be created at the UR when it tries to decode each user symbol coherently while the decoding quality at legitimate users is not affected. • To evaluate the secrecy performance and investigate the benefits of the proposed scheme, an analytical expression of a lower bound on the uplink/downlink ergodic secrecy sum rate (ESSR) is derived. We demonstrate that a positive ESSR can be guaranteed by the proposed scheme with the least utilization of resources to complete an uplink/downlink communication. To gain more insights, an asymptotic scaling law of the ESSR is determined at high ρ where ρ is the transmit signal to noise ratio (SNR). It can be clarified that the proposed scheme can achieve an uplink/downlink scaling gain of ln ρ over two time slots of communication. Furthermore, the simulation results confirm the accuracy of the derived formulas and the superiority of the proposed scheme over the benchmark schemes. • We obtain an optimal value of the uplink sharing power coefficient that maximizes the ESSR for an arbitrary constant value of downlink power coefficient to investigate the impact of IUI on the eavesdropping capability of the UR. First, we prove that the ESSR is a unimodal maximal function, then, the optimal value is obtained by carrying out an iterative algorithm based on the golden search method. B. ORGANIZATION The reminder of the paper is organized as follows. In section II, we present the proposed cooperative TWR NOMA scheme with its design structure and channel modeling. The secrecy performance evaluation is investigated in section III and verified by simulation results in section IV. Then, the paper is concluded in section V. II. SYSTEM MODEL AND DESIGN Consider the security issue of a cooperative TWR NOMA wireless network that consists of one base station, Bs, the near user, NU and far user, FU, 1 that communicates with Bs via an AF-UR employing TWR and analog network coding (ANC) [50] techniques as shown in Fig.1. All these nodes are equipped with a single antenna and operate in HD mode of operation. It is assumed (as in [17]) that the link between Bs and FU is absent due to heavy shadowing loss and only the AF-UR is responsible for delivering information between Bs and FU. However, the UR is only trusted at the service level while it is not allowed to decode any confidential information at the data link processing level. This means that the UR is only permitted to perform AF protocol TWR that involves reliable channel state information (CSI) feedback and estimation as well as signal amplification and forwarding (e.g., it is assumed that full CSI is owned by the transmitting nodes via pilot estimation processes prior to information transmission [48] 2 ). To this end, the UR may try to intercept confidential information exchanged between nodes during 1 Two user NOMA model has been adopted by the third generation partnership project long-term evolution (3GPP-LTE) to minimize large inter-user interference and complex analysis inherent to multi-user NOMA model for practical implementation of cooperative NOMA networks, e.g., see [3]. 2 We will discuss a two-step CSI pilot estimation in subsection D. uplink/downlink communications for its own sake. Hence, it is necessary to minimize the information leakage at UR. 3 To do this, FU simultaneously exploits transmitting its NOMA uplink signal as a noise signal to increase IUI at the UR and impair its eavesdropping capability. From a practical perspective, the proposed scheme can be vastly founded in some applicable cellular networks. The intended network can consist of a Bs which is capable of exchanging information with a pair of two types of residential and roadway users. The Bs is directly linked to the residential user whereas it communicates with the roadway user via an UR located near the road signs. Each type of user has a hierarchical secrecy demand against the UR which can be curious to decode its received signals before forwarding them. Thus, the transmission rates are dynamically determined by the transmit nodes to maintain those secrecy demands. The communications' channels are assumed to experience independent and identical distributed (i.i.d) quasi-static fading, i.e., channels' coefficients remain constants within a resource block of time but independently vary from block to another. Each uplink/downlink and relaying transmission is bounded by a transmit power budget of P while an additive white Gaussian noise (AWGN) is considered at each receiving node with mean equals to zero and variance equals to N 0 . Considering channel reciprocity, let the channel coeffi- RF and h NF , respectively, follow circularly-symmetric complex Gaussian random variables with zero means and variances equal to . This means that = \|h i \| 2 follows an exponential distribution with normalized average channel gains λ i i.e., λ i , i ∈ {BN , BF, RN , RF, NF}. Two communication phases (two time slots) are needed to complete information uplink/downlink transmission. This 3 Here, we consider the AF relaying protocol in the two-way scheme due to the following reasons: 1) AF relaying protocol is easy to implement where ANC can be applied efficiently to mix analog signals and reduce resource utilization; and 2) at low SNR, the AF relaying protocol can achieve better secrecy sum rate whatever the distance between Bs and UR is, e.g., see [46,50]. Furthermore, by exploiting the HD TWR, a positive secrecy capacity can be strictly achieved as we will see in the next section. can be described in the following two subsections where the system parameters used in this paper are summarized in Table 1. A. FIRST PHASE In this phase, Bs transmits simultaneously its downlink symbols x ND and x FD of NU and FU with power sharing coefficients of α and 1 − α, respectively, straightforwardly to NU and UR where E \|x ND \| 2 = E \|x FD \| 2 = 1. Meanwhile, FU transmits its uplink symbol which represents a background noise symbol at the UR, i.e., x FU , with a power sharing coefficient where E \|x FU \| 2 = 1. It is noted that NU shares the same uplink power budget, P, with FU with a power sharing coefficient of 1 − . Different from the cooperative AN-jamming schemes considered in the literature, e.g., see [46], where the AN relies on pseudo-random signals, FU generates its uplink signal as a noise background signal of deterministic waveform to the UR, e.g., x FU , which can easily decoded by NU relying on its captured characteristic during CSI estimation. Following the NOMA principle, Bs transmits the signal P intended for its uplink communication as a background noisy signal at the UR. Thus, the received signal at UR and NU can be respectively expressed as where P is the transmit power,n R , n N ∼ CN (0, N 0 ), N 0 is the thermal noise variance of the receiving nodes. Without loss of generality, we assume that UR and NU are more close to Bs than FU in the sense that λ BN λ RF and λ BR λ RF . Thus, the SIC decoding order starts from x B towards x F . Particularly, the UR carries out a complex joint maximum likelihood (ML) detection 4 to decode its received composite signal and achieve better eavesdropping capability in a two-step decoding algorithm. In the first step, the UR tries to decode x FD by treating both x ND and x FU as interference then it subtracts x FD from its observation before the attempt of decoding x ND and x FU . In the second step, after a successful wiretapping of x FD , the UR performs SIC one more time to decode x ND by treating x FU as a noise before acquiring x FU . In the composite signal, x ND can indeed represent an additional interference when decoding x FU which can present an over-estimated capability of eavesdropping at the UR. Thus, the achievable rates for decoding x FD , x ND and x FU at the UR can be respectively expressed as where ρ P N 0 is the transmit signal to noise ratio (SNR) of the transmit power budget P. Recall that the uplink and downlink transmit power is bounded by P as P uplink ≤ P and P downlink ≤ P. Depending on the previous knowledge of the CSI about x FU , NU can subtract x FU from its observation such that the achievable rates for decoding x FD and x ND at NU can be respectively expressed as 4 It is assumed that R can perform a complex joint ML detection to separate and remove IUI and decode the information signals correctly [8]. If the UR can perform more intelligent signal decoding techniques, such as the joint separation decoding technique, to indiviually separate the intended signals, the secure transmission may fail, and thus, the injection of a friendly jammer should be carried out to impair the decoding quality at the UR while the other nodes can decode their intended signals separately. As such, we have considered in the first phase the overestimated capability of eavesdropping at the UR which provides a performance lower bound for the proposed system [8], [48]. B. SECOND PHASE In this phase, the UR forwards an amplified version of its received signals to do its main function without analyzing the entire information contents. Meanwhile, NU transmits its uplink symbol x NU to Bs with a power allocation coefficient of 1 − . By exploiting the HD characteristic of the untrusted TWR, the transmission of the uplink signal containing x NU is completely secured so that x NU can not be eavesdropped by the UR as it is not able to transmit and receive at the same time. Thus, the received signal at Bs and FU can be respectively given by ANC and prior estimation cancellation term where g BR + g RF +1/ρ denotes the amplification factor of the UR and n B ∼ CN (0, N 0 ) and n F ∼ CN (0, N 0 ), where N 0 is the thermal noise variance of the receiving nodes, denote the AWGN at Bs and FU. Since Bs has a prior knowledge of the self-amplified version of its transmitted signal, it can cancel out the term from its observation by using ANC and prior estimation cancellation techniques. Without loss of generality, it is assumed that the received uplink signal power of x NU is larger than that of x FU due to its close proximity to Bs. 5 Therefore, the decoding sequence at Bs is considered to start from After performing the NOMA SIC decoding technique, the achievable rates for decoding x FU and x NU at Bs can be respectively expressed as On the other hand, FU has a prior knowledge of its selfretransmitted signal, as well as, the interference signal from NU, so that it can easily cancel out the term h 2 P (1 − ) from its observation by using ANC technique. Recall that NU should be previously able to decode x FD according to (6) before decoding its own downlink signal which means that the achievable rate for decoding x FD at FU should be no more than C x FD N . From (6) and (9), the achievable rate for decoding x FD at FU can be expressed as where are the signal to interference noise ratio (SINR) for decoding x FD at NU and FU, respectively. C. ERGODIC SECRECY SUM RATE (ESSR) To characterize the statistical effectiveness of the proposed scheme, the ESSR is adopted as a secrecy performance metric. The ESSR is defined as the rate beyond which a secure communication rate can be available. The ESSR and a lower bound on the ESSR are mathematically given by where {A} + max (0, A), for arbitrary A and (a) is based on Jensen's inequality as in [8]. Remark 1: The proposed scheme can guarantee a positive secrecy sum rate. This is enabled by exploiting the HD characteristic of the TWR which reveals the term C x NU B is out of the eavesdropping capability. Different from [46], [48], the proposed scheme confirms achieving a secure performance mode to the premier uplink symbol, i.e., x NU , while completing an uplink/downlink communication in only two time slots. D. CSI REQUIREMENTS To obtain the required CSI prior to the uplink/downlink transmission process, a channel training mechanism is adopted at the transmitting nodes comprised of a two-step training mechanism with a transmission of six pilots. Firstly, Bs needs to estimate g BN , g BR , g RN and g RF in order to adjust the achievable rates for x ND and x FD while FU needs to estimate g BN , g BR , g RF and g NF in order to adjust the achievable rate for x FU and the proper value of . Then, NU needs to estimate g BN in order to adjust the achievable rate for x NU according to the power budget, i.e., (1 − ) P. The training steps are described as follows: III. ESSR PERFORMANCE ANALYSIS In this section, we provide a detailed analysis on the secrecy performance of the proposed system in terms of the ESSR. In particular, we derive analytical expressions for lower bound and asymptotic lower bound of the ESSR of the proposed scheme which represent a sufficient approach to determine the secrecy benefits of the proposed scheme. Furthermore, we obtain the optimal value of the power sharing coefficient of the uplink transmission, i.e., , to maximize the ESSR relying on the iterative golden search algorithm [51] given a fixed value of the power coefficient of the downlink transmission, i.e., α. A. A LOWER BOUND ON THE ESSR To proceed forward, we use the definition of average rate (ergodic rate), i.e., E (C X ) E X (log (1 + x)), for a nonnegative random variable X as [52] where F X (x) and f X (x) are the cumulative density function (CDF) and the probability density function (PDF) of the random variable X, respectively. Theorem 1: A closed-form approximate expression for the lower bound of the ESSR, i.e., C lower ESSR , can be given by (17) at the bottom of the next page, where C BR = λ BR α, C RF = λ RF , C BN = λ BN α and λ k , k = 1, . . . , 5 are given in Appendix A. Proof: See Appendix A. The above theorem provides an efficient approach to scale the secrecy performance of the proposed scheme in terms of some evaluations of simple functions. To give more insights about the impact of the system parameters and the feasibility of proposing TWR scenario upon the system to improve both reliability and security issues, we investigate the asymptotic scaling behavior of the ESSR of the proposed system when ρ grows to infinity. To deal with, the approximations e 1/x ≈ 1 + 1/x and Ei (−1/x) ≈ − ln x can be used in (17) [48] with cooperative jamming and a scaling gain 1 2 ln ρ over the same system without cooperative jamming whereas it can offer the same uplink ESSR scaling gain of 1 4 ln ρ over the scheme proposed in [18]. This means that the proposed system can achieve an overall uplink/downlink ESSR scaling gain of 1 8 ln ρ over the adaptive uplink/downlink one-way relaying NOMA network proposed in [48] with the least utilization of resources. B. THE OPTIMAL VALUE OF THE UPLINK/JAMMING COEFFICIENT * In this subsection, we focus on finding out an optimal value of that maximizes the ESSR (e.g., C lower ESSR ) of the proposed system with arbitrary fixed values of Bs's power sharing coefficient, i.e., α, and uplink/downlink transmit power budget, i.e., P. 6 6 Unfortunately, a more general jointly optimal power allocation problem of ( ,α) can be considered to maximize the ESSR of the proposed scheme which is beyond this work. In particular, it is desired to determine an optimal value * that satisfies the following optimization problem * = arg max C lower ESSR , such that { , α} ∈ [0, 1] , In fact it is very difficult to obtain * analytically due to a certain composite structure of C lower ESSR . Instead, we divide the main problem into a problem of a simple structure of C lower ESSR based on a desired secrecy performance mode. Then, we investigate that C lower ESSR has at most one maximal critical point under a certain condition on ∈ [0, 1]. Then, we find out the optimal value * by the utilization of the golden search algorithm for finding a maximum of a function. First, we investigate that C lower ESSR combines an algebraic sum of monotonously increasing and decreasing functions of , then, we obtain * from the combined function as follows. Based on the expression of C lower ESSR in (15), we consider the condition where a secrecy performance mode for all x ND , x FD , x NU and x FU symbols can be achieved, i.e., {A} + A. This leads to the following optimization problem * where C lower Theorem 2: Following the methodology of obtaining C lower ESSR in (15), C lower ESSR is a sum of two monotonically decreasing and increasing functions of and we have the following modified optimization problem * = arg max C lower ESSR , such that α ∈ [0, 1] , P constant, Proof: See Appendix C. Remark 3: It is noted that the determination of the maximum critical point inside [max (0, 1 ) , min (1, 2 )] depends basically on both the sum of the increasing rate of E (C I ) and the decreasing rate of E (C D ) with respect to . In other words, there exists only one maximum point * that can be found out within [max (0, 1 ) , min (1, 2 )] according to the rate of the sum of E (C I ) and E (C D ). Regarding (17), it is very difficult to obtain an analytical expression for * with respect to the parameters of C lower ESSR . Instead, we propose an iterative optimized algorithm based on the golden search method [51] to obtain a very convergent value of * . The algorithm is summarized in the following steps as shown in Algorithm 1. Algorithm 1 Iterative Algorithm for Determining * Based on the Golden Search Method Input: α, ρ, g BR , g RF , g BN , u 1 = 10 −3 , N iter. = 0 and a maximum tolerance ε = 10 −4 . 1: Calculate: 1 , 2 , In this section, we provide numerical results to validate the analytical results of our enhanced secrecy performance scheme for TWR based cooperative NOMA network with UR via Monte Carlo simulations with 10 4 iterations. In particular, we discuss the secrecy performance point of view of the proposed scheme in terms of the state of art schemes that concern the untrusted relaying based NOMA and OMA networks. In terms of the uplink/downlink ESSR performance, we compare our proposed scheme with three benchmark schemes described as follows: 1) The adaptive uplink/downlink cooperative jamming NOMA based network proposed in [48]: In this scheme, Bs uses the NOMA principle to send a downlink superimposed signal, i.e., x ND √ Pα + x FD √ P (1 − α), to communicate with NU directly and FU by the aid of a one-way HD UR. Meanwhile, FU emits an adaptive structured jamming signal during its idle state to overcome information leakage at the UR. This adaptive jamming signal has the same symbol structure as the NOMA symbol structure and it is well-known to the corresponding receivers. Then, the UR uses the AF technique to forward its received signal. 7 Meanwhile, Bs sends another information signal to NU. In the uplink communication, both NU and FU sends their information signals to Bs and the UR, respectively. Meanwhile, NU emits in parallel a superimposed adaptive jamming signal with its information signal. Then, the UR amplifies and forward its received signal. Meanwhile, NU sends another information signal to Bs. It has been verified that the proposed system in [48] outperform the proposed system in [17,18] without adaptive jamming signals. 2) The uplink/downlink NOMA based network with destination aided AN-jamming proposed in [53]: Different from the previous proposed scheme [48] Fig. 2 the detection capability of the TWR system under PAR1 by discussing the probability of miss detection (PMD) of the symbol x FU when the UR employs the joint complex ML detection algorithm. In Fig. 2, the PMD of Bs and the UR are plotted versus ρ when the joint complex ML detection is employed for a predetermined values of g BR and g RF . It is evident that the PMD of Bs decreases rapidly and faster than the UR when the analytical i.e., or equivalently the condition αρ 2 g BR −ρ (g BR + g RF )−1 > 0 is met and the PMD gap increases with ρ. This means that and α should be carefully designed in order to achieve a positive secrecy rate for x FU . To start a comparative study on the secrecy performance between the proposed and the benchmark schemes. The lower bound of the ESSR (i.e., C lower ESSR ) of the proposed and the benchmark schemes are computed firstly by summing up the uplink and downlink secrecy rates. In Fig. 3, the analytical and simulation results C lower ESSR for the proposed and benchmark schemes are plotted under PAR1 versus ρ. It is evident that C lower ESSR of proposed scheme converges to the scale of lnρ in high ρ regime whereas benchmark 1 scheme converges to the scale of 7 8 ln ρ in high ρ per two time slots of an uplink/downlink communication. Furthermore, it is evident that benchmark 2 reaches a floor value at high ρ. This is because the transmitted AN-jamming signal x z an unknown structure at the receiving nodes, degrades the reception quality regardless of confusing the eavesdropping capability of the UR. Benchmark 3 scores the worst C lower ESSR performance among the comparative schemes. This is because it needs at least six time slot resources to complete an uplink/downlink communication. This emphasize that the proposed scheme yields the highest C lower ESSR with the least number of resources of a completed uplink/downlink communication. To illustrate the impact of variation of the system parameters on the C lower ESSR of the proposed scheme, the derived formula in (17) and the simulation analysis of C lower ESSR are plotted in Fig. 4 for two different channel gain settings, i.e., namely, the previous PAR1 settings, when λ i = 0.5 (i.e., denoted as PAR2), i ∈ {BN , BF, RN , RF, NF}) and when λ RF = 0.3 and all λ i = 0.5, (i.e., denoted as PAR3), i ∈ {BN , BF, RN , NF}). It is observed that the slope of the C lower ESSR curve in both scenarios converges the scale of lnρ in medium to high range of ρ. This emphasizes that the proposed scheme can achieve ESSR scaling gain of lnρ. Moreover, it is evident that the derived formula of C lower ESSR in (17) agrees well with its corresponding simulation result which verifies the asymptotic convergence of this analytical formula. Fig. 5 depicts the optimal value of the uplink/jamming sharing coefficient ( * ) of the proposed C lower ESSR by using the method of golden search algorithm with ρ = 10. It is evident that there exists one and only one maximum value of within ∈ [ 1 , min (1, 2 )] when the secrecy performance of x ND , x FD , x NU and x FU symbols can be achieved as C lower ESSR is a unimodal maximum of . Considering the impact of g RF on the performance, Fig. 5 is plotted for two different λ RF settings, i.e., λ RF = 1 shown in Fig. 5a and λ RF = 0.5 shown in Fig. 5b, while λ i = 0.5 for i ∈ {BN , BF, RF, NF}) where we consider that 1 > 0, 2 > 1. Although the increasing of g RF makes the symbol x FU vulnerable to be eavesdropped by the UR, it can greatly increase the IUI within the composite signal received by the UR, and therefore, it can impair the eavesdropping capability of the UR and improve the overall lower bound of the ESSR, e.g., C lower ESSR . VOLUME 8, 2020 B. DISCUSSION AND FUTURE WORK In this subsection, further discussion is added to clarify the impact of different system parameters on the secrecy performance and investigate how the proposed scheme can be extended to more general systems of practical considerations. It is worthy noting that the proposed scheme relies basically on imposing unordered composite signal to increase IUI and impair the decoding quality at the UR. Thus, system parameters should be carefully designed to unleash the benefits of deploying TWR technique into the UR to achieve the desirable level of secrecy performance in the end to end uplink/downlink communication process. On one side, it can be observed that g RN has no impact on the secrecy performance (i.e., or equivalently the ESSR) of the proposed scheme as NU has a prior knowledge about both x B and x F signals to extract its downlink signal, i.e., x ND , whereas its uplink signal, i.e., x N , cannot be eavesdropped by the UR due to its HD limitation. On the other side, g RF has a crucial impact on the ESSR of the proposed scheme. It is cleared that large value of g RF makes the uplink signal x F more vulnerable to be eavesdropped by the UR whereas small value of g RF sacrifices the interference level at the UR when it tries to decode the superimposed x B signal for its own sake. Furthermore, g BR (i.e., λ BR ) is an important parameter of determining 1 and 2 which should be carefully designed to maintain the desired secrecy performance of x ND , x FD , x NU and x FU . In particular, for a specific α, should be designed within the range of max (0, 1 ) < < min (1, 2 ). Additionally, λ BN should be also designed in the sense that the spatial location of NU is closer to Bs than the others in order to increase the ESSR. However, if those certain parameters cannot be controlled, an external injection of a friendly jamming signal should be devised or even switching the UR to perform OWR technique [48] with cooperative jamming. The proposed scheme can be extended to multi-user multiantenna systems. In particular, the multi-user system can provide higher degrees of freedom and diversity gain to enhance the ESSR by a joint user and jamming scheduling design [55]. In multi-antenna systems, joint beamforming and power optimization with cooperative jamming can be applied to further achieve better ESSR in delay-tolerant secrecysensitive scenarios. Considering global CSI availability at Bs, the proposed scheme can serve to schedule random users according to their current locations from Bs. In particular, a circular zone centered at Bs with a coverage radius equals to the distance between Bs and the UR can be invoked in the scheduling process. Thereafter, the user located within this zone can be scheduled as NU, otherwise, the user is scheduled as FU. And therefore, mobile users can decide to switch back and forth to the UR according to their current location inside and outside the zone. Another direction of practical implementation can be extensively applied to the proposed scheme with imperfect CSI where novel multi-pair, multi-user and multi-antenna joint selection strategies can be performed to maintain different secrecy targets for users of different secrecy performance levels [56]. In general, integrating those strategies into the proposed scheme can significantly enhance the ESSR. V. CONCLUSION In this paper, we proposed an enhanced secrecy performance scheme of cooperative uplink/downlink NOMA networks against untrusted relaying. To save resources and prevent eavesdropping, TWR and IUI techniques were exploited at the UR to intensify the exchange of information signals and confuse the eavesdropping capability at the UR, respectively. To reveal the merits of the proposed scheme, we derived an analytical expression for the lower bound of the uplink/downlink ESSR and verified it by simulation results. The results demonstrated that the proposed scheme can achieve an improvement in the uplink/downlink ESSR than the benchmark schemes with the least utilization of resources. Furthermore, we obtained an optimal value for the uplink power sharing coefficient by holding an iterative algorithm based on the golden search method. APPENDIX A PROOF OF THEOREM 1 To find out a closed form expression for C lower ESSR , we firstly have to derive mathematical formulas for αρg BR +1 , then, we can obtain the desired formulas as follows. By substituting by β (g BR + g RF + 1/ρ) − 1 2 , X FU and X NU can be rewritten respectively as Based on the above, the CDF of X ND can be expressed as where P(.) is the probability function. Hence, E C x ND R can be obtained as Consequently, E C x FU R can be simply obtained as E C x ND R by interchanging λ BR by λ RF and ρ by . Furthermore, the CDF of Y FD can be expressed as and E C x FD R can be obtained as After applying some algebraic manipulations, the method of undetermined coefficients as used in [57]- [59] and using [ [54], 3.352. [2][3][4][5], we arrive at In this regard, the CDF of X FU ρ g BR g RF (2g BR + g RF +1/ρ) can be accordingly expressed as x, ρ, )) , is the modified Bessel's function of the second kind. It is noted that we use VOLUME 8, 2020 [[54], 3.324.1] and the substitution ρy − 2x = t in (A. 7) where the condition y > 2x/ ρ should be satisfied. Recall that we focus on the lower bound of the ESSR, then, K 1 (x) can be upper-bounded by K 1 can be simply obtained as and γ x FD F , whereas the lower bound for the minimum tends to a lower bound. Thus, the CDF of X FD can be expressed by the following probability According to (12), P 1 can be obtained as e whereas P 2 can be obtained after substituting for β in (12) as Substituting into (A.10), we can get the CDF of X FD as x, ρ, )) . (A.12) Recall that we search for a lower bound of the ESSR in the sense that we can making the use of the relation K 1 (x) ≤ 1/x when ρ → ∞ to simplify the analysis. Then, E C x FD F can be expressed as where step (d) follows by letting (1 − α − αx) = 1/u and Finally, the CDF of X NU ρg BR g RF +2g BR + g RF +1/ρ can be evaluated as where we can making the use of the relation can be simply obtained as 16) where in step (g) follows after performing some algebraic manipulations, λ 4 ( ) = (1 − ) λ BN and λ 5 ( ) = 1 λ BR − 1 λ RF . This completes the proof. APPENDIX B PROOF OF COROLLARY 1 At high transmit SNR, based on (15) and (17) can be regarded to scale as a constant with the ever increase of ρ as Y FD , Y ND , Y FU and X NU approach a constant value with ρ → ∞, respectively. Furthermore, by utilizing the approximations e 1/x ≈ 1 + This completes the proof. APPENDIX C PROOF OF THEOREM 2 Recall that the secrecy performance of x ND , x FD , x NU and x FU can be achieved. It is evident from (4) and (8) αρg BR +f 2 ( ) , where f 1 ( ) = g BR g RF + 1 + + 1/g RF ρ and f 2 ( ) = ρg RF + 1. Since we consider the condition where a secrecy performance mode is achieved, i.e., when {A} + A, the term E C x FD F − E C x FD R is a monotonically increasing function of when f 1 ( ) increases slower than f 2 ( ). Otherwise, the term E C x FD F − E C x FD R 0, e.g., no secrecy performance for x FD can be achieved. In other words, the condition f 1 ( ) − f 2 ( ) = g BR g RF + (1 − ρg RF ) + 1 g RF ρ < 0 must be hold to guarantee that a secrecy performance for x FD can be achieved. Furthermore, the condition (1 − ρg RF ) < 0, i.e., or equivalently g RF > 1/ρ, can guarantee that the term E C x FD F − E C x FD R is a monotonically increasing function of . Consequently, the term E C x FD F − E C x FD R is a monotonically increasing function of with positive secrecy performance when both g RF > 1/ρ and > ρg BR +1 g RF ρ(ρg RF −1) are satisfied. Next, we discuss the monotonicity of the term . It is evident from (5) and (12) Finally, it is evident from (11) that the term E C x NU B 1 2 log 1 + (1− )ρg BN (g BR + g RF +1/ρ) ρg BR g RF +2g BR + g RF +1/ρ is a monotonically decreasing function of . Combining the above discussion and using the fact that the sum of monotonically increasing (or decreasing) functions is also an increasing (or decreasing) function, it is evident that C lower ESSR is a sum of two monotonically decreasing and increasing functions of and the first part of the proof is completed. Let Recall that E C x NU B is a monotonically decreasing function of which can be rewritten after some algebraic manipulations as E (C D ) ≡ log 2 −D 1 + D 2 + D 3 , where D 1 , D 2 and D 3 are arbitrary coefficients independent of . Thus, E (C D ) is a strictly decreasing function of ∈ [0, 1]. Furthermore, E (C I ) can be further rewritten after some algebraic manipulations as E (C I ) ≡ log 2 I 1 3 +I 2 2 +I 3 +I 4 I 5 4 +I 6 3 +I 7 2 +I 8 +I 9 , where I 1 to I 9 are arbitrary positive coefficients independent of , then, E (C I ) has negative roots and it comprises a sum of monotonically increasing functions of with positive secrecy performance of x ND , x FD and x FU if and only if > ρg BR +1 g RF ρ(ρg RF −1) and < g BR (αρg BR −1)−1/ρ g RF . Thus, E (C I ) is a strictly increasing function of ∈ [max (0, 1 ) , min (1, 2 )], where 1 = ρg BR +1 g RF ρ(ρg RF −1) and 2 = g BR (αρg BR −1)−1/ρ g RF . Consequently, C lower ESSR ≈ E (C D ) + E (C I ) has at most one maximal critical point of , ∈ [max (0, 1 ) , min (1, 2 )] and the second part of the proof is completed.	2020-12-03T09:01:28.837Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "6b79802f8e0bb20ae2ca98946a5c19ef4f68ab7e", "oa_license": "CCBY", "oa_url": "https://ieeexplore.ieee.org/ielx7/6287639/6514899/09275305.pdf", "oa_status": "GOLD", "pdf_src": "IEEE", "pdf_hash": "99496c79bca9cd5de3a17b7ee0ab343a9b978bd5", "s2fieldsofstudy": [ "Engineering", "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
201203656	pes2o/s2orc	v3-fos-license	Detecting Genotype-Population Interaction Effects by Ancestry Principal Components Heterogeneity in the phenotypic mean and variance across populations is often observed for complex traits. One way to understand heterogeneous phenotypes lies in uncovering heterogeneity in genetic effects. Previous studies on genetic heterogeneity across populations were typically based on discrete groups in populations stratified by different countries or cohorts, which ignored the difference of population characteristics for the individuals within each group and resulted in loss of information. Here, we introduce a novel concept of genotype-by-population (G × P) interaction where population is defined by the first and second ancestry principal components (PCs), which are less likely to be confounded with country/cohort-specific factors. We applied a reaction norm model fitting each of 70 complex traits with significant SNP-heritability and the PCs as covariates to examine G × P interactions across diverse populations including white British and other white Europeans from the UK Biobank (N = 22,229). Our results demonstrated a significant population genetic heterogeneity for behavioral traits such as age at first sexual intercourse and academic qualification. Our approach may shed light on the latent genetic architecture of complex traits that underlies the modulation of genetic effects across different populations. INTRODUCTION Most human traits are polygenic and their phenotypes are typically influenced by numerous genes and environmental factors, and possibly by their interactions, e.g., genotype-environment (G × E) interaction (Plomin et al., 1977;Mackay, 2001;Reddon et al., 2016). These traits have been termed as "complex traits, " which are distinguished from Mendelian traits that are shaped by a single or few major genes (Lander and Schork, 1994). Genome-wide association studies (GWAS) have successfully discovered thousands of associations between single-nucleotide polymorphisms (SNPs) and complex traits, which have revolutionized our understanding of the polygenic architecture of complex traits (Stranger et al., 2011;Goddard et al., 2016;Visscher et al., 2017). Subsequently, in order to increase the power and precision to identify more causal variants, there have been numerous follow-up studies using meta-analyses of GWAS summary statistics or mega-analyses of multiple GWAS by combining diverse data sources that usually span across different nations or populations (Torgerson et al., 2011;Nagel et al., 2018). However, many human complex traits [e.g., height and body mass index (BMI)] are substantially different between diverse populations (Guo et al., 2018). For instance, the mean height across European nations generally increases with latitude (Robinson et al., 2015). Although across-population differences in the mean values are often observed for the phenotypes of complex traits, the underlying genetic and environmental bases remain largely unknown (Robinson et al., 2015). One way to understand such phenotypic heterogeneity lies in uncovering genetic differentiation for the traits captured by common variants across populations (Falconer and Mackay, 1996). Some studies (Maier et al., 2015;Robinson et al., 2015;Yang et al., 2015;Tropf et al., 2017) have focused on examining population genetic differentiation for several anthropometric, behavioral and psychiatric phenotypes, using whole-genome statistical methods such as applying bivariate genomic restricted maximum likelihood (GREML) (Lee et al., 2012) to estimate genetic correlations between populations from the United States and Europe for height and BMI (Yang et al., 2015) or determining interaction of genotype by seven sampling populations for behavioral traits by a GREML approach (Tropf et al., 2017). They reported significant evidence for interaction of genotype by populations in behavioral phenotypes (education and human reproductive behavior) and BMI (Tropf et al., 2017). The analytical method and designs used in their studies were based on discrete groups, which ignored the difference of population characteristics for the individuals within each group. Furthermore, the population groups used in their studies were classified according to their country of origin, thus the results were likely to reflect heterogeneity across countries due to country-specific factors (e.g., trait definition and measurement (van der Sluis et al., 2010;Evangelou et al., 2011;Manchia et al., 2013), cultural and societal difference and socio-economic status). In addition, genetic measurement errors (e.g., due to the genotyping platform or imputation quality) across different cohorts within country may further cause confounding with genuine genetic heterogeneity across populations (Tropf et al., 2017). Principal component (PC) analysis provides a powerful tool to characterize populations and the first few PCs are typically used to control population stratifications in large-scale GWAS (Novembre and Stephens, 2008). PCs allow us to cluster individuals that are genetically similar to each other. Unlike discrete variables such as cohort and country, PCs are continuous variables that can differentiate individuals even within a cohort or a country according to their underlying genetic characteristics. Here, we introduce a novel concept of genotype-by-population (G × P) interaction where population is defined by the first and second PCs. It is of interest to test if different genotypes respond differently to the gradient of the first or second PC for complex traits using a whole-genome reaction norm model (RNM) , which has been recently introduced and allows fitting continuous environmental covariates, i.e., PCs in this study. RNM has been well established to estimate G × E interaction in agriculture (Gregorius and Namkoong, 1986;Jarquín et al., 2014) and ecology (Nussey et al., 2007). Furthermore, in this study we used the data source of UK BioBank (UKBB), which is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals across the United Kingdom, aged between 40 and 69 at recruitment (Sudlow et al., 2015;Bycroft et al., 2018). Therefore, in our G × P interaction model applied to UKBB, the population characteristics for individuals are fully utilized and the findings are less likely to be confounded with country-specific factors or genetic measurement errors as mentioned above. The aim of the study is to explore if there exists significant G × P interaction, which is also referred as genetic heterogeneity (heterogeneous genetic effects) across populations, for a wide range of complex traits. To do so, we applied the whole-genome RNM with PCs as continuous covariates to investigate G × P interactions for more than one hundred phenotypes using the UKBB data. The significant G × P interaction detected in this study may shed light on the latent genetic architecture of complex traits that underlies the modulation of genetic effects across different population backgrounds. Data and Quality Control (QC) Our study was based on the UKBB data which contains approximately 500,000 individuals sampled across the United Kingdom (Bycroft et al., 2018). UKBB's scientific protocol and operational procedures were reviewed and approved by the North West Multi-centre Research Ethics Committee (MREC), National Information Governance Board for Health & Social Care (NIGB), and Community Health Index Advisory Group (CHIAG). Research Ethics approval was obtained from University of South Australia Human Research Ethics Committee (HREC). According to the ethnic background (data field 21000), there are currently 472,242 individuals of the white British ancestry and 17,038 individuals of any other white ethnic background (not with British or Irish ethnicity) in the UKBB participants. In order to match the sample size between the white British and the other white ethnic individuals, we randomly selected 17,000 individuals from the white British group, totaling 34,038 admixed European populations considered in this study. As the information of the first and second ancestry PCs is efficient to infer genetic ancestry and geographical origin with a high accuracy in Europeans , we examined a two-dimensional scatter plot of PC1 and PC2 provided by the UKBB of the 17,000 white British and the 17,038 other white ethnic subjects ( Figure 1A). It is shown that the white British group is situated within the group of the other white Europeans and we named the white British group as POP1 (N = 17,000). As shown in Figures 1B,C, we used a geometric method by which we constructed a rectangle with maximums and minimums of PC1 and PC2 of the white British group as four sides and then group the individuals of the other white Europeans inside this rectangle, named as POP3 (N = 9,809). The rest of the other white Europeans except POP3 were named as POP2 (N = 7,229). FIGURE 1 \| Two-dimensional scatter plots of PC1 and PC2 with red points representing white British individuals and blue points representing other white ethnic individuals from the UKBB. The white British group named as POP1 is situated within the group of the other white Europeans (see panel A). As shown in panels (B,C), we used a geometric method by which we constructed a rectangle with maximums and minimums of PC1 and PC2 of POP1 as four sides and then group the individuals of the other white Europeans inside this rectangle, named as POP3. The rest of the other white Europeans except POP3 were named as POP2. As POP1 and POP3 are very close in terms of PCs, the individuals in the two data designs POP1 + POP2 and POP2 + POP3 have similar population structures while POP1 + POP3 was a negative control as there was little population difference among this combination (see panel D). Our primary interest was to investigate G × P interaction where population was classified by ancestry PCs. For this purpose, we used three designs of combinations of the three groups, i.e., POP1 + POP2 (Figure 1B), POP2 + POP3 (Figure 1C), and POP1 + POP3 ( Figure 1D). To avoid any potential bias due to an imbalance in the sample size across populations, we made sample size consistent across POP1 and POP2 in the design of POP1 + POP2 by randomly selecting 7,500 individuals from the 17,000 white British individuals, which were used as POP1 in the downstream analyses. We extracted genetic data including around 92 million imputed SNPs across autosomes from the UKBB for all the individuals of POP1, POP2 and POP3. Stringent QC was applied to the combined data across POP1, POP2 and POP3. The QC criteria were to exclude (1) all duplicated and non-autosomal SNPs, (2) SNPs with INFO score <0.6, (3) SNPs with call rate <0.95, (4) individuals with missing rate >0.05, (5) SNPs with Hardy-Weinberg equilibrium p-value < 0.0001, (6) SNPs with minor allele frequency <0.01, and (7) ambiguous SNPs with A/T or G/C alleles. We also retained HapMap3 SNPs only as they are reliable and robust to bias in estimating SNP-heritability and genetic correlation (The International HapMap 3 Consortium, 2010;Bulik-Sullivan et al., 2015;Tropf et al., 2017). Hereafter, 1,133,957 common SNPs remained for the G × P analyses. Moreover, we excluded one individual randomly selected from any pair with a genetic relationship >0.05 (see section "Statistical Models") to avoid bias due to confounding by shared environment among close relatives. After the QC, the sample sizes of POP1, POP2, and POP3 were reduced to 7,487, 6,913 and 7,829. UKBB Phenotypes For current UKBB resource, we have access to 496 variables whose data types are categorical (multiple), categorical (single), continuous, integer, date, text and time. Here, we focused on the variables of categorical (multiple), categorical (single), continuous and integer types, and categorized each variable as one of four value types: continuous, binary, ordered categorical and unordered categorical variable (Millard et al., 2019; Supplementary Table S1). Where a data field is measured at several time points we use the first occurrence only. It was noted that qualifications (data field 6138), a categorical (multiple) trait, was reorganized according to the underlying system (Guggenheim and Williams, 2016). Briefly, the original and unordered seven categories were reclassified and ordered as (1) none, (2) O-levels or CSEs, (3) A-levels, NVQ, HND, HNC or other professional qualification, and (4) college or university degree. Then the continuous, binary and ordered categorical variables were selected and used as the main phenotypes in G × P interaction analyses. Among the 199 variables, we selected 128 variables as the main phenotypes (Supplementary Table S2) in our proposed model to estimate G × P interactions where population difference was inferred from the first and second PCs. The other variables were used to control confounding effects owing to sex, age, year of birth, genotype batch, and assessment center (basic confounders adjusted for all the main phenotypes; the first 20 PCs were also used as basic confounders to account for population stratification) and Townsend deprivation index, smoking status, alcohol consumptions and many other variables (additional cofounders adjusted for some relevant phenotypes) or excluded if they were not likely to affect any of the main phenotypes (see the note of Supplementary Table S2). It is noted that in this paper, for the covariates used as fixed effects in the models to correct mean difference across confounding factors, we used the term "confounders" to distinguish the covariates used in the RNM methods, i.e., PC1 and PC2 (see Section "Statistical Models"). A Linear Mixed Model Without Considering G × P Interaction (Baseline Model) A standard linear mixed model assuming no G × P interaction can be written as: where y is an n × 1 vector of phenotypes with n being the sample size, µ is an n × 1 vector for fixed effects, g is an n × 1 vector of total genetic effects of the individuals with g ∼ N(0, Aσ 2 g ) and e is an n × 1 vector of residual effects with σ 2 g , where σ 2 g is the variance explained by all common SNPs and σ 2 e is the residual variance. In the GREML context (Yang et al., 2010(Yang et al., , 2011, A is a genomic relationship matrix (GRM) and I is an identity matrix. GRM can be estimated based on common SNPs across the genome and the elements of GRM can be defined as (VanRaden, 2008;Yang et al., 2010;Lee and Van der Werf, 2016): where L is the number of all common SNPs (L = 1,133,957 in this study), x il denotes the number of copies of the reference allele for the lth SNP of the ith individual, x l denotes all the numbers of copies of the reference allele across all the individuals, andp l denotes the reference allele frequency of the lth SNP. The variance-covariance matrix of the observed phenotypes (V) is: The SNP-based heritability, the proportion of the additive genetic variance explained by the genome-wide SNPs over the total phenotypic variance, is then referred as: The phenotypes with significant SNP-based heritability from this baseline model will subsequently be investigated for G × P interaction. G × P RNM Method In cases where G × P interaction exists across populations, the baseline model cannot account for heterogeneous genetic effects. We therefore applied RNM methods to detect heterogeneity across populations using the UKBB data. RNM and multivariate RNM (MRNM) have been demonstrated to perform better than the current state-of-the-art methods when detecting genotype-covariate and residual-covariate interactions in terms of simulation studies on type I error rate and power analyses . Here, we focus on G × P interaction by considering PCs as covariates in the RNM: where y, µ, g and e are the same defined in the baseline model above, g 0 and g 1 are n × 1 vectors of the zero-and first-order random regression coefficients, respectively, c is an n × 1 vector of covariate values of the n individuals (for which we used PC1 and PC2 values in this study). In the RNM, the random genetic effects, g, are regressed on the covariate gradient (reaction norm), which can be modeled with random regression coefficients, g 0 and g 1 . This G × P RNM accounts for phenotypic plasticity and norms of reaction in response to different populations (represented by PC values) among samples. The mathematical properties of variance-covariance structure between g 0 and g 1 allow us to verify whether estimates of the parameters are reasonable or not. Specifically, estimated values should be within a valid parameter space: The estimates which violated one of above criteria were excluded for follow-up analyses. We obtained a p-value to detect G × P interaction using a likelihood ratio test (LRT) that compared the goodness of fitness of two models (GREML and G × P RNM), penalizing the difference in the number of parameters between them. We further tested if the significant G × P interactions were orthogonal (independent without confounding) to residualpopulation (R × P) interactions, i.e., residual heterogeneity across populations . Similarly, the R × P interaction can be detected by an R × P RNM: where e 0 and e 1 are n × 1 vectors of the zero-and firstorder random regression coefficients when residual effects, e, are regressed on the covariate, c, i.e., an n vector of PC1 or PC2. Furthermore, a full RNM model with both G × P and R × P interactions can be expressed as: Since the G × P and R × P models are nested within the full model, LRT comparing the full and R × P or G × P model with an appropriate degree of freedom can determine the significance of orthogonal G × P or R × P interaction . More details about RNM can be found elsewhere . For the analyses showing a significant G × P interaction, we used rank-based INT phenotypes to check explicitly if the significance was due to phenotypic heteroscedasticity or normality assumption violation (Robinson et al., 2017). The bias of RNM/MRNM estimates due to non-normality of phenotypic values can also be remedied by applying the rank-based INT . In short, the pipeline of our G × P analysis method is briefly as below: firstly, exclude the phenotypes with no significant heritability by GREML; secondly, for the remained phenotypes, exclude the ones with no significant result by LRT comparing RNM and GREML considering basic and additional confounders of fixed effects; thirdly, for the remained phenotypes, exclude the ones with no significant result after further considering robustness to normality assumptions of phenotypic values. We also presented a flowchart showing the pipeline of our G × P analysis on the design of POP1 + POP2 (Figure 2). All models described above (i.e., GREML, bivariate GREML, RNM, MRNM) can be fitted using software MTG2 (Lee and Van der Werf, 2016). Spurious Signals Due to Selection or Collider Bias We used the UKBB data that have only a 5.5% response rate, i.e., selection. Consequently, the resulting sample may not be representative of the UK population as a whole and the selection may be associated with some of the phenotypes in the UKBB, causing selection or collider bias (Swanson, 2012;Munafò et al., 2018). To test whether the G × P interaction effects detected by our method was genuine or spurious due to selection or collider bias, we conducted a series of simulation studies with phenotypes differentially selected for POP1 (white British) and POP2 (other white Europeans). If two or more phenotypic variables simultaneously influence the probability of participation of individuals in a study, then investigating associations between those variables in the selected sample may induce collider bias (Munafò et al., 2018). Therefore, we further considered the same selection model but including two traits to evaluate collider bias effects on the detection of G × P interaction across POP1 and POP2. The statistical models to test selection and collider bias can be found in Supplementary Text S1 or Munafò et al. (2018). Estimating SNP-Based Heritability for 128 Phenotypes We first applied the standard GREML model to estimate h 2 SNP for the 128 phenotypes across POP1 + POP2, POP2 + POP3 and POP1 + POP3, respectively. The phenotypes with significant h 2 SNP (Supplementary Tables S3-S5) were further investigated for G × P interaction effects using our G × P RNM approach. Genetic and Residual Correlations Between Phenotypes and PCs The main response (y) and environmental covariates (c) are not always uncorrelated, for which multivariate RNM accounting for (genetic and residual) correlations between y and c should be used . We examined if there were non-negligible genetic and residual covariances between the main phenotypes and covariate (PC1 or PC2) for the complex traits with significant heritabilities (Supplementary Tables S6-S8). All genetic and residual covariances estimated by bivariate GREML were not significantly different from zero, and thus we used univariate RNM to detect the G × P interaction effects with covariate PC1/PC2 for those phenotypes. G × P Interaction For POP1 + POP2, we fit the data of the 70 phenotypes with significant h 2 SNP by modeling the G × P RNM with covariates PC1 and PC2, respectively (Supplementary Tables S9, S10). We excluded those estimates, which were not within the valid parameter space (see Statistical models), from the followup statistical test analyses, resulting in 29 and 32 traits remaining for PC1 (Supplementary Table S9) and PC2 analyses (Supplementary Table S10). We examined if there was significant G × P interaction and obtained p-values based on LRT comparing the fit to the data of the G × P RNM and null model. Significance level was determined by Bonferroni multiple testing correction: 0.05/140 = 3.57E−4 for the 70 phenotypes with covariates PC1 and PC2. Supplementary Figure S1 show that significant G × P interactions were found for ten complex traits which are related to blood pressure (pulse rate, automated reading), bone-densitometry of heel (heel BMD T-score, automated; heel broadband ultrasound attenuation, direct entry; heel QUI, direct entry; heel BMD), diet (lamb/mutton intake), sexual factor (age at first sexual intercourse), sleep (sleep duration), smoking (ever smoked) and education (qualifications). For each of the ten traits, we further considered a multiple covariate model that fit PC1 and PC2 jointly (Supplementary Table S11). However, G × P interactions were less significant than those obtained using the single covariate model fitting PC1 or PC2 separately (Supplementary Figure S2), otherwise, the estimates were out of the valid parameter space. This was probably due the fact that there was collinearity between G × P interactions from PC1 and PC2. In addition to the basic confounders for which the main phenotypes were initially adjusted (see Materials and methods), we further considered additional trait-specific confounders that Table S12). We examined the distribution of phenotypic values after controlling additional confounders of the six traits with significant G × P interactions (Supplementary Figure S3) and could not rule out the possibility that the interaction signals were due to non-normality (e.g., residual heteroscedasticity). We conducted the same analyses for the six traits using rank-based INT phenotypes (Table 1), which could control type I error rate due to a skewed and non-normal distribution of residual values . Indeed, phenotypic heteroscedasticity was remedied when using rank-based INT for the phenotypes of six traits as shown in Supplementary Figures S4-S9. For age first had sexual intercourse and qualifications that were shown to have significant G × P interactions, we further tested if the G × P interactions were orthogonal to R × P interactions, i.e., residual heterogeneity (see section "Materials and Methods"). Using the rank-based INT phenotypes adjusted for basic and additional confounders, we carried out an R × P model and a full model in which both G × P and R × P were fitted jointly. Subsequently, we conducted LRT to obtain p-values, comparing the full and nested models. A significant p-value from LRT between the full and R × P model indicates that G × P interaction is orthogonal to R × P interaction (see section "Materials and Methods" and Supplementary Table S13). For age first had sexual intercourse, although G × P or R × P interaction was significantly detected from the G × P or R × P model, it was shown that G × P interaction was not orthogonal to R × P (p-value = 0.88 for PC1 and 0.92 for PC2 in Supplementary Table S13). For qualifications, on the other hand, it was shown that the G × P and R × P interactions were statistically independent (p-value = 4.15E−05 for PC1 and 0.003 for PC2 in Supplementary Table S13). For POP2 + POP3, we conducted analyses using the same procedure as in the analyses of POP1 + POP2. The POP3 individuals are very close to those in POP1 in terms of ancestry PC, but their ethnicities are not white British as in POP1 (see section "Materials and Methods" and Figure 1). Thirteen phenotypes demonstrated a significant genetic heterogeneity for covariate PC1 or PC2 as shown in Supplementary Tables S14, S15. After controlling for additional trait-specific confounders and transforming by rank-based INT (Supplementary Table S16), the results for age first had sexual intercourse (p-value = 7.86E−05 for PC1) and qualifications (pvalue = 1.06E−15 for PC1) have demonstrated strong genetic heterogeneity signals (Table 2), which are consistent with our findings for POP1 + POP2. For qualifications, G × P interactions were significantly orthogonal to R × P interactions (p-value = 0.003 for PC1 in Supplementary Table S17). We also found significant results across POP2 + POP3 for anthropometric traits (waist circumference and weight) and diabetes diagnosed by doctor ( Table 2). However, these phenotypes were not discovered across POP1 + POP2 with significant G × P interaction signals. We also performed the same analyses on POP1 + POP3, which is not a diverse population group as POP1 + POP2 or POP2 + POP3, and thus was used as a negative control group (see section "Materials and Methods"). For several traits showing significant heterogeneous signals with covariate PC1 or PC2 after Bonferroni correction (see Supplementary Tables S18, S19), we further examined them by adding stringent confounders to correct for fixed effects and applying rank-based INT. The The phenotypes were adjusted by basic plus additional confounders of fixed effects and transformed by rank-based INT. The G × P interaction signals of age first had sexual intercourse and qualifications were remained significant even after applying rank-based INT phenotypes, however, the other traits were not significant anymore. The estimates which were not within the valid parameter space are marked as "Excluded." SE denotes standard error. DF denotes degree of freedom. For the categorical phenotype qualifications, there were various ways to convert the seven UKBB categories into a continuous or a binary measure (Okbay et al., 2016;Gazal et al., 2017;Lee et al., 2018). Following a previous study (Okbay et al., 2016), we transformed the multiple categories (data fields: 6138.0.0 to 6138.0.5) into an educational year measure (Supplementary Table S22). Based on this continuous phenotypic measure, we found significant genetic heterogeneity across POP1 + POP2 and POP2 + POP3 but no signal across POP1 + POP3 (Supplementary Table S23), which was consistent with our results obtained using four-level categories. We also examined G × P interactions for qualifications based on two types of binary measures (highest educational attainment versus other levels, and lowest educational attainment versus other levels) (Gazal et al., 2017). The results were consistent with those obtained using four-level qualifications, except that an unexpected significant signal across POP1 + POP3 for covariate PC1 was detected based on the binary measure of "college or university degree" versus other six categories (Supplementary Table S24). The Findings Are Robust to Selection or Collider Bias We examined the distribution of phenotypic values for age first had sexual intercourse and qualifications in which G × P interactions were consistently detected from both POP1 + POP2 and POP2 + POP3 (Supplementary Tables S25, S26). The distribution of age first had sexual intercourse is similar across POP1, POP2 and POP3. However, for qualifications, it is apparently shown that the subjects in POP2 and POP3 (other white Europeans) have higher qualification levels than those in POP1 (white British). Moreover, it is likely that the individuals in POP1 have higher educational levels than the general population of United Kingdom because individuals with higher educational levels are more likely to response to surveys from UKBB (Munafò et al., 2018). Our simulation studies testing for detecting spurious heterogeneity across POP1 and POP2 with multiple scenarios varying the level of selection odds ratios (see Supplementary Text S1 for details) have verified that (1) both G × P RNM and bivariate GREML are robust to the selection bias when using the same selection odds ratio across populations ( Table 3); (2) only bivariate GREML is robust against the selection bias when using different selection odds ratios across populations ( Table 3); (3) bivariate GREML is robust against the collider bias when estimating genetic correlation between POP1 and POP2, however, it generates biased estimation of genetic correlation between the two traits ( Table 4). It is noted that the level of selection odds ratios used in simulations is likely to reflect the real situation of qualifications, i.e,. different selection pressure between POP1 and POP2 in UKBB (see Supplementary Text S1 and Supplementary Table S27). For age first had sexual intercourse and qualifications, we also confirmed our findings using bivariate GREML, a robust approach against selection bias ( Table 5). As confirmed by the bivariate GREML, it was not likely that the findings for qualifications were spurious because of selection and collider bias. This was also evidenced by the fact that G × P RNM detected a significant interaction signal from POP2 + POP3, noting that POP2 and POP3 were similarly distributed for qualifications (see Supplementary Table S26). Similarly, the findings for age first had sexual intercourse were mostly robust whether using RNM or bivariate GREML except that there was no signal for POP1 + POP2 when using the bivariate GREML, probably due to the lack of power. It was noted that the phenotypic distributions of age first had sexual intercourse were very similar across POP1, POP2 and POP3 (Supplementary Table S25). Different odds ratio combinations (OR POP1,Y andOR POP2,Y ) generated phenotypic values in POP1 + POP2 with different selection bias levels. Type I error rates based on 100 simulation replicates were examined by G × P RNM and bivariate GREML respectively. The genetic correlations of the phenotype between POP1 and POP2 were estimated by bivariate GREML. The results by G × P RNM indicated high type I error rates for selection scenarios of OR POP1,Y = 1, OR POP2,Y = 2 and OR POP1,Y = 2, OR POP2,Y = 3. But for same selection pressures (OR POP1,Y = 1, OR POP2,Y = 1 and OR POP1,Y = 2, OR POP2,Y = 2), G × P RNM can effectively control false positive rate. Type I error rates assessed by bivariate GREML were controlled for all scenarios and the estimated genetic correlations were not significantly different from one for all selection scenarios. Here, we consider 0.05 as a significance level for controlling type I error rate. SE denotes standard error. DISCUSSION Previous results (Maier et al., 2015;Robinson et al., 2015;Yang et al., 2015;Tropf et al., 2017) were more likely to reflect heterogeneous genetic effects across nations or cohorts rather than populations as those designs were evidently confounded with country-specific factors (e.g., trait definition and measurement, cultural and societal difference). In this study, we focused on populations and proposed the new concept "genotype-population interaction" in which population is defined by the first and second ancestry PCs (each individual has a unique PC value). Using the RNM with whole-genome data from the UKBB, we have demonstrated significant G × P interaction effects for qualifications and age first had sexual intercourse across populations. Our findings corroborate the results in Type I error rates based on 100 simulation replicates were examined through estimated genetic correlations of the phenotype Y between POP1 and POP2 by bivariate GREML. Type I error rates under the null hypothesis that genetic correlation of 1 implies no interaction were controlled for these combinations (<5%), and meanwhile, significant negative genetic correlations between the two simulated phenotypes Y and Z demonstrated strong collider bias signal in selected POP1 + POP2. Here, we assume that the phenotype Z involves a sum of collider bias effects across all other traits on the main response Y. Here, we consider 0.05 as a significance level for controlling type I error rate. SE denotes standard error. Tropf et al. (2017) who reported that behavioral phenotypes (education and human reproductive behavior) have significant G × E interactions across populations. For anthropometric phenotypes, height and BMI, our G × P RNM model did not detect any significant interaction signals (Yang et al., 2015). However, the analyses of another two anthropometric traits (waist circumference and weight) have demonstrated significant genetic heterogeneity across the POP2 + POP3 group (other white Europeans). Actually, the results by Tropf et al. (2017) across seven populations have also revealed significant G × E interaction for BMI although the heterogeneity is not strong as for education and reproductive behaviors. Robinson et al. (2015) The phenotypes were adjusted by basic plus additional confounders of fixed effects and transformed by rank-based INT. The bivariate GREML results for qualifications indicated a significant genetic heterogeneity between POP1 and POP2 (p-value = 8.09E−04), and between POP2 and POP3 (p-value = 7.85E−04), but showed no genetic heterogeneity between POP1 and POP3. These results were consistent with our findings from the G × P RNM. For age first had sexual intercourse, the bivariate GREML detected a significant heterogeneity between POP2 and POP3 (p-value = 3.14E−05), however, there was no interaction signal between POP1 and POP3 (as expected). Unexpectedly, the bivariate GREML failed to find genetic heterogeneity across POP1 + POP2 although RNM provided a significant signal. SE denotes standard error. P-value was obtained through a Wald test under a null hypothesis that genetic correlation equals to 1. also reported that, for BMI, environmental differences across Europe masked genetic differentiation. Thus, these findings may be consistent for some anthropometric phenotypes when using diverse European ancestry populations. The previous results (Maier et al., 2015;Robinson et al., 2015;Yang et al., 2015;Tropf et al., 2017) were based on data collected from multiple countries. Therefore, various trait definitions in phenotypic measure and genetic measurement errors across countries may generate artificial heterogeneity. In our study, however, we used the UKBB data that have less cross-country factors and confounders. The phenotypic definitions and measurement of complex traits in the UKBB samples are well standardized and calibrated. Moreover, UKBB utilized uniform standards of imputation and quality control for genotype data. Therefore, our results may provide more reliable estimations of G × P interaction effects across populations. From the POP2 + POP3 analyses, we also found a significant G × P interaction for diagnosis of diabetes that is a binary response variable. As the RNM has not been explicitly verified for binary traits, we also used bivariate GREML to estimate the genetic correlation between POP2 and POP3 for this disease trait and found no significant signal for genetic heterogeneity (estimate is 0.7988, SE = 0.2044, p-value = 0.3249). This might be due to the fact that there was no genuine interaction effects or that the bivariate GREML was simply underpowered. For the two binary measuring ways of qualifications (lowest educational attainment versus other levels, and highest educational attainment versus other levels), we also used bivariate GREML to examine genetic correlations between POP1, POP2 and POP3 (Supplementary Table S28). The results for the binary phenotype of "none of the above" versus other six educational categories demonstrated significant genetic heterogeneity between POP1 and POP2 (p-value = 5.58E−05) and between POP2 and POP3 (p-value = 7.59E−05) but no significant signal between POP1 and POP3 (p-value = 0.0619), which were consistent with those obtained from the main analyses. For the binary data of "college or university degree" versus other six categories, the bivariate GREML indicated a marginally significant heterogeneity between POP1 and POP2 (p-value = 0.035) and no significant signal between POP2 and POP3 (p-value = 0.494), and POP1 and POP3 (p-value = 0.94). The reason that the genetic heterogeneity became weaker or disappeared is probably due to the fact that the bivariate GREML has less power compared to the RNM approach, and the phenotype categories reduced from four to two levels. Our results imply that causal variants at multiple loci may not be universal but rather specific to populations for some complex traits. The results on qualifications across POP1 + POP2 suggested that G × P interaction might be a reason for attenuation of SNP-based heritability when using data from different populations (see Supplementary Text S2 and Supplementary Table S29), which are agreed with Tropf et al. (2017). This missing or hidden heritability issue (Witte et al., 2014) can produce lower predictive power of polygenic risk scores from large GWAS (usually generated from meta-analyses of different populations) compared with single homogenous population since the reference heritability obtained from the meta-analyses among several populations is smaller than that obtained from single homogenous population (de Vlaming et al., 2017). Therefore, our findings suggest that large homogeneous population data sources (e.g., around 400,000 white British individuals in the UKBB) should be used to conduct polygenic risk prediction for some specific traits such as human behaviors. The current methods used for estimating G × E (or G × P) interactions, e.g., random regression (RR)-GREML (Jarquín et al., 2014) and GCI-GREML (Tropf et al., 2017), require that the main response should be stratified into multiple discrete groups according to covariate levels even for a continuous covariate (Maier et al., 2015). However, the arbitrary grouping ignores the difference of covariate values for the individuals within each group, and results in some loss of information. In contrast, the RNM allows us to fit a continuous covariate representing individuals uniquely (e.g., PC) in the model and produces unbiased estimates . In our results, bivariate GREML which labels the individuals into two discrete groups (POP1 and POP2) failed to find genetic heterogeneity for age first had sexual intercourse (Supplementary Table S27), while RNM detected significant G × P interaction across POP1 + POP2 (see Table 1). It may imply that G × P RNM is more powerful as it uses individual-level information represented by PC across populations, while bivariate GREML ignores such information within each stratified group. However, on the other hand, RNM may suffer from the selection bias when using different selection odds ratios across populations ( Table 3) while bivariate GREML is robust against such selection and collider bias (Tables 3, 4). It is noted that bivariate GREML requires pre-defined population labels (e.g., self-reported ethnicities). Residual-covariate interaction may result in heterogeneous residual variances across different covariate values, thus it is necessary to examine and distinguish genotype-covariate and residual-covariate interactions . Our results (Supplementary Tables S13, S17) provided cogent evidence of G × P and R × P interaction effects, which are (partially) independent without confounding, across populations for qualifications. However, for age first had sexual intercourse, there was no evidence showing that G × P interaction was orthogonal to R × P interaction from LRT comparing the full and nested models. Therefore, we could not rule out the possibility that the significant signal was mainly because of residual heterogeneity across populations. In order to disentangle G × P interaction from R × P interaction, the magnitude of G × P interaction should be large (e.g., qualifications) or sample size may have to be increased. There are several limitations in this study. Firstly, we examined G × P interaction across populations using three data designs (POP1 + POP2 and POP2 + POP3 as primary data, and POP1 + POP3 as a negative control), in which population is referred to the first and second ancestry PCs. As POP1 and POP3 are very close in terms of PCs, the individuals in the two primary groups POP1 + POP2 and POP2 + POP3 have common population structures (Figure 1). But both groups involve in different white ethnic backgrounds, i.e., POP1 may be closer to native British and POP2/POP3 is more likely to be descended from recent immigrants from many other European nations. Therefore, for our data designs, we cannot rule out the possibility that G × P interaction was confounded with immigrationspecific factors such as socioeconomic attainment, social relations and cultural beliefs (Drouhot and Nee, 2019). We also notice that, in the UKBB data source, there are numerous samples with other ethnicities (e.g., Indian, Caribbean, and African), thus future studies using our approach may aim to detect genotype-ethnicity interaction, which may uncover challenges for investigations into the genetic architecture of phenotypes across various ethnicities. Secondly, population defined by PCs in this study or by discrete groups in others (Yang et al., 2015;Tropf et al., 2017) includes both environmental and genetic information for individuals, thus the G × P interaction may not merely embody G × E interaction but also contains confounded genotype-by-genotype (G × G) interaction across populations. It may become a new challenge in the future to distinguish G × E and G × G in studies of genetic heterogeneity across populations. Thirdly, the sample size for people with other white ethnicity in UKBB (i.e., the sum of POP2 and POP3) is not large, thus the study may lack power for phenotypes with small SNP-based heritability such as behavioral traits. The phenotypes without significant heritability in the current samples were not investigated for G × P interaction, however, if boosting statistical power for those phenotypes, there may be new findings for heterogeneity across populations. Fourthly, the simulations on selection bias have demonstrated that the G × P RNM is not robust for data across populations with different selection odds ratios (see Table 3). Thus our approach is more preferable and restricted to data without selection bias or with the same selection pressure for populations. Finally, for genotypic information used in this study, we only examined common SNPs (minor allele frequency > 0.01). However, a recent study (Wainschtein et al., 2019) reported that the missing heritability for height and BMI may be explained by rare genetic variants accessed from whole-genome sequence data. Therefore, can rare population-specific variants increase our understanding of genetic heterogeneity across populations? Further research is required to answer this question. In conclusion, the main findings in our study demonstrated a significant population genetic heterogeneity for behavioral traits (age first had sexual intercourse and qualifications). Our study provided a paradigm shift tool in investigating genetic heterogeneity across populations. The new concept of G × P interaction with the use of ancestry PC is more plausible in explaining the genetic architecture of complex traits across heterogeneous populations. The G × P interaction effects on age first had sexual intercourse and qualifications were found by a powerful approach based on technically homogeneous data (free of genetic measurement errors and cohort/country confounding factors), and these findings were validated in both data designs POP1 + POP2 and POP2 + POP3. The analyses performed in this study can be applied to dissect the genetic architecture of complex traits and diseases across populations, and the results from these analyses will provide important information and suggestion for studies of polygenic risk prediction across Europeans. DATA AVAILABILITY STATEMENT Publicly available datasets were analyzed in this study. This research has been conducted using the UK Biobank Resource. UK Biobank (http://www.ukbiobank.ac.uk) Research Ethics Committee (REC) approval number is 11/NW/0382. Our reference number approved by UK Biobank is 14575. AUTHOR CONTRIBUTIONS CY and SL conceived and initiated the project. SL supervised and directed the study. CY and GN quality-controlled the data. CY analyzed the data and drafted the manuscript. JW provided key statistical advice. All authors revised the manuscript and approved the final version of the manuscript.	2019-08-23T06:03:58.964Z	2019-07-30T00:00:00.000	{ "year": 2020, "sha1": "e99c62102729bea80a74e680d55ff845095fcece", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fgene.2020.00379/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f372251a44dc24a89c2887c589dd36e7adbc9489", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology" ] }
259586047	pes2o/s2orc	v3-fos-license	Agrosilvopastoral system as a potential model for increasing soil carbon stocks: a century model approach ABSTRACT Agrosilvopastoral systems have been used as sustainable production models that can promote soil organic carbon (SOC) storage. However, there are no simulation studies with the Century model to estimate the SOC accumulation capacity in the long term, analyzing the effects of management and climate change in integrated crop-livestock-forest (ICLF) systems. This study aimed to simulate soil C dynamics in two chronosequences of land-use composed of native vegetation (NV), degraded pasture (DPA) and ICLF system in the Cerrado of Minas Gerais, in addition to designing future scenarios to verify the potential of SOC accumulation through climate change. The results showed that the Century model reliably simulated the SOC stocks in the two chronosequences evaluated. The model predicted an increase in SOC stocks at two sites by converting the DPA system (46.04 and 42.38 Mg ha-1) into ICLF systems (54.94 and 51.71 Mg ha-1). The Century also predicted that a 20 mm decrease in rainfall and a 2 °C increase in temperature in the tropical regions studied could reduce the SOC stocks more expressively in degraded pastures, while agroforestry systems could show a smaller reduction in SOC stocks. In addition, the results showed that replacing degraded pastures into agrosilvopastoral systems, especially in clayey soils, contributes to increasing SOC stocks. Thus, agroforestry systems are potentially viable to maintain the sustainability of agriculture in the face of climate change. INTRODUCTION Increase of greenhouse gases (GHG) emissions in recent decades has been the subject of extensive studies on climate change and, in particular, the carbon dioxide (CO 2 ), which is emitted into the atmosphere when native vegetation areas are converted into agriculture, which are identified as one of the main factors in the agricultural sector, responsible for the GHG emissions (Carvalho et al., 2014;Fujisaki et al., 2015;Durigan et al., 2017;Cerri et al., 2018). One of the strategies to mitigate climate change through soil carbon (C) accumulation is to identify soils that are low in C stocks, such as eroded soils and in stages of degradation, and convert them into sustainable agricultural systems (Lal, 2019;Lorenz et al., 2019).The conversion of degraded soils to sustainable production systems is an important management strategy to increase the potential for capture, storage, and sequestration of atmospheric C, mitigating GHG emissions (Minasny et al., 2017;Yang et al., 2019).Therefore, diversified and intensified systems, such as agroforestry systems, which integrate crop, livestock and forest in the same area, have the potential to promote CO 2 sequestration and to accumulate soil organic carbon (SOC) in depth with greater efficiency than conventional agricultural systems (Lorenz and Lal 2014;Coser et al., 2018;Ruiz et al., 2020). As the potential for soil C accumulation is directly related to climatic conditions (temperature and rainfall), the type of soil (texture and mineralogy), and the type of vegetation cover (Brandani et al., 2015), the application of models that value the capacity of soils to accumulate C can complement studies about soil organic matter (SOM) dynamics.From this perspective, it is possible to compare modeled data from different edaphoclimatic conditions and management strategies with long-term experimental observations or chronosequence studies, aiming to adjust the model and make the data more reliable for scale applications. The Century model has been used to evaluate the dynamics of SOM and nutrients in the long term, providing tools for analyzing the effects of management and climate change on productivity and sustainability in different agroecosystems (Parton et al., 1988).The model simulates the active (representing soil microbes and microbial products), slow (including resistant plant material derived from the structural pool and soil-stabilized microbial products arisen from the active pool), and passive (very resistant to decomposition and includes physically and chemically stabilized SOM) compartments that are directly affected by edaphoclimatic factors (for example, temperature, humidity and soil texture), litter chemical composition (lignin/N, C/N) and handling practices that control the decomposition rates and determine the flow of C between the compartments of the SOM (Metherell et al., 1993).In addition, the Century model simulates the microbial surface compartment related to litter decomposition on the soil surface.Plant residues above and below ground are subdivided into structural compartments related to the cell wall of plant residues, and metabolic, referring to the intracellular content of plant cells, depending on the lignin/N ratio in the residue.Increases in this proportion result in more residues, being added to the structural sets, which present slower decomposition rates (Parton et al., 1988).Thus, the Century model was created to simulate the dynamics of SOM in agroecosystems in temperate regions (Paustian et al., 1992;Parton and Rasmussen, 1994;Smith et al., 1997), but several studies show its applicability to tropical areas (Cerri et al., 2004(Cerri et al., , 2007;;Bortolon et al., 2011;Brandani et al., 2015;Zani et al., 2018). The objective of this study was to measure and to simulate the soil C dynamics in two chronosequences of land use intensification composed of native vegetation (NV), degraded pasture (DPA) and crop-livestock-forest integration systems (ICLF) in the Brazilian Cerrado, in addition to designing future scenarios to evaluate the SOC stocks in the face of climate change. Study areas The first study area is located at Barra Farm (16° 38' 44.02" S and 43° 42' 43.77" W) in the municipality of Francisco Sá -MG, in the northern mesoregion of Minas Gerais State, and is located in an area between the Cerrado and Caatinga biomes (Figure 1).The predominant Cerrado phytophysiognomy is "Cerrado sensu stricto". The average altitude of the area is 590 m and the municipality's annual rainfall for the past 30 years has been 1,003 mm.According to Köppen's classification system, the climate is Aw (Tropical savannah climate) with a rainfall regime characterized by two well-defined seasons, with average temperature oscillating between 21 and 28 °C (Figure 2).The soil in the study area was classified as Cambissolo Háplico eutrófico (Santos et al., 2018) with medium texture (Table 1), which corresponds to a Cambisol (IUSS, 2015). After converting the native Cerrado vegetation (NV1), the experimental area was first cultivated with Urochloa brizantha cv.Marandu (DPA1), and for about 13 years it remained without continuous management for maintenance of the plant stand homogeneity, fertilization, pest and disease management, and pre-defined stocking rate.The agrosilvopastoral system (ICLF1) was implemented in October 2012, where the genetic material of eucalyptus (Eucalyptus urograndis) was planted in double rows and forage sorghum (Sorghum bicolor) was grown between the lines.At the beginning of 2014, forage sorghum was planted along with the pasture, Urochloa brizantha cv.Marandu, and after its cultivation, the area was conducted as a silvopastoral system (eucalyptus and pasture).The arrangement of (3 × 2) + 14 m was adopted, and the figure 3 shows the chronosequence of the experimental area.(1997). Rev Bras Cienc Solo 2023;47:e0220136 The second area is located at the Moura Experimental Farm (18° 44' 52.03" S and 44° 26' 53.56" W) in the municipality of Curvelo -MG, central mesoregion of Minas Gerais State and is in a transition area between the Cerrado and Atlantic Forest biomes (Figure 1).The predominant Cerrado phytophysiognomy is "Cerradão". According to the Köppen classification system, the region's climate is Aw, corresponding to a tropical savanna climate with concentrated rains in the summer (October to April) and high temperatures, while winter consists of a dry period (May to September) with lower temperatures.The annual rainfall rate in the municipality over the past 30 years was 1,064 mm, with an average temperature between 20 and 26 °C (Figure 2).The soil in the experimental area was classified as a Latossolo Vermelho distrófico típico (Santos et al., 2018) with clayey texture (Table 1), which corresponds to a Ferralsol (IUSS, 2015). After converting the native Cerrado vegetation (NV2), the experimental area was initially cultivated with Urochloa decumbens Stapf pasture (DPA2), for about 20 years, and remained without continuous management for maintenance of the plant stand, fertilization, pest and diseases management, and pre-defined animals stocking rate.In 2014, the site was characterized by an area of exposed soil and the presence of invasive plants, when the agrosilvopastoral system (ICLF2) was installed.Eucalyptus urograndis (clone 144) was intercropped with corn (SHS hybrid 7920) and forage of marandu grass (Urochloa brizantha cv.Marandu).The arrangement of 12 × 3 m was adopted, with a 1.5 m separating the eucalyptus from the intercropped corn and forage. The Marandu grass experimental unit (MAR) was installed in December 2014 using the same planting recommendations and cultivation described for Urochloa brizantha cv.Marandu in the ICLF2 system.The pasture area (DPA2) consisted of brachiaria grass (Urochloa decumbens Stapf) pasture and was previously used by dairy cattle and left without defined management and without maintenance fertilization for about 20 years, thus presenting a pasture characterized by low productivity, exposed soil and infestation of invasive plants.The area of native vegetation (NV2), classified as "Cerradão", was located adjacent to the experimental units.The chronosequence and land-use are described in figure 3. Rev Bras Cienc Solo 2023;47:e0220136 Soil physical and chemical analysis Four soil trenches (1 × 1 × 0.5 m) were opened in different land-uses.Soil samplings were carried out in 2016 and 2018 at 0.00-0.20 m soil layer, and the samples were collected randomly in the tree lines (rows) and in the pasture (alleys).The samples were passed through 2 mm sieves, and the residues of plants, roots and seeds were removed manually.Determining bulk density, pH, clay content, silt and sand was carried out according to the methodology proposed by Claessen (1997). To determine the total C content (Table 1), the soil samples were previously air-dried, homogenized, ground, and passed through 0.150 mm sieves and, later, analyzed by dry combustion in an elemental analyzer (Carlo Erba / CHN-1110) coupled to a mass spectrometer (Thermo Scientific/ Delta Plus).Soil C stocks in ICLF1, ICLF2, MAR, DPA1, and DPA2 systems were calculated for the same soil mass as their respective native vegetation (NV1 and NV2), according to the methodology proposed by Ellert and Bettany (1995) and Moraes et al. (1996). Simulation of changes in soil carbon stocks The Century model, version 4.0, was used to simulate changes in soil C stocks at 0.00-0.20 m soil layer in two chronosequences under different management practices, and the standard values of the fix.100 parameters were kept unchanged.Three compartments of soil organic matter (active, slow and passive) were evaluated, each representing fractions of soil organic matter with different potential decomposition rates.These compartments are directly related to edaphoclimatic factors, chemical litter composition such as lignin/N, C/N and management practices.These factors control the decomposition rates of organic material and determine the C and N flow between the SOM's compartments (Metherell et al., 1993). Parameterizations for SOM evaluation were performed in the top 0.20 m, according to the Century model.The simulation yield variables evaluated were soil C stocks (somsc) and SOC compartments: active (som1c(2); which represents soil microbial biomass and microbial products, free light fraction), slow (som2c; resistant plant material to decomposition derived from structural compartment and microbial products stabilized in soil derived from active and surface microorganisms, soil occluded light fraction) and passive (som3c; very resistant to decomposition and includes physically and chemically stabilized SOM, soil humic substances, heavy fraction ) (Metherell et al., 1993). Initializing the Century model To initialize the model, data on climate, soil texture, soil density, and pH and N input were inserted, as described by Parton et al. (1993).In this study, climatic data (average maximum and minimum monthly temperature and precipitation) for the period from 1988 to 2018 were used.The data sources were obtained by the National Institute of Meteorology (Inmet, 2019), whose weather stations, Montes Claros-A506 Station (16° 68' 63.16" S and 43° 84' 37.63" W) and , were close to the experimental farms, as well as historical information from the Meteorological Database for Teaching and Research -BDMEP, obtained by Inmet.The pluviometric index and average temperature of the last 30 years are shown in figure 2. The input data of soil texture, pH, and bulk density are described in table 1. To initialize the compartments of the SOM model, Century was provided with the history of land-use and the management practices of the chronosequences of Francisco-Sá-MG and Curvelo-MG.An equilibrium condition (7000 years) was performed on the model for the Cerrado forest until 1995, (corresponding year 1), (Chronosequence 2, Curvelo) and 1998, (corresponding year 1), (Chronosequence 1, Francisco Sá), when the native vegetation was removed for the initial conversion to pastureland (Figure 3).After conducting pasture without management operations for 13 and 20 years in Francisco Sá and Curvelo, Rev Bras Cienc Solo 2023;47:e0220136 respectively, the areas were classified as degraded pasture.Then, in both places the degraded pasture was converted to an agrosilvopastoral system.In Curvelo, there was also a simulation of the conversion of degraded pasture into a well-managed pasture of Marandu grass. The soil C data is derived from an initial several thousand-year "equilibrium" simulation using average precipitation and temperature data that was read from the site specific.100file. The model was calibrated with SOC stocks measured under native vegetation of Cerrado sensu stricto (Chronosequence 1) and a second calibration was also performed for native vegetation of Cerrado "Cerradão" (Chronosequence 2).The output variable "somsc" was used because it most closely represents our measured soil C stocks.Calibration of the Century model was first performed for the chronosequence 1.After simulations, another calibration was performed for the chronosequence 2. The calibration process consisted of iteratively running the model, inspecting the output and changing the default biomass production parameters "prdx" for tropical trees or grasses until the "somsc" matched the measured soil C stocks.For Eucalyptus urograndis tree, above-ground biomass production in g m -2 was calculated from production data obtained by Santana et al. (2016) for this species in the northern region of Minas Gerais and parameterized in the crop.100file to run the Century model. Future scenarios To simulate the impacts of different long-term management practices, for each of the evaluated chronosequence, the model Century runs were extended for 100 yrs after current scenarios were completed.Another "site.100"file was created for future scenarios.Scenarios were modeled for a 2 °C increase in temperature, according to IPCC scenarios (2018), and a decrease in monthly precipitation of 20 mm, in line with the downward trend in precipitation in the regions observed by Marengo (2007) and Cunha et al. (2019).Cunha et al. (2019) studied the Integrated Drought Index (IDI), which combines a meteorology-based drought index and an index based on remote sensing, to assess drought events from 2011 to 2019 in Brazil and verified the occurrence of droughts and droughts severe throughout Brazil and especially in semi-arid regions.Marengo (2007) pointed out that there may be a 20 % reduction in rainfall in the Southeast region.Thus, an annual reduction of 20 % was calculated for the climatic precipitation data of the last 30 years, and the average monthly reduction values were close to 20 mm.The data was altered in the "site.100"file to analyze the response of SOC stocks after the simulation of climate changes. Statistical analysis Statistical analyses of the observed and modeled quantitative data were performed according to the methodology proposed by Smith et al. (1997) to assess the adequacy of the Century model to SOC stocks measured as a function of management practices and the time released since land-use conversion.These metrics included were root mean square error (RMSE), Pearson correlation coefficient (r), coefficient of determination (R 2 ), mean difference between observed and simulated values (M), coefficient of residual mass (CRM) and modeling efficiency (EF). RESULTS The two chronosequences were chosen to represent different soil clay contents, climatic conditions and phytophysiognomies of the Brazilian Cerrado.As expected, SOC stocks were higher in areas of native vegetation. Chronosequence 1 -Francisco Sá/MG The Century model reliably simulated the SOC stocks of the chronosequence 1 as the values were very close to those determined in the laboratory (Figure 4a and figure 5a). After the native vegetation's removal and subsequent introduction of Urochloa brizantha cv.Marandu (DPA1), with 13 years without grazing management, SOC stocks decreased from 65.57 to 46.04 Mg ha -1 after ten years (Figure 5a).The SOC stocks will continue to decline if the DPA1 system remains without proper management since stocks will reach 32.27 Mg ha -1 at the end of the simulation after 100 years (2098 -Corresponding year). However, the simulation was different when the DPA1 system was converted to ICLF1 in 2012, with the introduction and proper management of Marandu grass in 2014.Four years after the introduction of the integrated production system and with current management practices, Century simulated that SOC stocks would increase from 54.94 Mg ha -1 after 18 years to 76.10 Mg ha -1 after 100 years (2098 -Corresponding year), exceeding the equilibrium values of NV1, 65.57Mg ha -1 , prior to 1998 (Figure 4 and figure 5a). The future scenario of chronosequence 1 can be seen in figures 5b and 5c.We observed that the adoption of ICLF 1 system with good soil management practices can increase soil C stocks in levels close to that found in NV1 after 30 years (2027 -Corresponding year), to exceeding these values in later years.However, the DPA1 system will lead to a reduction in soil C stocks over time. The future scenario with a decrease in rainfall of 20 mm per month showed a reduction in C stocks, when compared to the initial simulated SOC stocks without future scenarios (Figures 5a and 5b).In the ICLF1 system, soil C stocks ranged from 52.52 Mg ha -1 after 30 years (2027 -Corresponding year) to 60.57Mg ha -1 after 100 years (2098 -Corresponding year).On the other hand, soil C stocks in DPA1 system ranged from 27.99 Mg ha -1 after 30 years to 20.50 Mg ha -1 after 100 years. Likewise, in the future scenario increasing the average temperature in 2 °C (Figure 5c), there was a reduction in SOC stocks when compared to the initial simulated SOC stocks without future scenarios (Figures 5a and 5c).In the ICLF1 system, SOC stocks showed values from 57.35 Mg ha -1 after 30 years (2027 -Corresponding year) to 68.60 Mg ha -1 after 100 years.However, for the DPA1 system, the observed values ranged from 25.35 Mg ha -1 after 30 years to 19.64 Mg ha -1 after 100 years (2098 -Corresponding year).The simulations of the active, slow and passive compartments modeled by Century showed differences between the systems.In chronosequence 1 (Figure 6a), the active and slow compartments of the DPA1 system decreased over time.However, when converting it to the ICLF1 system (Figure 6b), its values increased considerably.For example, in the DPA1 system, the active and slow fractions reduced from 0.79 Mg ha -1 and 39.08 Mg ha -1 after 10 years to 0.41 Mg ha -1 , and 8.55 Mg ha -1 at the end of the simulation, after 100 years (2098 -corresponding year), respectively.Different ) pattern was observed for the passive fraction, which remained stable over time.However, in the ICLF1 system, there was an increase in the same fractions (active and slow) that ranged from 1.72 and 30.22 Mg ha -1 after 18 years to 1.67 Mg ha -1 and 49.49Mg ha -1 at the end of the simulation, after 100 years, respectively.The Century model estimates were consistent with the changes observed in the field in the SOC stocks of the chronosequence 2 (Figure 4b and figure 5d).After removing the native vegetation (NV2) and introducing the Brachiaria decumbens pasture (DPA2), maintained for 20 years without management and with low-productivity, the SOC stocks decreased from 87.74 Mg ha -1 in year 1 (1995 -Corresponding year) to 42.38 Mg ha -1 after 23 years (2018 -Corresponding year).The model predicted a decline in SOC stocks over time, with values of 33.28 Mg ha -1 at the end of the simulation, after 100 years (2095 -Corresponding year).However, in 2015, part of this system was converted into an area with ICLF2, combining Urochloa brizantha 'Marandu' with Eucalyptus urograndis, and another part in monoculture of Urochloa brizantha 'Marandu' (MAR).According to the results of the simulation, the conversion to these systems with maintenance of current management practices would promote an increase in SOC stocks in ICLF2 and MAR, ranging from 51.71 and 48.11Mg ha -1 after 23 years to 72.07 and 61.57Mg ha -1 after 100 years, respectively (Figure 5d). The introduction of good soil management practices promoted an increase in SOC stocks in the ICLF2 system in greater proportions compared to the other systems evaluated, and this increase is gradual over time.However, the simulation showed that the DPA2 system reduced C stocks over time. Future scenarios for the chronosequence 2 can be seen in figures 5e and 5f.Firstly, with a decrease in rainfall of 20 mm per month, we found an increase in SOC stocks (Figure 5e) in ICLF2 and MAR systems, ranging from 67.48 and 60.20 Mg ha -1 after 30 years to 78.00 and 64.64 Mg ha -1 after 100 years, respectively.However, for DPA2 system, there was a decline over time, with values of 39.09 Mg ha -1 after 100 years.Secondly, in the future scenario, increasing the average temperature by 2 °C, there was a reduction in SOC stocks, compared to the initial simulated values in the evaluated systems (Figures 5d and 5f).For ICLF2 and MAR, values ranged from 59.48 and 52.13 Mg ha -1 after 30 years to 69.37 and 58.22 Mg ha -1 after 100 years, respectively.However, for t DPA2 system, there was a decline in SOC stocks over time, with values of 28.90 Mg ha -1 after 100 years. The simulations of the active, slow and passive compartments of Curvelo/MG chronosequence are shown in figures 6c, 6d and 6e.In the DPA2 system (Figure 6c), the active and slow fractions decreased from 0.78 and 17.87 Mg ha -1 after 20 years to 0.79 and 9.74 Mg ha -1 at the end of the simulation, after 100 years, respectively.When the conversion of the DPA2 system to ICLF2 occurred, the values of the active and slow fractions increased from 2.03 and 26.56 Mg ha -1 after 22 years to 2.79 and 42.21 Mg ha -1 after 100 years, respectively (Figure 6d). Increased values in the active and slow compartments were also evident in the conversion of DPA2 system to MAR, which ranged from 1.70 and 22.64 Mg ha -1 after 22 years to 2.24 and 33.77Mg ha -1 after 100 years (Figure 6e).It is noted that the values of these fractions were higher in the ICLF2 system than in MAR.In addition, we observed that the passive fraction values were reduced when NV2 system was changed to DPA2, varying from 42.63 Mg ha -1 in year 1 to 23.65 Mg ha -1 after 20 years of simulation and a slight increase with the introduction of the ICLF2 and MAR systems. Statistical results The results of the statistical tests used to evaluate the simulations in the two chronosequences are described in table 2. The measured and simulated SOC stocks, respectively, showed a high correlation (r = 0.95; p<0.05) for chronosequence 1 and (r = 0.97; p<0.05) and chronosequence 2 (Figure 7).The result of the simulations produced values of R 2 <1.0, meaning that the deviation from the average of the simulations of the simulated values is less than that observed in the measurements.The CRM values ranging from -0.03 and -0.06 allowed us to infer about the success of the simulation process.The results for RMSE (ranging from 5.66 and 7.28) indicated that we found a small difference between the measured and simulated values.The modeling efficiency (EF) ranging from 0.75 and 0.97 provides a comparison of the efficiency of the chosen model with the efficiency in describing the data as the average of the observations.Its positive value indicates that the simulated values describe the trend in the measured data better than the average of the observations.The M values calculated close to zero (-1.64 and -3.08) between observations and simulated scenarios showed that the consistent error was small. DISCUSSION The Century model accurately simulated the SOC changes influenced by the history of land-use of the two chronosequences evaluated in the Brazilian Cerrado.Our results confirmed that the model was efficient and capable of assertively simulating variations in soil C stocks in the chronosequences of land-use evaluated.Our findings agree with several studies that used the Century model to assess the impacts on SOC stocks in different chronosequences and soil management practices.Using the Century model, Cerri et al. (2004Cerri et al. ( , 2007)), Tornquist et al. (2009), Bortolon et al. (2011), Brandani et al. (2015), Silva-Olaya et al. ( 2017) and Zani et al. ( 2018) reliably reflected SOC trends in studies of agricultural chronosequences and changes in soil management in Brazil. The management history influenced the SOC stocks of the two studied chronosequences, when the soil under DPA was converted into a more conservationist-integrated farming system such as ICLF1 and ICLF2 (Figure 4).The litter, originating from the natural deforestation of the eucalyptus and the influence of the root system of the pastures, deposited on the soil by these systems, contributes to the inputs of SOM and increase of the SOC stocks over time.2018) simulated the SOC stocks and found that the addition of vinasse (sugarcane residue) to the soil increased the SOC stocks at the expense of the system without vinasse application, confirming the importance of the entry of residues for the increment of SOC stocks in agricultural management systems.Similarly, Brandani et al. (2015), used the Century model to evaluate the SOC dynamics in conventional sugarcane systems, with waste burning, and the use of conservationist management without burning waste.These authors observed that, at the end of the simulation, the conservationist system maintained SOC stocks at levels equal to or higher than native Cerrado vegetation, while conventional practices, such as burning residues, would cause SOC stocks reduction to levels below 60 % of native vegetation levels. The model simulated that the deforestation of native Cerrado and the implementation of pastures, which due to the lack of adequate management practices, were in a degradation stage, culminating in a decline in SOC stocks (Figure 5).When converting the DPA1 and DPA2 pasture into the ICLF1 and ICLF2 system, respectively, there was an increase in SOC stocks, the same was verified for the MAR system, but with lower values.In the chronosequence 1, at the end of the simulation, the SOC of the ICLF1 system (showed higher values than the native Cerrado (NV1; Figure 5a).This also occurred in the active, slow, and passive SOM compartments.As the active compartment has a very fast turnover rate, its monitoring can be useful as an alert to changes in soil quality.Similar results were found by Cerri et al. (2007) who, assessing the accuracy of the Century model in estimating SOC changes in 11 chronosequences in forest condition for pastures in the Brazilian Amazon, predicted that deforestation and conversion to well-managed pastures would cause an initial decline in SOC stocks (0.00-0.20 m) followed by a slow rise to levels higher than the native forest. In another study by Oelbermann and Voroney (2011), the Century model was adequate to simulate SOC stock changes in tropical and temperate monoculture and agroforestry systems.The model predicted, over time, a decline in SOC stocks for the monoculture system and an increase in SOC in agroforestry systems.According to the authors, this result was associated with a higher input of SOM from tree pruning and litter.In our evaluations modelled by Century, an increase in SOC stocks was observed for the active, slow, and passive compartments, when DPA system was converted into ICLF1, ICLF2 and MAR (Figure 6).This is related to the conservationist soil management practices that increase the content of SOM, due to greater floristic diversity, litter input, and presence of roots in the soil, mainly by forage grass, contributing to an increase in SOC stocks over time. The active, slow and passive compartments of NV1 and NV2 are different since they present different phytophysiognomies and edaphoclimatic conditions (Figure 2).The floristic composition of native vegetation is quite different, which may contribute to the differences in the carbon fractions of the active, slow and passive compartments.Furthermore, these NVs are an example of the interference of climate in the decomposition dynamics of SOM.The NV1 rainfall is distributed over a few months of the year, with several months with dry period.This can cause stress in the microbiota community and increase the mineralization rates of SOM when the soil has moisture again, and even the consumption of the most protected organic matter in the soil, such as the slow and passive compartments, may occur. Under these conditions, easily decomposable compounds are consumed (active soil compartment) and small populations of k-strategist microorganisms survive and slowly consume the most resistant and stable organic matter in the soil.Microbial activity continues to decrease, part of the carbon is physically protected in the micropores, while another part, which is chemically protected, such as humic substances (passive compartment), can be acted upon by k-strategist microorganisms, releasing carbon into Rev Bras Cienc Solo 2023;47:e0220136 the atmosphere.In stable systems like NVs, this is compensated during the annual cycle by slow and continuous decomposition by k-strategists, resulting in a small net change in SOM over time.However, in conventional agricultural systems, soil tillage contributes to the reduction of slow and passive soil compartments (Brady and Weil, 2013;Vries and Shade, 2013;Jansson and Hofmockel, 2020). In the scenarios of future precipitation reduction (Figures 5b and 5e), we found that the permanence of the DPA1 and DPA2 systems would cause a reduction in SOC stocks, while the introduction of the ICLF1, ICLF2 and MAR systems would promote increases in SOC stocks.According to Roe et al. (2019), the introduction of trees in agricultural systems promotes benefits for increasing water flow in the system.For example, the presence of deep roots increases the water's infiltration capacity and the litter deposition increases the levels of organic matter in the soil, which together improve the soil structure and the stability of the aggregates, preventing water loss through the evaporation process. The presence of trees in agricultural systems has an important role in the production and regulation of temperatures and freshwater flows on a regional and even global scale (Ellison et al., 2017).Our simulations with future scenarios for increasing regional temperature (Figures 5c and 5f) showed that the permanence of the DPA1 and DPA2 systems would cause a reduction in SOC stocks, decreasing the soil carbon reservoir over time, while the change of land use to ICLF1 systems, ICLF2 and MAR would promote increases in COS stocks.Strategy for land-use change from degraded areas to agricultural systems containing trees and reforestation associated with strategies to reduce forest deforestation are important for adaptation and mitigation strategies in the face of climate change.In addition, Brazil is among the ten countries with the greatest potential for GHG mitigation in the land use sector, which can help achieve the 1.5 °C temperature target proposed in the Paris Agreement (Roe et al., 2019). According to Houghton et al. (2015), adopting sustainable strategies, such as reforestation, using degraded lands with the introduction of forest species, and containing deforestation, has great potential to prevent global warming above 2 °C.According to these authors, the management of tropical forests becomes important, since they maintain large carbon stocks in their plant biomass and in the soil.The scenarios of reduced rainfall and increased average temperature in the regions of Francisco Sá and Curvelo/MG corroborate with the data obtained in the two weather stations (Figure 2), where there was a tendency to decrease precipitation and increase the region's average temperature over the past 30 years.This global trend is in line with projections made by the IPCC (2018) and by several studies (Schewe et al., 2014;Sedláček and Knutti, 2014;Schleussner et al., 2016a,b;Rogelj et al., 2018). Agricultural systems with a greater diversity of floristic species stimulate the growth of a more abundant and diversified heterotrophic decomposing community with the capacity to increase SOC stocks and contribute to the mitigation of climate change (Buzhdygan et al., 2020).A similar idea is expressed by Oelbermann and Voroney (2011), considering that the systems with greater diversity of floristic species show a high input of SOM, contributing to the increase of SOC stocks in the long term and, consequently, of the active, slow and passive C compartments of the soil. A study performed by Oelbermann et al. (2017) also obtained reliable results when applying the Century model to analyze long-term SOC in monoculture and intercropped systems with corn and soybean crops.The authors observed that SOC stocks declined when production was under monoculture with conventional soil management, after which there was an increase in SOC when implementing the intercropped system.In addition, an increase in SOC stocks was observed in the active and slow fractions of the intercropped systems to the detriment of monocultures.According to Lal (2018), the adoption of sustainable soil management practices, with the introduction of trees to the agricultural system, implies increasing of SOM and promoting the C sequestration from the atmosphere for fractions that are physically and chemically protected in the soil, contributing to the mitigation of GHG emissions. In Brazil, approximately 60 % of pasturelands are in some stage of degradation (Andrade et al., 2014;Oliveira et al., 2017).The replacement of these degraded areas, with low SOC stocks, by more sustainable management systems, such as the ICLF, presents itself as an alternative to monoculture systems in promoting improvements in soil quality and increasing SOC stocks in the short, medium and long-term (Conceição et al., 2017). Our simulations showed that the replacement of DPA1 by the ICLF1 system (Figure 5a) showed an increase in SOC stocks that can compensate, in the long run, the losses of C that occurred by converting native vegetation to pasture.In addition, the simulation revealed a greater contribution by SOC in the conversion of DPA2 into the ICLF2 system than in the MAR system, at the end of the simulation (Figure 5d).This can be explained by the greater input of litter and tree roots and greater biomass production by the root systems of the Marandu grass in the soil, which provides, according to Lal (2018), accumulation of SOM for a good performance of the system productivity, allowing improvements in the physical, chemical and biological properties of the soil to reduce the inputs of agricultural products. The entry of organic waste, with a low C:N ratio, such as litter, root senescence, and crop residues, can reduce the priming effect of native SOM in agroforestry systems, contributing to the increase in SOC stocks (Cardinael et al., 2018).When organic waste with a high C:N ratio is added to the soil, microorganisms, in search of energy and nutrients, decompose the native SOM, releasing stabilized C in the passive compartment of the atmosphere. According to Pezzopane et al. (2021), integrated systems with the presence of trees have a high productive potential and capacity to remove atmospheric C and mitigate GHG emissions, where E. urograndis with eight years and density of 167 trees per hectare showed a total biomass of 9.175 Mg ha -1 yr -1 and C stocks in the stem of 4.125 Mg ha -1 yr -1 .Müller et al. (2009), using different species in silvopastoral system with 10 years and population density of 105 plants hectare, estimated the stem biomass of 2.481 Mg ha -1 yr -1 and C stocks of 1.117 Mg ha -1 yr -1 for E. grandis, and biomass accumulation of 1.28 Mg ha -1 yr -1 and C stocks of 0.58 Mg ha -1 yr -1 for U. brizantha.In the same way, Pezzopane et al. (2020) found biomass accumulation data for the species of Urochloa brizantha cv.BRS Piatã in integrated systems with Eucalyptus urograndis, containing 333 trees per hectare, ranging from 0.79 to 1.14 Mg ha -1 cycle -1 of forage dry mass.According to the authors, forage accumulation in integrated systems was similar to the intensive monoculture system.This denotes that the entry of light into the integrated system, with the presence of trees, is crucial for the proper management of these systems and for obtaining forage with adequate productivity. According to previous studies, the potential of C accumulation in the trees of integrated systems is related, among other factors, to the species, the population density and the entry of light into the system.In addition, integrated systems, as reported by Pezzopane et al. (2021), have the potential to sequester C, which, depending on the destination of wood, can store the C in the biomass for long periods.Santana et al. (2016), evaluating the productivity of E. urograndis in silvopastoral system and monoculture, found the highest average volume of wood per tree, around 0.2228 m 3 (111.4m 3 ha -1 ) when compared to monoculture (0.1895 m 3 per tree).The authors also argue that well-managed integrated systems have a high potential for recovering degraded pastures, as they are an indicator of soil quality recovery. Regarding the simulation results for SOC stocks (Figures 5 and 6), they also can be related to C and N inputs in the soil, as well as turnover rates of SOM.Thus, considering the difference in the clay content (Table 1), the more clayey soil in chronosequence 2 showed less SOC losses than chronosequence 1, associated with sustainable agricultural practices that can contribute to greater accumulation in SOC stocks, as well as in the probable Rev Bras Cienc Solo 2023;47:e0220136 dynamics of the formation and stabilization of new soil aggregates.In clayey soils, the organic material is better protected from decomposition by microorganisms because it is linked to humo-clayey complexes or sequestered inside the aggregates (Six et al., 2000).Therefore, the results of this simulation indicate that clayey soils for these land uses are priority situations for allocating resources to increase carbon fixation in the soil. CONCLUSION Century model can be applied to evaluate the chronosequences of the land-use intensification with agrosilvopastoral systems in the Cerrado region.The SOC stocks increased over time in the conservationists' systems, including well-managed pastures. However, the integrated farming systems were the best alternatives for SOC accumulation in relation to pasture in monoculture.So, the land-use change from degraded pasture to agroforestry systems would not only recover SOC stocks, but their stocks can reach higher values than native Cerrado areas after 100 years of simulation.The model also predicted that the decrease in rainfall and an increase in temperature by 2 °C in the studied region could cause a decline in SOC stocks, especially in degraded systems, while agrosilvopastoral systems can be more resistant to reducing SOC stocks over time. Figure 1 .Figure 2 . Figure 1.Location of the study areas in the municipalities of Francisco Sá and Curvelo, Minas Gerais State, Brazil. Figure 3 . Figure 3. Chronosequence of land-use and brief description of the management practices used in the integrated systems located at the municipalities of Francisco Sá and Curvelo, Minas Gerais State, Brazil. Figure 4 . Figure 4.Measured and simulated values of soil C stocks at 0.00-0.20 m soil layer.Chronosequence evaluated in the municipality of Francisco Sá (a) and Curvelo (b), Minas Gerais State, Brazil.The averages followed by the same letter do not differ between land-uses "lower case" and between simulated vs measured "upper case" using the t test at 10 % probability.NV1 and NV2: Native vegetation, DPA1 and DPA2: Degraded pasture, ICLF1 and ICLF2: Integrated Crop-Livestock-Forest systems, MAR: Monoculture of Marandu grassd at the municipalities of Francisco Sá and Curvelo, Minas Gerais State, Brazil. Figure 6 . Figure 6.Carbon stocks in the Slow, Passive and Active compartments of soil organic matter.Chronosequence evaluated in the municipalities of Francisco Sá (a, b) and Curvelo (c, d, e), Minas Gerais State, Brazil.NV1 and NV2: native vegetation; DPA1 and DPA2: degraded pasture; ICLF1 and ICLF2: Integrated Crop-Livestock-Forest systems; MAR: Monoculture of Marandu grass. Figure 7 . Figure 7. Measured versus simulated soil C (a,b) in the municipality of Francisco Sá and Curvelo, respectively, in areas under native vegetation, degraded pastures, crop-livestock-forest integration systems and pasture monoculture in the Brazilian Cerrado, n=4. Table 1 . Soil properties at 0-20 cm in the two chronosequences of land use in the municipalities of Francisco Sá and Curvelo, Minas Gerais State, Brazil Table 2 . Statistical tests applied for comparison between simulated and observed values of soil C stocks in the two chronosequences located at Francisco Sá (n = 5) and Curvelo -MG (n = 7), in the Cerrado of Minas Gerais State, Brazil (1) r: sample correlation coefficient; R 2 : coefficient of determination; CRM: coefficient of residual mass; RMSE: root mean square error; EF: modeling efficiency; M: mean difference between observed and simulated values.	2023-07-11T15:46:35.596Z	2023-03-10T00:00:00.000	{ "year": 2023, "sha1": "bed7a98e05f24496f3108d3dfcdf6e9ae2b67c80", "oa_license": "CCBY", "oa_url": "https://www.rbcsjournal.org/wp-content/uploads/articles_xml/1806-9657-rbcs-47-e0220136/1806-9657-rbcs-47-e0220136.pdf", "oa_status": "GOLD", "pdf_src": "Dynamic", "pdf_hash": "acb73ae8bb14c75ba1021972ebec8d19e0813b31", "s2fieldsofstudy": [ "Environmental Science", "Agricultural and Food Sciences" ], "extfieldsofstudy": [] }
125270213	pes2o/s2orc	v3-fos-license	Emergent organization in a model market We study the collective behavior of interacting agents in a simple model of market economics originally introduced by N{\o}rrelykke and Bak. A general theoretical framework for interacting traders on an arbitrary network is presented, with the interaction consisting of buying (namely, consumption) and selling (namely, production) of commodities. Extremal dynamics is introduced by having the agent with least profit in the market readjust prices, causing the market to self--organize. We study this model market on regular lattices in two--dimension as well as on random complex networks; in the critical state fluctuations in an activity signal exhibit properties that are characteristic of avalanches observed in models of self-organized criticality, and these can be described by power--law distributions. I. INTRODUCTION Application of the methods of statistical physics and nonlinear science to different problems in economics has been an active area of research in so-called econophysics [1][2][3][4]. This is in part prompted by an interest in characterizing and understanding the various mechanisms that operate in a market. By virtue of its structure a market is a good example of an evolving complex dynamical system, being composed of a large number of interacting agents that can be an individual, a group or a firm. The market forms a network, with the nodes being the agents while the trading forms the links, with buying and selling activities giving both direction and weight. The network paradigm has been very useful in understanding interactions in a variety of complex dynamical systems, and the role of network topology in modifying the system dynamics has been of interest in earlier studies [5]. In a market, there are constraints relating to demand and supply or to available money under which each agent wishes to maximize profits. An important aspect of the study of such constrained complex systems is to understand the nature of fluctuations in the collective behavior that arises from the dynamics of many interacting agents. It has been shown [6] that the distributions of different quantities such as price differences and returns have probability distributions that are non-Gaussian. The need to comprehend and characterize the mechanisms that operate in a market-in particular stochastic fluctuations, chaotic variations and nonlinearity-have seen applications of statistical mechanics to many economic models and form the basis of predictions in financial markets [6][7][8][9][10][11]. Power-law distributions in economic systems are ubiquitous, dating to the early work of Pareto [12] and studied extensively since the work of Mandelbrot [13,14]. Given the large number of interacting agents financial markets are quintessentially complex systems that are continuously evolving. An early hypothesis for the emergence of power-laws in such systems has been that of self-organized criticality (SOC) [15][16][17][18][19] which has been applied extensively to various natural phenomena. Systems exhibiting SOC are characterized by slow driving and instantaneous dissipation events thus having separation of time-scales, and the system reaches its steady state, which is an attractor, without tuning of an external parameter. Applications have ranged from studies of earthquakes [20] to species evolution [21], forest-fires and epidemics [22], neuronal dynamics [23] as well as to abstract entities in number theory [24]. Indeed one of the early applications of SOC was to study fluctuations in an economic model [25]. A highly simplified market model of economic behaviour, with agents interacting on a one-dimensional lattice, was introduced by Nørrelykke and Bak [26] (NB). Each agent in the market produces a good at a variable price that can be sold to a neighbouring agent (say on the left) in order to trade with the agent on the right. Differences in the demand and supply of goods leads to each agent finally making a profit, but as trading continues, the agent with the smallest profit changes the product price in order to improve earnings. NB showed that while SOC is attained, the model has a non-stationary attractor, in contrast to the usual attracting statistically stationary critical state in most sandpile type models that show SOC. In the present work we consider that an agent buys products and sells goods from/to more than one other in the market: the trading interactions thus form a complex network. This generalizes the NB model by incorporating a feature of real markets, and our interest is in examining how the properties change with the complexity of the underlying connections. Furthermore we consider that agents may have different incomes, leading to differences in the level of expenditure according to the priority and capacity of each agent. The network itself becomes weighted as a consequence; the interaction strengths can arXiv:1608.03521v1 [q-fin.GN] 11 Aug 2016 differ for each link. We have examined the effect of some simple choices of weights on the system dynamics and our numerical results suggest that the SOC features of the one-dimensional (1D) Nørrelykke and Bak model carry over to higher dimensions, both for regular networks as well as for random complex networks with nonlocal interactions. In Section II of this paper, we present a general framework for the NB model of interacting agents on a spatially embedded complex network. The evolution rules are also discussed here, along with a brief description of the various interaction topologies considered in this work. The results of our simulations are presented in Section III, which is followed by a summary and discussion in Section IV. II. THE GENERALIZED INTERACTING MARKET We generalize the NB model as follows. Agents interact by trade, namely the buying and selling of goods. Each agent produces a single commodity that is sold to a set of customers at a certain price, and goods are purchased from a set of suppliers. The number of suppliers and customers can vary from agent to agent, and clearly this forms a general directed interaction network. If the ith agent has K i suppliers, the utility function can be written as [26] where the functions c is the cost (or the so-called discomfort) and the d j 's correspondingly account for the "comfort" associated with the quantities of commodities produced q i and consumed q ij , respectively. Typically the function c is convex while d is concave, and since these are nonlinear functions, a power-law form is a suggestive choice, we take c(q) = q 2 /2 and d(q) = 2 √ q [26,27] in our simulations and analysis below, although the assumption that all agents have the same level of comfort associated with every good they consume, d j = d is not a necessary restriction. The money constraint that operates in the market, where p i is the price of one unit good produced by the ith agent, suggests that for every agent, the total earning balances the total expenditure. Agents are aware of the prices charged by their suppliers, and thus the amount that should be produced, and the amount intended to be purchased can be calculated by optimizing the utility function for the given money constraint. In order to accommodate the variability in the number of suppliers and consumers for any given agent as well as to examine the effect of nonlocal interactions, both of which are prevalent features of a real market scenario, we extend the NB model to introduce an expenditure matrix A. The elements a ij of A represent the weights of interactions or the fraction of earnings that agent i spends on buying a quantity q ij of the goods produced by agent j, namely, Clearly, for each agent j a ij = 1, and a ii = 0. The matrix elements of A are choice parameters that specify the expenditure structure, i.e., what fraction of the total money earned is to be spent on buying a particular commodity. On optimizing the utility function for the ith agent, Eq. (1), we find that the quantity to be produced is where P ij = p i /p j and the superscript p denotes 'product'. The amount of intended want (superscript w) for this agent is The net want or demand of the product of the ith agent is denoted q W i , and is given by The total number of terms in the summation is K i , the total number of customers for the ith agent. Since the net demand of a product does not depend on the net supply, the minimum of these two quantities is traded, Each agent earns p i q t i amount of money, of which the fraction b ij is spent in buying goods from suppliers, hence contributing to their total earnings. The difference between the supply and demand of the goods affects the balance between earning and expenditure of an agent and therefore there may be a nonzero profit, where b ij = q w ij /q W j . We keep the same additional assumption introduced earlier [26,28], that an agent has enough credit to buy the permissible quantity (so that the trade would not be affected due to shortage of money) and that at the end of each cycle, no liquid money is withheld by agent (to avoid memory effects in profit calculation). A. Market Dynamics For a general directed network of N agents, it is necessary to modify the NB update rules in [26] appropriately. 1. Each agent is assigned a random initial price p i for their product. This price is chosen uniformly from the unit interval [p, p+1], with p > 0. The elements a i,j are chosen at random, but are fixed throughout process. 2. The quantities to be produced and consumed are computed for every agent, as well as the profit, which is calculated after computing the quantity of goods traded. 3. The agent with the least profit is identified and the price of her goods is reduced by a uniform random factor η ∈ [0, η max ]. 4. This process is repeated, starting from step 2, and each time step is assumed to correspond to one (trading) day. We consider a variety of network topologies in higher dimensions. As a first example, we take a 2-dimensional square L×L lattice with N = L 2 agents. Periodic boundary conditions are imposed, and each node or agent has two suppliers and two customers. We can construct different types of directed square lattices based on the location of supplier sites (see Fig 1). The suppliers can be at two of the following possible sites (with respect to the agent under consideration): right (R), left (L), top (T), and bottom (B). In this convention, the position of the two suppliers could be at any one of the following six possibilities: RT, LT, LB, RB, LR, and TB. With these local structures, different directed square lattices can be constructed, but we focus on the following three, namely (a) Manhattan lattice: starting from any node in the lattice, if we traverse a loop of unit area then the suppliers' positions for all the nodes involved in the loop follow a cyclic permutation of {RT, RB, LB, LT} [29]. (b) F lattice: alternate agents have suppliers at LR and TB positions respectively, i.e., alternate agents can buy goods from left and right or up and down neighbors. (c) A topology can be constructed such that the two suppliers are at one of the following combinations: RT, LT, LB, or RB. We also consider a directed Erdős-Rényi network [30] spatially embedded in one dimension with a constraint that each agent has at least one supplier. This is a random complex network and each node can have a variable number of suppliers and customers. However each agent trades since there is at least one neighbour per site. The avalanche properties exhibited by the random network (discussed later) are robust even if this condition is relaxed. The average number of suppliers and customers in the network are equal and related to total number of nodes as K = K = N α, where α is the probability to form a link between two nodes. For large N , the degree distribution is binomial while it is Poisson when N is small. On a spatially embedded network, as a function of time the locus of the agent with the lowest profit (the "loser") moves at random. This can be described as a discrete random walk, where x denotes the position coordinate, the jump step is ξ. The distance between successive losers' positions is a random variable characterized by probability distribution P (ξ). The risk of incurring minimum profit is transferred from one agent to another due to one of the two following possible reasons: a) A supplier of the loser can be at risk due to local interactions. In order to recover from the least profit situation, when the loser reduces the price of her product, the supplier's production estimate increases [see Eq. (4)] and this may lead to overproduction. b) Alternately, any agent (including the suppliers of the loser at the previous time step) can be in the global minimum profit position at the present time step. III. RESULTS Our numerical results show that P ( ξ) obeys the following distribution where ξ is the norm of ξ and L is linear extent of the lattice. In Fig. 2 we show the cumulative probability distribution F (ξ) for regular lattices in 2D. The behavior of F (ξ) is independent of the choices of ξ, namely, the component or the norm. Throughout our numerical studies, we use initial price interval [10,11], η max = 1%, and the total evolution time is 10 6 steps. Data is discarded up to 10 5 time steps to avoid transient effects. Numerical results also suggest that for a fixed lattice, F (ξ) vs ξ graph looks similar to Fig. 2, when computed at different values of the choice parameter a (not shown). On the other hand, in a random complex network, our numerical results show that long range spatial correlation feature is destroyed due to existence of non local interactions. However, the avalanche properties that are signature of self-organized criticality have been observed numerically as discussed later. The dynamics of this market model is driven by a strategy in which agent lowers the price of the product intending to improve profit. Consequently, the evolution of profit over time shows an effective exponential decaying behavior, where the decay rate or inverse characteristic time k has an inverse dependence on N . The leading behavior of k varies as where the angular brackets · denote an ensemble or time average [26]. Therefore to get a time independent profit distribution, the profits are scaled to obtain a stationary profit, f c (t) = f c . Since the system is driven by a price change mechanism in which agents lower their prices gradually, it turns out that there is a deflationary trend. In the steady state, the profit attains a stationary value, f c . A threshold value, say f 0 , is set and starting with the rescaled profits of all agents being above the threshold f 0 , a losing agent can initiate an avalanche by changing the prices of her goods so as to increase her profit. Consequently, in the successive updates, the profit of some agents may fall below f 0 . At each time step the number of agents with profit below f 0 constitutes an activity signal y(t). Clearly this is a stochastic variable, and in order to analyze such fluctuations, we consider the portion of activity signal separated by successive zeros or quiescent periods as an avalanche event, namely, the condition that y(t) = y(t + T ) = 0 and y(t ) = 0 for t < t < t + T . This event can be characterized by an observable {X} such as the cluster size S S = t+T t =t y(t ), (13) or duration T that denotes the sum of activities and time spent between two successive zeros respectively. It has been observed that for critical avalanche processes [31] the probability distribution of X shows a power-law behavior given as where τ X is the scaling exponent. The two observables S and T are related as S ∼ T γ ST , and this gives a relationship between the scaling exponents, Figures 3 and 4 show P (S) for different market topologies. As the cluster size distribution exponents in 2D and random complex network don't show significant variation, it seems that the structural detail of network does not affect the exponent of power law. Further, on a random network, the duration exponent is τ T = 1.46 ± 0.04 and it is found to be of the same order in the 2D lattices. However, the critical behaviour is observed at different values of the threshold f 0 for all the topologies considered here, thus suggesting that the critical profit is indeed network dependent. As fluctuations in avalanche activity can be understood as a critical branching phenomenon and for mean field branching process [32] (MFBP) the exponents are exactly known to have values τ S = 3/2 and τ T = 2. Clearly, this model belongs to a different universality class. IV. SUMMARY AND DISCUSSION Here we have studied a simple economic model that generalizes a one-dimensional market model introduced by Nørrelykke and Bak to account for an underlying network structure that is a more realistic representation of trading relationships. Introduction of the expenditure matrix is an striking feature of our model since it allows addressing the variability in expenditure structure of an agent in a real market system. We have numerically investigated several interaction networks that range from different regular lattices in two dimension to a random complex network with variable number of suppliers and customers. Our studies show that in all these types of networks the existence of critical avalanche properties is a manifestation of self-organized criticality. The differences in structural properties lead to different nonlinear local interactions, and thus the resulting exponents that characterize the statistical properties of various quantities differ from the simple one-dimensional market. From past studies it is worth noting that many extremal driven models exist that are known to exhibit long-range spatial correlations. Examples include the Bak-Sneppen (BS) model that describes an evolving ecology of interacting species [21], the animal mobility model [33] in which an animal moves to the closest site that has the largest prey resources, reflecting the maximization of foraging benefits and minimization of cost (this is being driven by optimal search strategies), and the Prisoner's Dilemma game that models interacting members of a population [34]. The emergence of long-range spatial and temporal correlation is a direct consequence of the extremal driving mechanism. The extent to which this is a feature of real markets is a question of considerable interest.	2016-08-11T09:18:18.000Z	2016-08-11T00:00:00.000	{ "year": 2016, "sha1": "5f9b8e423f5e1ee1fc0d56134ef00ff8c65db0e6", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1608.03521", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "5f9b8e423f5e1ee1fc0d56134ef00ff8c65db0e6", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Economics", "Physics" ] }
203827897	pes2o/s2orc	v3-fos-license	Editorial Comment to A case in which bladder cancer invaded the ureteral orifice and was resected via photodynamic diagnosis‐assisted transurethral resection involving orally administered 5‐aminolevulinic acid aminolevulinic acid (5-ALA). Photodiagn. Photodyn. Ther. 2016; 13: 91–6. 7 Filbeck T, Roessler W, Knuechel R, Straub M, Kiel HJ, Wieland WF. 5aminolevulinic acid-induced fluorescence endoscopy applied at secondary transurethral resection after conventional resection of primary superficial bladder tumors. Urology 1999; 53: 77–81. 8 Grimbergen MC, van Swol CF, Jonges TG, Boon TA, van Moorselaar RJ. Reduced specificity of 5-ALA induced fluorescence in photodynamic diagnosis of transitional cell carcinoma after previous intravesical therapy. Eur. Urol. 2003; 44: 51–6. 9 Hendricksen K, Moonen PM, der Heijden AG, Witjes JA. False-positive lesions detected by fluorescence cystoscopy: any association with p53 and p16 expression? World J. Urol. 2006; 24: 597–601. 10 Palou J, Salvador J, Millan F, Collado A, Algaba F, Vicente J. Management of superficial transitional cell carcinoma in the intramural ureter: what to do? J. Urol. 2000; 163: 744–7. Supporting information Editorial Comment Editorial Comment to A case in which bladder cancer invaded the ureteral orifice and was resected via photodynamic diagnosis-assisted transurethral resection involving orally administered 5-aminolevulinic acid Watanabe et al. 1 reported an interesting case of bladder cancer in the intramural ureter exhibiting red fluorescence with the use of orally administered 5-aminolevulinic acid-mediated photodynamic diagnosis (ALA-PDD). The tumor was resected with negative margins, which did not show red fluorescence by ALA-PDD. The present study reports that an exact diagnosis and precise resection of the bladder tumor in the intramural ureter are possible with the use of ALA-PDD. Faba et al. 2 reported a good oncological outcome for conventional transurethral resection of bladder cancer without T1 or carcinoma in situ in the intramural ureter. ALA-PDD can be considered in patients suspicious for bladder cancer in the intramural ureter, though long-term follow-up for local or upper urinary tract recurrence is needed in the present case, and accumulating information on such cases is necessary. False-positive results should be noted during ALA-PDD for bladder cancer. 3 Oblique illumination and inflammation can lead to false-positive results, which are often observed at the bladder neck, trigone, and around the orifice. 4 Because ureteral stenosis can occur after resection of the orifice (11.6%), 2 unnecessary surgery should be avoided. Draga et al. 5 reported that the learning curve for surgeons performing transurethral resection under PDD was proportional to the decrease in the number of false positives up to 12-18 months after the initial PDD procedure. Thus, some experience with ALA-PDD and careful observation are necessary to perform ALA-PDD for bladder tumors in the intramural ureter. The present case also reveals the potential of ALA-PDD for diagnosis of upper urinary urothelial carcinoma (UTUC). Kata et al. 6 reported the potential of ureteroscopy using 5-ALA, and that ALA-PDD for UTUC can identify tiny lesions and carcinoma in situ, which is missed under conventional white light. 5-ALA can be administered using either an oral or transurethral route. On the other hand, hexaminolevulinate, which is used in Europe for PDD of non-muscle-invasive bladder cancer, is administered via intravesical instillation only. Therefore, ALA-PDD is a promising diagnostic tool for various kinds of cancers including UTUC, and trials of ALA-PDD in the treatment of such cancers are being performed. The use of ALA-PDD for bladder cancer has recently been approved in Japan and performed in various institutions. We should report the effectiveness of ALA-PDD from Japan, and transurethral resection with ALA-PDD should be approved as a treatment for bladder carcinoma outside of Japan as emphasized by Watanabe et al. in their keynote message.	2019-09-19T09:15:53.922Z	2019-09-16T00:00:00.000	{ "year": 2019, "sha1": "1a565ed1cbeee9cf1dce59171807612bf39f24ce", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/iju5.12114", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4e99fd5f363aaebda7a5501670548f8cef4f41da", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
53017405	pes2o/s2orc	v3-fos-license	Discourse Embellishment Using a Deep Encoder-Decoder Network We suggest a new NLG task in the context of the discourse generation pipeline of computational storytelling systems. This task, textual embellishment, is defined by taking a text as input and generating a semantically equivalent output with increased lexical and syntactic complexity. Ideally, this would allow the authors of computational storytellers to implement just lightweight NLG systems and use a domain-independent embellishment module to translate its output into more literary text. We present promising first results on this task using LSTM Encoder-Decoder networks trained on the WikiLarge dataset. Furthermore, we introduce"Compiled Computer Tales", a corpus of computationally generated stories, that can be used to test the capabilities of embellishment algorithms. Introduction Narratives can be analyzed to consist of at least two layers: plot-what is told-and discoursehow it is told. Usually, computational systems first generate the events of the plot, and then decide how to render these in text (Gervás, 2009). This means that a tight coupling exists between the knowledge bases that are used for plot generation, and the NLG modules for discourse rendering. The result is that it is hard to share these modules between systems, while the implementation of a custom module to generate literary discourse requires significant effort and linguistic expertise. Indeed, our subjective feeling is that often it is possible to recognize repeating textual patterns when reading several stories created by the same system. To alleviate this problems we suggest a new NLG task, textual embellishment (TE), with the goal to automatically increase the lexical and syntactic complexity of a text. Textual embellishment can be understood as the inverse task to textual simplification (TS), which has been researched for at least two decades (Shardlow, 2014). Recent TS systems are trained on vast corpora using machine learning techniques and work independent of domains or handcrafted rules. These results open the intriguing possibility for an equally general resource for TE. This could allow the authors of storytelling systems to safe time by implementing only thin NLG modules and perform subsequent embellishment. We would like to strongly caution that TE most likely will prove to be a more difficult task then TS. While the latter typically results in removing information in a text, the former might lead to an automated adding of information which can introduce semantic contradictions. However, on a syntactic level TE ideally just results in periphrastic constructions with no added information. Related Work Traditionally, narrative generation systems like Meehan (1977); Lebowitz (1985) were devised to generate macro-level plot and character elements without connecting them into a comprehensive story in natural language. Later work has sought to combine plot and NLG into a unified system (Callaway and Lester, 2002). These full pipelines generate story elements and employ rule-based language models that produce naturally sounding narratives. While these approaches make use of text embellishment, the disadvantage of these rule-based approaches is that rules for text enrichment have to be conceived of in advance by the system's architect. The work presented here, in contrast, seeks to learn a natural language rep-resentation from human data to automatically embellish priorly generated narratives. This approach can be located in the field of natural language processing. Within this area of research several related subfields can be identified, including text summarisation, statistical machine translation, and most notably, TS. Both, text summarisation and simplification, seek to extract the relevant information of a phrase and lose all linguistic embellishment that is deemed unnecessary for its understanding. Our work has a complementary objective, which is enriching text while maintaining its original meaning. This generative task is related to recent developments in computer vision using Generative Adversarial Networks for problems like image super resolution (Ledig et al., 2017). Therein similar challenges are faced as new information has to be "hallucinated", requiring particular caution in content sensitive domains. Within TS, Shardlow (2014) distinguishes between syntactic and lexical simplification. While the former approach seeks to facilitate readability by reducing the complexity of sentence structure, the latter seeks to replace words deemed difficult for a target audience by words that are easier understandable. Previous work has focused on both tasks individually (Siddharthan, 2006;Paetzold and Specia, 2017), while recent approaches addressed syntactic and lexical simplification simultaneously (Wang et al., 2016;Zhang and Lapata, 2017). These latter systems cast text simplification as a monolingual machine translation problem and borrow insights from automatic natural language generation (Wen et al., 2015). Therein, the task of simplifying text is framed as a translation problem between "complex English" and "simple English". The recent success of Deep Learning systems in "neural machine translation" has increased the employment, specifically of the LSTM encoder-decoder architecture (Sutskever et al., 2014). These models make use of the Long Short-Term Memory neural network architecture (Hochreiter and Schmidhuber, 1997) to solve a sequence to sequence task. Therein, a mapping from a source sequence to a target sequence is learned both of which may be of variable length . To that goal, an encoder LSTM generates a sequence of hidden representations using the source, which is then used by a second de-coder LSTM to generate the target sequence word by word, whereof each is conditioned on all previous outputs. Here, we follow the same approach but address the opposite problem to TS, translating from "simple" to "complex" English, building on the success of previous neural network approaches. Discourse Embellishment For the trained network, two LSTM layers per encoder and decoder were chosen, since Wang et al. (2016) demonstrated that this is sufficient for sorting, reversing and reordering operations in a textual simplification task. The network was set up and trained using Harvard's OpenNMTtf framework (Klein et al., 2017). Parameters were selected as suggested by Zhang and Lapata (2017): Each layer contained 512 hidden units with weights uniformly initialized to [-0.1, 0.1], learning was performed with a 0.2 dropout rate (Zaremba et al., 2014) and Luong attention was used by the decoder (Luong et al., 2015). If not stated otherwise, training was always performed for 24 epochs, with a learning rate of 1 and a simple gradient descent optimization, where gradients where clipped when their norm exceeded 5 (Pascanu et al., 2013). After 18 epochs a decay rate of 0.25 was applied each epoch. Furthermore, pre-trained 300 dimensional GloVe vectors were used to initialize word embeddings (Pennington et al., 2014), and the vocabulary size was 50,000. Story Corpus The performance of a discourse embellishment system should ideally be judged on domainspecific texts. To the best of our knowledge no corpus of computationally generated stories has been compiled so far. Since no common resources for NLG generation have been deployed, the stories that have been generated by different systems over the decades differ markedly in their language. To reflect this diversity we decided for a breadth-focused approach while setting up Compiled Computer Tales (CCT), a corpus of computationally generated stories. Since the aim of this corpus is to be used as a qualitative test set, and not as training data for machine learning algorithms, we collected at most three stories from as many systems as available to us, instead of compiling many stories from few systems. Because it is unfeasible to deploy all individual systems, we instead opted for using stories that have been reported in scientific publications, although this runs the danger of biasing the corpus towards highquality exemplars. 1 The resulting corpus can be found online 2 and we cordially invite researchers to expand this necessarily incomplete collection. At the moment of writing, it contains 14 stories from 8 storytellers, ranging from early systems like "Novel Writer" (Klein et al., 1973) to recent ones like "Simlextric" (Riegl and Veale, 2018). It is formatted in a way that allows separation based on system, story, paragraph (45 in total) or sentence (290 in total); depending on the task at hand. We also provide a python script that can be used to perform such splits, as well as common preprocessing steps like tokenization or named entity anonymization. Sentence Based: Lexical Embellishment As with the more common TS task, we trained our network on a sentence-aligned dataset. The largest simplification dataset is, to the best of our knowledge, WikiLarge, which contains 296,402 automatically aligned sentences from the ordinary and simple English Wikipedias in its training set. To reduce vocabulary, named entities in the dataset were anonymized (Zhang and Lapata, 2017). Unlike for simplification, for the present task the network was trained to generate original ('complex') English based on simple English input. The performance of the system is evaluated using the common BLEU metric (Papineni et al., 2002), which measures the degree to which generated embellished text differs from the language employed in the original Wikipedia. Training is continuously evaluated on a held-out evaluation set using the Moses toolkit (Koehn et al., 2007), and BLEU stagnates after around 18 epochs. The final performance on the held-out test set is a BLEU of 56.02 on the tokenized data, which is a reasonable performance 3 . To judge the performance of this model on CCT, the corpus had to be split in sentences, tokenized, and named entities had to be anonymized in a manner comparable to WikiLarge. Since no goldstandard of embellished tales exists, quantitative evaluation of embellishment quality is not possible. Comparing the embellished version with the original ones shows that the network mostly learned to correctly reproduce its input (BLEU 92.13). If differences that are due only to outof-vocabulary words and minor formatting differences are ignored, then roughly 83% of the sentences are simply reproductions of the input. Around 5% of the generated sentences do not bear any resemblance to the corresponding input, which we consider to happen when the network overfitted a certain type of input. 2% of the generated sentences miss individual words. The remaining 10% contain syntactically correct lexical substitutions, which can be considered cases of lexical embellishment. Some examples of felicitous embellishment are the mappings: Because of this → consequently tried → attempted wanted to → sought to bossy → overbearing; while examples of failed embellishment are: sinful → drinking lioness → cat ubiquitous → familiar towards princess → towards LOCATION@1. Paragraph Based: Degradation Sentence aligned training enabled the network to perform lexical embellishment, however, no syntactical embellishment like concatenation or parenthetical insertion was observed. It stands to reason that such sentence-level reordering can be better picked up by learning from a corpus that aligns whole paragraphs, where several sentence are simplified/embellished at a time. A promising candidate is the document aligned Wikipedia corpus compiled by Kauchak (2013). After the necessary preprocessing and splitting steps the resulting dataset contained 134,233 paragraphs in the training set. Training a network on this dataset using the same setup was not successful. The model achieved a BLEU score of 3.55 on the held-out test set, which is a clear sign of its failure. A manual inspection of the dataset revealed, that the employed alignment of paragraphs is rarely meaningful, since paragraphs of the same ordinal number often fail to contain semantically related information, let alone simplified/embellished versions of each other. In fact, further literature review did not reveal work on ei-ther paragraph or document aligned simplification, as well as no published results using the document aligned dataset. This lead us to suspect that the encountered problems are not of trivial nature, and abandon this direction of inquiry. Pair Based: Syntactical Embellishment An inspection of the WikiLarge dataset revealed that the sought-for syntactical reorderings can also be found there. In particular, the sentencealignment does not necessarily result in a bijective mapping from complex to simple sentences, but sometimes in a 1:2 mapping, when complex sentences are split in two. This lead us to the hypothesis that the network described in section 3.2 should already be able to perform syntactical embellishment, when presented with two input sentences. To test this we pre-processed the CCT corpus by concatenating, with a space, two consecutive sentences. This resulted in sentence-pairs that in most cases had the potential to be combined, because they shared subjects or were causally related. In some cases, syntactical combinations were not meaningful, especially when sentence pairs crossed paragraph or story boundaries as a result of the unsophisticated concatenation operation. Comparing the embellished version with the original ones shows that the network is doing an acceptable job in keeping original and embellishment related (BLEU 68.20). Approximately 56% of the generated pairs were combined by the network into one sentence. In 42% of the cases the network did not combine the pairs, in which case it usually kept the first sentence and removed the second. In the remaining 2% the networks output was not related to the input. The combination of sentences was performed by the network in different ways. Most commonly, it conjoined them using a comma and the conjunction and, or replaced the period sign with a comma, while changing the following letter to lower case. We also observed cases where the pronouns who, which or where were employed. Another mode of combining sentences employed by the network was by using participles: . . . she robbed PERSON@2 of her illusions . She said : " PERSON@3 took . . . " → . . . she robbed PER-SON@2 of her illusions , saying : " PERSON@3 took . . . ". The most interesting case appeared to involve an inclusion: PERSON@1 saw the affair . PERSON@1 was jealous → PERSON@1 was jealous of the affair. In a previous run the same sentence was embellished using nominalisation: PERSON@1 saw the affair with jealousy. For more context, a comparison between an original and a pair-embellished story can be found in the appendix. Conclusion NLG systems in computational storytellers are commonly dependent on the systems' knowledge base and require idiosyncratic, hand-coded rules if complex language output is desired. Based on this observation we propose to extend the discourse generation pipeline with a TE step that takes as input simple English sentences and performs a monolingual translation into lexically and syntactically more complex English. This approach has the benefit of allowing the authors of storytellers to save time by implementing only simple custom text generation modules, and employ a domainindependent embellishment module to translate into more sophisticated text. The LSTM Encoder-Decoder approach explored in this paper is not ready for productive use as is, mainly because it does not always fail gracefully, i.e. produces well-formed and semantically appropriate language when embellishment fails. However, it shows promising results by demonstrating that the same trained network is capable of producing interesting lexical as well as syntactical embellishment of varying kind. We are confident that the presented results can be improved by employing techniques that have proven beneficial in a simplification setting, like e.g. the reinforcement learning proposed by Zhang and Lapata (2017). Due to the nature of the corpus we used for training, only those entries could contribute to the performance of syntactical embellishment that contained two sentences on the simple English, and one on the normal English side. At the same time, the language employed in the Wikipedia can be expected to be markedly different from the one usually aimed for by storytelling systems, which has an impact on lexical embellishment performance. Hence, further improvements can be expected from using more-or better suited-corpora, like e.g. Newsela (Xu et al., 2015). Another promising option would be to explore the viability of embellishment/simplification datasets based on an align-ment of simple English versions of novels with their original counterparts. To exceed sentencepair based processing, paragraph aligned versions of text would prove to be valuable resources. Apart from the above theoretical and technological contributions we also presented a first version of Compiled Computer Tales, a corpus of computer generated stories. This corpus can be used to test the capabilities of embellishment algorithms, as well as for historiographic inquiries. We cordially invite collaboration to complete its coverage. PERSON@1 needed a place to live and PER-SON@2 had plenty of it . PERSON@1 found PERSON@2 at an underground lair . PER-SON@1 rented accommodation from her . She paid PERSON@2 what she owed . LOCA-TION@1 could not achieve bossy PERSON@1 's lofty goals . She refused to honour PERSON@2 's commitments to her , so PERSON@1 ripped off rich PERSON@2 's best ideas . PERSON@1 PERSON@2 evicted PERSON@3 from LOCA-TION@1 's home . At a smoke-filled back room PERSON@3 met PERSON@4 . LOCATION@1 assiduously curried favor with dictatorial Oscar after cheated PERSON@1 evicted PERSON@2 from LOCATION@2 's home . PERSON@2 told eager Wilde a pack of lies . PERSON@1 said : " Dolores wrote propaganda to promote your cause . " His attitude hardened toward LOCATION@1 . He openly disrespected PERSON@1 because earlier she took everything that PERSON@2 had . PERSON@1 tried to tune out loudmouthed PER-SON@3 's voice . LOCATION@1 PERSON@1 wrote PERSON@2 off as a loser , so he coldly dismissed PERSON@2 and turned away . It was at the red carpet when PERSON@2 found LOCA-TION@2 . PERSON@1 started a new job for influential Rina after unsatisfied PERSON@2 told PERSON@1 to get out and not come back . PER-SON@3 took full advantage of her . She pulled the wool over PERSON@1 's eyes . She said : " PERSON@2 was a real suck-up to aristocratic PERSON@3 . " LOCATION@1 could not reach the bar set by bossy LOCATION@2 . She was very disappointed in her , so " Get out ! You 're fired " said PERSON@1 . It was at a recording studio when PERSON@2 found PERSON@3 . LOCATION@1 PERSON@1 recruited PERSON@2 into her ranks after PER-SON@3 asked her to clear out her desk and leave . PERSON@2 took the spotlight from lackadaisical Dolores . PERSON@1 withheld due payment from lazy Maura . PERSON@2 criticized sinful Dolores in public . She said : " PERSON@1 showed no shame in sucking up to influential PER-SON@2 . " She broke with her and went her own way . What do you think ? Can PERSON@1 and PERSON@2 ever mend their relationship ? PERSON@1 needed a place to live and PER-SON@2 had plenty of it , and PERSON@1 found PERSON@2 at an underground lair . PER-SON@1 rented accommodation from her and paid PERSON@2 what she owed . LOCA-TION@1 could not achieve overbearing PER-SON@1 's lofty goals and refused to honor PERSON@2 's commitments to her , so PER-SON@1 ripped off rich PERSON@2 's best ideas . PERSON@1 PERSON@2 evicted from LOCA-TION@1 's home , and at a smoke-filled back room PERSON@3 met PERSON@4 . LOCA-TION@1 assiduously curried favor with dictatorial Oscar after cheated PERSON@1 evicted PER-SON@2 from LOCATION@2 's home , who told eager a pack of lies . PERSON@1 said : " Dolores wrote propaganda to promote your cause " . He openly disrespected PERSON@1 because earlier she took everything that PERSON@2 had , and PERSON@1 tried to tune out loudmouthed PER-SON@3 's voice . LOCATION@1 PERSON@1 wrote PERSON@2 off as a loser , so he coldly dismissed PERSON@2 and turned away at the red carpet when PERSON@2 found LOCATION@2 . PERSON@1 started a new job for influential Rina after unsatisfied PERSON@2 told PERSON@1 to get out and not come back . She pulled the wool over PERSON@1 's eyes , saying : " PER-SON@2 was a real suck-up to aristocratic PER-SON@3 . " LOCATION@1 could not reach the bar set by overbearing LOCATION@2 , and she was very disappointed in her , so " Get out ! You 're fired " said PERSON@1 , at a recording studio when PERSON@2 discovered PERSON@3 . LO-CATION@1 PERSON@1 recruited PERSON@2 into her ranks after PERSON@3 asked her to clear out her desk and leave . PERSON@1 withheld due payment from lazy Maura . She said : " PER-SON@1 exhibited no shame in digestion up to influential PERSON@2 " she broke with her and went her own way . What do you think ?	2018-10-18T14:29:50.000Z	2018-10-18T00:00:00.000	{ "year": 2018, "sha1": "2c40e81ece8d4b2291648d94d0bbf5417d926e40", "oa_license": "CCBY", "oa_url": "https://www.aclweb.org/anthology/W18-6603.pdf", "oa_status": "HYBRID", "pdf_src": "Arxiv", "pdf_hash": "96e236ebcd3157e68f7129e7c3e3802bf4fcc11e", "s2fieldsofstudy": [ "Computer Science", "Linguistics" ], "extfieldsofstudy": [ "Computer Science" ] }
267464829	pes2o/s2orc	v3-fos-license	Novel application of the ferroptosis-related genes risk model associated with disulfidptosis in hepatocellular carcinoma prognosis and immune infiltration Hepatocellular carcinoma (HCC) stands as the prevailing manifestation of primary liver cancer and continues to pose a formidable challenge to human well-being and longevity, owing to its elevated incidence and mortality rates. Nevertheless, the quest for reliable predictive biomarkers for HCC remains ongoing. Recent research has demonstrated a close correlation between ferroptosis and disulfidptosis, two cellular processes, and cancer prognosis, suggesting their potential as predictive factors for HCC. In this study, we employed a combination of bioinformatics algorithms and machine learning techniques, leveraging RNA sequencing data, mutation profiles, and clinical data from HCC samples in The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and the International Cancer Genome Consortium (ICGC) databases, to develop a risk prognosis model based on genes associated with ferroptosis and disulfidptosis. We conducted an unsupervised clustering analysis, calculating a risk score (RS) to predict the prognosis of HCC using these genes. Clustering analysis revealed two distinct HCC clusters, each characterized by significantly different prognostic and immune features. The median RS stratified HCC samples in the TCGA, GEO, and ICGC cohorts into high-and low-risk groups. Importantly, RS emerged as an independent prognostic factor in all three cohorts, with the high-risk group demonstrating poorer prognosis and a more active immunosuppressive microenvironment. Additionally, the high-risk group exhibited higher expression levels of tumor mutation burden (TMB), immune checkpoints (ICs), and human leukocyte antigen (HLA), suggesting a heightened responsiveness to immunotherapy. A cancer stem cell infiltration analysis revealed a higher similarity between tumor cells and stem cells in the high-risk group. Furthermore, drug sensitivity analysis highlighted significant differences in response to antitumor drugs between the two risk groups. In summary, our risk prognostic model, constructed based on ferroptosis-related genes associated with disulfidptosis, effectively predicts HCC prognosis. These findings hold potential implications for patient stratification and clinical decision-making, offering valuable theoretical insights in this field. INTRODUCTION Liver cancer is a significant global contributor to cancer-related mortality, and its incidence is anticipated to rise considerably by 2025 (Llovet et al., 2021;Ganesan & Kulik, 2023).HCC predominates as the primary liver cancer, comprising 90% of cases, making it the most prevalent pathological type (Forner, Reig & Bruix, 2018).Prominent risk factors for HCC encompass cirrhosis, hepatitis B and C infections, and alcohol abuse (Anwanwan et al., 2020).For early-stage HCC patients, surgical resection stands as the preferred therapeutic approach, though the 5-year recurrence rate is alarmingly high, reaching 70% (Liu et al., 2021).The last decade has witnessed a transformative wave in the realm of advanced HCC treatment, marked by the introduction of molecularly targeted drugs (e.g., kinase inhibitors), angiogenesis inhibitors (e.g., bevacizumab), and immune checkpoint inhibitors (e.g., nivolumab), which have kindled fresh hope for patients (Lee, Guan & Ma, 2022).Nevertheless, the benefit of these treatments is constrained to a limited patient pool, and the specter of drug resistance, recurrence, and metastasis looms large (Oura et al., 2021;Tang et al., 2022).To recap, the prognosis for HCC remains bleak.Hence, there is an urgent and imperative need to explore novel approaches for screening, diagnosis, and therapy to enhance HCC prognosis.The development of a risk prognosis model holds the potential to stratify patients, conferring substantial importance in the realms of survival prediction and clinical decision-making. In early 2023, a novel mode of cell demise, termed disulfidptosis, was unveiled, setting it apart from conventional forms like apoptosis, cuproptosis, and ferroptosis.Disulfidptosis is characterized by an anomalous collapse of disulfide bonds within the actin cytoskeleton, stemming from an accumulation of cystine within the cell (Liu et al., 2023).The starvation of glucose in cells with heightened SLC7A11 expression can incite disulfide-induced cell death.Furthermore, researchers noted that glucose transporter inhibitors triggered disulfidptosis in tumor cells with elevated SLC7A11 expression, consequently impeding tumor growth (Liu et al., 2023).Numerous genes connected to disulfidptosis have been pinpointed, including glycogen synthetase (GYS1) and various genes involved in mitochondrial oxidative phosphorylation (e.g., NDUFS1, NDUFA11, NUBPL, and LRPPRC), whose deactivation synergizes with glucose deprivation to provoke cell disulfidptosis (Liu et al., 2023).This discovery of a novel mechanism wherein disulfide bonds precipitate cytoskeletal collapse unveils potential targets for modulating this mode of cell death and devising innovative cancer treatment strategies (Machesky, 2023). Like disulfidptosis, ferroptosis represents an alternative mode of cell demise, characterized by its iron-dependent nature, and it plays a pivotal role in tumorigenesis and tumor progression.Throughout the evolution of tumors, ferroptosis serves a dual purpose, both promoting and restraining tumor growth (Huang et al., 2023).Furthermore, it exerts a substantial impact on the efficacy of chemotherapy, radiotherapy, and immunotherapy in cancer patients (Yuan et al., 2021;Wang et al., 2019).Notably, it has been observed that the metabolic reconfiguration of tumors is intrinsically tied to their sensitivity to ferroptosis.In particular, tumor cells exhibiting heightened metabolic activity display a heightened susceptibility to ferroptosis (Chen et al., 2021;Friedmann, Krysko & Conrad, 2019).Consequently, manipulating tumor metabolism to trigger ferroptosis emerges as a promising therapeutic avenue for inducing cancer cell demise, particularly in cases where traditional treatments falter.Several signaling pathways in the liver oversee the orchestration of ferroptosis (Chen et al., 2022).In the context of advanced liver cancer therapy, the molecularly targeted drug Sorafenib has exhibited anti-tumor effects linked to ferroptosis (Li et al., 2021).ATF4 and YAP/TAZ, however, can foster resistance to Sorafenib in liver cancer by impeding ferroptosis (Gao et al., 2021).Conversely, targeting the ferroptosis inhibitor NRF2/GPX4 axis can bolster Sorafenib-induced cell death, thereby improving the efficacy of drug therapy (Wang et al., 2021).Intriguingly, research has unveiled that inducing ferroptosis in mice does not inhibit the onset of HCC but rather elicits immunosuppression (Conche et al., 2023).In conclusion, the specific regulatory effect of ferroptosis on the occurrence and progression of HCC remains inconclusive, and the specific mechanism involved remains to be fully elucidated. Owing to the limited number of disulfidptosis-related genes (DRGs), we performed a correlation analysis between ferroptosis-related genes (FRGs) and DRGs to identify FRGs associated with disulfidptosis.This investigation aimed to elucidate the connection between ferroptosis, disulfidptosis, and the prognosis of HCC.Consequently, we integrated multiple DRGs-FRGs by scrutinizing publicly available sequencing datasets, thereby constructing a risk prognostic model for HCC.Furthermore, we assessed the drug sensitivity of this model to establish a theoretical foundation for the stratified clinical management of HCC patients.Figure 1 depicts the flowchart of this study. Data acquisition We retrieved clinical information, survival data, gene mutation details, and RNA sequencing data (RNA-seq) from The Cancer Genome Atlas (TCGA) database, encompassing a total of 415 samples, comprising 365 HCC samples and 50 normal samples.Subsequently, we acquired validation cohorts for HCC, GSE14520, from the Gene Expression Omnibus (GEO) database, and ICGC data from the International Cancer Genome Consortium (ICGC) database.The GSE14520 cohort included 242 HCC samples, and the ICGC cohort included 260 HCC samples, none of which contained normal liver tissue samples.To obtain 259 FRGs, we accessed the FerrDb website (http://www.zhounan.org/ferrdb/legacy/operations/download.html).We sourced 10 DRGs from a prior study (Liu et al., 2023).Subsequently, a Spearman correlation analysis between DRGs and FRGs was carried out, and FRGs with nonzero correlation coefficients and p < 0.05 were considered to be associated with DRGs.Finally, 174 DRG-FRGs were picked out. Analysis of differentially expressed genes We aligned RNA-seq data of HCC samples from the TCGA cohort with the set of 174 DRG-FRGs.We employed the "limma" package in the R software to analyze the differentially expressed genes (DEGs) in the RNA-seq data from both HCC tissue and non-tumor tissue samples.\|log fold change (FC)\| > 1, the gene for adjusted p < 0.05 was screened out to obtain DE-DRG-FRGs (Sun et al., 2022).Log FC > 1 represents genes upregulated in tumor tissues, while Log FC < -1 represents genes downregulated.Heatmap and volcano plot were generated to show the DE-DRG-FRGs.For univariate Cox regression analysis, we utilized the R "forestplot" package and identified DE-DRG-FRGs with a p-value less than 0.01, which were considered independent prognostic factors associated with overall survival (OS) in HCC (He, Yang & Jin, 2022).To explore the protein-protein interactions (PPI) among DE-DRG-FRGs associated with OS, we constructed a network using the STRING website (http://string.embl.de/)(Wang et al., 2021). Enrichment analysis of DEGs We conducted KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses to elucidate the biological processes and signaling pathways enriched in the DE-DRG-FRGs using the R "clusterProfiler" package (Lian et al., 2021).To investigate the potential associations of DE-DRG-FRGs with liver disease, we performed Disease Ontology (DO) analysis using the "DOSE" software package (Wang et al., 2023). Unsupervised cluster analysis We employed the R software package "ConsensusClusterPlus" for unsupervised clustering analysis, utilizing OS-associated DE-DRG-FRGs as the basis (Zeng et al., 2022).To assess Full-size  DOI: 10.7717/peerj.16819/fig- 1 cluster differentiation, we utilized Principal Component Analysis (PCA), Uniform Manifold Approximation and Projection (UMAP), and t-Distributed Stochastic Neighbor Embedding (t-SNE).Survival analysis of different clusters in HCC was conducted using the R "survival and survminer" package.HCC samples from the GSE14520 and ICGC cohorts were also performed unsupervised clustering and survival analysis as validation cohorts. Analysis of biological behavior and tumor microenvironment (TME) on phenotypes of DRG-FRGs "c5.go.symbols" and "c2.cp.kegg.symbols"gene sets were downloaded, and the R software "GSVA" package was used to perform the Gene Set Variation Analysis (GSVA) to explore the differences in molecular function, biological behavior, and signaling pathways of different phenotypes based on DRG-FRGs.Online website TIMER 2.0 (http://timer.cistrome.org/)including diverse algorithms (Jiang et al., 2021) of TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL, and EPIC was used to conduct analysis of immune cell infiltration, and a heatmap to display the differences.Single-sample gene set enrichment analysis (ssGSEA) plays a role in calculating the enrichment score that indicates the level at a gene is enriched in a particular gene set.ssGSEA was performed using R software packages "GSVA" and "GSEABase" to calculate the concentration score of 23 immune cells in different clusters (Xiong et al., 2023). The ESTIMATE algorithm is a method of calculating tumor purity (Jiang et al., 2021).The R software "ESTIMATE" package was used to analyze the immune scores and stromal scores in different clusters to explore the infiltration of immune cells and stromal cells in TME.Furthermore, the infiltration data of immune cell in HCC were obtained from the online website CIBERSORT (Liu et al., 2023) (https://cibersortx.stanford.edu/),and the R "ggpubr" and "limma" packages (Dong et al., 2023) were used to visualize the differences in clusters. Construction and validation of a risk prognostic model based on DRG-FRGs The TCGA cohort was used as the training cohort, and GEO and ICGC cohorts as the validation cohorts.We used DE-DRG-FRGs to construct a risk prognostic model to predict immune features, survival time, and drug sensitivity in HCC patients.First, we have previously performed univariate Cox regression analysis to screen out DE-DRG-FRG associated with OS as model candidate genes.Subsequently, to avoid collinearity and overfitting, the least absolute shrinkage and selection operator (LASSO) were conducted with the R "glmnet" package (Xu et al., 2023) to complete the compression of the coefficients and eliminate genes with zero coefficient (Lin et al., 2023).Finally, we chose the genes with non-zero coefficients to carry on multivariate Cox regression analysis, and the regression coefficients of each gene were obtained.The risk score was a linear combination of multivariate Cox regression coefficients and gene expression levels.The calculation formula of RS was as follows, RS = regression coefficient 1 Ã gene 1 expression + regression coefficient 2 Ã gene 2 expression + … + regression coefficient n Ã gene n expression. According to this formula, the RS of HCC samples in the TCGA cohort and the validation cohort GSE14520 and ICGC were calculated, respectively.Based on the median RS, the HCC samples in the TCGA, GEO and ICGC cohort were all divided into high-and low-risk groups.In R software, Kaplan-Meier analysis was performed using the "survminer" package to compare survival differences in the high-and low-risk groups across the three cohorts (Liu et al., 2021).The accuracy of the risk model in predicting survival was evaluated with the receiver operating characteristic (ROC) curves plotted using the R software "timeROC" package (Song et al., 2022).Risk plots were mapped with the R "pheatmap" package (Zhang et al., 2023) to evaluate health status, risk score distribution, and expression of model genes in the two risk groups.In addition, gene correlations were visualized using the R "corrplot" package (Zhang et al., 2023), and the Kaplan-Meier survival analysis on six signature genes were performed.RS differences for different clinical features were plotted with the R "ggpubr" and "limma" packages, and survival curves for two risk groups under different clinical features were plotted.Based on clinical features including age, gender, pathological grade, T (tumor size), stage and RS, univariate and multivariate Cox regression analysis were carried out.Finally, nomogram model with a corresponding calibration curve were conducted with the R "rms" package (Zou et al., 2023) to predict HCC survival by combining multiple clinical features and RS. Decision curve analysis and model comparison The R "timeROC" package was used to draw ROC curves to assess the predictive efficacy of the nomogram model.R "ggDCA" package was used to conduct decision curve analysis (DCA) to measure the net clinical benefit of the nomogram model (Clift et al., 2023). The ROC and survival curves of multiple HCC prognostic models were plotted with the R software "timeROC" and "survminer" packages, respectively. Analysis of gene mutation and tumor mutation burden Somatic mutations of HCC were downloaded from cBioPortal (http://www.cbioportal.org/)database and R "maftools" package was used to analyze the incidence of somatic mutations in the two risk groups (Wang et al., 2023).Tumor mutation burden (TMB) is referred as somatic mutations numbers in the tumor genome exon coding region after germline mutations are removed.The "TMB" function in the R software "maftool" package was used to calculate the TMB of high-and low-risk groups, subsequently the TMB differences between the two groups were analyzed.Finally, the R "survminer" package was used to draw the survival curves of patients with different TMB levels. Analysis of tumor microenvironment The analysis on the infiltration features of 23 immune cells in the two risk groups was also performed with ssGSEA.Also on the TIMER 2.0 website, the infiltration features of immune cells were analyzed and visualized with a heatmap.The scatterplots displaying the association between model genes and immune cell infiltration were downloaded using the online website TIMER (Peng et al., 2022).Similarly, R software "ggpubr" and "limma" packages were used to visualize the difference of immune cell infiltration between the two risk groups. Analysis of response to drug therapy The Genomics of Drug Sensitivity in Cancer (GDSC) (https://www.cancerrxgene.org/) is a website to obtain the drug sensitivity information files.The R software "pRRophetic" package (Ding et al., 2023) was used to calculate the half maximal inhibitory concentration (IC50) to analyze the predictive ability of the risk prognostic model for hundreds of antitumor drugs sensitivity.IC50 is used as an evaluation index of drug sensitivity, which was negatively correlated with drug sensitivity (Wang et al., 2020).IC50 differences between the two risk groups were visualized with R software "ggplot2" package. Analysis of cancer stem cell infiltration Cancer stem cells are subsets of cells with stem cell-like characteristics that play an important role in tumor proliferation, metastasis, and recurrence.In the TCGA cohort, DNA methylation-based stemness score (DNAss) and RNA-based stemness score (RNAss) were acquired from the UCSC Xena browser (Wang et al., 2022) (http://xena.ucsc.edu/)website.Subsequently, the correlation of DNAss, RNAss and risk score was analyzed. Single-cell analysis The online website TISCH (Yao et al., 2023) (http://tisch.comp-genomics.org)was used to analyze the expression of model genes in single cells of HCC tissues. Analysis of immune checkpoints and antigen presentation The immune checkpoint (IC) is an inhibitory pathway of the immune system, which is closely related to human immune status.HLA is the expression product of the human major histocompatibility complex (MHC), which is found on the surface of many immune cells and contributes to antigen presentation.The R software "limma" package was used to analyze the difference between ICs and HLA expression in high-and low-risk groups. Validation of signature genes in databases The protein staining results of 6 DRG-FRGs in liver cancer tissues and liver normal tissues were obtained the Human Protein Atlas (HPA) (Chen et al., 2023) (https://www.proteinatlas.org/)database.The mRNA expression of genes in different cell lines of HCC was analyzed in the Cancer Cell Line Encyclopedia (CCLE) (http://www.broadinstitute.org/ccle) database (He et al., 2023). Immunohistochemistry (IHC) Tissue paraffin sections were placed in xylene and heated for 15 min for dewaxing.Hydration treatment was used with different concentrations of alcohol (absolute ethanol, 85%, 75%).Then sections were put into EDTA antigen retrieval buffer (PH9.0) and microwave for 23 min.After natural cooling, it was placed in PBS and washed three times on a decolorization shaker for 5 min each.Add primary antibody (1:400~1:100, different antibody ratios are different) and incubate overnight in a humidified box at 4 C.In the secondary day, PBS was used to wash off the primary antibody, and the secondary antibody was added.Subsequently, these sections were incubated at a room temperature for 50 min.The secondary antibody was washed off with PBS.The sections were developed with DAB.After completing color development, the nucleus was stained with hematoxylin. Statistical analysis Overall statistical analysis and charts making were completed on R software version 4.2.1 (R Core Team, 2022).The differences between the two groups were compared using the Wilcoxon rank test.The Spearman test was used to test the correlation between two variables.Heatmaps, volcano maps, and violin plots were drawn with the R software "pheatmap", "ggplot2" and "ggpubr" packages, respectively.The Kaplan-Meier curves and log rank test were carried out to analyze the OS differences between groups. Identification and analysis of differentially expressed ferroptosis genes associated with disulfidptosis Differentially analysis showed that there were 72 DE-DRG-FRGs in HCC tissues and non-tumor tissues (Fig. 2A).Among these, 71 genes exhibited up-regulation, while only one gene demonstrated down-regulation in the tumor tissue (Fig. 2B).GO enrichment analysis showed that DE-DRG-FRGs were mainly enriched in the biological activities of response to chemical and oxidative stress inside and outside the cell (Fig. 2C).KEGG enrichment analysis highlighted the primary enrichment of DEGs in signaling pathways involving autophagy, mTOR, and hepatocellular carcinoma, among others (Fig. 2D).DO analysis uncovered associations of DE-DRG-FRGs with various malignancies, including those of the nervous system and the female reproductive system (Fig. 2E).Univariate Cox regression analysis indicated that 16 out of the 72 DE-DRG-FRGs were independently linked to OS in patients with HCC (Fig. 2F).Among these, the expression of 10 genes exhibited a positive correlation with OS, while six genes displayed a negative correlation with OS (Figs.S1A-S1P).The PPI network illustrated the correlation of 16 OS-associated DE-DRG-FRGs and their respective contribution to prognosis (Fig. 2G).In summary, our study identified 72 DEGs between HCC tissues and non-tumor tissues, of which 16 were associated with OS. Unsupervised cluster analysis Utilizing the 16 OS-related DRG-FRGs, we conducted unsupervised clustering analysis.This analysis identified K = 2 as the optimal number of clusters, classifying HCC patients in the TCGA cohort into two distinct clusters, denoted as Cluster A and B (Figs. 3A and 3B).Subsequent verification through PCA, t-SNE, and UMAP analyses confirmed a substantial differentiation between these two clusters (Fig. 3C, Figs.S2A and S2B).Survival analysis indicated that Cluster B exhibited improved OS benefits compared to Cluster A (Fig. 3D).We also conducted cluster analyses in the GSE14520 and ICGC validation cohorts (Figs.S2C, S2E, S2F-S2H).To elucidate the underlying mechanisms responsible for the survival disparity between the two clusters, we investigated variations in immune features.Heatmap analysis employing multiple algorithms revealed higher immune cell infiltration scores in Cluster A (Fig. 3H).ssGSEA further confirmed that Cluster A exhibited a superior immune status compared to Cluster B (Fig. 3I).CIBERSORT analysis highlighted substantial differences in immune cell infiltration features between the two clusters (Fig. S2I).Cluster A displayed significantly higher immune scores and ESTIMATE scores, with no significant difference in stromal scores (Fig. 3E).Cluster A had lower tumor purity, potentially accounting for the shorter survival time observed (Zhang et al., 2017). In addition, the GO GSVA result showed that Cluster B was significantly enriched in molecular biological activities such as lipid metabolism, amino acid metabolism, and iron ion binding (Fig. 3F).KEGG GSVA result showed that Cluster B was significantly enriched in signaling pathways related to amino acid and lipid metabolism (Fig. 3G).In summary, our unsupervised clustering analysis identified two distinct clusters in HCC, characterized by significant differences in both prognosis and the tumor immune microenvironment. Construction and validation of risk prognostic model based on DRG-FRGs Initially, a univariate Cox regression analysis identified 16 DRG-FRGs associated with OS as candidate genes.Subsequently, employing penalty and compression coefficients, 11 out of the initial 16 candidate genes were selected (Figs.4A and 4B).Finally, multivariate Cox regression analysis was performed, and 6 DRG-FRGs (FTH1, G6PD, PML, SLC1A5, SLC7A11, STMN1) were included in our risk prognostic model (Fig. 4C).The risk score was calculated using the following formula, RS= (−0.178977181838147)Ã FTH1 + 0.203421627375275 Ã G6PD + (−0.537653732806195)Ã PML + 0.14649163633218 Ã SLC1A5 + 0.145028457083383 Ã SLC7A11 + 0.190272995477894 Ã STMN1.Risk scores were computed in two validation cohorts using the same formula.Within the three cohorts, samples were stratified into high-and low-risk groups based on the median RS.Survival analysis demonstrated a substantially superior OS outcome in the TCGA cohort's low-risk group compared to the high-risk group (p < 0.001) (Fig. 4D).Corresponding results were observed in the other two validation cohorts, GSE14520 (p < 0.001) and ICGC (p = 0.004) (Figs.4F and 4H).The area under the curves (AUC) for predicting survival time at 1, 3, and 5 years were 0.786, 0.704, and 0.683, respectively (Fig. 4E).These values indicate the high predictive accuracy of the risk model.The AUCs of ROC curves plotted for the GSE14520 and ICGC cohorts further demonstrated the favorable predictive power of our risk model for survival (Figs.4G and 4I).In the low-risk group, higher expression levels of FTH1 and PML were observed, whereas in the high-risk group, higher expression levels of G6PD, SLC1A5, SLC7A11, and STMN1 were evident.Additionally, higher RS and a greater number of deaths were both concentrated in the high-risk groups (Fig. 4J). Figure 4K showed the correlation of the 6 DEG-FRGs.Univariate Cox regression analysis discovered that 6 DRG-FRGs were all independently related to OS (Fig. 4L).Specifically, the expression levels of FTH1 and PML were positively correlated with survival, whereas G6PD, SLC1A5, SLC7A11, and STMN1 displayed a negative correlation with survival (Figs.S3A-S3F).This was consistent with the distribution of DRG-FRGs in the two risk groups.In conclusion, the risk prognosis model effectively differentiated the prognoses of high-and low-risk groups and provided accurate predictions for the survival of HCC. Evaluation of the risk prognostic model based on DRG-FRGs Both univariate and multivariate Cox regression analyses both showed that RS was an independent prognostic factor in HCC (Figs. 5A and 5B).Soon afterwards, we successfully construct a nomogram model containing risk score and clinical features (age, sex, pathological grade, T, and stage) to predict the survival of HCC (Fig. 5C).The calibration plot illustrated a high degree of consistency between the predicted and observed survival (Fig. 5D).An analysis of risk score distributions within distinct clinical feature groups revealed significantly higher risk scores in cluster A, grade 3-4, T3-T4, and stage III-IV (p < 0.05), with no notable variation observed in terms of age and gender (Figs.S3G-S3L).The Kaplan-Meier curves revealed that RS was a powerful prognosis factor in HCC patients with different ages, genders, grades, T, and stages (Figs.6A-6J).In addition, HCC patients with different clinical features also had better survival in the low-risk group. We subsequently assessed and compared the predictive capacity of our nomogram model.The area under the curve (AUC) values for 1-, 3-, and 5-year survival in ROC curves indicated that our nomogram model outperformed other clinical features (age, gender, T, and stage), with the RS following closely (Figs.6K-6M).The 1-, 3-and 5-year DCA curves also indicated our nomogram model has superior ability to make clinical decisions (Figs. 6N-6P).To compare predictive ability of our model with other prognosis models in HCC, we also plotted ROC curves.AUCs for 1-, 3-and 5-year OS discovered that our risk prognostic model for HCC had better predictive ability than other models (Liu et al., 2019;Tian et al., 2021;Zhang et al., 2020) (Figs.5E-5H).In addition, Kaplan-Meier curves showed that our risk model could most significantly distinguished survival differences in high-and low-risk groups (Figs.5I-5L).Collectively, these findings indicate that our risk prognostic model possesses strong predictive capabilities for HCC survival. Analysis of gene mutation and tumor mutation burden To assess the utility of the 6 DRG-FRGs in developing diagnostic, prognostic, and recurrence models, we investigated genetic alterations using the online cBioportal database.Our analysis revealed that 6.2% of patients exhibited mutations in these six genes, with G6PD displaying the most substantial mutation frequency, followed by FTH1, which featured notable amplifications and deep depletions (Fig. 7A).In the TCGA cohort, the high-risk group exhibited an 89.89% incidence of gene mutations, significantly surpassing the 81.46% observed in the low-risk group.Notably, TP53, CTNNB1, and TTN were the genes with the highest mutation frequencies in both risk groups (Figs.7B and 7C).TMB serves as a promising biomarker for predicting the response to immune checkpoint inhibitors (ICIs).Tumors with elevated mutation burdens typically yield more neoantigens, rendering them more immunogenic and, consequently, more likely to respond favorably to ICIs.As illustrated in Fig. 7D, the TMB level was markedly higher in the high-risk group compared to the low-risk group (p < 0.05), suggesting that the high-risk group might exhibit a more robust response to ICIs.Survival analysis revealed that low TMB had longer survival time than high TMB (Fig. 7E).To explore the co-effect of risk score and TMB on prognosis of HCC patients, we divided patients into four groups for Kaplan-Meier survival analysis.The results revealed that patients with both low RS and low TMB had the longest survival time (Fig. 7F).In summary, the analysis of gene mutations and tumor mutation burden unveiled distinctions in gene mutations and potential variations in the tumorigenesis and progression of HCC between the high-risk and low-risk groups. Analysis of immune features and antigen presentation To elucidate the mechanisms contributing to the divergent survival probabilities in the two HCC risk groups, we conducted an analysis of immune cell features.Analysis of immune cell features revealed a more pronounced infiltration of various immune cells in the high-risk group, including dendritic cells (DCs), macrophages, Th1 cells, and Th2 cells (Fig. 8A).CIBERSORT analysis demonstrated that the mRNA expression levels of memory B cells, activated CD4 memory T cells, Tregs, T cells gamma, T cells follicular helper, and M0 macrophages were notably elevated in the high-risk group compared to the low-risk group.In contrast, resting memory CD4 T cells, naive B cells, monocytes, and resting mast cells exhibited significantly higher expression levels in the low-risk group (Fig. 8B). The heatmap depicted marked disparities in the abundance of various infiltrated immune cells between the two risk groups, as determined by multiple algorithms (Fig. 8C).HLAs play a critical role in antigen presentation and are thus positively correlated with the efficacy of immunotherapy (Schaafsma et al., 2021).The high-risk group exhibited higher expression levels of both class I and class II HLAs, suggesting a potentially more favorable response to ICIs (Fig. 8D).ICIs are a pivotal approach to anti-tumor therapy, primarily functioning by inhibiting the interaction between ICs on the surface of tumor cells and IC receptors.The analysis demonstrated higher expression levels of ICs in the high-risk group, suggesting a greater likelihood of the effectiveness of ICIs in this group (Fig. 8E).These six signature genes were found to be correlated with the infiltration of immune cells.Specifically, FTH1 displayed positive associations with macrophage, dendritic cell infiltration, B cells, and neutrophils, but not with CD8+ T cells and CD4+ T cells (Fig. S4A).SLC7A11 expression exhibited positive correlations with neutrophil and dendritic cell infiltration, while no significant associations were observed with the other four types of immune cells (Fig. S4E).The expression of G6PD, PML, SLC1A5, and STMN1 were positively related to the infiltration level of six types of immune cells (Figs. S4B-S4D and S4F).In conclusion, these findings indicate disparities in the immune features of the TME between the high-and low-risk groups.Gene set enrichment analysis Figure 9 illustrates significantly enriched biological activities and signaling pathways.GO GSEA indicated that the high-risk group exhibited significant enrichment in molecular functions, including immunoglobulin production, leukocyte-mediated immunity, membrane invagination, production of molecular mediators of immune response, and immunoglobulin complex (Fig. 9A).Conversely, the low-risk group primarily displayed enrichment in processes such as alpha amino acid catabolism, cellular amino acid catabolism, lipid oxidation, monocarboxylic acid catabolism, and monooxygenase activity (Fig. 9B).KEGG GSEA identified the high-risk group as predominantly enriched in signaling pathways related to the cell cycle, the interaction between cytokines and cytokine receptors, DNA replication, neuroactive ligand-receptor interaction, and ribosome (Fig. 9C).In contrast, the low-risk group was primarily enriched in pathways associated with drug metabolism cytochrome P450, complement and coagulation cascades, fatty acid metabolism, peroxisome, and retinol metabolism (Fig. 9D).In summary, the primary enriched biological processes and signaling pathways in HCC exhibited significant differences between the high-and low-risk groups. Drug sensitivity analysis We conducted an analysis of IC50 values, revealing a significant difference in IC50 between the two risk groups.IC50 values for drugs including 5-Fluorouracil, docetaxel, and paclitaxel were notably elevated in the low-risk group, whereas drugs like Sorafenib, Axitinib, and Oxaliplatin exhibited higher IC50 values in the high-risk group (Figs. 10A-10P).In summary, a noteworthy disparity in the sensitivity of HCC to antitumor drugs is evident between the high-and low-risk groups. Analysis of cancer stem cell infiltration We examined the variance in tumor stemness characteristics between the two risk groups using DNAss and RNAss.Our analysis results revealed that DNAss exhibited no significant association with the risk score (R = 0.1, p = 0.056).In contrast, RNAss displayed a positive correlation with the risk score (R = 0.38, p = 7e−14), indicating elevated tumor stemness in the high-risk group and a reduced degree of tumor cell differentiation (Figs.11A and 11B). Single-cell analysis of signature genes The findings indicated that HCC tissues were primarily composed of malignant cells, with fibroblasts as the secondary component.FTH1, G6PD, PML, SLC1A5, SLC7A11, and STMN1 exhibited their highest expression levels in macrophages, B cells (in three cases), hepatic progenitor cells, and endothelial cells (Figs. 11C-11H).In summary, the distribution of the six signature genes of the model within HCC tissues displayed variations. Validation of signature genes in databases Immunohistochemical data for both liver normal tissue and tumor tissue was obtained from the HPA database.While accurately discerning differences in gene expression between liver normal and tumor tissues might be challenging, preliminary findings revealed elevated expressions of G6PD, STMN1, and PML in HCC tissues in comparison to normal liver tissues.Notably, protein expression data for SLC7A11 was unavailable.However, there was no significant disparity in the expression of FTH1 and SLC1A5 (Fig. 12A).Moreover, we observed variations in the expression levels of the six signature genes across different HCC cell lines (Fig. 12B).In summary, the expression of three out of the six signature genes exhibited significant differences between HCC tissues and normal liver tissues in the HPA database.Validation of signature genes in protein level Immunohistochemical analysis of 24 pairs of HCC tissues and their respective adjacent tissues revealed that there were no statistically significant differences in the expression of FTH1 and SLC1A5 between the tumor tissues and adjacent counterparts (Figs. 13A and 13B,Figs. S5A and S5B).However, the staining intensity of the signature proteins-PML, G6PD, SLC7A11, and STMN1-in the tumor tissues was notably stronger than that in the paired adjacent tissues (Figs.13C-13F, Figs.S5C-S5F). DISCUSSION Primary liver cancer continues to pose a significant challenge with one of the lowest 5-year survival rates (21%) among malignancies.HCC predominates in the pathological classification of liver cancer.Early-stage HCC often presents with inconspicuous clinical symptoms, leading to diagnoses at an advanced stage in 50% of patients.While the emergence of molecularly targeted drugs and immunotherapy has provided a newfound advantage for advanced HCC patients, the scope of survival benefits remains constrained.Consequently, the imperative to explore novel prognostic biomarkers and therapeutic approaches has intensified.In addition to the tumor growth inhibition observed with Sorafenib in HCC, targeting ferroptosis has demonstrated its capacity to induce cancer cell death and impede tumor growth across various malignancies.Modulating the expression of cysteine desulfurase (NFS1) to induce ferroptosis has shown potential in curtailing the progression of lung adenocarcinoma (Alvarez et al., 2017).Erastin, an inducer of ferroptosis, heightens the sensitivity of lung cancer cells to cisplatin, while bolstering Erastin's presence inhibits ovarian cancer invasion (Guo et al., 2018;Basuli et al., 2017).The combined application of sulfadiazine (SSZ) and piperine (PL) significantly elevates ROS levels, thereby inducing ferroptosis in pancreatic cancer cells (Yamaguchi, Kasukabe & Kumakura, 2018). Exploring a recently uncovered form of cell death, disulfidptosis, remains an uncharted frontier in various malignancies.As a response, our study has developed a prognostic model for HCC, anchored in ferroptosis-related genes linked to disulfidptosis. We conducted an analysis of RNA-seq data obtained from HCC samples sourced from the TCGA, GEO, and ICGC databases.Subsequently, an unsupervised cluster analysis was applied to these HCC samples based on the transcriptome expression levels of 16 DE-DRG-FRGs associated with OS.Our investigation aimed to delineate disparities in survival probability and immune cell infiltration between the resulting clusters.The findings revealed that cluster B exhibited improved OS outcomes compared to cluster A. Moreover, cluster A demonstrated higher levels of immune cell infiltration, immune score and ESTIMATE score.Notably, cluster A displayed heightened infiltration of immune cells such as myeloid-derived suppressor cells (MDSCs), macrophages, and regulatory T cells.MDSCs (Gabrilovich, 2017), tumor-associated macrophage (TAM) (Cassetta & Pollard, 2018) and Treg cells (Tanaka & Sakaguchi, 2017) are recognized for their roles in promoting immunosuppression during tumorigenesis and disease progression.Consequently, it was conjectured that the shorter survival duration observed in cluster A might be attributed to its more pronounced immunosuppressive TME.The composition of the TME holds a pivotal role as a mediator affecting immune escape, tumor growth, invasion, and resistance to immunotherapy (Bagaev et al., 2021).An inhibitory TME has been associated with unfavorable prognoses in cancer patients. A risk prognostic model was subsequently developed using LASSO and Cox regression analysis, based on six DRG-FRGs: FTH1, G6PD, PML, SLC1A5, SLC7A11, and STMN1.FTH1, responsible for encoding ferritin heavy subunits, emerged as a pivotal mediator of ferroptosis in bladder cancer cells induced by baicalin.Moreover, overexpressing FTH1 was observed to attenuate the anticancer effects of baicalin in both in vitro and in vivo settings (Kong et al., 2021).G6PD primarily functions in generating NADPH, a crucial electron donor for biosynthesis reactions, and is closely intertwined with cell growth and death processes (Yang et al., 2019).Notably, G6PD exhibits abnormal elevation in various cancer types, with its aberrant activation associated with the proliferation and invasion of several malignant tumor types (Yang, Stern & Chiu, 2021).PML expression is contingent upon the cell cycle and possesses the ability to activate TP53 or Rb/E2F pathways, thereby inhibiting apoptosis (Martin-Martin et al., 2016).Liying Han and colleagues reported a significant increase in SLC1A5 expression in glioblastoma tissues compared to low-grade gliomas (Han et al., 2022).Notably, the knockdown of SLC1A5 significantly curtailed the proliferation and invasion of glioma cells.Furthermore, the targeting of SLC1A5 substantially impeded the growth and proliferation of lung cancer cells (Hassanein et al., 2013).Previous studies have documented the overexpression of SLC7A11 in various cancer types, with a demonstrated role in tumor promotion.Cancer cells can augment tumor growth by upregulating SLC7A11 expression and inhibiting ferroptosis through diverse mechanisms (Koppula, Zhuang & Gan, 2021).SLC7A11 is also a disulfidptosis-related gene.Elevated SLC7A11 expression introduces vulnerabilities to cancer cells, rendering them notably sensitive to glucose or glutamine deficiencies.Consequently, it becomes easier to trigger disulfidptosis during glucose deprivation (Liu et al., 2023;Koppula, Zhuang & Gan, 2021;Lee & Roh, 2022).STMN1, an unstable phosphorylated microtubule-associated protein, is recognized as an oncogene.Its heightened expression is closely linked to unfavorable prognoses in multiple cancer types.The downregulation of STMN1 has been observed to impede the growth of gallbladder cancer cells and mitigate the Warburg effect (Wang et al., 2021).Elevated STMN1 expression in lung squamous cell carcinoma correlates with vascular invasion, decreased sensitivity to paclitaxel, and unfavorable prognostic outcomes (Bao et al., 2017).In a similar vein, Yaming Li and colleagues demonstrated that targeting STMN1 can mitigate chemotherapy resistance and metastasis in triple-negative breast cancer (Li et al., 2018).Our study affirms the efficacy of the risk prognostic model, constructed based on these six genes, in accurately predicting the survival of patients with HCC. KEGG enrichment analysis revealed that genes in the high-risk group were predominantly associated with pathways involving the cell cycle, cytokine and cytokine receptor interaction, as well as DNA replication.Cell cycle dysregulation is one of the mechanisms underlying tumorigenesis and is intricately linked to metabolic reconfiguration and immune evasion (Liu, Peng & Wei, 2022).The division of tumor cells involves a mutation that enables the cell cycle to advance, inhibiting withdrawal.Numerous malignant tumors increasingly depend on residual cell cycle control mechanisms to avert genomically unstable reproduction.Thus, targeting cancer's reliance on cell cycle control pathways has emerged as a promising therapeutic strategy (Matthews, Bertoli & de Bruin, 2022).Many studies have focused on the role of cytokines and cytokine receptors as potential targets for tumor intervention, including the inhibition of the tumor-promoting functions of IL-1β, IL-6, and TNF.Various antagonists targeting inflammatory cytokines or their receptors have been employed in patients with advanced cancer, potentially leading to short-term disease stabilization (Propper & Balkwill, 2022).In the cell cycle, a dividing cell must accurately replicate its DNA once to preserve genome integrity.Disruptions in this process can readily lead to the onset of diseases like cancer (Boyer, Walter & Sorensen, 2016).Tumors are characterized by sustained proliferation, evasion of apoptosis, and genomic instability (Macheret & Halazonetis, 2015).DNA replication stress could drive cancer progression and is considered a hallmark of the disease.Overall, the poorer prognosis observed in the high-risk group may be attributed to disturbances in the cell cycle, the production of tumor-promoting cytokines, and unstable DNA replication. To the best of our knowledge, this is the inaugural investigation into the potential utilization of ferroptosis genes linked to disulfidptosis in the prognosis and tumor immunity of Hepatocellular Carcinoma.Nevertheless, our study is not without limitations. Firstly, the restricted number of disulfidptosis-related genes posed a limitation.Our prognostic model was constructed using ferroptosis-related genes linked to disulfidptosis, which may limit the specificity of our description of the correlation between disulfidptosis-related genes and the prognosis of HCC.To address this, further exploration necessitates the inclusion of additional disulfidptosis-related genes, large-scale clinical trials, and validation of molecular functions and mechanisms through in vitro and in vivo studies.Secondly, despite our validation involving two cohorts, substantial heterogeneity persisted among tumor samples characterized by diverse regional and ethnic attributes, and even within the individual samples.Nevertheless, notwithstanding these limitations, it is indisputable that our model holds clinical significance in predicting the tumor microenvironment and the survival of patients with HCC. Figure 4 Figure 4 Identification of signature genes and prognosis analysis.(A) LASSO coefficient profile plots of the 16 DRG-FRGs.(B) Regression algorithm using LASSO, 10 cross-validation method was used to select the optimal parameter (lambda).(C) Multivariate Cox regression analysis for six signature genes and corresponding regression coefficients.(D) The risk score, survival status, and gene expression distribution of HCC in the high-and low-risk groups in the TCGA cohort.(E) Correlation plot of six signature genes in the TCGA cohort.(F) Univariate Cox analysis showed that six signature genes were associated with OS. (G-I) Survival analysis of high-and low-risk groups in the TCGA, GEO, and ICGC cohorts.(J-L) Receiver operating characteristic (ROC) curves were used to analyze the predictive power of the prognostic model at 1, 3, and 5 years in the TCGA, GEO, and ICGC cohorts.DRG-FRGs, ferroptosis-related genes associated with disulfidptosis-related genes; OS, overall survival.Full-size  DOI: 10.7717/peerj.16819/fig-4 Figure 5 Figure 5 Construction of the nomogram model and comparison of the prognostic models.(A) Univariate Cox regression for RS and clinical features in the TCGA cohort.(B) Multivariate Cox regression for RS and clinical features in the TCGA cohort.(C) The nomogram for predicting 1-, 3-, and 5-year OS of HCC in the TCGA cohort.(D) The calibration curves for predicting 1-, 3-, and 5-year OS of HCC.(E-H) ROC curves for 1-, 3-, and 5-year OS prediction of HCC in different prognostic models.(I-L) Survival analysis of high-and low-risk groups in different prognostic models.RS, risk score; OS, overall survival; ROC, receiver operating characteristic.Full-size  DOI: 10.7717/peerj.16819/fig-5 Figure 6 Figure 6 Survival analysis of HCC with different clinical features and comparison of the predictive power of the nomogram model and other clinical features.(A-J) Survival analysis of HCC with different clinical features in the TCGA cohort.(K-M) ROC curves for 1-, 3-, and 5-year OS prediction of nomogram model and other clinical features in the TCGA cohort.(N-P) Decision curve analysis (DCA) of the nomogram model and other clinical features in the TCGA cohort.OS, overall survival; ROC, receiver operating characteristic.Full-size  DOI: 10.7717/peerj.16819/fig-6 Figure 7 Figure 7 Gene mutation and tumor mutation burden (TMB) analysis of the prognostic model.(A) Mutation analysis of six signature genes in the TCGA cohort.(B and C) Mutation landscape of high-and low-risk groups in the TCGA cohort.(D) Comparison of TMB in high-and low-risk groups in the TCGA cohort.(E) Survival analysis of high-and low-TMB in the TCGA cohort.(F) Survival analysis of TMB and risk score in the TCGA cohort.Full-size  DOI: 10.7717/peerj.16819/fig-7 Figure 8 Figure 8 Immune infiltration analysis of the prognostic model.(A and B) Infiltration score of immune cells in high-and low-risk groups in the TCGA cohort.(C) The heatmap of immune cell infiltration in high-and low-risk groups in the TCGA cohort based on multiple algorithms.(D) Expression of HLA in high-and low-risk groups in the TCGA cohort.(E) Expression of immune checkpoints in high-and lowrisk groups in the TCGA cohort.HLA, human leukocyte antigen.Ã p < 0.05; ÃÃ p < 0.01; ÃÃÃ p < 0.001 Full-size  DOI: 10.7717/peerj.16819/fig-8 Figure 9 Figure 9 Gene set enrichment analysis (GSEA) of the prognostic model.(A and B) GO enrichment analysis in high-and low-risk groups in the TCGA cohort.(C and D) KEGG enrichment analysis in high-and low-risk groups in the TCGA cohort.Full-size  DOI: 10.7717/peerj.16819/fig-9 Figure 10 Figure 10 Drug sensitivity analysis of the prognostic model.(A-P) Analysis of different drugs in highand low-risk in the TCGA cohort.Due to the large variety of drugs, only partial charts of drugs were shown.Full-size  DOI: 10.7717/peerj.16819/fig-10 Figure 11 33 Figure 12 Figure 11 Cancer stem cell analysis and single-cell analysis of the prognostic model.(A and B) Cancer stem cell analysis in high-and low-risk groups in the TCGA cohort.(C-H) Single-cell analysis of six signature genes in HCC tissues.Full-size  DOI: 10.7717/peerj.16819/fig-11	2024-02-06T17:04:11.409Z	2024-02-02T00:00:00.000	{ "year": 2024, "sha1": "2b1e9b7942c93b417625422977c0a0af409e61d7", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "3da8128892deffe953ffc50e1bc19d9472d95267", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
55519364	pes2o/s2orc	v3-fos-license	The Effect of Green Marketing on Purchase Decision with Brand Image as Mediating Variable The purpose of this study is to explain the influence of marketing strategy on purchase decision through brand image as mediation. To test the empirical model, this study used Structural Equation Modeling (SEM) analysis tool. Other analysis tools used include AMOS 21.0, SPSS 16.0 and Microsoft Excel 2007. The number of samples in this study were 140 users of Tupperware in Tasikmalaya. The results of this study indicate that the dimensions of marketing strategy that are green product, green price, green place and green promotion have positive effect on purchase decision and brand image, and brand image mediate the influence of marketing strategy dimensions on purchase decision. INTRODUCTION Background Environmental and health problems that are directly and indirectly caused by human activities, whether in the fields of science and technology, agriculture, economics and business, have become central issues in all circles.Concern and awareness of the environment and health, has changed the perspective of life and lifestyle of human and business actor.This is shown in the change of business approach pattern which starts to direct the business with the approach of business activity based on environmental sustainability. Environmental or green marketing is a new focus in business ventures that is a strategic marketing approach that is beginning to emerge and become the attention of many parties from the late 20 th century.This condition requires marketers to be more careful in making decisions that involve environment (Byrne, 2003). In addition, companies use the term green marketing, in an effort to get a chance to achieve corporate goals.This is evident in the concerns of businesspeople on environmental and health issues with the increasingly environmentally concerned market (Laroche, et al., 2000). If environmental issues (pollution, species protection, and recyclable products) have an important sense for consumers in choosing products, and if a company in the market is the only one to offer with an environmental marketing mix among its competitors, then the company must have a high strategic competitive advantage (Cravens, 2000). One of companies that apply the concept of green marketing as its marketing strategy is Tupperware.Tupperware is a company of quality plastic products.Tupperware products are made from top quality plastic materials, do not contain toxic chemicals and have meet the standards of some world agencies such as the US FDA (Food and Drug Administration), European Food Safety Authority (Europe), Japan Food Safety Commission (Japan), so in addition to safe use repeatedly for food and beverage but also environmentally friendly (tupperware.co.id).Tupperware products that are quite expensive does not discourage the public interest to buy healthy and environmentally friendly products.Tupperware products are in great demand by various circles, both unmarried and married people. Based on the phenomenon, the researcher wants to know whether the marketing mix based on green marketing concept includes product, price, distribution, and promotion has influence on purchasing decision of Tupperware products for people in Tasikmalaya. Problem Formulation Based on the background of the problem described above, can be formulated research questions as follows: 1. Does green marketing have an influence on purchase decision?2. Does brand image mediate the relationship between green marketing and purchase decision? Research Objective To find out and analyze: 1. Does green product have an influence on purchase decision?2. Does green price have an influence on purchase decision?3. Does green place have an influence on purchase decision?4. Does green promotion have an influence on purchase decision?5. Does brand image mediate the relationship between green product and purchase decision?6.Does brand image mediate the relationship between green price and purchase decision?7. Does brand image mediate the relationship between green place and purchase decision?8. Does brand image mediate the relationship between green promotion and purchase decision? Research Function The results of this study are expected to provide benefits for the development of science, especially in the field of marketing management.And also this research is expected to be used as reference material for further research. The results of this study are expected to be inputs and development materials for the management of Tupperware as a source of information and formulate appropriate marketing strategies in dealing with corporate problems, especially related to the formation of high consumer purchase decision.Pride and Farrel (1993) defined green marketing as an effort to design, promote, and distribute non-destructive products.Charter (1992) defines green marketing as a holistic, strategic management process responsibility that identifies, anticipates, satisfies and meets the needs of stakeholders for a reasonable member of the prize, which does not cause harm to humans or the health of the natural environment. LITERATURE REVIEW AND HYPOTHESES DEVELOPMENT Charter (1992) defined green marketing as a holistic, management process strategic responsibility that identifies, anticipates, satisfies, and meets the needs of stakeholders to give a reasonable appreciation, which does not cause disadvantage to humans or the health of the natural environment. The importance of green marketing by Mc. Taggart, Findlay and Parkin (1992) in Polonsky (1994) refers to the economic principle, which states that the economy is a form of one's expenditure in trying to satisfy unlimited desires by using limited resources.Thus, according to Polonsky (1994), it can mean that we have limited resources on earth, with an unlimited desire of the world, so if applied in the company, green marketing can be seen as a marketing activity by utilizing limited resources to meet the satisfaction of the consumer desire so as to achieve the company's sales goals. The solution can ensure the company's participation in understanding the needs of the society and as a company's opportunity to achieve excellence in the industry (Murray & Montanari, 1986in Lozada, 2000). Thus it can be assumed that companies that market their products with environmental characteristics will have a competitive advantage compared to companies that market without responsibility to the environment, this is an attempt to satisfy the needs of their customers as in Mc.Donalds by replacing shell packing with wax paper due to increased consumer concerns related to polystyrene and ozone reduction (Gofford, 1991;Hume 1991). Several existing research have found that governments and organizations have already done an approach on environmental marketing from a number of perspective deviations, from responsibility of comfort to the pressure on emission level constraints, which all can encourage consumers to adopt items with various levels of green characteristics from various marketing mix approaches (Hawken, et al., 1999 andKalafatis, et al., 1999). Green Product Swastha and Irawan (1996) said that product is a complex nature that can be touched or cannot be touched, including wrapping, color, price, company achievement, and retailers received by the buyer to satisfy the desires and needs. Increasing product variety in markets that support sustainable development can undertake the basics of product management as follows: 1. Product can be made from recyclable materials.2. Product can be recycled or reused.3. Product is efficient, which conserves water, energy, or gasoline use, saves money, and minimizes the impact of product on the environment.4. Product packaging is responsible.5. Product does not contain substances that damage health in humans and animals.6. Use green labels that enhance product offerings.7. Product is organic, many consumers are willing to purchase organic products at premium prices that offer quality assurance.8. Service that rents or lends products, such as library.9. Product is certified that clearly meets the responsibility criteria on environment. Labeling of products has a purpose in conveying information to consumers on product attributes (Kotler, 2003).In a study in the United States, Britain, Australia, and South Africa that consumers interpret the information of environmentally-friendly products on different packaging labels to what the marketers meant, for example in a case that the consumer means the product has been labeled environmentally friendly, other consumers are in doubt because the product that promotes environmentally friendly does not in detail inform the criteria of environmentally friendly quality.(Polonsky, et al., 2002). Green Price Price is the value of goods and services measured by the amount of money.Based on that value, a person or company is willing to release goods or services owned to other parties.Inside the firm, price of a good or service is a determination for market demand.Price can affect the position of company competition.Price decision should never be made by chance.On a common product, price reduction can increase sales, while on products that carry prestigious images, price increases will increase sales because high-priced products will show a person's achievement. According to a survey taken by GHI along with the Roper organization that 82% of Americans said they were willing to pay more for products that could have a better environmental impact (Voss, 1991).Many retailers have put on more environmentallyfriendly products at a cost beyond the consumer's ability (Reitman, 1992).Consumers' desire to pay a certain amount of money for environmentally-friendly products is more due to their concern for environmental issues (Laroche, et al., 2001). The conceptual model proposed by Ottman (1992), Voss (1991), Reitman (1992) concerning consumers' willingness to pay with premium prices for environmentallyfriendly products is contrary to research by Capelins and Strahan (1996) explaining that consumers desire to pay with premium prices on environmentally-friendly products is only ranging from 5% to 10% of the price for conventional products (Capelins and strahan, 1996).Furthermore, Polls (2002) described that consumers generally believe that environmentally-friendly products have a high price.This is supported by the study of Polls (2003) in the United Kingdom who found that the premium price of an environmentallyfriendly product is negatively related to the consumer's choice on environmentallyfriendly products. Green Place Place reflects the company's activities that make the product available to target consumers.Part of the distribution task is to choose an intermediary to be used in a distribution channel that physically handles and lifts the product through the channel, meaning that the product can reach the intended market on time. Location also consistently must be considered in accordance with the intended image.Location must be different from competitors.This can be obtained by in-store promotions and creating attractive displays or using recyclable materials to emphasize environmental excellence and other advantages. Green Promotion Promotion reflects activities that communicate product excellence and persuade consumers to buy it.So, this promotion is a component used to provide and influence the market for the company's products. Promoting a product and service to gain market can be done with advertising, public relation, sales promotion, direct marketing, and on-site promotion.Intelligent green product sellers will be able to emphasize the credibility of environmentally-friendly products by using sustainable marketing as well as communication tools and practices (Queensland Government, 2002).The key to success of green marketing is credibility.By not exaggerating the environmental excellence of the product or forming unrealistic expectations on the customer, the communication of environmental excellence can be done through credible figures (Haryadi, 2009). Purchase Decision According to Engel, et al. (1995), consumer behavior is activities that are directly involved in getting, consuming, and depleting products and services including processes that precede and follow the action.The process of decision-making by consumers is influenced by three factors: consumers as individuals, environmental influences, and marketing strategies conducted by the company.The influence of consumers as individuals in decision making includes consumer needs, consumer perceptions of product characteristics, demographic factors, lifestyle, and personal character.Environmental influences include culture (social norm, religion, and ethnic group), social class, and kinship.The marketing strategy that affects consumers in decision making is the marketing mix of products evaluated by consumers (Mursyid, 2003). Assael (1992) divided the decision-making process conducted by consumers into five stages: the introduction of needs, information processing, assessing the existing brands, purchasing, and assessment after making a purchase.Stages of decision making process of product purchase decision by consumers can be seen in Figure 1.Psychological factors consist of motivation, perception, learning, and consumer attitude.Motivation is an encouragement to perform an action.Perception is how consumers interpret the stimulus that comes either in the form of image, place, or an object.The logical expansion of the process of motivation and perception is learning.Learning is a process of exploring or extending knowledge based on past experience.According to Engel, et al. (1995), attitude is an overall evaluation that enables people to respond to a given object.The purpose of marketing strategy is to meet the customer need and satisfaction.In order to meet the customer need and satisfaction then we must know the consumer preference first.A product can be acceptable depends on the response of and the way the consumer receives the product.Marketing stimuli consists of primary stimuli caused by the product itself, secondary stimuli caused by symbol, image, and information about the product (Assael, 1995). Brand Image Brand is the name, term, sign, symbol, or design or combination of such matters, intended to identify the good and service of a person or group of sellers and to distinguish them from competitor products (Kotler, 2009: 258).Brand image is the overall consumer perception of the brand, judged by the understanding of information from the brand.Therefore, brand image should be able to be identified by consumers and evaluate products and services, reduce risk cost, ensure whether the customer needs are met, and give consumers a satisfaction from product or service differentiation (He, et al. 2013: 2).Further, Kotler (2005: 215) Postpurchase Behavior image as a set of beliefs, ideas, and impressions that a person has for a brand, the attitude and action of the consumer towards a brand is determined by the brand image. Previous Research Environmental pollution is increasing rapidly throughout the industrialization of products that is harmful to the environment.When the hazardous components of the product become one of the factors affecting consumer purchase decision, the business world starts to produce environmentallyfriendly products or green products and make green product policy (Uydacı, 2002: 113). Boztepe ( 2012) conducted a survey on 540 green consumers in 2012.Targeted individuals include respondents aged between 18-65, who were aware of green products and who had purchased green products in the past.According to this study, consumers consider green products as people who minimize the impact on the environment (e.g., energy-efficient, recycled, natural, or organic).Only 30% of consumers consider green practices on product labels and word of mouth is the main source of information about green products for consumers (Ayzel, 2012). A lower price caused by cost saving will encourage consumers to buy environmentally-friendly products.When product demand is responsive, lower price will be a more successful strategy for the company.The positive nature of the product on the environment can be used as an element of competitive advantage.In the case of higher product price, it is very important to give a promotion that distinguishes green products and also there must be consumers who are ready to pay more for the green products. In this case, what matters is the price level (Emgin and Turk, 2004).Ayzel conducted a survey on 540 green consumers in 2012.According to this study, consumers who have bought green product feel blocked because it is considered too expensive.Price is the main reason consumers do not buy green products (Boztepe, 2012). The choice of where and when to make the product is always available that can give a significant influence on the customer.Very few customers only want to buy the product because of the environmentally-friendly only.Sellers who want to achieve success in selling environmentally-friendly product should position their products widely in the market so that it can be more recognizable (Queensland Goverment, 2002). Location also consistently must be considered in accordance with the intended image.Location must be different from competitors.This can be obtained by instore promotion and by creating attractive display or using recyclable material to emphasize environmental excellence and other advantages.Song, et al. (2001) in Ariawan ( 2005) stated that changes in the competitive environment and pressures faced by the organization, a synergy must be built by combining firms, distribution channels, and flexible engineering capabilities.Some research has shown that the development of channel relationship quality is the results obtained from the synergy that is built among the company's capabilities and resources and expertise rooted in the concept of product marketing channel success. A number of previous studies have analyzed the relationship between brand image and consumer attitude.Almuarief (2016) conducted a study on 140 questionnaires in Yogyakarta.According to this research, there is a positive and significant influence of brand image on purchase decision of Ades packaged drinking water.This means that the better the brand image the better and many consumers who do purchase decision. RESEARCH METHODS The research method used in this research is survey research method, according to Sugima (2008: 135): "Research by proposing statements to people or subjects and recording the answers to be analyzed critically".This survey method is part of descriptive research and causal research. Research Object The object of research is green marketing, brand image, and purchase decision.Research respondents are Tupperware users in Tasikmalaya. Type and Source of Data The type of data in this study is divided into 2 parts, namely: 1) Primary data That is data on green marketing, brand image, and purchase decision obtained directly from the field through interview and filling questionnaire by selected respondents. 2) Secondary data That is data collected from other parties, existing or available data which are then reprocessed for a particular purpose, this data is in the form of history and condition of market, literature, articles, and scientific papers considered relevant to the topic of green marketing, brand image, and purchase decision. The data collection technique used in this study consists of: 1) Field Research That is the research conducted directly on the object of research, so it can be known the actual condition by the ways as follows: a. Interview that is data collection activities and facts by holding direct questions and answers with respondents regarding green marketing, brand image, and purchase decision.b.Questionnaire that is spreading questions on related issues to selected respondents to find out their response to green marketing, brand image, and purchase decision. 2) Library Research Library research is a method of data collection conducted by studying the literature related to green marketing, brand image, and purchase decision so that it can be used as the basis of analysis. Population and Sample Population in this research is users of Tupperware product in Tasikmalaya.The size of the population cannot be accurately known so it is infinite.Sampling used judgment sampling/purposive sampling where the sampling is based on personal consideration of the researcher concerned (Sugiama, 2008).In this way of sampling, the researcher attempted to find confidence fist that the individual chosen as the sample is the right individual. Determination of the sample size is based on the opinion of Hair (1995: 444) that is for the survey research, the minimum sample size is 100.To further ensure the accuracy in this study, the questionnaires were distributed to 200 respondents. Analysis Tool Before conducting data analysis, it is necessary to conduct the validity and reliability test of the distributed questionnaires.The technique used is structural equation model analysis aiming to estimate some separate regression equations but each has a simultaneous or simultaneous relationship.In this analysis, there may be some dependent variables, and this variable is possible to be an independent variable for other dependent variables. DISCUSSION Table 1 shows the indicators used in each of the variables, loading factor, reliability based on Cronbach's Alpha value.The results of the analysis show that there are four indicators that have loading factor value < 0.60 that are X15, X20, X21, and X22.Then the four indicators were excluded from the model.The results of the analysis in Table 2 also show that all AVE values are higher than the quadratic correlation between constructs on all relationships between variables.Therefore, it can be concluded that the test results show that all constructs used have good validity and can be used in the next analysis phase. Model Fit Test Goodness of fit test shows that a model is fit to the data used in the research.This can be seen from the existing criteria of Chi-Square, Probability, CMIN/DF, GFI, AGFI, TLI, CFI, and RMSEA.According to Hair, et al. (2010), goodness of fit test is acceptable or the model is categorized fit if at least 5 criteria are met.The result of goodness of fit test can be seen in table 3 below: Table 3. Results of Discriminant Validity Test Based on the statistical results of SEM analysis in model-goodness-of-fit test, obtained six criteria included in good category, and two criteria are included in marginal category.Based on these results, the model in this study as a whole can be categorized as a very good model or fit model category. Hypothesis Testing Hypothesis testing is done by looking at the critical value or ttable that is by comparing the value ttable with tcount in the study or comparing the p value with 0.05.The hypothesis is said to be accepted if the value of ttable < tcount or p < 0.05.Based on the t distribution table, the value of ttable in this study is the number of sample of 125 and the significance level of 5% (0.05) is equal to 1.979.While, for the value of tcount on the relationship between variables of research can be seen in table 4. DISCUSSION OF RESEARCH RESULT H1: Green product has a Positive Effect on Purchase Decision Based on table 4, it is known that the value of C.R. (2.39875) > ttable (1.977) and p (0.01494) < 0.05.Therefore, the null hypothesis which states that regression weight is equal to zero can be rejected and this means the effect of green product on purchase decision is positively significant.Thus, the hypothesis stating that green product positively affects purchase decision is accepted. According to respondents' answers, they feel the product used in accordance with the desire and expectation of consumers that is to use environmentally friendly products.They feel comfortable and secure when using products that have been issued by Tupperware.According to respondents' answers, they greatly appreciate the products that have been provided or created by Tupperware.Thus, green product that has been created by the company can be felt directly by them.Respondents also consider the green product in making purchase decision.Thus, it affects them directly in making purchase decision. These findings also reinforce the notion that an environmentally friendly product strategy implemented by a company can lead to an overall perception to better benefit the brand, thereby supporting common green marketing approach (Hartmann, 2005: 21). H2: Green Price has a Positive Effect on Purchase Decision Based on table 4, it is known that the value of C.R. (2.39783) > ttable (1.977) and p (0.01186) < 0.05.Therefore, the null hypothesis which states that regression weight is equal to zero can be rejected and this means the effect of green price on purchase decision is positively significant.Thus, the hypothesis stating that green price positively affects purchase decision is accepted. In general, respondents said that they were satisfied with the price set by the company, because they could see and feel themselves better quality than other products they have purchased.Respondents also said they did not feel hesitant in making purchase decision because with the same price tariff consumers could choose products that all use environmentally friendly concepts that suit their tastes.So that consumers felt the affordability of the price to buy Tupperware products. This means that the green price predetermined by the company can directly influence the purchase decision.The results of this study are in line with the statement of Emgin and Turk (2004) Based on the results in this study, means that the green place that Tupperware has done can directly affect the creation of purchase decision.The results of this study are in line with the statement of Queensland Government (2002), sellers who want to achieve success in selling environmentally friendly products should position their products widely in the market so that they can be more recognizable and can drive purchase decision. H4: Green Promotion has a Positive Effect on Purchase Decision Based on table 4, it is known that the value of C.R. (2.29562) > ttable (1.977) and p (0.02171) < 0.05.Therefore, the null hypothesis which states that regression weight is equal to zero can be rejected and this means the effect of green promotion on purchase decision is positively significant.Thus, the hypothesis stating that green promotion positively affects purchase decision is accepted. According to respondents' answers, they knew the promotions made by Tupperware through the implied messages of every use of product material issued by Tupperware. The results of this study are in line with Shrum, et al. (1993) conducting a study on 3,690 people.According to the study, it is showed that women who tend to buy green products are more skeptical on the trust in advertising. H5: Brand Image has a Positive Effect on Purchase Decision Based on table 4, it is known that the value of C.R. (3.51365) > ttable (1.977) and p (0.0000) < 0.05.Therefore, the null hypothesis which states that regression weight is equal to zero can be rejected and this means the effect of green brand image on purchase decision is positively significant.Thus, the hypothesis stating that brand image positively affects purchase decision is accepted. H6: Brand Image Mediates the Relationship between Green Product and Purchase Decision The test of the mediation influence between intervening variables and dependent variable was done by Sobel's formula calculation.Based on the results of these calculations, the value of Z count of 2.28 is greater than Z table of 1.96 with significance of 0.05, so it can be concluded that the brand image mediates the causal relationship between green product and purchase decision. H7: Brand Image Mediates the Relationship between Green Price and Purchase Decision The test of the mediation influence between intervening variables and dependent variable was done by Sobel's formula calculation.Based on the results of these calculations, the value of Z count of 2.19 is greater than Z table of 1.96 with significance of 0.05, so it can be concluded that the brand image mediates the causal relationship between green price and purchase decision. H8: Brand Image Mediates the Relationship between Green Place and Purchase Decision The test of the mediation influence between intervening variables and dependent variable was done by Sobel's formula calculation.Based on the results of these calculations, the value of Z count of 2.09 is greater than Z table of 1.96 with significance of 0.05, so it can be concluded that the brand image mediates the causal relationship between green place and purchase decision. H9: Brand Image Mediates the Relationship between Green Promotion and Purchase Decision The test of the mediation influence between intervening variables and dependent variable was done by Sobel's formula calculation.Based on the results of these calculations, the value of Z count of 2.28 is greater than Z table of 1.96 with significance of 0.05, so it can be concluded that the brand image mediates the causal relationship between green promotion and purchase decision. CONCLUSION AND SUGGESTION Conclusion Based on the results of the discussion in the previous above, it can be drawn some conclusions as follows: 1. that the better the green product, green price, green place, green promotion, and brand image then Tupperware purchase decision in Tasikmalaya will be better too. Suggestion The suggestions that can be given are as follows: 1. Green product, green price, green place, and green promotion have strong influence on purchase decision.Therefore, the products must be designed according to the needs of consumers, developed with the best, always follow the existing development, and given superior service to consumers.2. The process of image development is not easy.One of the triggers that can quickly form an image is product quality, fast service, signage, and more.The company should always make continuous improvement on factors that can improve the company image. Figure 1 . Figure 1.Consumer Decision Making Process Figure Figure 2. Full Model of Structural Equation Table 2 . Results of Discriminant Validity Test .	2018-12-05T13:13:55.403Z	2017-07-11T00:00:00.000	{ "year": 2017, "sha1": "92976477eff8e073eba9145a70ad22d4cc343b33", "oa_license": "CCBY", "oa_url": "http://jos.unsoed.ac.id/index.php/jame/article/download/971/710", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "92976477eff8e073eba9145a70ad22d4cc343b33", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Business" ] }
254564016	pes2o/s2orc	v3-fos-license	Ringing and echoes from black bounces surrounded by the string cloud In the string theory, the fundamental blocks of nature are not particles but one-dimensional strings. Therefore, a generalization of this idea is to think of it as a cloud of strings. Rodrigues et al. embedded the black bounces spacetime into the string cloud, which demonstrates that the existence of the string cloud makes the Bardeen black hole singular, while the black bounces spacetime remains regular. On the other hand, the echoes are the correction to the late stage of the quasinormal ringing for a black hole, which is caused by the deviation of the spacetime relative to the initial black hole spacetime geometry in the near-horizon region. In this work, we study the gravitational wave echoes of black bounces spacetime surrounded by a cloud of strings under scalar field and electromagnetic field perturbation to explore the effects caused by a string cloud in the near-horizon region. The ringing of the regular black hole and traversable wormhole with string cloud are presented. Our results demonstrate that the black bounce spacetime with strings cloud is characterized by gravitational wave echoes as it transitions from regular black holes to wormholes, i.e. the echoes signal will facilitate us to distinguish between black holes and the wormholes in black bounces surrounded by the string cloud. I. INTRODUCTION Recently, the LIGO and Virgo interferometers have made significant progress in the observation of gravitational waves (GWs) [1][2][3][4][5][6].In addition, the Event Horizon Telescope has also made a breakthrough in the imaging of black hole shadows [7,8].These results validate the predictions of general relativity (GR) about black holes (BH).It also allows physicists to test new physical features beyond GR [9][10][11][12][13][14], such as the existence of event horizons in compact objects.Gravitational wave spectroscopy plays a crucial role in the examination of new physical features beyond general relativity [15,16].For the gravitational wave signal generated by the binary merger, its late stage always decays in the form of the ringdown.It can usually be described using a superposition of complex frequency damping exponents, which are called quasinormal modes (QNMs) [17][18][19].The detection of QNMs can serve as a tool to test GR predictions.Therefore, this makes gravitational wave detectors (LIGO/Virgo and LISA, etc.) expected to detect some new physical features in the future, such as gravitational wave echoes and so on.Gravitational wave echoes are an important observable for probing the spacetime near the event horizon of the black hole.In addition, gravitational wave echoes are closely related to the unique characteristics of compact objects. Under the framework of general relativity, with the perturbation of black hole spacetime, it must be accompanied by the emergence of quasinormal modes.Because as long as a black hole is perturbed, it re-sponds to the perturbation by emitting gravitational waves, and the evolution of gravitational waves can be divided into three stages [20,21]: first, a relatively short initial burst of radiation; then a longer damped oscillation, which depends entirely on the parameters of the black hole; and finally the exponentially decays over a longer period of time.Note that the three stages refer to the postmerger gravitationalwave signal.Among these three stages, people are generally most concerned about the middle quasinormal ringing stage.The QNMs of black holes have attracted extensive attention [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41].Although there are many indirect ways to identify black holes in the universe, gravitational waves emitted by perturbed black holes will carry unique "fingerprints" that allow physicists to directly identify the existence of black holes.In particular, Ref. [42] proposes that gravitational wave echoes can be used as a new feature of exotic compact objects.Later, when people studied QNM in various spacetime backgrounds, gravitational wave echoes were analysed in the late stage of quasinormal ringing .These works make GWs echoes very important in studying the properties of compact objects.In Ref. [72], the author found a new mechanism to produce the gravitational wave echoes in the black hole spacetime.Bronnikov and Konoplya [73] found that the echoes appeared in the black hole-wormhole transition when studying the quasinormal ringing of black hole mimickers in brane worlds.In Ref. [74], the authors studied the time evolutions of external field perturbation in the asymmetric wormhole and black bounce spacetime background, they observed echoes signals from the spacetime of asymmetric wormholes and black bounce.Especially, Churilova and Stuchlik in Ref. [75] studied the quasinormal ringing of black bounce, and they found the gravitational wave echoes signal during the regular black-hole/wormhole transition.We need to pay attention that not all compact objects can show echoes signals in the late stage of quasinormal ringing.Cardoso et al. [76] pointed out that the precise observation of the late stage of quasinormal ringing allows us to distinguish different compact objects.Therefore, in our work, we plan to explore whether the string cloud will destroy the gravitational wave echoes signal in the black bounce spacetime.We hope to provide some direction for probing black bounces with strings cloud experimentally after obtaining its relevant basic properties. String theory points out that the fundamental blocks of nature are not particles but one-dimensional strings.Therefore, a generalization of this basic idea is to think of it as a cloud of strings.On the other hand, the black hole in general relativity usually has singularities, which forces theoretical physicists to constantly try to avoid the occurrence of singularities.A black hole without singularities is called a regular black hole (RBH).Bardeen was the first theoretical physicist to propose regular black hole [77].Ayon-Beato et al. interpret it as a black hole solution for the Einstein equations under the presence of nonlinear electrodynamics [78].Letelier proposed a black hole solution in 1979, which is surrounded by the string cloud [79].The string cloud is a closed system, therefore its stress-energy tensor is conserved.Subsequently, black holes with strings have attracted a lot of attention [80][81][82].Sood et al. proposed an RBH surrounded by the string cloud, but the string cloud makes this black hole solution no longer regular [83].It would be very fascinating if string cloud would not insert singularities in the RBH.Simpson and Visser proposed a type of regular black hole known as black bounces [84].The difference between this solution and the standard RBH is that it is achieved by modifying the black hole area, and it allows a nonzero radius throat in r = 0.Many studies have been done on black bounces including analysis of their properties [85][86][87][88][89][90][91][92].Recently, Rodrigues et al. embedded the Simpson-Visser spacetime into a string cloud [93].They demonstrate that the Simpson-Visser spacetime is still regular even if the string cloud exists.In this work, our goal is to study the effect of the presence of string cloud on the GW echoes of black bounces spacetime and explore what gravitational effects are caused by string cloud. Our work is organized as follows.In Sec.II, we briefly review the black bounces in a cloud of strings.In Sec.III, we discuss the scalar field and electromagnetic field perturbations for black bounces in a cloud of strings.In Sec.IV, we outline the time-domain integration method as well as the WKB method.In Sec.V, we present the quasinormal ringing and gravitational wave echoes of the scalar field and electromagnetic field perturbations to black bounces in a cloud of strings.Sec.VI is our main conclusion of the full text.In this work, we use the units G = = c = 1. II. A BRIEF REVIEW OF THE BLACK BOUNCES IN STRINGS SLOUD To gain black bounces in a cloud of string, Rodrigues et al. [93] considers the following Einstein equations where where T SV µν denotes the stress-energy tensor related to the Simpson-Visser spacetime, and the information about the non-minimum coupling between the string cloud and the Simpson-Visser spacetime is included in the stress-energy tensor T N M C µν .Furthermore, T CS µν in Eq. ( 1) represents the stress-energy tensor of the string cloud, which can be written as where ρ represents the density of the string cloud. T CS µν must satisfy the following conservation laws (4) By solving the above Einstein field equations, Rodrigues et al. obtain the following black bounces with the string cloud [93] where If a = 0, this spacetime can be reduced to the Letelier spacetime, and this spacetime can be reduced to the Simpson-Visser spacetime when L = 0.If L = 1, this spacetime will have no event horizon, so the range of the string parameter L is 0 < L < 1.In addition, the value of the parameter a has a critical value The black bounce with the string cloud will correspond to a different spacetime for different a: i) regular black hole with string cloud for 0 < a < a c ; ii) one-way wormhole with string cloud for a = a c ; iii) traversable wormhole with string cloud for a > a c . III. MASTER WAVE EQUATION The covariant equations of scalar field perturbation can be written as considering the black bounces surrounded by the string cloud we studied, we can get Since the spacetime we are studying is spherically symmetric, we can achieve separation of variables through the following ansatz where R is the function of radial coordinate r and the parameter a, which has been defined in equation ( 6), and Y lm (θ, φ) are the spherical harmonic function. After separating the variables and using the properties of spherical harmonics, we can simplify equations ( 9) to the following form where tortoise coordinate r * can be defined by Moreover, the effective potentials for scalar field perturbation can be written as The motion equation of the electromagnetic field in the curved spacetime background can be written as where A µ being the four vector potential, and Since spacetime has spherical symmetry, we have where the term on the left has odd parity (−1) l+1 , and the term on the right has even parity (−1) l .Substituting the above equation into ( 14), we can get where V elec (r) denotes the effective potential of the electromagnetic field perturbation, In Fig. 1, we present the effective potential of the scalar field perturbation for different a with M = 0.5, l = 1, L = 0.1 and for different L with M = 0.5, l = 1, a = 0.1 as the function of the tortoise coordinate r * .Here we are studying l = 1 mode of scalar field perturbation mainly because the peak value of l = 0 mode is too small.From Fig. 1, we can see that the effective potential is the single peak, which indicates that the black bounce in a cloud of strings at this time is the black hole spacetime with the string cloud.These results show that the effective potential is very sensitive to changes in L, but not particularly sensitive to changes in a. In Fig. 2, we show the effective potential of black bounces surrounded by the string cloud under electromagnetic field perturbation.We can observe that when the value of parameter a is less than its threshold a c (when M = 0.5, L = 0.1, the value of the threshold a c is 1.11111), the effective potential is the single peak (blue solid line on the left panel for a = 1.1), and when the value of parameter a is greater than its threshold a c , the effective potential has two peaks.Moreover, one can see that the change of a has almost no effect on the peak value of the effective potential.But as a increases, the depth and width of the effective potential decrease, and it eventually becomes a single peak effective potential (green solid line on the right panel for a = 1.7).Although we have not given the effective potential image of a larger than the threshold under scalar field perturbation, we have verified that similar behavior can appear under scalar field perturbation. In Fig. 3, we give the effect of parameter L on the effective potential under electromagnetic field perturbation.We can see that the effective potential behaves similarly to the influence of the parameter a, but the depth of the potential well is very shallow. IV. THE METHODS In this section, we introduce numerically solving the wave equation for black bounces in a cloud of strings to obtain the time-domain profiles.We use the light cone coordinates then Eq. ( 11) can be written as We adopt the discretization scheme suggested by Gundlach and Price et al. [94,95] . Moreover, we use the Gaussian initial pulse [96][97][98] for two null surface, i.e. For the frequency domain, we use the WKB method to calculate the QNM frequencies.Schutz and Will first used the WKB method to calculate the quasi-normal scale of black holes in 1985 [99], and they subsequently extended it to the third-order WKB method with higher accuracy [100].Moreover, Konoplya extended it to the sixth-order [101,102].When using the Padé approximation [103,104], WKB method can even be generalized to the more accurate thirteenth order.The higher-order WKB method take the form [105] where K denotes half-integer values.The correction term A k (K 2 ) depends on the derivative of the effective potential at its maximum value. V. QUASINORMAL MODES AND ECHOES OF BLACK BOUNCES SURROUNDED BY THE STRING CLOUD A. Quasinormal modes of the regular black hole surrounded by the string cloud Here we first study the case of 0 < a < a c , that is, the regular black hole with the string cloud.In TABLE I, we give the fundamental QNM frequencies (l = 1, n = 0) of scalar field perturbation for black bounces in a cloud of strings with M = 0.5.One can see that when L is fixed and a is changed, both the real and imaginary parts of the QNM frequencies decrease with the increase of a, which implies that its actual oscillation frequencies decrease with the increase of a, while a decrease in its damping rate means that its decay time becomes longer as a increases.When a is fixed and L is increased, both the real and imaginary parts of the QNM frequency are also decreased, which indicates that L and a have similar contributions to the QNM frequencies for the scalar field perturbation to black bounces in a cloud of strings. In Fig. 4, we show the time-domain profiles of the scalar field perturbation (left panel) for different a with M = 0.5, l = 1, L = 0.1, and the timedomain profiles of the electromagnetic field perturbation (right panel) for different a with M = 0.5, l = 1, L = 0.1.In Fig. 4, the blue solid line represents a = 0.1, the black solid line represents a = 0.6, and the red solid line represents a = 1.1.The corresponding effective potential is given in Fig. 1.One can see that the decay time of quasinormal ringing is the longest when a is larger, which indicates that its damping rate should be smaller for the larger a.In other words, the imaginary part of its quasinormal modes frequency is smaller.Such a result is a good validation of our results shown in TABLE I and II, i.e. the imaginary parts of the quasinormal mode frequencies decrease as a increases. In Fig. 5, we present the time-domain profiles of the scalar field perturbation (left panel) for different L with M = 0.5, l = 1, a = 0.1, and the timedomain profiles of the electromagnetic field perturbation (right panel) for different L with M = 0.5, l = 1, a = 0.1.We can clearly see the quasinormal ringing after the initial pulse, which represents the unique " fingerprint" of black bounce in a could of strings.Furthermore, we can find that the contribution of L to the quasinormal ringing for black bounces in a cloud of strings is similar to that of a, but the quasinormal ringing is more sensitive to L than a.As we expected, because the effective potential is a single peak and the existence of the black hole event horizon, there is no gravitational wave echoes signal here.In addition, late-time tails are also shown after quasinormal ringing. B. Echoes of the wormhole surrounded by the string cloud For black bounce in a could of strings, when the parameter a increases, it can change from a black hole to a wormhole.It should be noted that when a = a c , the space-time we are studying becomes a one-way wormhole with the string cloud, where the QNM has similar behavior to the regular black hole with the string cloud, and has no other distinctive characteristics in quasinormal ringing.Therefore we do not give the corresponding results.Now, let's study the case of a > a c , which is the traversable wormhole with the string cloud.In Fig. 6, we present the effective potential and corresponding GWs echoes of the scalar field perturbation to black bounces in a cloud of strings with M = 0.5, l = 1, L = 0.1, a = 1.112.One can see that when a is increased slightly above the threshold a c (a c = 1.11111 for M = 0.5, L = 0.1), there are two peaks in the effective potential.It implies that the black bounce in a could of string at this time has become a wormhole spacetime.Due to the large distance between the two peaks of the effective potential, the two peaks can scatter waves independently.Therefore, the gravitational wave will be reflected by both peaks and will be repetitively reflected in the potential well, while a fraction of the wave also passes through the potential barrier.This allows observers to see the gravitational wave echoes.Note that Ref. [107] shows the event horizon with quantum nature will also reflect gravitational waves so that the echoes appear.From the right panel of Fig. 6, one can see clear gravitational waves echoes after the initial quasinormal ringing.Since the potential well at this time is wider, the time for the gravitational wave to reach another peak from one peak will be relatively long.Therefore, we see a long time interval between the first gravitational wave echo signal and the initial quasinormal ringing. As a increasing, we can see from Fig. 7 and Fig. 8 that the peak value of the effective potential hardly changes, but its potential well width becomes smaller and smaller.This means that the time required for gravitational waves to reach another peak from one peak becomes shorter so that the gravitational wave echo signal appears sooner after the initial quasinormal ringing, and the time interval between gravitational wave echoes is smaller when a is larger.We only made a qualitative analysis of time delay between gravitational wave echoes, while Refs [42,76] conducted a quantitative study on it, which proved that time delay has the logarithmic dependence on the width of the cavity.In Figs. 9, 10, and 11, we show the GWs echoes of electromagnetic field perturbation to black bounces in a cloud of strings.Time-domain profiles for electromagnetic field perturbations near the threshold a c (a c = 1.11111 for M = 0.5, L = 0.1) show the distinct echoes signal.But the echoes signal seems to become less clear as a increases.If a continues to increase, the echoes will become characteristic quasinormal ringing with a power-law tail, as shown in Fig. 12 (right panel).Such similar characteristics also exist for scalar field perturbations to black bounces in a cloud of strings, as shown in Fig. 12 (left panel).We also study the effect of the string cloud parameter L on the quasinormal ringing of the wormhole surrounded by the string cloud in Fig. 13.We can observe that there are weak echoes signal for the quasinormal ringing corresponding to the red solid line, but not for other cases.Perhaps the answer can be found in the effective potential.From Fig. 3, we can see that the string cloud parameter L has a very significant impact on the effective potential.Its increase causes the effective potential to change from unimodal to bimodal and then unimodal again.It is the double-peak effective potential (red solid line) in Fig. 3 that causes the gravitational wave to be captured in the potential well, so that the echoes signal appear. VI. CONCLUSION In this work, we studied the gravitational wave echoes for the black bounces surrounded by the string cloud.The distinctive feature of the black bounces with a cloud of strings is that when parameter a reaches a certain threshold a c , it can transform from a black hole to a wormhole, which is characterized by the emission of gravitational wave echoes signals. For the regular black hole (0 < a < a c ) with a cloud of strings, due to the existence of the event horizon, we did not find the gravitational wave echoes.This is consistent with the fact that Schwarzschild black holes have no gravitational wave echoes in the framework of general relativity. For wormholes (a > a c ) with a cloud of string, we obtained clear gravitational wave echoes signals after initial ringing.We demonstrate that the two peaks of the effective potential are the necessary conditions for the generation of gravitational wave echoes, and the shape of the potential well plays a decisive role in the gravitational wave echoes.When the parameter a is closer to the threshold a c , the width of the potential well is wider, making it easier for us to observe the gravitational wave echoes signal after the perturbations.As the parameter a increases, the width of the potential well becomes smaller and smaller so that the time interval between gravitational wave echoes becomes smaller and smaller until the echoes disappear.This may cause great difficulties to detect the black bounces surrounded by the string cloud experimentally through the gravitational wave echoes. Furthermore, we find that a has a very small effect on the peak of the effective potential, but the increase in string cloud parameter L has a very significant effect on the peak of the effective potential.In the process of L increasing, the effective poten-tial also changes from the single peak to the double peaks, and then to the single peak again.Although the effective potential exhibits the double peaks, the potential well is so shallow that perturbations can easily escape from the potential well.Therefore, we can only observe the weak echoes signal.On the other hand, by comparing with the work of Churilova and Stuchlik [75], we find that the strings cloud has the following effects on the black bounces spacetime: (i) It extends the parameter range of black bounces spacetime keeping as a regular black hole; (ii) The presence of the string cloud depresses the peak of the effective potentials barrier; (iii) It reduces the real oscillation frequency of gravitational waves and reduces the damping rate of gravitational wave signals. It should be noted that the parameter L related to the strings will not affect the appearance of the echoes.As long as the appropriate parameter a is selected, we can observe the echoes, but the existence of the strings makes the threshold a c larger. We discussed the gravitational wave echoes of the black bounces surrounded by the string cloud under the scalar field and electromagnetic field perturbation.The behavior of the gravitational wave echoes under the two kinds of perturbations are very similar, as a result we believe that the similar behavior can also be continued in the Dirac field perturbation [108] or the gravitational perturbation.It might also be very interesting to examine it in future work. FIG. 6 . FIG.6.The effective potential and gravitational wave echoes of the scalar field perturbation to black bounces in a cloud of strings with M = 0.5, l = 1, L = 0.1, a = 1.12. FIG. 7 . FIG. 7. The effective potential and gravitational wave echoes of the scalar field perturbation to black bounces in a cloud of strings with M = 0.5, l = 1, L = 0.1, a = 1.13. FIG. 8 . FIG.8.The effective potential and gravitational wave echoes of the scalar field perturbation to black bounces in a cloud of strings with M = 0.5, l = 1, L = 0.1, a = 1.14. FIG. 9 .FIG. 10 . FIG.9.The GWs echoes of electromagnetic field perturbation (left panel) and semilogarithmic plots for the GWs echoes of electromagnetic field (right panel) to black bounces in a cloud of strings with M = 0.5, l = 1, L = 0.1, a = 1.12.	2022-10-25T01:16:05.562Z	2022-10-23T00:00:00.000	{ "year": 2023, "sha1": "d452cec112f561832fbed635aa452abca6602dd0", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1140/epjc/s10052-023-11382-5.pdf", "oa_status": "GOLD", "pdf_src": "ArXiv", "pdf_hash": "d452cec112f561832fbed635aa452abca6602dd0", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
16854879	pes2o/s2orc	v3-fos-license	A Rare Fungal Infiltration of Lungs in a Healthy Young Girl Cunninghamella bertholletiae is an opportunistic fungal organism found in soil and is a rare cause of human disease. The few reported cases of C. bertholletiae have involved immune compromised hosts. We report a case of C. bertholletiae in an otherwise healthy patient presenting with persistent high-grade fever and pulmonary infiltration resistant to antibiotics. The organism is isolated through bronchoscopy and responded to broad spectrum antifungal. This is the first case of C. bertholletiae reported in the literature in an immune competent individual. Introduction Cunninghamella bertholletiae is an opportunistic fungal organism of the order Mucorale. It is found in soil and is a rare cause of human disease [1]. The few reported cases of the fungus have predominantly involved immune compromised hosts and have been associated with very poor outcomes [2][3][4][5][6]. We report a case of C. bertholletiae infiltrating the lungs in an otherwise healthy girl presenting with high-grade fever. The patient was treated with broad spectrum antifungal with successful outcome. Case Report An eighteen-year-old unmarried young girl was admitted with fever for fifteen days. She had mild cough and mucoid expectoration with no haemoptysis. The fever was continuous, high grade (varying between 103 • to 105 • F), with mild chills and rigors off and on (nonspecific). No sore throat, urinary, or bowel problem was reported. Anorexia and weight loss was insignificant. There was no history of tuberculosis or treatment for any major illness in past. History of poisoning was absent. Menstrual history was normal. She was none responding to broad spectrum antibiotics like clarithromycin and amoxicillin-clavulanic acid combination. On examination she was mildly anemic but no cyanosis, lymphadenopathy, jaundice, or any organomegaly could be found. Chest examination revealed only occasional crepts bilaterally. In chest X-ray PA view, lowdensity peripheral infiltrates were seen bilaterally ( Figure 1). On routine investigation, hemoglobin was 10.5 gm%, total leucocytes count was 8000 per cubic mm & differential count was P-59, L-36, M-1, and E-4. Urine routine examination was normal. Widal and PBF for malarial parasite were negative. Sputum for AFB was negative thrice and pyogenic was sterile. Serum HIV was negative. Ultrasonography for abdomen and thorax was normal. On fibro-optic bronchoscopy no lesion could be seen. CT scan sections show peripherally placed bronchiectatic patches ( Figure 2). The bronchoalveolar lavage sample on culture revealed unusual fungus Cunninghamella bertholletiae (Figure 3). Patient was put on fluconazole empirically at the dose of 150 mg twice a day with a prompt response and was discharged in three days. Discussion C. bertholletiae is a rare cause of zygomycosis in humans often associated with trauma and immune suppression. C. bertholletiae is the only species of the genus known to cause 2 Case Reports in Pulmonology disease both in humans and animals [1][2][3][4][5][6]. Humans may be infected through inhalation of airborne fungal spores, however protection against mucor infection is provided by the normal phagocytic and neutrophil function, the exact mechanism of which is not known [7]. Experimental and clinical observations have suggested multiple predisposing factors including acidosis, diabetes mellitus, renal failure, corticosteroids, cytotoxic and antibiotic therapy, leucopenia, hematopoietic malignancies; and depressed phagocytosis. Pulmonary mucormycosis occurs very rarely in healthy individuals. The typical clinical setting consists of an immunologically compromised patient with persistent fever and a progressive pulmonary infiltrate for which no etiological agent has been found [7]. In this case, no immune suppression or predisposing factor was present, otherwise the clinical setting is same for C. bertholletiae infection as described above. Patient presented with high-grade fever and lung infiltrations that failed to respond to usual effective antibiotics. The organism was isolated through bronchoscopy. Patient responded to fluconazole promptly within three days and was discharged on long-term antifungal therapy. To our knowledge, this is the first case of pulmonary infiltration of C. bertholletiae in an otherwise healthy person reported in the literature. The mucor infections are rare and unknown in healthy individuals and because of difficulty in isolating the organism [6] may go unsuspected and hence undiagnosed. Infection with C. bertholletiae is known in immune compromised where it is difficult to treat [2,3,5,6]. In this case, the host is immune competent and it responded to usual antifungal drug promptly. Probably the immunity (the unknown mechanism) acts in synergism with antifungal agent against the organism that happened to anchor the host somehow when seemingly resistance was low.	2016-05-12T22:15:10.714Z	2011-09-21T00:00:00.000	{ "year": 2011, "sha1": "253407f36856045750772a694dfa130c41226672", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/cripu/2011/917089.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "82f79dcfa63fb4826588b569b1fb02750dc07aad", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
225423219	pes2o/s2orc	v3-fos-license	Impact of a quality improvement project to reduce the rate of obstetric anal sphincter injury: a multicentre study with a stepped‐wedge design Objective To evaluate the impact of a care bundle (antenatal information to women, manual perineal protection and mediolateral episiotomy when indicated) on obstetric anal sphincter injury (OASI) rates. Design Multicentre stepped‐wedge cluster design. Setting Sixteen maternity units located in four regions across England, Scotland and Wales. Population Women with singleton live births between October 2016 and March 2018. Methods Stepwise region by region roll‐out every 3 months starting January 2017. The four maternity units in a region started at the same time. Multi‐level logistic regression was used to estimate the impact of the care bundle, adjusting for time trend and case‐mix factors (age, ethnicity, body mass index, parity, birthweight and mode of birth). Main outcome measures Obstetric anal sphincter injury in singleton live vaginal births. Results A total of 55 060 singleton live vaginal births were included (79% spontaneous and 21% operative). Median maternal age was 30 years (interquartile range 26–34 years) and 46% of women were primiparous. The OASI rate decreased from 3.3% before to 3.0% after care bundle implementation (adjusted odds ratio 0.80, 95% CI 0.65–0.98, P = 0.03). There was no evidence that the effect of the care bundle differed according to parity (P = 0.77) or mode of birth (P = 0.31). There were no significant changes in caesarean section (P = 0.19) or episiotomy rates (P = 0.16) during the study period. Conclusions The implementation of this care bundle reduced OASI rates without affecting caesarean section rates or episiotomy use. These findings demonstrate its potential for reducing perineal trauma during childbirth. Tweetable abstract OASI Care Bundle reduced severe perineal tear rates without affecting caesarean section rates or episiotomy use. Introduction An obstetric anal sphincter injury (OASI), graded as a third-or fourth-degree perineal tear, is a severe complication of vaginal childbirth. 1 Long-term outcomes of OASI van 584 include chronic pain, sexual dysfunction and urinary or anal incontinence. 2 OASI rates are increasing in many countries. In the English National Health Service (NHS), reported OASI rates tripled among primiparous women over a decade, from 1.8% in 2000 to 5.9% in 2011, 3 with similar trends in many other countries. [4][5][6][7] The rise in OASI rates is likely to be linked to improved recognition of tears, changes in the characteristics of women giving birth as well as to changes in practice, such as an increased use of a 'hands-poised/hands-off' approach, opposed to a 'handson' approach to protect the perineum, 8-10 a reluctance to perform an episiotomy 9 and gaps in the training of midwives and obstetricians. [11][12][13] Evidence from studies carried out in Scandinavian countries suggests that training to improve intrapartum techniques with focus on slowing down the birth of the head can significantly decrease OASI rates. [14][15][16][17][18] However, similar studies carried out elsewhere did not confirm these results. 19,20 A multidisciplinary team of national UK experts, supported by national professional organisations, developed a 'care bundle', which is a set of interventions likely to improve outcomes when implemented together. This OASI Care Bundle includes information provision to women during the antenatal period, manual perineal protection and use of mediolateral episiotomy when clinically indicated at 60-degree angle. The care bundle also included the requirement that the perineum should be carefully checked following birth, including a per rectum examination, to improve detection of perineal injuries and instigate prompt repair. A quality improvement project was initiated to implement the OASI Care Bundle following a stepped-wedge design in 16 maternity units across the NHS in England, Scotland and Wales. The implementation strategy was informed by a detailed 'theory of change' that highlighted the need for ongoing project team support to participating units and local communications and awareness campaigns. 21 In this paper, we report the impact of the quality improvement project on OASI rates as well as on caesarean section rates and use of episiotomy. Study design and participants The OASI Care Bundle was evaluated in 16 maternity units between 1 October 2016 and 31 March 2018, using a multicentre study with a stepped-wedge design. All women who had singleton live births were eligible for inclusion. The participating units were located in four regions with four units in each region. A stepwise region-by-region rollout was instigated. At each step, the maternity units in a region started at the same time. The regional roll-out minimised contamination across units and facilitated the delivery of the skills development module and site visits by the project team. The eligibility and section criteria for the participating units have been previously reported. 21 Briefly, 91 units that had expressed an interest in participating in a pilot study in 2015 were eligible for inclusion. For each of the four regions, units were purposively selected from different areas in the region, aiming to include units of various sizes and types (obstetric-led, alongside midwifery unit and freestanding midwifery unit) in each region. Women who expected to give birth after the introduction of the care bundle were given a sheet that explained that the care bundle did not affect their choice about how or in what position they would like to give birth. As a consequence, manual perineal protection was not used when a woman had different preferences and the per rectum check following birth was only performed with consent. Sequential roll-out The order of the four regions in the stepwise region-by-region roll-out was determined by a member of the project team (IGU) using computer-generated random numbers before the start of the roll-out. The maternity units and their local clinical champions were informed of their allocation 2 months before the start of the roll-out period in their region to allow for preparation time. The roll-out took place in three phases. For each region, a 'baseline phase' was used to determine the OASI rates before implementation of the care bundle. The care bundle was implemented in the maternity units during a 3-month 'transition phase'. An 'evaluation phase' was used to determine the OASI rates after implementation. The duration of the baseline and evaluation phases depended on the place in the order of the sequential roll-out (see Supplementary material, Figure S1). Births that took place during the transition phase were excluded from the analysis. The roll-out of the OASI Care Bundle started in the first region in January 2017. It has to be noted that the OASI Project was retrospectively registered on the ISCTRN database in September 2017. However, the project team engaged in an extensive publicity campaign throughout 2016 and the study protocol, describing the project in detail, was available on the Royal College of Obstetricians and Gynaecologists website from January 2017. Intervention The OASI Care Bundle was developed following recommendations from existing UK guidelines 22 by a multidisciplinary team of national experts. The three components of the care bundle are listed in Figure 1 and a full description can be found on the project website. 23 The first component of the care bundle is a leaflet providing information about perineal trauma during childbirth, its risk The second component is the use of manual perineal protection for all singleton vaginal births, unless a woman objects or if her chosen birth position does not allow it (e.g. water birth or use of birthing stool). This component of the care bundle advises manual perineal protection using a specific technique ('Finnish grip'). 16 The third component is the use of episiotomy when clinically indicated, performed at a 60-degree angle at crowning. The recognised indications for episiotomy in this context are fetal distress, delayed second stage of labour, operative vaginal birth and cases when a severe perineal tear is judged to be imminent (e.g. thick inelastic perineum). For operative vaginal birth, episiotomy should be used for all forceps births, regardless of parity, and for all vacuum-assisted births in primiparous women. In multiparous women, episiotomy could be omitted for vacuumassisted births after considering the woman's OASI risk. The care bundle also includes the requirement to carry out a thorough perineal examination following all vaginal births, including a per rectum examination. 24 Whereas the first three components of the care bundle contribute to the primary prevention of OASI, the per rectum examination is a secondary prevention measure, which may have improved detection rates after the implementation of the care bundle. The implementation of the OASI Care Bundle in each unit was led by local midwives and obstetricians. These clinical champions received central multidisciplinary training on key elements of the care bundle at designated 'skills development days' at the start of the transition period. Project clinical leads visited all units during the study period to provide further training, support and advice. Clinical data Patient-level data were extracted from local electronic maternity information systems for 15 units in England, Scotland and Wales, and from the Scottish Morbidity Record 02 and Scottish Birth Record for one unit in Scotland. The maternity information systems, available in almost all English units, captured detailed demographic and clinical information related to maternity care and outcomes with the data entered by midwives and support staff. Scottish Morbidity Record 02 collects data submitted by maternity units to the Information Services Division Scotland since 1975 for all women admitted to Scottish maternity units. 25 Outcomes The primary outcome was OASI in women who had singleton live vaginal births with episiotomy as a secondary outcome. Another secondary outcome was caesarean section in all women who had a singleton live birth. Statistical analysis The study's sample size was calculated based on the approach proposed by Hussey and Hughes. 26 There were on average 912 vaginal births in a 3-monthly period ('step') for the participating units (with a range of 339-1617) between 1 April 2014 and 31 March 2015. The baseline OASI rate (3.2%) and the intra-cluster correlation (q 0.006) were calculated from English data for births that took place between 1 April 2013 and 31 March 2014. 27 Based on these numbers, and a 5% significance level, the statistical power of the study to detect a 25% reduction in OASI rate (from 3.2 to 2.4%) was estimated to be 0.92. We used multi-level logistic regression to estimate adjusted odds ratios (aOR) that represent relative differences in the odds of OASI before and after implementation of the care bundle. To adjust for the small number of clusters, we estimated the model using adaptive Gaussian-Hermite approximation to the likelihood. 28 The regression model included a linear term for calendar time in 3-monthly intervals to account for temporal confounding, a random effect to account for clustering at both region and unit levels 26 and individual case-mix factors (maternal age, ethnicity, body mass index, parity, mode of birth and birthweight). 3 The case-mix factors were categorised as listed in Table 2. Multiple imputation was used to deal with missing values for age (missing for 3.0% of women), ethnicity (12.8%), body mass index (9.7%), parity (1.2%), birthweight (0.6%) with statistical coefficients obtained from ten imputed data sets, pooled using Rubin's rules. The analysis was carried out following the intention-totreat principle, with births analysed according to whether they took place during the baseline or evaluation periods, irrespective of whether or not all aspects of the care bundle could be implemented. Differences with a P value <0.05 were considered to be statistically significant. Although not defined in the published protocol, subgroup analyses of the care bundle effect were carried out according to parity and mode of birth (spontaneous, forceps or vacuum-assisted). 21 The Wald test was used to test for significance of interactions. A first sensitivity analysis was performed to examine whether or not results were affected if time was included as a categorical variable (in 3-monthly period). A second sensitivity analysis estimated the impact of the care bundle using a model that included a random interaction between phase (baseline versus evaluation) and maternity unit with an unstructured covariance matrix that allowed the impact of the care bundle to vary between maternity units. 29 Patient and public involvement The OASI Project had patient and public involvement throughout inception, implementation and evaluation stages to ensure that care bundle development and implementation were informed by the perspective of women. The project was supported by an Independent Advisory Group, including lay representatives. The antenatal information sheet (first component of the OASI Care Bundle) was developed together with patient and public involvement groups in order to ensure that the material was appropriate. Patient and public involvement representatives were present at all skills development days. Results The characteristics of the 16 participating units are described in Table 1. In total, 80 339 singleton live vaginal births were included, 40 475 in the baseline phase (before the implementation of the care bundle) and 39 864 in the evaluation phase (after the implementation of the care bundle). Of the 40 475 singleton live births during the baseline phase, 27 668 (68.4%) were vaginal births and 12 807 (31.6%) were caesarean sections with some variation between the four regions (Table 1). Of the 39 864 singleton live births that took place during the evaluation phase, 27 932 (68.7%) were vaginal births and 12 472 (31.3%) were caesarean sections. There was no evidence of differences in the caesarean section rate in the baseline and the evaluation phase (aOR 0.96, 95% CI 0.89-1.02, P = 0.19). A total of 55 060 vaginal births were included in the analysis: 27 668 births (50.3%) in the baseline phase and 27 392 births (49.7%) in the evaluation phase ( Table 2). The median maternal age was 30 years (interquartile range 26-34 years) and 46% of the women were primiparous. Of the vaginal births, 79.3% were spontaneous and 20.7% were operative. The characteristics of the women included in the baseline and evaluation phases were similar. Table 3 shows that the OASI rate decreased significantly from 3.3% in the baseline phase to 3.0% in the evaluation phase (aOR 0.80, 95% CI 0.65-0.98). There was no statistically significant evidence for a time trend in OASI rates (linear trend, aOR 1.04 per 3-monthly interval, 95% CI 0.99-1.09, P = 0.16). Subgroup analyses showed that there was no evidence that the effect of the care bundle was different for primiparous women (decrease of OASI from 5.2 to 4.9%; aOR 0.81, 95% CI 0.65-1.00) and multiparous women (decrease from 1.7 to 1.5%; aOR 0.78, 95% CI 0.78-1.01; P for interaction 0.77). Neither was there evidence that the effect of the care bundle differed between women who had a spontaneous vaginal birth (OASI rate 2.6% before and 2.2% after implementation; aOR 0.75, 95%CI 0.60-0.93), those who had a forceps (OASI rate of 7.6% in both periods; aOR 0.88, 95% CI 0.69-1.14) and those who had a vacuum-assisted birth (OASI rate 2.7% before and 2.6% after implementation; aOR 0.82, 95%CI 0.54-1.25; P for interaction 0.31). The episiotomy rate decreased slightly from 25.1% in the baseline phase to 24.5% in evaluation phase, but this decrease was not statistically significant (aOR 0.99, 95% CI 0.88-1.12, P = 0.90). A sensitivity analysis with calendar time included as a categorical variable did not alter the estimated decrease in the OASI rate results following care bundle implementation (aOR 0.81, 95% CI 0.65-0.996, P = 0.046). Neither did we find a substantial change in the estimated decrease in the OASI rates in another sensitivity analysis including a random interaction between phase and maternity unit in the model (aOR 0.79, 95% CI 0.63-0.996, P = 0.047). Main findings This study including 55 060 singleton live vaginal births found a reduction of 20% in the case-mix-adjusted risk of severe perineal injury after the introduction of the OASI Care Bundle. Subgroup analyses did not provide evidence that the care bundle had different effects according to parity or mode of birth. The implementation of the care bundle did not affect caesarean section or episiotomy rates. Strengths and limitations We used a stepped-wedge design to evaluate the effects of the care bundle. Stepped-wedge designs are pragmatic and often used when logistical constraints require a sequential roll-out. However, they are susceptible to selection bias and temporal confounding. 30,31 We aimed to mitigate these biases by including all eligible births and by controlling for a time trend in the analysis using both a linear term in the main analysis as well as a categorical variable in the sensitivity analysis to represent calendar time. In our study, we randomised the order of the roll out of the OASI Care Bundle in four regions. This implies that there were only four randomisation units. As a consequence, it cannot be simply assumed that the characteristics of the women who gave birth before the implementation of the care bundle were similar to those of women who gave birth after the implementation. However, the characteristics of the women included in the baseline and evaluation phases were similar as well as the mode of birth and the babies' birthweight, which provides support for the validity of the comparison. In addition, the impact of the implementation of the care bundle that we report is adjusted for a time trend and a range of case-mix factors (age, ethnicity, body mass index, parity, birthweight and mode of birth), also including additional sensitivity analyses exploring different model assumptions. The development and implementation of the care bundle was supported by the two relevant national professional bodies in the UK, representing obstetricians and midwives, and promoted multidisciplinary teams to work together. The intervention was multifaceted and informed by a detailed theory of change. 21 Women were involved in all stages of the quality improvement project, which ensured that the implementation of the care bundle supported women's choices of birth position and the importance of communication during labour, but their involvement in the design of the care bundle itself was limited. Our quality improvement project was designed as a pragmatic study only using routinely collected clinical data. Therefore, we could not measure the 'coverage' (compliance rate for all eligible births) and the 'fidelity' (extent to which the care bundle was applied as intended) of the intervention, which limits our ability to determine to what extent the effect of the OASI Care Bundle can be further increased through enhancing its uptake. We could not control for some of the main risk factors for OASI, most notably shoulder dystocia, epidural use and length of second stage of labour. However, shoulder dystocia accounts for <1% of births and it is unlikely that the rates of other major risk factors changed before and after implementation of the care bundle. Therefore, it is unlikely that these risk factors had a major confounding effect on our results. Interpretation A 'package of interventions' to reduce the rate of OASI was developed for the first time in Norway, in response to observations that the rates of OASI were consistently lower in Finland than in other Scandinavian countries. 14,16,17 The difference between these countries was attributed to the ongoing and consistent practice of slowing down the birth of the head using a specific manual perineal protection technique, the so-called 'Finnish grip', 8 and episiotomy when clinically indicated, explicitly avoiding a median cut. An observational study in five Norwegian units using a structured training programme to implement this approach showed a reduction in the OASI rates from 4-5% to 1- 2%. 16,17 Subsequently, this approach was implemented in Denmark, 32,33 Holland, 20 England 34 and the USA. 19 The results of the Danish studies matched those seen in Norway, but the reductions in OASI rates observed in the other countries were smaller, and not statistically significant because of small sample sizes. Possible explanations for these differences include the extent to which the intervention to reduce OASI rates was fully adopted by the clinical teams 35 as well as methodological issues, including improved detection rates of perineal and anal injuries after implementation of the intervention. 36 One might argue that the reduction in OASI rates that was found after the implementation of the OASI Care Bundle is relatively small and that the extra effort linked to the implementation process may outweigh the benefits. However, our OASI Care Bundle also requires a careful check of the perineum following birth, ensuring accurate diagnoses, which may have increased the OASI detection rate after the implementation of the care bundle. Therefore, the reduction of OASI rates that we found after implementation of the OASI Care Bundle is likely to be an underestimate of its true effect. Our study was not powered to study the effect of the OASI Care Bundle in specific groups. However, the lack of subgroup differences in our study is in line with the results of studies from Norway and Denmark with respect to parity 16,32,33 and studies from the Netherlands and Denmark with respect to mode of birth. 20,33 Concerns have been raised about the impact of interventions similar to the OASI Care Bundle on episiotomy and caesarean section rates. For example, increases in episiotomy rates were observed for subgroups of women or some participating units in Norway and Denmark, 14,16,17,33 but not in the Netherlands 20 or the USA. 19 Our study did not find any indication that the implementation of the care bundle affected caesarean section or episiotomy rates, albeit that the episiotomy rates that we observed (25.1% in the baseline phase and 24.5% in the evaluation phase) were higher than the corresponding episiotomy rate of 21.9% reported by the National Maternity and Perinatal Audit for vaginal singleton cephalic births at term between April 2016 and March 2017 in England, Scotland and Wales. Our results represent the effect of a combination of interventions. It has been argued that interventions such as manual perineal protection should not be considered in isolation but always as part of a combination of interventions that can be implemented together because the causes 37 This would explain, in addition to their limited statistical power and heterogeneity, why metaanalyses of randomised controlled trials of manual perineal protection 37,38 did not find evidence of a protective effect. Conversely, a single-centre study in the UK of 10 370 vaginal births evaluating the impact of an OASI prevention programme, including encouraging an upright birthing position, effective communication to slow down birth of the fetal head, and a hand placed on the head to judge speed of birth, but without manual perineal protection, found a reduction in OASI rate from 2.8 to 1.7%. 39 Study authors argue that the key to success of their programme is that it not only focuses on how the second stage of labour is conducted but includes a multifaceted campaign increasing awareness and engagement among healthcare professionals, which echoes the theory of change underpinning the approach used in our study. Conclusion These findings from a large-scale quality improvement project across the NHS in England, Scotland and Wales demonstrate the potential of the care bundle to improve perineal care during childbirth. Disclosure of interests All authors have completed the Unified Competing Interests form (available on request from the corresponding author). NS reports grants from National Institute for Health Research and personal fees from London Safety and Training Solutions Ltd, during the conduct of the study, and grants from Sanofi Pasteur and King's Health Partners, outside the submitted work. IGU, PB, NS, LS, VN, AH, RT and JvdM report a grant from the Health Foundation Scaling Up Improvement Programme during the conduct of the study. RF has nothing to disclose. Completed disclosure of interests forms are available to view online as supporting information. Contribution to authorship The study was conceived and designed by all authors. IGU and JvdM performed the statistical analyses. RT, RF, LS, NS, VN and AH assisted with the interpretation of results. IGU, PB and JvdM wrote the manuscript, with input from all other authors. The joint senior authors (RT and JvdM) have made an equal contribution to this study and manuscript.	2020-08-13T10:07:20.039Z	2020-08-09T00:00:00.000	{ "year": 2020, "sha1": "c427578348833915970708a6f0d1431a01984c34", "oa_license": "CCBYNC", "oa_url": "https://obgyn.onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1471-0528.16396", "oa_status": "HYBRID", "pdf_src": "Wiley", "pdf_hash": "a48b68674cf7c78d54bbf53d3abfdab957166912", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
218969014	pes2o/s2orc	v3-fos-license	Transperineal ultrasound to predict vaginal deliveries Department of Obstetrics, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo, SP, Brazil; Medical Course, Municipal University of São Caetano do Sul (USCS), Bela Vista Campus, São Paulo, SP, Brazil Correspondence to: Prof. Edward Araujo Júnior, PhD. Rua Belchior de Azevedo, 156 apto. 111 Torre Vitoria, São Paulo, SP CEP 05089-030, Brazil. Email: araujojred@terra.com.br. Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article did not undergo external peer review. Comment on: Peng F, Yu Y, Sun Y, et al. Using transperineal ultrasound to predict labor onset. Ann Transl Med 2019;7:718. The mechanical processes involved in the different stages of labor have been long known (1). The classic ways to access these processes, like abdominal palpation, digital evaluation of cervical dilatation and fetal head station and position, have helped millions of newborns to be delivered in a safe way. But some deliveries still challenge our capability as clinicians, trying to determine the need for an intervention and the best moment to perform it. In this scenario, the development of new tools, adequate for application in day to day use and that can identify and differentiate these patients, would be extremely important. In the last 10 years, there has been an increasing number of publications evaluating the role and utility of intrapartum ultrasound. Multiple techniques, linear and angular, have been described to evaluate cervical dilatation, head descend and position. These ultrasonographic techniques have proven to be more reliable than digital examination (2,3) in accessing important parameters, and to some extent, distinguishing between those women destined for spontaneous vaginal delivery and those destined for operative delivery (4). Furthermore, there is now evidence that ultrasound in labor can predict to some extent the outcome of operatory vaginal delivery (5). Studies using ultrasound during the second stage of labor have shown good results while trying to predict the mode of delivery. In the other direction, some articles have tested the measurement of head-perineum distance before the start of contractions to predict success in vaginal delivery (6). Although the results were inspiring, we still need further investigation before we can apply these as a standard evaluation in clinical care. The measurement of the subpubic arch angle (SPA) using three-dimensional ultrasound (3DUS) as a parameter of the bony pelvis was accessed during the third trimester, between 34 and 36 weeks, to try to predict vaginal delivery and the necessity of assisted versus spontaneous vaginal delivery (7). Unfortunately, the author was not able to find an association between the SPA and mode of delivery. However, there was an association between this parameter and the duration of the second stage of labor. The changes in bony pelvic have also been tested using 3DUS after delivery and compared to pelvic X-rays (8). The novel method described proved to be a reliable way to evaluate the width of symphysis pubis (WSP), superior pubic ligament length and pubic symphysis height. Peng et al. (9) tried to combine all these techniques in a new approach. Pregnant women with 39+ weeks gestation were evaluated before going into labor, and four variables were acquired: progression distance (PD), angle of progression (AoP), SPA and WSP. The data was analyzed aiming to differentiate which group was more prone to go into labor. In the study, the authors concluded the PD and AoP were not suitable predictors of labor onset in late gestation. On the other way, SPA and WSP might have a role as possible predictors of labor, and help in diagnostic accuracy for the start of active labor. But why should we worry about that? Eventually the majority of then will go into labor. A recent multicenter trial compared labor induction versus expectant management in low risk pregnancies, and opposite to what we classically would expect, found that induction resulted in a lower frequency of cesarean delivery, without significantly altering Editorial adverse perinatal outcome (10). But making all these women go into labor is not cost free. A cost analysis sought to examine the cost-effectiveness of applying induction to all nulliparous term gestations in the United States (11). The authors came to an estimate of additional 2 billion dollars per year in healthcare costs if that was to be done. So, after all it seems that it is important to know if women are prone to go into labor or not after 39 weeks. So, going back to Peng et al. (9), the paper is not conclusive about its results. The methodology still needs some improvement and, as the authors state in the discussion, the sample was small and the groups of patients should be better distributed to clear any bias. But considering the cost impact we could have if we knew which patients are going into labor soon and which are not, the study opens new ground to be evaluated by other studies, so that we can better understand the application of these novel methods in addressing the onset of labor. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.	2020-05-21T09:12:31.494Z	2020-05-01T00:00:00.000	{ "year": 2020, "sha1": "4a685936fa43223410ef4e0d6cb7af6c1a070b6c", "oa_license": "CCBYNCND", "oa_url": "https://atm.amegroups.com/article/viewFile/41942/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "45b1bd239765d4769c8eccc312c214e49f1b58df", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
229602826	pes2o/s2orc	v3-fos-license	Compare of Clustering School Operational Aid Using Fuzzy Cluster Means and Fuzzy Geographically Weighted Clustering Method The use of appropriate Cluster method will support the distribution of School Operational Aid (BOS) fund. Clustering is needed to classify the amount of School Operational Aid (BOS) funds with other influential variables with the aim as the consideration in making policy on the distribution and amount of School Operational Aid funds. Compare of method Fuzzy Cluster Means and Fuzzy Geographically Weighted Clustering were used. The variables used in this study were the School Operational Aid (BOS) funds, total coaching costs, and total regency/city management costs in Central Java Province. The best result of the clustering process was Fuzzy Geographically Weighted Clustering use cluster 3. INTRODUCTION School Operational Aid (BOS) funds are government programs that are used to provide non-personnel operating costs for education units as a compulsory education program. One of the factors that influenced the success of the BOS program was the management of funds and all the resources in the program. Good management of BOS funds is a school's success through a systematic collaborative process from planning, implementation, to evaluation (Fitri, 2014). Each Regency and City Ministry of Education and Culture in Central Java has a different student population in primary, junior and senior high school, in addition to the different geographical conditions in Central Java Province causing different funding to build educational facilities. Fuzzy C-Means (FCM) is a clustering method to minimize objective functions in the clustering process, while other clustering methods generally try to minimize variations within a cluster and maximize variation between clusters (Sari & Suranti, 2016). The advantage of using Fuzzy C Means algorithm is that it is always convergent or capable of clustering (with a quadratic convergence level), does not require complicated calculation operations, and the computational burden of light training so that convergence can be achieved more quickly depending on the amount of data and cluster to be achieved. The Fuzzy C-Means method has several weaknesses, including requiring the number of groups and the matrix of group membership predetermined (Ramadhan et al., 2015). The initial group membership matrix was randomly initialized which caused the Fuzzy C Means method has inconsistency problems Fuzzy C Means is also relatively sensitive to initialization, without good initialization this can produce fewer cluster values than previously determined (Ji et al., 2014). Fuzzy C-Means has several limitations that are very sensitive to initial solutions (initialization), constrained by local minimums and noise-sensitive, automatic central initialization methods to reduce the computational complexity of Fuzzy C Means by improper centers of the actual dataset class (Kannan et al., 2012). One solution to overcome the weaknesses of the Fuzzy C-Means method, can be done by using the analysis of Fuzzy Geographically Weighted Clustering (FGWC) which was first introduced by (Mason & Jacobson, 2006). Fuzzy Geographically Weighted Clustering is an integration of Classical Fuzzy Clustering methods use geographically elements. Fuzzy Geographically Weighted Clustering includes geographical elements in its analysis so that the clusters formed will be sensitive to environmental effects and affect the central cluster values to create a cluster that is "geographically aware" (Sara, 2018). Fuzzy C-Means algorithm still has weaknesses in the initialization stage. To overcome weaknesses and limitations in the FCM algorithm, the FGWC algorithm is used to determine clusters that have a geographical effect therein. The use of an appropriate cluster method supports the need for information dissemination in the form of groups or clusters to improve coordination and integration of the distribution of School Operational Aid (BOS) funds. The main data used is data on revenue or aid funds that have been received by schools in the Regency/City of Central Java Province. Previous studies regarding Fuzzy Cluster Means research have been carried out, including conducted research on the selection of optimum clusters on Fuzzy C-Means with a case study on the grouping of Regencies/Cities in Central Java based on Human Development Index Indicators. Hogantara (2011) concludes that, schools in the city of Semarang accept BOS, but the government and the community are still weak in supervision. (Wasono,R. et al.,2018), conducted a study of the spatial effects of BOS using a spatial analysis which concluded that there was no linkage of the distribution of BOS funds for districts and cities in Central Java. Research on Fuzzy Geographically Weighted Clustering has been carried out by Sara (2018) regarding the grouping of People's Welfare Indicators in Central Java Province with the results of the study forming 3 optimum clusters with each different characteristic where FGWC analysis produces more significant values and fulfills assumptions compared to classic fuzzy clustering. Based on the above background, this research will present the grouping of School Operational Aid (BOS) funds, total coaching costs, and total district/city management costs in Central Java Province using Fuzzy C-Means and Fuzzy Geographically Weighted Clustering methods. METHOD The data used in this study are data obtained from the Ministry of Education and Culture for the 2018 period. In this study, the observation units are regencies and cities in Central Java Province. The variable used is based on the Constitutional Court Minutes No. 13 / PUU-VI / 2008. The full explanation can be seen below: Algoritma: The FCM algorithm is as follows: 1. Determining: a) Matrix X sized n x m, with n = the number of data to be clustered; and m = the number of variables., b) Number of clusters to be formed = C (≥ 2). c) Rank (weighting) = W (> 1) d) Maximum iteration. e) Termination criteria = ξ (very small positive value). f) Initial iteration, t = 1 and Δ = 1. 2. The initial partition matrix form of U 0 is as follows: (initial partition matrix is usually chosen randomly) 3. Calculating the cluster center V for each cluster: 4. Fixing the degree of membership of each data in each cluster (fixing the partition matrix), as follows: Determining the termination criteria which is the change of the partition matrix in the current iteration with the previous iteration as follows: If , the iteration is stopped; however, if , then it increases the iteration (t = t + 1) and returns to step 3. RESULTS AND DISCUSSION The distribution pattern of BOS (School Operational Aid) in Central Java Province is explained in the figure as follows: Based on the BOS data, it can be seen that the districts/cities in the BOS distribution are divided into 4 colors, the color of the location is getting darker, the BOS is getting higher. It can be seen that districts / cities that have the darkest color BOS are Banyumas, Banjarnegara, Purworejo, Karanganyar, and Surakarta City that get the biggest BOS funds among other cities and regencies, regions that receive the smallest BOS funds are Pemalang, Pekalongan, Purbalingga, Kendal, Temanggung, Boyolali, Jepara, Kudus, Pekalongan City, and Salatiga City. Result-1 The grouping process using the FCM algorithm is done by testing a variety of many clusters, the results are as follows: From Table 2 above it can be seen that the minimum Root Mean Squre Error (RMSE) index in many clusters = 4. The smaller the RMSE, the greater the success rate of the grouping process. So that the best results from the grouping process on the data is to use cluster 4. Algorithm: 1. The first thing to do is determine the following: 3. Calculate the center of the cluster so that the center of the cluster is obtained as follows: 4. Calculates the value of the objective function for the first iteration, and when the objective function value does not meet the specified criteria, the next iteration is performed with a new membership matrix. 5. Calculates the change in the u_ik membership matrix 6. When the epsilon or error value has reached the expected error, the iteration process is stopped. In this study, iteration was carried out 131 times to get the epsilon value fulfilled with an objective function of 7,490. So that the results obtained from the cluster using Fuzzy C-Means are presented in the following Table (more complete in the excel file). Result 2 The grouping process using the FGWC algorithm is done by testing a variety of many clusters, the results are as follows: In the concept of fuzzy clustering, a member can be a member of several clusters at once according to their degree of membership. In the clustering process always look for the best solution for the defined parameters. To determine the optimal number of clusters it is necessary to have a validity index measurement. Partition Coefficient (PC) is a method that measures the number of overlapping clusters. In measuring the validity index using the PC index, the most optimal cluster is determined based on the greatest PC value (Sara, 2018). Classification Entropy (CE) is a method that measures fuzziness and cluster partitioning. The most optimal cluster is determined based on the smallest CE value. According to (Sormin, et al, 2015) the CE index evaluates the randomness of data in clusters whose values are in the range [0.1] so that if the value gets smaller it approaches 0 then the cluster quality becomes better. The Separation Index uses the minimum distance separator for partition validity. The optimum number of clusters is indicated by the minimum S index value (Sara, 2018). So that the best results from the grouping process on the data is to use cluster 3. And the results obtained from the cluster using FGWC are presented in the following Table (more complete in the excel file). Result 3 Comparison of clustering results using the FCM method with FGWC Testing multivariate normal distribution is done by looking at the correlation between mahalanobis distance and chi square values in the data. The results of multivariate normal distribution testing in the FWGC and FCM analysis are as follows: The above Table is the result of a multivariate normal distribution test, with a significance level of 95%. In the FCM analysis test there is no relationship between mahalanobis distance and chi square values in the data. In this case, the data on FCM is not multivariate normally distributed and on FGWC data, it is normally multivariate. Homogeneity testing of multivariate data is done by Lavene's Test, with the following results: Based on the Table above, each p-value in the FCM and FGWC analysis is the same, that is 0.000 which is less than a = 95% so the testing decision rejects Ho. Because Ho is rejected, there are differences in the variance-covariance matrix in the data, which means that the data are heterogeneous in nature, so the second assumption is not fulfilled. One Way Manova Test and Comparison between FCM and FGWC The last step is to conduct a one-way manova test, which is obtained to determine differences in the characteristics of each cluster. In this one way manova test uses a significance level of 95%, and the following results are obtained: The above Table is the result of one way manova test, because the second assumption is that the homogeneity of the variance-covariance matrix is not fulfilled, so the one way manova analysis test uses Pillai's Trace. FCM and FGWC analyzes have the same value of 0.000 less than 0.05, and both show significant results. The characteristics of a good cluster according to (Hidayatullah, 2014) are multivariate normally distributed data and have different characteristics from one another (heterogeneity). Discussion The clustering using the method of Fuzzy C Means shows the best results when using cluster 4 with the value of Root Mean Square Error (RMSE) of 0.335 and the best results FGWC method , the grouping process on the data is to use cluster 3. This can be interpreted that the FGWC analysis is able to fulfill the characteristics of a good cluster compare FCM. CONCLUSION The data used in this study are data obtained from the Ministry of Education and Culture for the 2018 period. In this study, the observation units are regencies and cities in Central Java Province. The variable used is based on the Constitutional Court Minutes No. 13 / PUU-VI / 2008. The clustering using the method of Fuzzy C Means shows the best results when using cluster 4 with the value of Root Mean Square Error (RMSE) of 0.335 and the best results FGWC method , the grouping process on the data is to use cluster 3. This can be interpreted that the FGWC analysis is able to fulfill the characteristics of a good cluster compare FCM.	2020-12-29T00:01:04.721Z	2020-04-30T00:00:00.000	{ "year": 2020, "sha1": "9c07dcf47f6535557e95e16a18dca03a4c50394e", "oa_license": "CCBYNCSA", "oa_url": "http://sunankalijaga.org/prosiding/index.php/icse/article/download/527/501", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "d237b07d95d177bd5d19914a5e139198a84730b1", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
7358869	pes2o/s2orc	v3-fos-license	Intravenous dexmedetomidine as an adjunct to subarachnoid block: A simple effective method of better perioperative efficacy In a bid to improve regional anesthesia techniques, many drugs have been tried as sedative agents in patients undergoing lower abdominal surgeries under subarachnoid block.[1-4] All these agents have their own integral merits and demerits, and none of them can be considered as an ideal agent for sedation during spinal anesthesia. Therefore, the search for supplementing regional anesthesia with sedative agents seems to be unending. Introduction In a bid to improve regional anesthesia techniques, many drugs have been tried as sedative agents in patients undergoing lower abdominal surgeries under subarachnoid block. [1][2][3][4] All these agents have their own integral merits and demerits, and none of them can be considered as an ideal agent for sedation during spinal anesthesia. Therefore, the search for supplementing regional anesthesia with sedative agents seems to be unending. Studies have compared propofol and midazolam for achieving faster onset and longer duration. [5] However, patients receiving propofol were three times more likely to have hypotensive episodes which limited the role of propofol as a sedative agent, especially in cardiac patients. Newer alpha-2 agonist dexmedetomidine has emerged as a wonderful drug in anesthesia practice since last one and a half decade. [6] Very few studies have been done with dexmedetomidine as a sedative agent to supplement subarachnoid block. As such there is a paucity of literature on the effect of dexmedetomidine on overall block characteristics of regional anesthesia. This limited literary evidence encouraged us to design a double-blinded randomized prospective to assess the effect of intravenous (I.V.) dexmedetomidine on spinal anesthesia with regard to duration of sensory and motor block, quality of sedation as well as for any observed side effect. Material and Methods The Hospital Ethical Committee approved the prospective, double-blinded randomized study and a written informed consent was taken from sixty patients of physical status American Society of Anaesthesiologist (ASA) Grades I and II, of age lesser than 60 years, scheduled for lower abdominal surgeries amenable under spinal anesthesia. Patients allergic to drugs used in regional anesthesia, ASA Grades III-V, patients on β-blocker and Ca 2+ channel blocker, pregnant patient, obese patient, and patients for cesarean section were excluded from the study. The study was conducted during 2012-2015 at our tertiary care center. The sample size for the study was evaluated to be sixty, which was generated using a sample size calculator. Considering a difference of 30 min in postoperative analgesia, a sample of 28 was considered adequate for the study keeping α-error at 0.05 and power of the study at 80%. However, we took thirty patients in each group for better validation of results. The study participants were randomly divided into two groups of thirty patients each (n = 30) using sealed envelope technique. In this technique, anyone envelope was picked up by the patient from a box which contains sixty envelopes in which thirty D Group and thirty C Group were mentioned. According to picked up the envelope, the drugs was given to the patients by a senior resident of our unit without disclosing the fact neither to patients nor researcher. Group D (n = 30) patients received a loading dose of 1 mcg/kg of I.V. dexmedetomidine by infusion pump over 10 min followed by a maintenance dose of 0.6 mcg/kg/h till the end of surgery whereas the Group C (n = 30) received an equivalent quantity of normal saline (NS) as loading and maintenance dose I.V. by infusion pump and served as control. After the arrival of the patient in the operation theater, I.V. line was secured with two 18-gauge cannula. Through one cannula ringer lactate infusion (10 ml/kg) infusion was started and baseline vitals were recorded and through the other cannula infusion of respective study, drug solutions were given in respective groups. Baseline parameters were observed and recorded. Following this, spinal anesthesia was administered with a 25-gauge Quincke's needle at L3-L4/L2-L3 interspace using standard midline approach. Vitals were recorded immediately after the subarachnoid block. Sensory blockade was checked by using pinprick technique at 1, 2, 5 min after giving spinal anesthesia and then at every 5 min till 30 min and then at every 15 min till recovery of block to S1 level. Motor blockade was assessed by modified Bromage scale at 1, 2, and 5 min after giving spinal anesthesia, then at every 5 min till 30 min and then every 15 min till full recovery of motor level by asking the patient to move and flex legs with prior information to operating surgeon during intraoperative period. The level of sedation was evaluated both intra-and post-operatively on the basis of sedation scale used in one of the previous studies. [2] Intraoperatively, sedation scale was evaluated at 1, 2, and 5 min after giving spinal anesthesia, then at every 5 min till 30 min and then every 15 min till discharge from postanesthesia care unit. Side effects during intra-and post-operative were also observed and recorded and treated accordingly. Intraoperative side effect such as hypotension and bradycardia were detected by the continuous monitoring of blood pressure (BP) and heart rate (HR). The systolic BP (SBP) <90 mmHg was the cut off point to consider Hypotension and HR < 60 beat/min was the cut off point for bradycardia. Postoperative pain intensity was assessed using a 10 point visual analog scale (VAS) on which 0 indicated no pain and 10 indicated the worst pain imaginable. Postoperative nausea, vomiting, and shivering were also observed till the discharge of the patients. Hypotension (SBP <90 min), bradycardia (HR <60/min), and postoperative complications like nausea and vomiting were managed appropriately. Any hypotension with SBP <90 mmHg was managed with a fluid bolus of 300-500 ml NS. If such hypotension did not respond to this fluid administration, then injection mephentermine 5 mg I.V. was administered. If hypotension did not respond to two repeat doses of mephentermine, then dopamine infusion was started to maintain the BP. Any incidence of bradycardia with HR <50/min was treated with atropine 0.6 mg I.V. For postoperative pain, tramadol 100 mg intravenously was given if VAS >3 and ondansetron 4 mg intravenously was used to treat Postoperative nausea and vomiting (PONV).Statistical analysis was done using Statistical Package for the Social Sciences Version 20 for windows (IBM Corp, Armonk, New York). All continuous variables were analyzed using Student's t-test. Categorical variables were analyzed using Chi-square test and ordinal variable like Ramsay sedation scale was analyzed using Mann-Whitney U-test. P < 0.05 was considered statistically significant and P < 0.001 was considered highly significant. Results All patients (n = 60) completed the study. There was no statistically significant difference in two groups with regards to demographic profile including patient's age, gender, weight, ASA physical status I and II, and the duration of surgery [ Table 1]. The onset of sensorimotor block was earlier in dexmedetomidine group (9.6 ± 5.2 min for sensory block and 2.3 ± 0.9 min for motor block (P < 0.001) as compared to control group (9.8 ± 2.8 min for sensory block and 5.1± 2.0 min for motor block), which was statistically significant (P < 0.05). The duration of sensory blockade was significantly prolonged in dexmedetomidine group (341.7 ± 20.8 min) as compared to control group (329.6 ± 22.1 min) (P < 0.001). The mean time for two dermatomal regression of sensory blockade was significantly prolonged in dexmedetomidine group (115.5 ± 8.7 min) compared to control group (95.8 ± 11.4 min) (P < 0.001). The regression time to reach the modified Bromage scale to "0" was significantly prolonged in dexmedetomidine group (278.0 ± 11.0 min) as compared to control group (250.0 ± 14.8 min) (P = 0.001) [ Table 2]. Intraoperative Ramsay sedation scores were significantly higher in dexmedetomidine group (mean -3.4 ± 0.7, range -2-4) as compared to control group (mean -2.9 ± 0.3, range -2-4] (P < 0.001). More than 30% of patients in the dexmedetomidine group had sedation score of 4 whereas more than 90% of patients in the control group had sedation score of 2-3 [ Table 3]. There was significant statistical difference noted in the hemodynamic parameters (BP and HR) in two groups. The lowest mean HR after subarachnoid block was significantly lower in dexmedetomidine group (51.2 ± 7.3) as compared to control group (68.2 ± 7.4) (P < 0.001). In this study, in 26.7% (8/30) cases had bradycardia that required atropine as compared to control group (2/30) [ Table 4]. The lowest intraoperative SBP after spinal block was significantly lower in dexmedetomidine group (97.6 ± 9.3) as compared to control group (103.9 ± 10.9) (P = 0.001) and there was significant difference in the number of patients requiring mephentermine for management of hypotension in both groups (33.3% vs. 10% in dexmedetomidine and control groups, respectively [P = 0.001]). Total I.V. fluids administered in dexmedetomidine group (2922 ± 516.2 ml) was significantly more as compared to control group (2240 ± 280 ml) (P = 0.012) [ Table 5]. The time of first request for postoperative analgesic was significantly prolonged in dexmedetomidine group (5.3 ± 1.8 h) as compared to control group (3.8 + 0.6 h) (P < 0.001) [ Table 6]. There was no significant difference in the incidence of postoperative nausea, vomiting, and shivering between both groups [ Table 7]. However, none of the patients in the dexmedetomidine group had postoperative shivering as compared to 10% in control group (P = 0.056). Discussion Different drugs such as epinephrine, phenylephrine, adenosine, magnesium sulfate, sodium bicarbonate, and neostigmine and alpha2 agonists have been used as adjuvants to local anesthetics to prolong the duration of spinal anesthesia. Among them clonidine, a α 2 agonist is widely used by oral, intrathecal, and I.V. routes as an adjuvant to prolong spinal anesthesia. [7] Dexmedetomidine is a more suitable adjuvant to spinal anesthesia compared to clonidine as it has more sedative and analgesic effects due to its more selective α-2A receptor agonist activity. [6] In this study, there is statistically difference in the onset as well as in the duration of sensory block in dexmedetomidine and control group. The onset of sensory block was earlier in dexmedetomidine group (9.7 ± 5.2 min) as compared to control group (9.8 ± 2.8 min). The duration of sensory blockade was significantly prolonged in the dexmedetomidine group (341.7 ± 20.8) as compared to control group (329.5 ± 22.1) (P < 0.001). The time for attaining the highest level of sensory block was comparable in dexmedetomidine (9.6 ± 5.2 min) and control groups (9.8 ± 2.8 min). The median highest cephalad level of sensory block T4 (T3-T8) was attained in 15 min in the dexmedetomidine and control groups which is almost similar to the observations by Whizar-Lugo et al. [8] In the present study, mean time for two dermatomal regression of sensory blockade was significantly prolonged in dexmedetomidine group (115.5 ± 8.8 min) as compared to control group (95.8 ± 11.4) (P < 0.001). These findings are in synchronization with observations of other similar studies in which significant prolongation in mean duration of sensory blockade in dexmedetomidine group was reported. [5,6] These effects can be explained on the basis of site of action of dexmedetomidine which is locus coeruleus and is mediated by hyperpolarization of noradrenergic neurons thus inhibiting noradrenaline release and inhibiting activity in descending medullospinal noradrenergic pathways. [6] Analgesic effects are mainly meted by α-2C and α-2A receptors present on the neurons of the superficial dorsal horn in lamina II, by inhibiting the release of pronociceptive transmitters namely substance P and glutamate and by hyperpolarization of spinal interneurons. These similar mechanisms also possibly explain the motor blockade augmentation effects. In this study, there was a significant difference in time taken for motor blockade to reach modified Bromage scale 3 in dexmedetomidine group as compared to control group. 2.3 ± 0.9 min in dexmedetomidine versus 5.1 ± 2.0 min in control group (P < 0.001). The regression time to reach the modified Bromage scale 0 was significantly prolonged in dexmedetomidine group (278.0 ± 11.0 min) as compared to control group (250.0 ± 14.8 min) (P = 0.001). Elcıcek et al. [9] and Hong et al. [10] also found that complete resolution of motor blockade was significantly prolonged in dexmedetomidine group, and the findings of present study corroborate these facts. Mean arterial BP shows biphasic variations with an initial transient rise with a reflex fall in HR brought about by stimulation of α-2B subtypes of receptors present in vascular smooth muscles. This is followed by fall in BP and HR due to inhibition of central sympathetic outflow and stimulation of presynaptic α-2 receptors cause decreased release of noradrenaline leading to further fall in the BP. [6] The mean intraoperative HR was significantly lower in dexmedetomidine group as compared to control group (P < 0.001). These findings are on expected lines as dexmedetomidine is known to cause bradycardia and hypotension. The results obtained in the present study with regards to HR almost matches those with other research studies. [9,11] However, in the present study, bradycardia owing to dexmedetomidine was very much treatable with I.V. bolus doses of atropine which can be considered as safety feature with this adjuvant. These hemodynamic effects; however, may be deleterious in patients with fixed stroke volume, on The consumption of atropine dosage can be considered as a normal entity as the incidence and management of the bradycardia almost coincides with the results of earlier studies. [8] However, few earlier studies did not find any significant difference in the incidence and management of bradycardia, both in dexmedetomidine and control group. [10] Similarly, the average intra-and post-operative mean arterial and diastolic BPs were significantly lower in the dexmedetomidine group as compared to control group. Elcıcek et al. [9] reported a significant decrease in mean arterial pressure after 20, 25, and 30 min after dexmedetomidine infusion as compared to control group. Contrary to above studies and the present study, Al-Mustafa et al. [11] reported no significant difference in mean arterial pressures in dexmedetomidine and control groups. In this study, there was a significant difference in the number of patients requiring mephentermine for the management of hypotension in both groups (33.3% vs. 10% in dexmedetomidine and control groups, respectively [P = 0.001]). This finding is in contrast to the study of Tekin et al. [12] who reported no significant difference between groups in the number of patients who received ephedrine to treat hypotension. In the present study, intraoperative Ramsay sedation scores were significantly higher in dexmedetomidine group as compared to control group. Ramsay sedation score was 2 in all patients in the control group and ranged from 2 to 5 in dexmedetomidine group in the study done by Al-Mustafa et al. [11] Hong et al. [10] noted that the median sedation scores during surgery were 4 in the dexmedetomidine group and 2 in the control group (P < 0.001), respectively. Ramsay sedation score significantly favorable (P < 0.0001) along with minimum hemodynamic responses to intubation (P < 0.05) and less oxygenation desaturation (P < 0.0001) in dexmedetomidine group than control group reported by Mondal. [13] Recovery and discharge times were 15 min longer in the dexmedetomidine group noted by Ahmed et al. [14] A significantly higher average sedation score in dexmedetomidine group was also reported by others. [13,15,16] Sedation characteristics of dexmedetomidine include a normal sleep pattern and calming effect on the patients who remain quiet but arousable and cooperative. Sedation is a desirable feature in regional anesthesia as it diminishes the anxiety associated with surgical thoughts to a large extent. Dexmedetomidine was found to be effective in providing postoperative analgesia in the present study. The time to first request for postoperative analgesic was significantly prolonged in the dexmedetomidine group (5.3 ± 1.8 h) as compared to control group (3.8 + 0.6 h) (P < 0.001). Similarly, Hong et al. [10] and Whizar-Lugo et al. [8] noticed that postoperative pain intensity was lower and the mean time to first request for postoperative analgesia was longer in the dexmedetomidine group compared to the control group (6.6 h vs. 2.1 h). Kaya et al. [17] in their study observed that dexmedetomidine increased the time to the first request for postoperative analgesia (P < 0.01) compared to midazolam and saline, and decreased analgesic requirements (P < 0.05). The use of dexmedetomidine as an adjuvant to local anesthetics, when used in conjunction with general anesthesia, have shown to lower intraoperative esthetic requirements, improved oxygenation, and prolonged postoperative analgesia. In this study, none of the patients in dexmedetomidine group had postoperative shivering as compared to 10% in control group (P = 0.056). Similar results were reported by other researchers in which they used dexmedetomidine by various routes. [15] The incidence of other adverse effects including PONV as well as other side effects did not show any significant difference as compared to observation reported by other studies Mittal et al. [18] and other. [19,20] Conclusion I.V. dexmedetomidine significantly prolongs the duration of sensory and motor block of bupivacaine spinal anesthesia. Dexmedetomidine-induced hemodynamic changes are transient but are responsive to pharmacological agents and I.V. fluid administration. Dexmedetomidine provides excellent sedation during surgery, and sedation scores reach normal within 15 min after stopping the drug. Dexmedetomidine is effective in providing significant intraoperative sedation, postoperative analgesia, and minimization of postoperative shivering. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.	2018-04-03T03:36:07.987Z	2017-04-01T00:00:00.000	{ "year": 2017, "sha1": "f5740709cfe27d8c8e34f83f2b3d60bcfaf5720c", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/joacp.joacp_367_15", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "f5740709cfe27d8c8e34f83f2b3d60bcfaf5720c", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
11980883	pes2o/s2orc	v3-fos-license	Exploring the adaptive experiences of children with parents of myocardial infarction: A Qualitative Study Background Cardiovascular diseases are the world’s leading cause of mortality. These diseases are rooted in an unhealthy lifestyle. In order to confront this subject, it is essential to identify several risk factors that contribute to heart disease (HD) in people with different attitudes, values, beliefs, expectations and motivations. This study was therefore an attempt to explain the adaptive experiences of children whose parents were involved in myocardial infarction since they were more likely subjected to get the so-called disease. Objective To identify the risk factors and to clear ambiguity using a qualitative research method from the experiences of people at risk of the above mentioned disease. Methods This qualitative study was a directed content analysis. Eighteen children (above 18 years old) of parents with a history of myocardial infarction participated, and were chosen with purposive sampling and the highest diversity. Data were collected through deep and semi structure interviews based on Protection Motivation Theory (PMT) from March to November 2015, and were analyzed along with their data collection and with usage of Lundman and Graneheim method. Interviews were conducted in non-stressful conditions with a place and time agreement. Results During content analysis process, 220 codes were extracted. After reviewing several times and summarizing, the codes were categorized based on similarity and proportion, and finally 12 subcategories and three categories were elicited including efforts to perform self-care in order to prevent HD, poor life style as a factor not to do preventive HD and health continuation with positive changes in life style. Conclusions Most participants, despite intending to do self-care behaviors to prevent HD, due to factors such as time constraint, high costs, laziness, impatience and prioritizing other life affairs, did not pay attention to their health. Therefore, providing the training programs with an emphasis on life skills can play an important role in reducing perceived response cost and promoting health. Introduction Forecasts suggest that by 2020, these diseases will cause more than 75% of deaths worldwide and if the upward trends continue, by 2030, annually, around 23.4 million people worldwide will die because of cardiovascular diseases (1). Cardiovascular diseases cause 47% of deaths in Iran (2). If control of risk factors is initiated in late adolescence, development of early stage atherosclerosis will be inhibited and will eventually lead to reduction or delay in incidence of the disease (3). With certainty, it can be said that for reducing morbidity and mortality of cardiovascular diseases, one of the most effective methods is implementing health promotion intervention and educational strategies (4). Choosing the health educational model and theory is the first step in the process of designing an educational plan (5). PMT has been used for assessing effective factors in motivation and eventually, behavior (6). This theory is a preventive one, considering the necessity to recognize the viewpoints of people at risk of HD, and further qualitative methods assist the health educators to deeply comprehend the behavioral and environmental motives from people at risk' views. PMT includes two processes of threat appraisal and coping appraisal and a fear construct that the output of two processes is protection motivation and behavior. Coping appraisal emphasizes on the coping response with the health threat and factors that decrease or increase probability of selecting adaptive responses. This cognitive meditational process consists of perceived self-efficacy, response efficacy, and response costs (7). Qualitative methods value participants' views so researchers put themselves in the context of people's real life by interviewing them (8). Therefore, the interview has the capacity of description, explanation and discovery of issues from participants' perspective (9). Hence, in order to recognize and perceive various dimensions and agents affecting the phenomenon of the social process of "preventive behaviors of people at risk of HD", methods that can distinguish ideally concrete aspects from abstract concepts are required. Understanding of the mentioned process for providing new knowledge and insights, and reloading the behavior of people whose parents are involved in myocardial infarction, because they are more at risk of this disease, it is necessary through qualitative research, until their life experiences, by using an inductive approach, can be defied and through a practical and effective educational plan, practical guidance provide can for their activity. Research design and participants This study was a directed content analysis qualitative research that was conducted based on PMT. Content analysis is beyond extracting factual content from context data, and can display themes and latent patterns from the content of participants' data (10). In this study, 18 children (over 18 years old) of parents with a history of myocardial infarction who, during last year, were hospitalized in hospitals affiliated to Shiraz University of Medical Sciences, were investigated. Selection criteria The inclusion criteria encompass having a parent, with history of MI, the capability to participate in interviews, aged over 18, and having at least one risk factor for HD. Exclusion criteria include having a history of HD and lack of tendency to participate in this research. Interviews and data collection Sampling was done during different hours and days of the week. In order to collect information, semi-structured and deep interviews based on PMT in different places such as hospitals, parks and health centers were used in 2015. Purposive sampling was conducted and continued until data saturation (9). Having introduced and explained the objectives of the study, the researcher conducted interviews lasting from 30 to 65 minutes in a non-stressful and private environment with a place and time agreement. . Interview questions were developed based on PMT constructs. For example, how capable do you see yourself in preventive behaviors? If you want to apply preventive behaviors of HD what would you lose? Minimal interference was done in interview trend and an attempt was made to keep the interview to the point. Data were analyzed through content analysis using the method proposed by Graneheim and Lundman and the transcript of the recorded interviews were written on the same day, and were used as the original data and reviewed several times to reach a general perception (11). Words, sentences or paragraphs that were considered as semantic units after abstraction and conceptualization were labeled by codes according to the concept behind them. The codes were compared according to their similarities and differences, and were classified with specified labels in more abstract classes. Finally, by comparing different classes with a deep, careful reflection, the content lying in data was introduced as the theme of study. The Helsinki codes were observed; the voluntariness of participation in the study, withdrawal from the study at any time, confidentiality of the information, receiving an introduction letter and legal permission (11). Research ethic The ethics committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran approved the study and oral consent was obtained (Code: 15646). Accuracy, reliability, conformability, credibility, and transferability To ensure the accuracy and reliability of data, the scientific precision criteria in qualitative research "Guba and Lincoln" were used (12). Conformability of the findings by observing revision, credibility by prolonged engagement of the researcher with the study samples and allocating enough time for data collection and dependability were provided by giving the interviews' transcripts and the extracted codes to other participants to be confirmed. For transferability of the findings, research processes were prepared clearly and in written form and their related computer files were saved to provide follow-up feasibility of research track and population feature for others. Also, sampling technique with maximum diversity helped transferability of the findings in this study. Results In total, interviews with 18 children of patients with a history of myocardial infarction led to a richness of information, data saturation and data replication. The age range of participants was 21-40 years and the mean age was 27±88 yrs. From the qualitative analysis and comparison of data, three categories and 12 subcategories emerged with categories including efforts to perform self-care for prevention of HD, poor lifestyle as a factor of neglect in doing HD preventive behaviors and sustaining health by positive changes in lifestyle (Table1). Efforts to perform self-care Self-care is a multidimensional structure that requires behaviors which are influenced by factors such as health beliefs, life situations and events. Subcategories of efforts to perform self-care included perform self-care, using stress reduction techniques, attempt to obtain more information, and the provision of appropriate family environment. Perform self-care: Some participants believed that they could relatively perform self-care and preventive behaviors of HD. Behaviors that these people stated for maintaining their health included avoiding junk food, weight control, exercises, walking and making a difference in their life by spending time in nature. "…During pregnancy I was very overweight, now I eat food like rice less and more fruits and vegetables... ". (int.8, female 25 yrs.). Using stress reduction techniques: In this study, stress was an issue that all participants had experienced because of life conditions, and believed that if this sense lasts too long it will endanger their physical and mental health. Some of these people tried to use some techniques to reduce their stress and mental tension."…I try to keep my distance from nagging and anxious people and instead go toward those who give positive energy…." (int.10, male 33 yrs.). Attempt to obtain more information: The majority of participants had the necessary information and knowledge regarding HD risk factors and some had sought further information in this regard and transferred it to the others, and believed that obtaining health Page 4909 information is easily possible from different ways. "… Honestly, I'm studying a lot about HD prevention …." (int.11, male 39 yrs.) The provision of appropriate family environment: All of the participants believed that valuing health depends on effective communication between family members, collaboration and cooperation in preventive behaviors and appropriate family situation. "…Whenever intimacy and communication between family members are more, mental concerns and the likelihood of sickness become less…." (int. 3, female 23 yrs.) Poor lifestyle as a factor of neglect doing HD preventive behaviors Subcategories of this category included lack of planning in life affairs, costs as a factor of neglect, inappropriate attitude to lifestyle and not enough effort to maintain health. Lack of planning in life affairs: In this study, most participants indicated time constrains due to job or educational conditions and also the occurrence of unexpected work as reasons not to do preventive behaviors, especially exercise and healthy diet. "… in summer, I had sport and club plans, but when my dad had a myocardial infarction, we had to take him to the hospital and I gave up my exercise…. " (int.16, female 34 yrs.). Costs as a factor of neglect: The participants believed that proper financial situation can prevent HD and have significant impact on valuing health. They reported high prices of nutritious food; seasonal fruits, check-ups and various exercises as effective factors of not making positive changes in lifestyle. "… I can't adhere to a healthy diet because of the high prices of healthy food…. " (int.12, male 25 yrs.). Inappropriate attitude to lifestyle: Poor attitude of some participants or their family members towards performing healthy behaviors, caused incidence of HD risk factors and dismissal of HD preventive behaviors. "… Some of my family members say that life is too short why should we bother ourselves?; this belief itself automatically has effect on the other members…." (int. 6, female 21 yrs.). Not enough effort to maintain health: The majority of participants mentioned laziness, fatigue, impatience, and preferring other affairs to exercise as the main barriers of doing preventive behaviors." …Sometimes I wonder if I stay at home and do sewing or cooking, would it be better for me than doing exercise? ..."(int. 5, female 40 yrs.). "…Regular walking is boring and monotonous for me...." (int.9, male 28 yrs.). Health continuation with positive changes in lifestyle "Lifestyle" is a collection of health-related factors; nowadays following a healthy lifestyle is the most important principle in achieving health and longevity which has significant influence on maintaining and promoting people ̓ s health and on disease control and prevention. Subcategories of this category included activity and exercise, healthy nutrition, tobacco cessation, having tranquility Activity and exercise: In terms of exercise and its effects on health and HD prevention, participants cited boost in morale, increased body resistance to disease, increase of human patience and endurance, weight control and prevention of arteriosclerosis. "… I go mountain climbing at least twice a week and do light exercise that raises my heart rate in the morning because it has a positive effect on my physical and mental health…." (int. 10, male 33 yrs.) Healthy nutrition: All participants believed that a healthy diet is the way to achieve physical health and longevity, although it is not ineffective in mental health. "… I do not eat fast food or byproducts at all because it is the only fat that remains in the body for at least six months. These foods result in shortening the human life expectancy…." (int.11, male 39 yrs.). Tobacco cessation: The majority of participants were educated, and so were aware of the consequences of smoking and hookah and also the positive effects of quitting them."…When my mother had plaque and open-heart surgery, we broke the hookah pipe. It keeps my mum's heart rate down. It stops my mum's heart rate from going up…." (int. 4, female 40 yrs.). Having tranquility: Participants considered tranquility as a factor in adherence to diet, regulation of heart beat and blood pressure, increased body resistance, disease prevention, as well as resistance to life's problems."…When I am calm and feel relaxed I care more about my health and behaviors that are associated with health…." (int.8, female 25 yrs.). Discussion To identify the result of this research, coping appraisal process of PMT has been used. This process consists of perceived self-efficacy, response efficacy, and response costs. The category associated with self-efficacy is "efforts to perform self-care in order to prevent HD". Self-care is a multidimensional structure that requires behaviors which are influenced by factors such as health beliefs, life situations and events (13). In this study, number of participants were more or less sure about their ability to perform self-care behaviors such as adhering to diet, walking, doing the exercise, checkup, especially in an emergency and avoiding tobacco. This concept points to self-efficacy which means confidence of the person in their ability to begin a new or difficult behavior. Some studies have investigated self-efficacy as an important factor in improving self-care and as a modifier of coronary artery disease (14). In a study conducted by Sung, negative correlation was found between self-care behavior and health maintenance with risk factors of cardiovascular disease (15). But in the study done by Chen and Al-Khawaldeh strong correlation has been proved between self-efficacy and self-care behavior (16,17). The results of a study by Khosravi Zade and colleague, showed that self-efficacy based training can lead to an increase in self-care behaviors such as reducing salt intake and weight control (18). Regarding stress and stress management, participants acknowledged that stress and psychological pressures are the most important factors in HD development and have an adverse impact on their self-care behavior. Parents ̓ heart disease, concerns about leaving them alone at home, economic problems, employment status and student conditions were stated as the reasons for participants' stress. In one study, it was shown that stress affects development of HD directly and its risk factors included poor nutrition, low physical activity, smoking and alcohol consumption indirectly (19). In this study, some participants tried to apply coping strategies such as relaxation exercises, being located in a quiet environment, spending time in nature, sociability with family, friends and positive people, giving positive suggestions to their own and adherence to religious values in order to be relaxed and control themselves. Improvement in stress management in the form of meditation and yoga are associated with reduction in risk factors of cardiovascular diseases such as weight loss, total cholesterol, cholesterol with high density (for men), triglycerides, Hba1c (for diabetics) and reductions in hostile feelings (20). Therefore, teaching stress management techniques and strategies to adolescents and youth, can promote their selfefficacy, mental health and consequently can reduce the risk of HD. Regarding knowledge acquisition of HD, although most participants had good information about preventive behaviors of HD because of existence of factors such as time constraint, laziness, impatience and prioritizing other life affairs, they acted poorly in terms of self-care due to the previously mentioned barriers. It can be justified since the majority of participants were educated, had good knowledge and information, but knowledge alone cannot have an impact on performance and as a result, cannot have an impact on self-care of individuals. This finding is consistent with the result of Kang's study (21). Concerning the subcategory of provision of appropriate family environment to prevent and control HD, factors that participants indicated as suitability of the family environment and were recognized as one of the most important factors regarding health were intimate relationship between family members and receiving psychological support from them. What is clear, is that the individual perception of availability of social support and personal satisfaction that one acquires in communication with others can make obstacles against disease and mental pressure. Cares and emotional connections created by social support can be very effective in health and Longevity (22). In Mendis and Cole's study it was also shown that receiving positive support from family, friends and peers can facilitate lifestyle changes (23,24). The results of that study are consistent with the results of our study. The category associated with perceived response cost construct is poor lifestyle as a factor of less attention to preventive behaviors. Lifestyle includes activities and attitudes that can affect a person's health. Appropriate food habits, stress management, physical activity and smoking cessation are the most important aspects of a healthy lifestyle (25). Continuous existence of healthy lifestyle factors are accompanied with reduction in risk of myocardial infarction and its mortality (26). In this category, one of the subcategories was lack of planning in life. Existence of factors such as time constraint due to job or educational conditions, having familial responsibilities, the occurrence of unexpected work and easy access to fast food were indicated as the most important factors of less attention to performing behaviors such as exercise and adhering to appropriate diet regime by the participants. The interesting point is that in the age group of youth, due to their certain cultural, social and economical situation which was student or employed and with most of their time spent away from home and according to the problems of transportation and traffic which stems from urbanization, they have to eat at least one meal a day away from home and consume a variety of convenience foods (27). In a study conducted by Keeler in female university students, it was revealed that time constraint for preparing healthy food and easy access to unhealthy food substances are barriers to a healthy diet (28). In other studies time constraint is also introduced as one of the factors of less attention to exercise and dietary adherence (29,30). Usually, the first and simplest reason for inactivity and lack of participation in exercises and consumption of unhealthy food is time constraint. The best recommendation for overcoming this obstacle is acquisition of time management skills. If these people are provided a good plan they will be able to exercise and prepare healthy food in their free time. In this category, one of the factors of neglect in doing preventive behaviors was cited as cost of doing exercise (31) and seasonal fruits. The studies showed that the consumption of fruit and vegetables is more expensive than other foodstuffs and the need to buy fresh fruit and vegetables every few days continuously is also a barrier of a healthy diet (32). Ashton's study showed the cost of unhealthy food is less than healthy food (30). Inappropriate attitude to life is another subcategory of poor lifestyle. The most important barriers related to this subcategory are, having myths and wrong beliefs in health maintenance styles such as achievement of tranquility by smoking and usefulness of solid oil. In this study, people with lower education and self-employment had positive attitude towards smoking and its effect, this subject suggests that low education levels have caused lower knowledge of the fatal complications of smoking. Conducted investigations also showed that participants, to achieve tranquility and reduce life stress tend to smoke, that confirms the results of the present study (33,34). Hence, in order to create awareness and improve attitude towards healthy lifestyle, and as a result, create positive behavioral changes, eliminating the leading cause of creating poor attitude and implementation of educational programs of life skills have an important role. Not enough effort to maintain health was another subcategory of poor life style category. In this regard, the most important factor mentioned was laziness and impatience. In Kumer's study laziness was stated as one of the obstacles of physical activity (35). The category associated with the response efficacy construct is health continuation with positive changes in lifestyle. From the participants' perspective, positive changes in lifestyle cause increased body resistance, longevity, disease prevention, health and vitality of the individual. Results of a prospective study conducted on young people aged 18-30 showed that healthy lifestyle changes are accompanied by risk reduction and unhealthy lifestyle changes are associated with an increased risk of atherosclerosis in middle age (36). Based on the results of one study, a small improvement in changes in lifestyle can lead to significant reduction in incidence of myocardial infarction (37). In general, the results of the literature reviews offer an overall view of the longitude relationship between physical activity and the incidence of noncontagious diseases and health problems. It seems that physical activity is a contributing factor to prevent agerelated diseases. Inactivity and unhealthy food habits are related to weight gain, obesity, and becoming overweight, and are a major cause of modern disease such as coronary heart disease or diabetes type 2 (38). Conclusions The result of this study showed that most of the participants were sure of their ability of preventive behavior about HD and knew about these positive effects. But because of issues like inappropriate attitude to life style, lack of planning in life affairs, didn't make such positive changes in their life. The practical importance of these results is that, based on our finding, since the unhealthy lifestyle is formed at an early age and remains stable during the next years, before consolidation and formation of these habits, necessary actions must be taken for following a healthy lifestyle, and parents should put the necessary education available for their children from early childhood, because the later attempts to change established habits will be very difficult and perhaps impossible.	2018-04-03T04:24:46.012Z	2017-07-01T00:00:00.000	{ "year": 2017, "sha1": "c0b754a193ab34a94033e040a5e2041bf0785d19", "oa_license": "CCBYNCND", "oa_url": "http://www.ephysician.ir/2017/4906.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "6bf993c523b37b792c7e2b476190c39e8e310bdd", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Psychology", "Medicine" ] }
230523742	pes2o/s2orc	v3-fos-license	Laboratory Measurements of Instrumental Signatures of the LSST Camera Focal Plane Electro-optical testing and characterization of the Vera C. Rubin Observatory Legacy Survey of Space and Time (LSST) Camera focal plane, consisting of 205 charge-coupled devices (CCDs) arranged into 21 stand-alone Raft Tower Modules (RTMs) and 4 Corner Raft Tower Modules (CRTMs), is currently being performed at the SLAC National Accelerator Laboratory. Testing of the camera sensors is performed using a set of custom-built optical projectors, designed to illuminate the full focal plane or specific regions of the focal plane with a series of light illumination patterns: the crosstalk projector, the flat illuminator projector, and the spot grid projector. In addition to measurements of crosstalk, linearity and full well, the ability to project realistically-sized sources, using the spot grid projector, makes possible unique measurements of instrumental signatures such as deferred charge distortions, astrometric shifts due to sensor effects, and the brighter-fatter effect, prior to camera first light. Here we present the optical projector designs and usage, the electro-optical measurements and how these results have been used in testing and improving the LSST Camera instrumental signature removal algorithms. INTRODUCTION The Vera C. Rubin Observatory, currently under construction on Cerro Pachón in northern Chile, is an 8-meterclass telescope that will conduct the 10-year Legacy Survey of Space and Time (LSST) using the 3.2 gigapixel LSST Camera. The four main science goals of this future wide, fast, and deep survey are to: study the nature of dark matter and dark energy, create a detailed catalog of the solar system, explore the transient optical sky, and study the structure and formation of the Milky Way. 1 The combination of a large field of view (3.5°) and fast cadence (a new field every 30 seconds) will allow the LSST Camera to image nearly a quarter of the sky in Send correspondence to Adam Snyder: E-mail: aksnyder@ucdavis.edu a single filter every three nights, resulting in an unprecedented amount of data that will be collected during the survey. The LSST Camera focal plane has a diameter of approximately 64 cm and is made up of a mosaic of 205 charge-coupled devices (CCDs) arranged into 21 science raft tower modules (RTMs) and 4 specialized Corner Raft Tower Modules (CRTMs) for wavefront sensing and guiding. An individual RTM contains all the required thermal, mechanical, and electronic connections needed to operate the CCDs and can itself be considered a self-contained camera. In addition to the science RTMs, there are four specialized corner raft tower modules (CRTMs) used for telescope guiding and to provide wavefront sensing capabilities for the telescope's active optics system. The custom-designed 16 megapixel LSST Camera CCDs, supplied by Imaging Technology Laboratories (ITL) and Teledyne e2v, are fully depleted high-resistivity bulk silicon sensors that are segmented into 16 separate sections, each with their own output amplifier electronics. In order for the Rubin Observatory project to achieve its stated science goals to the desired precision and accuracy, each CCD must meet a set of stringent electro-optical requirements (Table 1), determined by a number of past studies. [2][3][4] Additional sensor effects that must be characterized and, if necessary, corrected via hardware modifications or software algorithms in data reduction include source astrometric and shape distortions caused by pixel-area variations, electronic crosstalk, the brighter-fatter effect, and deferred charge during CCD readout. There are three levels of testing for each of the focal plane CCDs: sensor-level acceptance testing, raftlevel acceptance testing, and focal plane testing following integration of the RTMs into the cryostat. Sensor acceptance testing was performed in three steps prior to the assembly of the RTMs: acceptance testing by the CCD vendors, reprocessing of the vendor data at SLAC, and acceptance testing at the Brookhaven National Laboratory (BNL) using a single sensor cryostat and commercial electronics controller. 4 Raft acceptance testing was first performed at BNL after assembly of the RTMs, before each RTM was shipped to the SLAC National Accelerator Laboratory where the integration and testing of the LSST Camera is being performed. After receipt, the RTMs were re-verified before being safely stored to await installation into the cryostat and focal plane testing. 5 Section 2 begins with a brief summary of the LSST Camera focal plane testing equipment and procedures used at SLAC. Overviews of the usage of custom optical projectors to study electronic crosstalk and astrometric distortions due to sensor effects during a period of focal plane testing using a subset of RTMs installed into the cryostat are presented in Section 3 and Section 4 respectively. Finally, Section 5 consists of a summary of the results of the electro-optical characterizations presented in this study as well as a discussion of future improvements and extensions that are planned for the upcoming testing of the full LSST Camera focal plane. Focal plane testing refers to the testing of the CCDs after integration of the RTMs into the LSST Camera cryostat. This ongoing state of testing began with a period of operation of the focal plane subsystems with only a subset of RTMs installed, referred to as the partial focal plane testing period. During the data taking discussed here there were nine science-grade RTMs (4 consisting of ITL CCDs and 5 consisting of Teledyne e2v CCDs) and the four CRTMs installed. The partial focal plane testing period was important for verification of the operation of the RTMs within the focal plane, the refrigeration systems, the cryostat vacuum performance, the data acquisition software, and networking capabilities. An additional goal of this period was to identify electrooptical characteristics of the installed CCDs that would warrant further study for optimization or mitigation. The installation of the RTMs and electro-optical testing during the partial focal plane testing period was performed using a specialized test stand, referred to as the Bench for Optical Testing (BOT), in conjunction with a set of custom-built optical projectors designed to illuminate the focal plane with various light illumination patterns; this equipment will also be used for future testing of the full focal plane. Bench for Optical Testing The BOT test stand, designed and built at SLAC, is important for verification testing of major Camera subsystems prior to full assembly of the LSST Camera. The BOT Chassis, pictured in Figure 2, consists primarily of a large dark box enclosure that can be closed by a set of door panels to reduce background light to below 0.01 electrons per pixel per second. The LSST Camera cryostat is inserted into the mounting panel, located at the top of the BOT, such that the focal plane is pointed downwards, to facilitate the installation of the RTMs. Located within the BOT dark box are two Aerotech PRO225 stages, referred to as the XY-motorplatform, along with a mounting interface that is used to position equipment beneath the focal plane. A detailed description of the BOT mechanical design and design requirements is provided by Newbry et al. 6 The primary projector for electro-optical testing of the focal plane is the flat illuminator projector, shown positioned on the floor of the BOT dark box in Figure 3. The flat illuminator projector uses a near-IR enhanced Zeiss 25mm f/2.8 wide-angle lens to re-image the 1.5" exit port of an integrating sphere in order to smoothly and fairly uniformly illuminate the entire focal plane. This optical design results in images that are sufficiently flat (less than 5% signal variation across each CCD segment) to be used for measurements of the CCD non-linearity, full well, and gain from photon transfer curves. The light source for the flat illuminator projector is a Newport Fiber Optic Illuminator, containing a 150 W Quartz-Tungsten-Halogen lamp, that is delivered to the integrating sphere within the projector using a Newport single-branch glass fiber optic bundle connected to a Thorlabs 1" single-blade optical beam shutter. Two Thorlabs motorized filter wheels, connected in series between the shutter and the integrating sphere entrance port, are used to remotely control the light spectral bandpass and intensity. A set of Sloan Digital Sky Survey color filters (u, g, r, i, z, and Y) manufactured by Astrodon and a set of 6 narrow-band filters (10 nm full width at half maximum) centered at 480 nm, 650 nm, 750 nm, 870 nm, 950 nm, and 970 nm are installed in the first filter wheel. The second filter wheel holds a set of 6 masks with apertures of varying size that act as neutral density filters. A light baffle (not pictured in Figure 3) is attached in front of the flat illuminator projector lens to limit the angular extent of the projected beam to eliminate reflected light from the inner side of the cryostat flange, which extends beyond the surface of the focal plane. Electronic crosstalk measurements are made using a custom optical projector named the crosstalk projector ( Figure 4), that is designed to illuminate a single CCD with a pattern of large bright spots (80 pixels in radius) using collimated light. The decision to use an optical design that produced collimated light was made to reduce the impact of reflections of any projected bright spots that occur at the front and back side of the cryostat window, the surface of the CCD, and the back surfaces of the projector optics. Because collimated light will be perpendicular to these surfaces, the reflections of the bright spots will fall at the original location of the bright spot, rather than at different locations across the focal plane, which can interfere with the measurement of crosstalk. The light from the 1" exit port of an integrating sphere is passed through a pinhole, then through an aspheric lens, and finally through a metal mask to produce the desired pattern of bright spots. The mask used in the crosstalk projector was manufactured using precision wire electrical discharge machining and is painted matte black, to reduce its reflectivity. The light source is a QPhotonics 3 mW fiber-coupled 450 nm light emitting diode, connected by a single-mode fiber to a Thorlabs 1" single bladed optical beam shutter installed at the entrance port of the integrating sphere. When in use in the BOT, the crosstalk projector is mounted on a fixed stand on the XY-motorplatform, which is used to translate the projector in order to illuminate individual CCDs within the focal plane. The ability to project realistically-sized sources onto the focal plane prior to on-sky operation is provided by a custom optical projector named the spot grid projector. The spot grid projector uses a Nikon 105mm f/2.8 Al-s Micro-Nikkor lens to re-image the 1" exit port of an integrating sphere that has been masked with the desired optical pattern using an HTA Photomask photo-lithographic mask. A six-position Thorlabs motorized filter wheel is used to remotely select from a set of masks with various patterns and position the desired mask in front of the integrating sphere exit port. During the partial focal plane testing period the optical patterns included in the spot grid projector filter wheel were: a single spot, a grid of 24x24 spots, a grid of 49x49 spots, a thin slit to simulate satellite streaks, and two absorptive neutral density filter wheels, which can be used to mimic a low-level sky background. The spot grid projector shares the same 450 nm light emitting diode light source as the crosstalk projector and is also equipped with a Thorlabs 1" single-blade optical beam shutter. An additional feature of the spot projector is the ability to expose the focal plane to multiple illuminated mask patterns during a single exposure; this is accomplished by closing the shutter and rotating the filter wheel from one mask to another via remote command, before opening the shutter again, while continuing the CCD integration phase uninterrupted. When in use in the BOT, the spot grid projector is mounted on the XY-motorplatform using a manual adjustable z-stage. By adjusting the z-stage the spot grid projector can be positioned at the appropriate optical working distance to achieve good focus of the projected spots. The optical projectors described above were designed to be modular to support rapid replacement and installation in the BOT during testing and are all controlled remotely using the same Camera Control System (CCS) software used to monitor and operate the other focal plane subsystems. In addition to the flat illuminator projector, the crosstalk projector, and the spot grid projector there exists an additional set of optical projectors that will not be discussed in depth but remain important for electro-optical testing. The Camera Calibration Optical Bench (CCOB) Wide-Beam projector, designed and built by collaborators at the Laboratory of Subatomic Physics & Cosmology (LPSC) in Grenoble, France, is used to construct "synthetic flat field images" from a number of co-added images that can be used to make quantum efficiency and pixel response non-uniformity measurements. 6 Finally, a manually operated pinhole projector can be positioned underneath the focal plane to project images of printed pictures or physical objects for science outreach and publicity images, among other purposes. 7 Image Calibration The standard calibration for images taken during integration and testing of the LSST Camera CCDs includes an electronic offset correction, electronic bias correction, and dark current subtraction. The electronic offset correction is performed row by row; the mean signal of the serial overscan pixels in each is row (59 pixels total) is calculated, omitting the first 5 and last 2 overscan pixels, and then subtracted from the corresponding row of imaging pixels. The omission of the first 5 overscan pixels is done to reduce the contribution of serial deferred charge, while the omission of the last 2 overscan pixels is historically done as a result of large read noise in the final overscan pixels that was identified during earlier testing of individual LSST Camera CCDs. Electronic bias correction, using a superbias image constructed from a median stack of bias images, is necessary to remove additional spatial pixel non-uniformity across the image caused by the readout electronics and the rate of voltage clocking during readout. Dark current correction is done by subtracting an electronic offset and electronic biascorrected superdark image, constructed from the median stack of multiple dark images, which is then scaled to match the exposure time of the image to be calibrated. For the analyses presented here, 10 bias images and 10 dark images were used when constructing the superbias and superdark images for calibration. Gain values calculated from the photon transfer curve, which are in good agreement with gains calculated from 55 Fe soft x-ray hits, are used for gain calibration. In some cases, these gains are further adjusted using an algorithm to scale each gain value in order to minimize the discontinuity of the pixel signals of a flat field image across segment boundaries, calculated using a least-squares minimization. ELECTRONIC CROSSTALK Electronic crosstalk refers to an effect in CCDs that have multiple channels that are read out simultaneously whereby a bright source that is present on one CCD segment will cause a ghost image to appear in the other CCD segments in the corresponding location, due to the electronic couplings between the output electronics. The electronic crosstalk between two CCD segments can be characterized by the crosstalk coefficient c ij defined as the ratio of the induced signal on a "victim" segment j compared to the true signal of the "aggressor" segment i. Therefore to determine the true pixel signal of the victim segment V j one must subtract the pixel signal of the aggressor segment A i scaled by the crosstalk coefficient c ij from the measured pixel signals of the victim segment V j , The heavily segmented nature of the LSST Camera focal plane (16 segments per each of the 189 science CCDs, not including the specialized CCDs used in the CRTMs) necessitates the characterization of the electronic crosstalk in three different regimes: within a single CCD, between CCDs in the same RTM, and between CCDs in different RTMs. The electronic crosstalk is expected to be largest between segments located on the same CCD, referred to as intra-CCD crosstalk, where the primary electronic couplings are capacitive couplings between the on-chip readout electronics and off-chip trace-to-trace capacitive coupling within the cables connecting the CCDs to the external Readout Electronics Boards (REBs) that control the CCD operation and video channel output processing, including analog-to-digital conversion. 8 The exact designs of the on-chip electronic readout chains of the CCDs manufactured by ITL and Teledyne e2v are not the same; therefore, there is no expectation that the behavior of the intra-CCD crosstalk will be identical between the two types of CCDs. Crosstalk between segments on different CCDs located in the same RTM, referred to as intra-raft crosstalk could result from off-chip capacitive couplings that occur within or between REBs in a single RTM. A single REB is used to operate a row of three CCDs concurrently in an RTM; the same electronics are duplicated in three separate "stripes" that are shielded from each other. 9 The presence of measurable intra-raft crosstalk would likely require hardware mitigation to improve the shielding and isolation of the individual CCD electronics within each REB board. The final regime of crosstalk is between segments on CCDs located on different RTMs, referred to as inter-raft crosstalk. Because this regime of crosstalk would need to be caused by electronic couplings after the analog-todigital converters it is expected to be negligible. The goals of crosstalk measurements during the partial focal plane testing period were: verify the ability to measure crosstalk amplitude to the 10 −6 level, determine the intra-CCD crosstalk behavior in a subset of ITL and Teledyne e2v CCDs, and to show that there is no intra-raft and inter-raft crosstalk at a significant enough level to warrant additional hardware mitigation. Crosstalk Measurement Method Characterization of crosstalk for a single LSST Camera CCD, which has 16 segments, can be summarized by a 16x16 crosstalk matrix where each matrix element is the corresponding crosstalk coefficient c ij . A single exposure taken using the crosstalk projector, shown in Figure 6, results in an image with four bright spots illuminating four separate CCD segments. The four spots are positioned such that the victim regions for each spot do not coincide with other victim regions or aggressor spots allowing for the simultaneous measurement of the crosstalk caused by four aggressor segments, corresponding to four rows in the crosstalk matrix. Using the BOT XYmotorplatform, the crosstalk projector is dithered to allow for the measurement of the crosstalk coefficients for all 16 CCD segments, using a minimum of four images. In order to make measurements of crosstalk values with magnitudes on the order of 10 −6 , the background noise must be reduced by taking multiple exposures per dither position (5 exposures per position during the partial focal plane test period) and generating a set of co-added images, calibrated using the procedures outlined in Section 2.2. The spots produced by the crosstalk projector are not shaped as Gaussian functions or Airy disks; instead, the spots have a broad circularly symmetric center region approximately 80 pixels in radius, a full width at half maximum (FWHM) of approximately 150 pixels and no outer concentric rings. The width of the spots is set by the diffraction that occurs at the photomask holes and increases as a function of the distance between the projector and the CCD surface. Diffraction at the photomask holes also results in diffraction spikes that contribute to background scattered light in the crosstalk images. During the partial focal plane testing period, the mean signal within a 80 pixel radius of the center of each spot was approximately 100,000 electrons; there is some evidence that the electronic crosstalk is dependent on the aggressor signal level, or non-linear behavior, 10 which motivates future study of this effect during the future focal plane testing. The determination of each of the crosstalk coefficients is performed by fitting a crosstalk model to each victim region. Given a 200x200 pixel aggressor sub-image A i (m, n), where m and n are pixel indices, a corresponding 200x200 pixel victim sub-image V j (m, n) is modeled as where c ij is the crosstalk coefficient and the parameters x j , y j , and z j define a sloped plane that represents the possible contribution of any additional background scattered light. The over-determined system of equations defined by Equation 2 can be solved using an ordinary least-squares formalism. Figure 7 illustrates this method, comparing an aggressor sub-image to a victim sub-image and the best-fit model of the victim sub-image, defined by Equation 2. A low-level scattered light background from the crosstalk projector is visible in the victim sub-image; thus, it was necessary to include the additional x j , y j and z j parameters to model this background and improve the accuracy of the crosstalk measurements beyond that of a simple ratio of signals between the victim and aggressor region. This methodology results in parameter estimate errors for the crosstalk coefficient that are approximately 10 −6 or lower, for the majority of best-fit models. Crosstalk Characterization For visual clarity the crosstalk matrix results presented in this paper are plotted using a logarithmically binned color scale where each color bin represents a power of 10. Because electronic crosstalk can be both negative or positive, depending on the specific electronic couplings between segments, this color scale allows either positive or negative bins, symmetrically around 0. Crosstalk coefficient values whose amplitudes fall below a threshold of \|c ij \| < 5 × 10 −6 , which corresponds to 1 electron of induced victim signal for a 200,000 electron aggressor signal, are plotted as white. Absent data and the crosstalk coefficients c ii that lie on the matrix diagonal are plotted as black. The naming convention in this paper is to label each RTM by its location in the focal plane (e.g. R00, R01, etc.), to label each CCD by its location within its RTM (e.g. S00, S01, etc.) and to label each segment by its output amplifier number (1 through 16), where output amplifiers 1 through 8 lie on one side of the CCD mid-line break and output amplifiers 9 through 16 lie on the opposite side. The full 144x144 raft crosstalk matrix for ITL RTM R01 is shown in Figure 8, where the 16x16 sub-matrices along the diagonal represent the intra-CCD crosstalk and the off-diagonal sub-matrices represent the intra-raft crosstalk between segments on different CCDs. The order of magnitude characterization of the intra-CCD crosstalk for each of the 9 CCDs in the RTM is largely consistent. The largest crosstalk is positive, on the order of 10 −4 , and is measured between segments that are immediately adjacent neighbors on the same side of the mid-line break while crosstalk measured between ITL CCD segments on opposite sides of the mid-line break is negative with magnitudes on the order of 10 −5 and 10 −6 . Although the vast majority of the intra-raft crosstalk is below the threshold of \|c ij \| < 5 × 10 −6 , there are patterns of larger crosstalk coefficients along columns of the matrix, corresponding to specific victim segments on CCDs such as R01/S02 and R01/S21. This pattern is not believed to be physical crosstalk, but instead results from elevated read noise in specific victim segments that increases the error on the parameter estimate calculations involving these segments. No crosstalk results were obtained for a single aggressor segment on CCD R01/S00 due to a surface defect on the cryostat window that blocked the projection of a bright spot on this segment during the crosstalk acquisition. Figure 9 shows the full 144x144 raft crosstalk matrix for Teledyne e2v RTM R21. The intra-CCD crosstalk behavior of Teledyne e2v CCDs is consistent between single CCDs but shows a greater abundance of negative crosstalk between segments on the same side of the mid-line break and much lower crosstalk between segments on opposite sides of the mid-line break, compared to ITL CCDs. The intra-CCD crosstalk matrices are also much less symmetric for Teledyne e2v CCDs than for ITL CCDs. The periodic, diagonal patterns seen in the intra-raft crosstalk measurements are caused by residual diffraction effects from the crosstalk projector optics, that repeat as the projector is dithered across the focal plane. Similar patterns can also be seen in the intra-raft crosstalk results for the ITL case, although they are largely subdominant to the previously discussed horizontal noise patterns along columns of the matrix. From these results, it was concluded that there was negligible intra-raft crosstalk for ITL or Teledyne e2v RTMs. There are four cases of inter-raft crosstalk to consider, which were studied using a limited set of RTM pairings ( Table 2): crosstalk between two Teledyne e2v CCDs located on separate rafts, crosstalk between two ITL CCDs located on separate rafts, crosstalk between an aggressor ITL CCD and a victim Teledyne e2v CCD, and crosstalk between an aggressor Teledyne e2v CCD and a victim ITL CCD. In Figure 10 the distributions of crosstalk coefficients for each of the four cases are plotted and compared to a Gaussian distribution, calculated using a chi-square minimization. The standard deviation of the fitted Gaussian distributions is consistent with the standard error on the crosstalk coefficient parameter estimate, indicating that there is no measurable interraft crosstalk above measurement noise. The slightly larger widths of the distributions of inter-raft crosstalk ASTROMETRIC DISTORTIONS A number of sensor effects will modify the effective areas of the pixels in the rectilinear CCD array; the resulting spatial variations in pixel sizes can bias measurements of object astrometry, shape, and flux. These effects can be broadly categorized as static effects independent of pixel signal and dynamic effects that are dependent on pixel signal. 11 Well-known examples of causes of static pixel size variations in deep-depletion CCDs are tree-ring patterns and CCD edge effects. Tree-ring patterns are caused by circularly symmetric variations in dopant impurity concentrations that formed during the silicon boule growing process. 12, 13 CCD edge effects refer to changes to the transverse electric fields that define each pixel near the CCD perimeter, due to the influence of the guard drain voltage. The brighter-fatter effect is a well-studied example of a dynamic pixel size variation, where the repulsive effect of an increasing amount of collected charge within a pixel will decrease the effective pixel size resulting in bright sources that are systematically larger in width. 14 In addition to these previously studied effects, during the integration and testing of the LSST Camera CCDs a number of possible newly recognized causes of pixel size variations have been observed in flat field images, the most prominent being tearing patterns in Teledyne e2v CCDs. 15 Although this tearing pattern, referred to as "classical" tearing, has been largely eliminated by modifications to the operating voltages and clock timings of the Teledyne e2v CCDs, residual tearing patterns consisting of sharp gradients in pixel response near the edges of CCD segments, named "divisadero" tearing, remain. Examination of astrometric and photometric residuals from on-sky dithered images of star fields can be used to characterize the effect of pixel size variations resulting from many of these sensor effects. 16 During the focal plane testing period of the LSST Camera, the spot grid projector provided the capabilities to project a fake star field consisting of a grid of spots to perform similar characterization prior to the on-sky operation of the LSST Camera. This will allow for the possible development of mitigation schemes to be included in the data reduction pipelines, if necessary. Figure 11 shows an image of a single CCD that has been illuminated using the 49x49 spot grid mask installed in the spot grid projector. Because spots near the edges of the grid are further from the optical axis and are greatly affected by the vignetting caused by the commercial lens, it is necessary to use a logarithmic color scale with a linear scaling between ±10 electrons, in order to portray the range in magnitude of the spots in the entire grid. Spots located in the circular central region of the grid provide the largest signal-to-noise when measuring the astrometric and shape distortions caused by sensor effects. Determination of Astrometric and Shape Residuals Measurements of the changes to spot centroid position and shape distortions as a function of pixel location on a CCD are made by calculating the residuals between the measured properties of the observed spots and the properties of an ideal projected grid of spots that has been rotated and magnified. The residual vector ∆ r between the position of an ideal grid spot s and the position of the corresponding detected spot d is where the first term represents optical distortions from the projector, the second term represents deviations of the mask pattern from the ideal, the third term represents the distortions caused by sensor effects dependent on the pixel X/Y location of the spot on the CCD, and the final term is the error term. The optical distortion component ∆ r optics and mask pattern deviations ∆ r mask for an individual spot will be determined only by its column and row position in the spot grid. Therefore, by calculating the mean centroid residual ∆ r over a number of images of the spot located at different CCD pixel locations one can calculate this constant contribution to the spot's centroid residual: ∆ r = ∆ r optics + ∆ r mask . Given a sufficiently large set of measurements spanning a large region of the CCD, the sensor effect distortions terms and the error term are assumed to average to zero. Then, the distortions caused by sensor effects at a particular pixel location on the CCD surface will be ∆ r sensor s+∆ r = ∆ r − ∆ r where the location of the spot is at the CCD position s + ∆ r, the ideal position of the grid spot plus the mean centroid shift caused by the optics and mask variations. By dithering the projected 49x49 grid of spots across the CCD, measurements of the sensor effect distortions can be made at many different pixel positions using the same spot and at the same pixel position using different spots. The above formulation can also be applied to measurements of shape residuals of the spots, with only minor modifications. The shape measurements used in this study were intensity weighted second-moments I ij , where the i, j indices represent the x or y coordinate. The second-moment distortions caused by sensor effects is calculated from the residual ∆I ij,sensor s+∆ r = I ij − I ij (6) after subtracting the mean second-moment value, where the mean is over a set of measurements spanning a large region of the CCD. Astrometric Distortion Measurements The method presented in the previous section was used to measure centroid shifts and shape distortions caused by sensor effects on two CCDs installed in the LSST Camera focal plane: Teledyne e2v CCD R22/S11 and ITL CCD R02/S02. The error terms for the spot centroid and second-moment measurements were first estimated from the standard deviation of the distributions of these measurements taken for 1000 images of the spot grid positioned at the exact same XY-motorplatform position. The approximate value of these errors are also summarized in Table 3. The centroid and second-moment error terms for spots near the center of the spot grid are lower than for spots near the edge of the spot grid. There also is a slight asymmetry between measurements in the x and y directions, corresponding to the serial and parallel directions in the CCD pixel array. For each CCD, 1600 randomly dithered spot grid exposures were taken, where the X/Y positions of the XY-motorplatform used to dither the spot projector were determined by drawing from a uniform distribution centered at the center of each CCD and spanning ±5 mm. Every spot grid image acquisition was taken with an exposure time of 20 seconds to ensure that the spots located in the center of the grid achieved peak brightness of approximately 100 ke − and calibration of the images followed the procedure outlined in Section 2.2. Source identification and measurements were performed using version v18 0 0 of the Rubin Science Pipeline (https: //pipelines.lsst.io) to generate the source catalogs for each of the spot grid images. 17 The Rubin Science Pipeline uses an implementation of the Sloan Digital Sky Survey (SDSS) adaptive moments shape algorithm as one method to calculate the spot centroid and second-moments, 18 which was chosen as the method to be used for the analyses presented in the following sections. The mean spot centroid residual ∆ r for each of the spots in the grid representing centroid shifts caused by the projector optics and mask variations is shown in Figure 12 as a vector field. Here, the grid is centered at the center of the CCD, each spot is plotted at the pixel location where the spot is expected to be imaged, and the displacement vector direction and magnitude correspond to the direction and magnitude of the mean centroid residual. Spots located near the edges of the grid are most affected by the optical distortions, with many experiencing centroid shifts of several pixels, from the ideal grid position. Because of the precision of the photolithographic process used to manufacture the mask, it is expected that any centroid shifts due to mask variations will be subdominant to the optical distortions. Although not visible at the scaling used in Figure 12 there is some evidence for a systematic variation in the spacing between columns of the grid of spots on the photolithographic mask, likely resulting from the manufacturing patterning procedure. The total quantity of data, due to the large number of total spots in the grid and number of exposures, allows for calculations of centroid and shape residuals caused by sensor effects at many points on the CCD surface. The results are presented as 1-to-1 "pseudo-images"; at each CCD pixel coordinate, a Gaussian-weighted average (using a Gaussian with σ x = σ y = 2 pixels) of the residual measurements is calculated. Due to the size of the spot grid relative to that of the CCD and the extent of the random dither, the central region was well sampled using primarily the spots with the largest signal-to-noise ratio. The edge regions of the CCD, however, only have a small number of measurements associated with them taken with lower signal-to-noise and there are some regions of the pixel array where data was completely absent. Figure 13 shows the centroid shifts in the X and Y directions caused by sensor effects on Teledyne e2v CCD R22/S11. The largest centroid shift measured for this CCD is the centroid shift in the y-direction of sources that fall on the mid-line break implant. Although difficult to see at this resolution there is also some indication of the effect of tree-ring patterns at a very low level. A notable feature seen in both the x-direction and y-direction centroid shift results is a large dipole feature located near the center of the CCD. The dipole feature appears visually similar to the central patterns of the optical centroid shifts (Figure 12), although the mean centroid residual of each spot, meant to remove the optical distortion component has been subtracted. This may indicate that it is necessary to calculate the per spot mean centroid residual from images of the grid that have been dithered over a larger portion of the CCD, in order to remove the effect of larger scale structure in the optical distortion mapping. One possibility of a physical cause could be small pixel-area variations caused by strain on the silicon from the thermal, electrical and support connections of the sensor. Future testing of a larger number of CCDs of both types is necessary to determine if this dipole feature is inherent in Teledyne e2v CCDs or a result of a systematic effect in the residual calculations. Figure 14 shows the centroid shift in the X and Y directions caused by sensor effects on ITL CCD R02/S02. The tree-ring patterns are visible in both the x-direction and the y-direction centroid shifts, at a larger amplitude than in the Teledyne e2v CCD results, although still below ±0.1 pixels. The effect of the mid-line break on centroid shifts in the y-direction is also much less pronounced. These results are consistent with pixel signal variations in the flat field images of ITL CCDs and Teledyne e2v CCDs and are caused by differences in the manufacturing and the operating conditions between the two types of CCDs. It is also notable that the dipole feature observed in the Teledyne e2v CCD results is not visible in the ITL CCD results, which provide some evidence that the dipole feature is caused by some property of the Teledyne e2v CCDs. Accurate measurements of second-moment distortions caused by sensor effects are much more challenging due to the lower signal-to-noise; for this reason only the central region of the CCD has sufficient data and reduced Figure 13. Centroid shifts of objects in the x and y directions due to sensor effects on Teledyne e2v CCD R22/S11. Figure 14. Centroid shifts of objects in the x and y directions due to sensor effects on ITL CCD R02/S02. measurement error to show trends above the noise. The results for Teledyne e2v CCD R22/S11 are shown in Figure 15. The effect of the mid-line break on the I yy second-moment is the strongest feature observed, causing changes of approximately 0.1 pixels 2 . There are a number of horizontal streaks in both the I xx and I yy that appear to roughly follow the segment boundaries that are observed in shape distortion measurements, especially along the outer, higher noise regions of the shape distortion pseudo-images that may be signatures of shape distortions caused by divisadero tearing at the segment boundary. The second-moment distortion results for ITL CCD R02/S02, shown in Figure 16, reveal prominent disconti- Figure 15. Changes to the second-moments Ixx and Iyy of objects due to sensor effects on Teledyne e2v CCD R22/S11. nuities between the different CCD segments. The exact cause of these discontinuities is not known at this time, though one possibility is that it is related to the strong serial deferred charge effects that have been measured in ITL CCDs, but are absent in Teledyne e2v CCDs. 19 Once again, the mid-line break is visible in the I yy second-moment results, but at a smaller amplitude than in the Teledyne e2v results. CONCLUSIONS In this study, we have presented a number of studies related to electronic crosstalk and astrometric distortions that have been performed using a series of custom-built optical projectors during the partial focal plane testing period of the LSST Camera. The results of these studies will be important for informing improvements and expansions to the planned testing and analyses to be performed during the upcoming full focal plane testing period. The use of the custom-built crosstalk projector (Section 3) during the partial focal plane test of the LSST Camera has allowed for characterization of the electronic crosstalk at the 10 −6 level. The results presented in this study have shown that there is no detectable crosstalk between CCD segments located on different CCDs, either on the same RTM (intra-raft) or on different RTMs (inter-raft). The largest intra-CCD crosstalk is between immediately adjacent CCD segments located on the same side of the mid-line break and the amplitude of this crosstalk lies below the allowed maximum value of 0.002. Although there are several differences in the intra-CCD crosstalk behavior between ITL and Teledyne e2v CCDs, there is consistent behavior of the crosstalk within each type of CCD. During the upcoming full focal plane testing of the LSST Camera intra-CCD crosstalk measurements will be made for each of the 189 science CCDs and each of the CCDs within the CRTMs. Future crosstalk characterization will also include measurements of intra-CCD crosstalk made for a subset of CCDs at a larger range of aggressor signal values, by adjusting the exposure time used for each crosstalk projector image. This will allow for a greater understanding of any non-linear crosstalk behavior, that will be important for the development and testing of improved algorithms for crosstalk correction. The use of the custom-built spot grid projector has demonstrated the ability to study centroid shifts and shape distortions of sources caused by sensor effects prior to on-sky operation of the LSST Camera. This will allow for identification and characterization of sensor effects that may impact the measurement of source shapes and fluxes needed for weak lensing science. The results of the preliminary measurements presented in this study have shown the ability to measure the effect of tree-ring patterns and the mid-line break on source centroids and shapes. A number of features such as the dipole in Teledyne e2v CCD centroid shift measurements, the discontinuities between segments in ITL CCD second-moment measurements, and the horizontal streaks close to segment boundaries in Teledyne e2v CCD second-moment measurements have also been revealed that will require follow-up study to determine if these are physical features of the sensors. A number of improvements are planned for the upcoming full focal plane testing period: improving the dither pattern by drawing random dither positions from multiple Gaussian distributions positioned at different quadrants of the CCD (to more uniformly cover the CCD with the central spots of the grid), performing smaller dithers around specific regions of interest on a CCD that have shown large pixel response non-uniformities in flat field images, extending the residual analysis to include measurements of spot fluxes and the combined moment I xx + I yy , and increasing the number of CCDs of both manufacturers with a particular focus on CCDs that exhibit strong pixel response non-uniformities in flat field images. By replacing the 450 nm light emitting diode with light sources in other wavelengths, it will be possible to study the wavelength dependence of many of the sensor effects detailed here. The ability to project realistically sized sources is also planned to be leveraged to characterize brighter-fatter effect and signal-dependent deferred charge effects and to exercise the current correction algorithms in the data reduction pipelines.	2021-01-06T02:15:52.068Z	2020-12-14T00:00:00.000	{ "year": 2021, "sha1": "fffea2d474869ceb3df7503e3431b69c7aa4ca2f", "oa_license": null, "oa_url": "http://arxiv.org/pdf/2012.14484", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "fffea2d474869ceb3df7503e3431b69c7aa4ca2f", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Engineering", "Computer Science", "Physics" ] }
249934004	pes2o/s2orc	v3-fos-license	Temporal Changes of Clomiphene on Testosterone Levels and Semen Parameters in Subfertile Men Purpose Clomiphene citrate (CC) is prescribed off-label in men to improve testosterone and sperm parameters, but the duration of treatment needed to reach maximal benefit remains unclear. Our objective was to examine temporal effects of CC on total testosterone (TT) and semen analysis (SA) using longitudinal follow-up data in treated men. Materials and Methods We analyzed an IRB-approved database of men treated with CC (25 mg q.d. or 50 mg q.o.d.) from January 2016 through May 2021. We identified patients with 3, 6, 9, and 12 month follow-up data for TT and 3, 6, and 9 month follow-up SA. Mean absolute changes in TT and sperm concentration compared to baseline were calculated, along with 95% confidence intervals. Men with prior genitourinary procedures or hormone therapy were excluded. Paired t-tests were used to compare TT and sperm concentration at each time point to baseline (alpha=0.05). Results One hundered thirty-four men received CC, mean age 37.7 years (SD 6.7, range 24–52). TT at all follow-ups (3, 6, 9, and 12 months) were available for 25 men, and SA at 3, 6, and 9 months for 26 men. Baseline TT was 358±145 ng/dL and sperm concentration was 13±17.2 M/mL. Significant improvement in TT was identified at 3 months (62.7 ng/dL, 95% CI: 0.49–125.0, p=0.048), additional benefit at 6 months (181.8 ng/dL, 95% CI: 114.1–249.5, p<0.01), and plateau at 9 and 12 months. Improvement in sperm concentration was first observed at 9 months (20.7 M/mL, 95% CI: 10.2–31.2, p<0.01). Semen volume and sperm motility did not change. Conclusions Duration of treatment with clomiphene may impact testosterone and sperm concentration, and the historical 3 month milestone may be insufficient for clinical and research evaluation. Men taking CC may experience plateau in TT at 6 months and first benefit in sperm concentration at 9 months. INTRODUCTION www.wjmh.org scribed to hypogonadotropic men [2] in an attempt to improve serum total testosterone (TT; reference range: 200-1,000 ng/dL) and semen parameters through the modulation of luteinizing hormone (LH; reference range: 2-12 miU/mL) and follicle stimulating hormone (FSH; reference range: 1.6-9 miU/mL). Despite widespread prescribing of CC by reproductive urologists, discussion of CC is sparse within both the American Urological Association [3] and European Association of Urology [4] guidelines on male hypogonadism and infertility. Data supporting the use of CC vary widely: the largest randomized clinical trial by the World Health Organization (WHO) showed no improvement in pregnancy rates or sperm concentration (reference range >15 M/mL) with CC use compared to placebo at 6 months [5], whereas other retrospective studies have shown variable improvements in semen parameters with stricter initial sperm concentration and gonadotropin inclusion criteria [6,7]. Of note, few studies have evaluated outcomes of CC use in men beyond 3 months [8,9], and no temporal pattern of hormonal or seminal response has been identified. We sought to determine the magnitude of improvement in TT and sperm concentration in men taking CC for fertility optimization with long-term interval follow-up to investigate if and when patients may experience a plateau with CC monotherapy. MATERIALS AND METHODS We retrospectively evaluated men presenting to an academic andrology clinic for fertility evaluation who were prescribed CC (25 mg q.d. or 50 mg q.o.d.) from January 2016 through May 2021. We identified men with 3, 6, 9, and 12 month follow-up data for TT and 3, 6, and 9 month follow-up semen analysis (SA). Men with previous genitourinary surgeries, previous or concurrent hormone therapies, and patients without interval TT, LH, FSH, or SA were excluded from the analysis. Our primary endpoint was magnitude of improvement at each time point for TT and sperm concentration compared to baseline. We generated 95% confidence intervals (CIs) for each parameter at each time point. Paired t-tests were calculated to compare change in TT, sperm concentration, semen volume, and sperm motility (reference range: >40%) at each time point compared to baseline. RStudio v. 1.1.463 (RStudio, Inc., Boston, MA, USA) was used for statistical analy-sis, with p<0.05 considered statistically significant. Ethics statement All protocols, extractions, and analyses were approved by the UCLA Institutional Review Board (IRB#20-000710). The written informed consent was waived by the board owing to the retrospective design of the study. RESULTS CC was prescribed to 134 men seeking fertility optimization during the 5-year period. Mean age was 37.7 years (standard deviation [SD], 6.7 y; range, 24-52 y). Of these 134, data for TT at 3, 6, 9, and 12 months were available for 25 men, and SA at 3, 6, and 9 months were available for 26 men. Demographics of the analyzed patients are included in Table 1 and Table 2. In the subset of 25 men with long-term follow-up data, a statistically significant improvement in TT was identified at 3 months (62.7 ng/dL; 95% CI, 0.5-125.0 ng/dL; p=0.048), and additional benefit was seen at 6 months (181.8 ng/dL; 95% CI, 114.1-249.5 ng/dL; p<0.01), without additional statistical improvement at 9 or 12 months (Fig. 1A). Of note, these trends did not change when doing the same analysis on a subset of 40 men with only 3 and 6 month follow-up and in a subset of Values are presented as mean±standard deviation or number (%). LH: luteinizing hormone, FSH: follicle stimulating hormone, TT: total testosterone. All patients (n=26) had semen analyses at 3, 6, and 9 month follow-ups. 32 men with 3, 6, and 9 month follow-up. No patients reported adverse effects of changes in mood, blurred vision, or breast tenderness at any follow-up. Improvement in sperm concentration was first noted at 9 months of CC use (20.7 M/mL; 95% CI, 10.2-31.2 M/mL; p<0.01), without improvement at earlier time points (Fig. 1B). These trends were further confirmed when doing the same analysis on 49 patients with only 3 and 6 month follow-up data and 134 patients with only 3 month follow up data. At no point were there improvements in semen volume or sperm motility (Fig. 1C, 1D). Longitudinal effect of clomiphene citrate on testosterone CC has been shown consistently to improve both TT [10] and symptoms of hypogonadism [10,11] with a lim- Values are presented as mean±standard deviation or number (%). LH: luteinizing hormone, FSH: follicle stimulating hormone, TT: total testosterone. All patients (n=25) had TT labs at 3, 6, 9, and 12 month follow-ups. Differences between interval follow-ups and baseline total testosterone and semen parameters. Interval follow-up data for total testosterone were available for 3, 6, 9, and 12 months of treatment, while semen parameters were available for 3, 6, and 9 months of treatment. 95% confidence interval are shown. Paired t-tests were calculated at each interval time point with significance set at p<0.05. Statistical improvement compared to baseline. *Statistical improvement compared to baseline and compared to 3-month follow up. www.wjmh.org ited side effect profile [9]. AUA guidelines currently recommend CC to improve low testosterone in infertile men with the common practice of using 3 months as the primary endpoint in evaluating efficacy. Our study suggests men may experience the full benefit of CC with longer duration of use, as we observed maximal benefit in TT at 6 months of CC monotherapy before a plateau at the 9 and 12 month follow-up dates. There are some data in the literature to suggest benefit with longer durations of CC in hypogonadism. Krzsatek et al [8] found that a majority of hypogonadal men became eugonadal with at least 3 years of CC use; however, interval data was not provided in this study. Additionally, some studies have reported longitudinal data showing sustained TT response with CC alone; however, these studies do not specifically explore the temporal pattern of response and allowed for up-titration of CC when TT was not maintained at a certain threshold [9,10,12]. Taken together with our results, the data from these studies suggest that 6 months may be a more suitable endpoint in studying CC efficacy than the typical 3-month milestone historically evaluated in the scientific literature. Such data may be helpful in counseling patients regarding expectations for CC treatment duration during shared decision-making conversations in the clinical context. Longitudinal effect of clomiphene citrate on sperm parameters The efficacy of CC for sperm parameters in subfertile men remains less clear. Gundewar et al [13] reported that up to 24% of men may experience a paradoxical decline in sperm concentration during 3-month follow-up, with 17% of patients never recovering to baseline upon discontinuation of CC. Studying CC in male fertility has been stymied by a lack of knowledge on predictors of efficacy; multiple studies have reported that even baseline LH and FSH poorly correlate with sperm parameter improvement at 3-month follow-up [14]. Our data suggest that 3 months may not be a sufficient duration of treatment to experience a fertility benefit from CC. Our cohort of patients first showed statistical improvement in sperm concentration at 9 months. These data may be useful in setting timing expectations for couples trying to conceive, since 9 months (or longer) may be too burdensome a timeline before opting for intrauterine insemination or in vitro fertilization. Indeed, one of the reasons we observe a di-minishing sample size at longer durations of treatment with CC is that couples tend to move forward with advanced reproductive technologies after the frustrating experience of seeing no meaningful improvement in sperm parameters after 3 months of CC, which is typically the timing of first follow-up SA. The relationship between serum testosterone and sperm parameters continues to be an important topic of study in the male fertility research community. For example, dual therapy of CC with anastrozole has been studied with the idea that improving TT and the testosterone-to-estradiol ratio provides a lateral effect on fertility optimization [15]. Other groups have shown that optimizing TT may improve sperm retrieval rates in microsurgical sperm extraction in men with azoospermia [16,17]. While it remains unclear if CC alone is definitively effective in optimizing fertility parameters in subfertile men, longer intervals of treatment will likely be needed to answer this question. Limitations Our study is not without limitations. Data were limited by retrospective analysis and the inability to account for differences that may not be captured in the electronic medical record. This was a single-arm study without a control group and without randomization and was not powered to any single outcome. Patients were offered CC with a dosing regimen of 25 mg q.d. or 50 mg q.o.d., but no distinction was made between the dosing regimens in our retrospective analysis. In prior studies, one research group has evaluated response to CC titration from 25 mg q.o.d. to 50 mg q.o.d. and 50 mg q.d [12]. The same group identified an approximately 7% rate of tachyphylaxis defined as a return to a level within 50 ng/dL from baseline TT [18]. In our study, we found no patients who experienced tachyphylaxis by this definition. Our study was also limited by a small sample size due to inclusion criteria that eliminated patients who did not have all interval time assessments. We therefore could not account for patients who may have achieved benefit with CC at earlier intervals but either terminated therapy or were lost to follow-up. To address this question, we performed independent subset analyses on men with complete data for 3 and 6 months, and on men with complete data for 3, 6, and 9 months. As noted in the Results section, statistical significance did not change on these subset analyses despite the expanded sample sizes. CONCLUSIONS Overall, our study raises the question of what a suitable endpoint may be when studying CC monotherapy in the context of male subfertility and hypogonadism. We pose that 6 months of CC may be needed to achieve maximal benefit in TT while 9 months may be necessary to observe statistical benefit in sperm concentration. The findings from this work may serve as additional data for reproductive urologists to use to counsel men regarding the potential benefits of CC monotherapy for subfertility. Sigalos have no disclosures. Funding None. Data Sharing Statement The data required to reproduce these findings cannot be shared at this time due to personal information protection policy.	2022-06-23T15:23:52.758Z	2022-06-13T00:00:00.000	{ "year": 2022, "sha1": "846f307a33c42cb3f2feafe914170d8b2e18486a", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.5534/wjmh.220010", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4be8c328d3ff7807217d238b52ab24f6bf6441ca", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [] }
7092273	pes2o/s2orc	v3-fos-license	The hazards of death by smoking in middle-aged women Recent studies have found that the risk of death continues to increase among female smokers, as compared with women who have never smoked. We wanted to examine the effect of smoking on all-cause and cause-specific mortality and calculate the corresponding population attributable fraction (PAF) of mortality in the Norwegian women and cancer study; a nationally representative prospective cohort study. We followed 85,320 women, aged 31–70 years, who completed a questionnaire in 1991–1997, through linkages to national registries through December 2008. Questionnaire data included information on lifestyle factors, including lifetime history of smoking. Poisson regression models were fitted to estimate relative risks (RRs) with 95 % confidence intervals (CIs) adjusting for age, birth cohort, education, postmenopausal status, alcohol consumption and body mass index, all at enrollment. During a mean follow-up time of 14 years 2,842 deaths occurred. Compared with that of never smokers, current smokers had a mortality rate that was double (RR = 2.34; 95 % CI 2.13–2.62) from deaths overall, triple (RR = 3.30; 95 % CI 2.21–4.82) from cerebrovascular disease and myocardial infarction (RR = 3.65; 95 % CI 2.18–6.15), 12 times (RR = 12.16; 95 % CI 7.80–19.00) from lung cancer and seventeen times (RR = 17.00; 95 % CI 5.90–48.78) from chronic obstructive pulmonary diseases. The PAF of mortality due to smoking was 34 % (CI 30–39). In summary, one in three deaths among middle aged women in Norway could have been prevented if the women did not smoke. More middle-aged women, than ever before, are dying prematurely due to smoking in Norway. Introduction Worldwide, tobacco use causes annually more than five million deaths, or one death every 6 seconds. Unless current smokers stop smoking before or during middle age, this number is expected to rise to eight million or more by the year 2030 [1][2][3]. In spite of the overwhelming evidence of the detrimental effects of smoking on health [4,5], the tobacco use among women is rising globally and the age of initiation of daily smoking among women seems to have become as young as it is in men [6,7]. For women in Norway, the prevalence of daily smokers was 23 % in 1954, the peak was at 37 % in 1970 and then it stabilized at around 32 % for the rest of the century [8]. The decline among female smokers first started in the twentyfirst century [9]. As a consequence the lung cancer incidence and mortality rates have both been increasing for Norwegian women. In fact, the strongest increase in cancer incidence from the past 5-year period (2001)(2002)(2003)(2004)(2005) until the current one (2006-2010) occurred in lung cancer (16%) [10]. The four tobacco epidemic stages model suggested by Lopez et al. almost 20 years ago described the effects on mortality of the cigarette epidemic in Western countries such as the USA, UK and Australia. The rise and fall in female smoking and in smoking-attributed mortality usually lagged behind that in men by about 20-30 years [11]. Recent studies have found that for women the risk of death from cigarette smoking continues to increase resulting in a population attributable fraction (PAF) of mortality that is larger than previously reported [4,[12][13][14][15]. As pointed out by Oza et al. [16], estimating the number of deaths attributable to smoking is needed for priority setting and monitoring the disease burden associated with this risk factor in different populations. Today, the majority of middle-aged Norwegian women are either former or current smokers, who started to smoke in their teens [17]. We wanted to estimate the effects of cigarette smoking on all-cause and cause-specific mortality and estimate the proportion of deaths attributable to smoking in the Norwegian Women and Cancer (NOWAC) study, a nationally representative prospective cohort study. Study population The NOWAC study is a nationally representative prospective cohort study comprising a sample of the Norwegian middle-aged female population. The cohort profile has been previously described in detail [18,19]. Briefly, a random sample of women was selected from the central population register according to year of birth. These women were sent a letter of invitation to participate in the study, which also contained a questionnaire, and a prestamped return envelope. The National Data Inspectorate and the Regional Committee for Medical Research Ethics approved the study. All women gave an informed consent (http://site.uit.no/kvinnerogkreft). The inclusion criteria for the present study were women born from 1926 to 1965, who during 1991-1997 completed an initial questionnaire, containing questions on life style factors as smoking and alcohol consumption (N = 95,947). The overall response rate was 57 %. Women who emigrated or died (N = 22) before the start of follow-up were excluded. We excluded 10,605 women due to missing information on either smoking status (N = 2,224) or any of the co-variables (education, BMI, menopausal status and alcohol consumption) under study. Altogether 458 women with missing information had died during follow-up of which 64 (14 %) had missing information on smoking status. The analytical cohort comprised the remaining 85,320 women with complete information in the multivariable analyses. Compared with the women in the analytical cohort, the women that were excluded due to missing information were on average 2.7 years older, but were similar according to education, BMI and alcohol consumption (data not shown). Exposure information The baseline questionnaire included a detailed assessment of lifetime smoking history. The questionnaires asked if the women had ever smoked, and those answering ''yes'' were asked the number of cigarettes smoked daily at different ages. Subsequently, they were asked if they currently smoked on a daily basis. We categorized ever smokers according to current and former smoking status at enrollment. All women who were neither current nor former daily smokers were classified as never smokers. The baseline questionnaires also asked about years of education, height and current weight (allowing us to calculate body mass index, BMI, kg/m 2 ), menopausal status (pre-, peri-, postmenopausal), postmenopausal hormone therapy (yes, no), hysterectomy (yes, no), including alcohol consumption, all at enrollment. We calculated average consumption of alcohol in g/day based on the content of pure alcohol in different sorts of beverages and usual portion sizes in Norway among drinkers. Women who reported to be teetotalers and those answering ''seldom'' or ''never'' had their alcohol consumption set to zero. Follow-up and endpoints We followed the women through linkages to the Norwegian Central Population Register, utilizing the unique national birth number to identify all cases of deaths and emigrations. The end point in our study was death from any cause during follow-up, which ended December 31st, 2008. All causes of death were coded according to the original codes in the International Classification of Diseases, Ninth Revision (ICD-9) before 1996 and the original codes of the ICD-10 from 1996 and after. The deaths were classified into three main outcome categories: death from cancer (C00-C97), cardio-vascular diseases (CVDs) (I00-I99), respiratory diseases (J00-J99). The other deaths were collapsed in one group. Statistical analysis We used Poisson regression models (with attained age between cohort entry and exit as the underlying time variable) to obtain adjusted relative risks (RRs) with two-sided 95 % Wald confidence intervals (CI) that compared categories of smokers (current, former, ever) with never smokers treated as a fixed reference group. We fitted regression models by splitting the follow-up-time into 1-year intervals [20]. Risk time was counted from date of enrolment until censoring due to death, emigration or end of follow-up (December 31st, 2008), whichever occurred first. We estimated the age-and multivariable adjusted association between smoking status at enrolment and mortality overall and for 11 selected categories of disease outcome. To indicate what proportion of the deaths in the population that would not occur if smoking were eliminated we calculated the PAF as described in the WHO global report [4], using the formula: where the notation P e = the proportion of persons in the population exposed to the risk factor i.e. ever smokers and RR e = the RR in the exposed compared to the unexposed group; i.e. ever compared with never smokers. We calculated the two-sided 95 % CI's for the PAF's using the delta method [21]. All statistical tests were two-sided and were considered statistically significant at p B 0.05. The SASÓ software version 9.22 was used for all statistical analyses. Results Altogether 2,842 deaths occurred during the approximately 1.2 million woman-years of observation with a mean follow-up time of 14 years. Among the deaths, 1,800 (63 %) were due to cancer, 426 (15 %) due to CVD, 108 (4 %) due to respiratory diseases and 508 (18 %) to remaining causes. Overall, 65 % (n = 55,227) of the women reported to be ever (35 % current and 30 % former) smokers. Table 1 shows the distribution of selected characteristics according to smoking status at enrollment. Current smokers were younger, had fewer years of education, a lower BMI and consumed more alcohol than never smokers (Table 1). Both former and current smokers had, compared with never smokers, a significantly increased mortality rate from Alcohol consumption (g/day) b 2 (3.9) 3 (5.8) 4 (7.0) SD standard deviation, BMI body mass index a Given as mean (SD) b Among drinkers The hazards of death due to smoking 801 all three main outcome categories; cancer-, cardiovascular-and respiratory diseases. The association between smoking and the different mortality outcomes displayed in Table 2 was stronger for current than for former smokers. The highest increased mortality rate was from COPD (RR = 17.00; 95 % CI 5.90-48.78) when current was compared with never smokers (Table 2). Table 3 shows that the PAF of deaths among ever smokers was 34 % (95 % CI 30-39) compared with never smokers. The corresponding figure for lung cancer was 79 % (95 % CI 72-86) and for COPD 85 % (95 % CI 73-97) ( Table 3). Discussion Our study shows that compared with never smokers, both former and current smokers at cohort enrollment in the 1990s have an increased risk of dying overall, as well as dying from the three main outcome categories; total cancer, total CVD and total respiratory diseases. The increased risk was more pronounced for current than for former smokers indicating the effects of quitting smoking. One in three deaths in this female population may be attributed to smoking and could have been avoided if the women did not smoke. In our study, except for cancers not established to be smoking-related [5], current smoking was associated with an significantly increased risk of mortality from all categories of cause-specific mortality examined (lung cancer, other smoking-related cancers, circulatory diseases, MI, cerebrovascular diseases, COBP, and other respiratory diseases). We find a three-fold mortality rate among current smokers compared with that of never smokers for cerebrovascular diseases and MI, similar to those displayed in the Nurses' Health study [25]; and in the Million Women Study [13], while our mortality rate from COPD and from lung cancer was much lower than in the other two studies. The US study [25], had more than 12,000 deaths during a follow-up time of 24 years, while the UK study [13], had 66,000 deaths after 12 years. Both studies [25] had a RR of mortality from lung cancer of more than 20, while the mortality rate from COPD was 40 and 35, respectively. We expect that the deaths related to lung cancer and COPD, which have a long natural history, will increase when we have a longer follow-up time. Also, Kenfield et al. [26] showed in a later follow-up study that exposure assessed only at enrollment, underestimated the mortality risk due to smoking, compared with a second assessment during follow-up, especially for COPD and lung cancer. The PAF of mortality due to smoking among Norwegian women has previously been estimated to be less than 20 % when mortality rates from lung cancer were used as an indirect measure of cigarette smoking [4,12,29], when cohort studies from the mid-70 s were used [23] and when the smoking prevalence surveys published by the 25-29.9, C30 kg/m 2 ), menopausal status (pre-, peri-, postmenopausal, postmenopausal hormone therapy, hysterectomy), alcohol consumption (0, [0-4, C5 g/day), all at enrollment and birth cohort (1927-1936,1937-1946, 1947-1956, 1957-1964 The hazards of death due to smoking 803 Norwegian Central Bureau of Statistics were used [8,30] Thun et al. [12], estimated that in 2009, the PAF of mortality due to smoking was highest (26-30 %) among women aged 35-69 years, in The Netherlands, Denmark, Hungary and Canada. In our study, based on individual data on both exposure and outcome, the corresponding PAF was estimated to be 34 % in Norway in 2008, with the upper limit of the 95 % CI as high as 39 %. The life expectancy for Norwegian females was more than 83 years in 2008 [31]. The low background mortality rate for women, which is the denominator of our RRs, leads to greater PAF's of mortality due to smoking. Our finding of a high proportion of deaths that could have been avoided if nobody was smoking is in accordance with the rapidly increasing lung cancer incidence for Norwegian women. The age adjusted world lung cancer incidence rate more than doubled from 11.7 per 100,000 person-years in the 5-year period (1986-1990) immediately before the commencement of our study to 25.1 per 100,000 person-years for the current 5 year period including the year which our follow-up ended [10]. This increasing lung cancer incidence also tells us that the PAF of mortality due to smoking among Norwegian women has not peaked yet. As pointed out by Jha [3], the full effects of smoking can take 50 years to measure in individuals, and up to 100 years to measure in populations. Our study shows the PAF due to smoking among females already to be higher than the peak in the gender specific model recently developed by Thun et al. [12]. 5,18.5-24.9, 25-29.9, C30 kg/m 2 ), menopausal status (pre-, peri-, postmenopausal, postmenopausal hormone therapy, hysterectomy), alcohol consumption (0, [0-4, C5 g/day), all at enrollment and birth cohort (1927-1936,1937-1946, 1947-1956, 1957-1964 The most important strength of our study is that it is a nationally representative prospective cohort study allowing us to calculate the PAF of mortality due to smoking for middle-aged women. We know from our previous studies that the smoking exposure [17,32,33] and the cancer incidence [19] reflect known smoking patterns [8,9] and cancer incidence [10] for Norwegian women. Thus, we are confident that our cohort is representative of the Norwegian female population, born between 1926 and 1965, both according to exposure and outcome of this study. Other major strengths of our study are; we have a high proportion of both current and former smokers, virtually complete follow-up through the National population based registries and the possibility of examining the association with smoking according to both all-cause and cause-specific mortality. Another force is that we focus our PAF estimates on the comparison between ever versus never smokers. Thus, it is only never smokers that could possibly change smoking status during follow-up. Since very few Norwegians start to smoke after the age of 30 and the mean age at enrolment for our study is more than 40 years, we are confident that the possible changes in smoking status among the never smokers during follow-up did not influence our PAF estimates. Furthermore, we have detailed information on, and were able to control for alcohol consumption and BMI, which are established risk factors for mortality. Limitations One major limitation of this report is that we have a limited number of deaths. Furthermore, 9 % of the women had missing values for physical activity levels and therefore this variable was not included in the multivariable analyses as there was no difference in physical activity levels according to smoking status. As in our study, neither of the seven [13-15, 25, 26, 34, 35] recently published studies did find much effect of confounding when they compared the effect of smoking on mortality. Five of these studies were conducted in the US [14,15,25,26,34]; one in the UK [13], and one in China [35]. We found a small non-significantly increased risk of deaths from cancers not established to be associated with smoking. This may be due to chance or that breast cancer should be in the category for smoking-related cancers. According to the most recent IARC monograph cigarette smoking is possibly carcinogenic to the human breast [5]. After this monograph was published two large cohort studies, one from the US [36], and our own Norwegian study [37] based on more than 300,000 women, conclude that smoking initiation before the first childbirth increase the risk of breast cancer. Other limitations are that we have not validated selfreported information on height and body weight and that the current PAF formula does not provide a fully adjusted estimate of PAF due to lack of adjustment of the prevalence. We chose not to exclude women with prevalent disease as a previous study from the same cohort which examined physical activity and mortality found basically the same results when they did their analyses with and without women with prevalent disease at enrolment [38]. This has most likely deflated or RR estimates of mortality. Given the reduction in life expectancy associated with smoking we cannot rule out that part of the cause-specific associations is hidden or obscured by the competing causes of death with increasing age. We do not expect that the changes in BMI or alcohol consumption during follow-up would influence our results to any great extent. Nevertheless, we cannot rule out the possibility of residual confounding due to the above described factors, or other factors we did not measure. In summary, one in three deaths among middle aged women in Norway could have been prevented if the women did not smoke. More middle-aged women, than ever before, are dying prematurely due to smoking in Norway.	2016-05-12T22:15:10.714Z	2013-09-29T00:00:00.000	{ "year": 2013, "sha1": "0ce73ec4278fae54cec92be9f81301246b85dac3", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10654-013-9851-6.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "0ce73ec4278fae54cec92be9f81301246b85dac3", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
42102665	pes2o/s2orc	v3-fos-license	Dormant Polymers and Their Role in Living and Controlled Polymerizations; Influence on Polymer Chemistry, Particularly on the Ring Opening Polymerization Living polymerization discovered by Professor Szwarc is well known to all chemists. Some of the living polymerizations involve dormancy, a process in which there is an equilibrium (or at least exchange) between two types of living polymers, namely active at the given moment and dormant at this moment and becoming active in the process of activation. These processes are at least equally important although less known. This mini review is devoted to these particular living polymerizations, mostly polymerizations by the Ring-Opening Polymerization mechanisms (ROP) compared with some selected close to living vinyl polymerizations (the most spectacular is Atom Transfer Radical Polymerization (ATRP)) involving dormancy. Cationic polymerization of tetrahydrofuran was the first one, based on equilibrium between oxonium ions (active) and covalent (esters) dormant species, i.e., temporarily inactive, and is described in detail. The other systems discussed are polymerization of oxazolines and cyclic esters as well as controlled radical and cationic polymerizations of vinyl monomers. Introduction Professor Michael (Michał in Polish) Szwarc's contributions to the chemical science are not only related to polymers and to his discovery of living and (less known) dormant polymers [1]. Dormant polymers, reversibly formed from the active ones, are at the basis of several living/controlled polymerizations described in the present paper. Moreover, Professor Szwarc is also well known for his work on the bond energy (continued for some time in Syracuse) and on the electron transfer phenomena [2,3]. In 1956, during discussion with Samuel Weissman on the electron transfer in aromatic compounds, Szwarc asked Weissman about styrene (cit.) "Did you transfer electrons to styrene?" The answer he received was: (cit.) "No use, it polymerizes" [4]. Szwarc then realized that electron transfer in the case of styrene not only leads to polymerization, but also to creation of living active species which was indicated by the persistent red color. Since then, working with his students/coworkers, Moshe Levy, Ralph Milkovich, Joseph Jagur, Johannes Smid and several postdocs, he has answered all major questions concerning the anionic polymerization, and is acknowledged by all chemists to be the "father of the living polymerization". Thus, despite his other important contributions to chemistry, the discovery of the living and dormant polymers is rightly considered to be his greatest achievement. In the recently published "Perspectives" by Grubbs and Grubbs [5], it is amply shown that discovery Dormant Species in the Ring-Opening Polymerization (ROP) Probably, the first process that would be called today "living-dormant" was described by Flory already in 1944 for an anionic polymerization of ethylene oxide in the presence of an alcohol (Scheme 1) [27]. This process leads to the Poisson distribution of molar masses derived based on this polymerization [27,28]. As follows from Scheme 1, R'OH is the chain transfer agent that is present from the beginning of the polymerization (at first, as a low molar mass alcohol and later as macro alcohol). RO(CH2CH2O)CH2CH2OH is at the same time a chain transfer agent and a dormant, temporarily inactive polymer chain, reversibly converting in the reaction with macroanion R'O − into an active form-a living macromolecule. In several, later studied ring-opening polymerizations, living-dormant interconversions take place. However, species that look dormant may not necessarily be "completely inactive" and may retain partial activity as it has been documented in the polymerization of tetrahydrofuran (THF) initiated with triflic acid (trifluoromethane sulfonic acid, TfOH) [29], or in the polymerization of oxazolines initiated with alkylhalides [30]. In these systems, dormant macromolecules may have some activity yet many times less than the active ones and rightly could be considered as dormant. The equilibrium involving active and dormant macromolecules that takes place in the ROP of THF initiated by TfOH is given in Schemes 2-5 [31]. Due to the relatively slow ion-ester exchange rate (on the NMR time scale), both the covalent species and the ion pairs can be traced by 1 H-NMR ( Figure 1). Their relative concentrations strongly depend on the polarity of the solvent [31]. The conversion of the covalent species into the ionic ones proceeds with an anchimeric assistance (a neighboring group participation), and therefore is much faster than the reaction of the monomer with the covalent species (Scheme 3). This is a unimolecular opposed reaction (an ion pair is kinetically one species), and since its rate is sufficiently low, its rate constant was measured by 1 H-NMR [32]. It has been noticed that the direct covalent growth cannot take place, as it is forbidden by the symmetry rules. Covalent species may grow only by intermolecular conversion into ionic species, larger by one unit, when the macroester Scheme 1. An anionic polymerization of ethylene oxide in the presence of an alcohol. As follows from Scheme 1, R'OH is the chain transfer agent that is present from the beginning of the polymerization (at first, as a low molar mass alcohol and later as macro alcohol). RO(CH 2 CH 2 O)CH 2 CH 2 OH is at the same time a chain transfer agent and a dormant, temporarily inactive polymer chain, reversibly converting in the reaction with macroanion R'O´into an active form-a living macromolecule. In several, later studied ring-opening polymerizations, living-dormant interconversions take place. However, species that look dormant may not necessarily be "completely inactive" and may retain partial activity as it has been documented in the polymerization of tetrahydrofuran (THF) initiated with triflic acid (trifluoromethane sulfonic acid, TfOH) [29], or in the polymerization of oxazolines initiated with alkylhalides [30]. In these systems, dormant macromolecules may have some activity yet many times less than the active ones and rightly could be considered as dormant. The equilibrium involving active and dormant macromolecules that takes place in the ROP of THF initiated by TfOH is given in Schemes 2-5 [31]. Dormant Species in the Ring-Opening Polymerization (ROP) Probably, the first process that would be called today "living-dormant" was described by Flory already in 1944 for an anionic polymerization of ethylene oxide in the presence of an alcohol (Scheme 1) [27]. This process leads to the Poisson distribution of molar masses derived based on this polymerization [27,28]. As follows from Scheme 1, R'OH is the chain transfer agent that is present from the beginning of the polymerization (at first, as a low molar mass alcohol and later as macro alcohol). RO(CH2CH2O)CH2CH2OH is at the same time a chain transfer agent and a dormant, temporarily inactive polymer chain, reversibly converting in the reaction with macroanion R'O − into an active form-a living macromolecule. In several, later studied ring-opening polymerizations, living-dormant interconversions take place. However, species that look dormant may not necessarily be "completely inactive" and may retain partial activity as it has been documented in the polymerization of tetrahydrofuran (THF) initiated with triflic acid (trifluoromethane sulfonic acid, TfOH) [29], or in the polymerization of oxazolines initiated with alkylhalides [30]. In these systems, dormant macromolecules may have some activity yet many times less than the active ones and rightly could be considered as dormant. The equilibrium involving active and dormant macromolecules that takes place in the ROP of THF initiated by TfOH is given in Schemes 2-5 [31]. Due to the relatively slow ion-ester exchange rate (on the NMR time scale), both the covalent species and the ion pairs can be traced by 1 H-NMR ( Figure 1). Their relative concentrations strongly depend on the polarity of the solvent [31]. The conversion of the covalent species into the ionic ones proceeds with an anchimeric assistance (a neighboring group participation), and therefore is much faster than the reaction of the monomer with the covalent species (Scheme 3). This is a unimolecular opposed reaction (an ion pair is kinetically one species), and since its rate is sufficiently low, its rate constant was measured by 1 H-NMR [32]. It has been noticed that the direct covalent growth cannot take place, as it is forbidden by the symmetry rules. Covalent species may grow only by intermolecular conversion into ionic species, larger by one unit, when the macroester Due to the relatively slow ion-ester exchange rate (on the NMR time scale), both the covalent species and the ion pairs can be traced by 1 H-NMR ( Figure 1). Their relative concentrations strongly depend on the polarity of the solvent [31]. The conversion of the covalent species into the ionic ones proceeds with an anchimeric assistance (a neighboring group participation), and therefore is much faster than the reaction of the monomer with the covalent species (Scheme 3). This is a unimolecular opposed reaction (an ion pair is kinetically one species), and since its rate is sufficiently low, its rate constant was measured by 1 H-NMR [32]. It has been noticed that the direct covalent growth cannot take place, as it is forbidden by the symmetry rules. Covalent species may grow only by intermolecular conversion into ionic species, larger by one unit, when the macroester The complete set of equilibrating species in polymerization of THF initiated with triflic acid or its esters is schematically presented in Scheme 5 [33]. Polymers 2017, 9, 644 6 of 18 The complete set of equilibrating species in polymerization of THF initiated with triflic acid or its esters is schematically presented in Scheme 5 [33]. Scheme 5. The set of ion-ester equilibria in the polymerization of THF initiated with TfOH. In the polymerization of THF initiated by triflic acid the rate constants for the various conditions have been measured. Since the detailed discussion of the mechanism of an ionic and a covalent propagation and determination of the rate constants is out of the scope of this paper, the interested reader may find these information in [31][32][33]. Nevertheless, establishing those kinetic parameters allowed determination the times of an average macromolecule to exist in ionic and covalent state. Furthermore, it could be calculated how many monomer molecules are added at these periods of time. These findings are illustrated below. The record of life of a macromolecule that undergoes interconversions between active and dormant states can be visualized as sections marked on the line, where the lengths of these sections are equal to the time spent in a given state (Scheme 6). Thus, an average macromolecule propagates on ionic species for a certain time, adding X monomer molecules, then the ion-pair collapses into the covalent species. If these are sufficiently less reactive than the ionic species (kp >> kci), or unreactive at all, then the covalent species are dormant ones. Then, the macromolecule spends some time as an inactive (dormant) and recovers to the ionic state due to internal or external ionization (kci and/or kci'). Scheme 6. Periods of activity and inactivity (dormancy). In Scheme 7, it is shown how these active and inactive periods oscillate in the polymerization of THF in two solvents of different polarity (CCl4 and CH3NO2). Thus, even in CCl4, where a macromolecule is in a dormant state much larger than in an ionic state (124 s vs. 8.1 s), there is approximately 10 times larger number of monomer molecules introduced into the chain by ionic chain ends. This ratio is even more than 500 in CH3NO2 solvent. We have described the ionic polymerization of THF in some details, since it is the most general one (for ROP) and, moreover, the most thoroughly studied. Other polymerizations, e.g., polymerization of oxazolines or even the controlled radical polymerization, either proceed in the same way or are almost identical kinetically [34]. In the polymerization of THF initiated by triflic acid the rate constants for the various conditions have been measured. Since the detailed discussion of the mechanism of an ionic and a covalent propagation and determination of the rate constants is out of the scope of this paper, the interested reader may find these information in [31][32][33]. Nevertheless, establishing those kinetic parameters allowed determination the times of an average macromolecule to exist in ionic and covalent state. Furthermore, it could be calculated how many monomer molecules are added at these periods of time. These findings are illustrated below. The record of life of a macromolecule that undergoes interconversions between active and dormant states can be visualized as sections marked on the line, where the lengths of these sections are equal to the time spent in a given state (Scheme 6). Thus, an average macromolecule propagates on ionic species for a certain time, adding X monomer molecules, then the ion-pair collapses into the covalent species. If these are sufficiently less reactive than the ionic species (k p >> k ci ), or unreactive at all, then the covalent species are dormant ones. Then, the macromolecule spends some time as an inactive (dormant) and recovers to the ionic state due to internal or external ionization (k ci and/or k ci' ). The complete set of equilibrating species in polymerization of THF initiated with triflic acid or its esters is schematically presented in Scheme 5 [33]. In the polymerization of THF initiated by triflic acid the rate constants for the various conditions have been measured. Since the detailed discussion of the mechanism of an ionic and a covalent propagation and determination of the rate constants is out of the scope of this paper, the interested reader may find these information in [31][32][33]. Nevertheless, establishing those kinetic parameters allowed determination the times of an average macromolecule to exist in ionic and covalent state. Furthermore, it could be calculated how many monomer molecules are added at these periods of time. These findings are illustrated below. The record of life of a macromolecule that undergoes interconversions between active and dormant states can be visualized as sections marked on the line, where the lengths of these sections are equal to the time spent in a given state (Scheme 6). Thus, an average macromolecule propagates on ionic species for a certain time, adding X monomer molecules, then the ion-pair collapses into the covalent species. If these are sufficiently less reactive than the ionic species (kp >> kci), or unreactive at all, then the covalent species are dormant ones. Then, the macromolecule spends some time as an inactive (dormant) and recovers to the ionic state due to internal or external ionization (kci and/or kci'). Scheme 6. Periods of activity and inactivity (dormancy). In Scheme 7, it is shown how these active and inactive periods oscillate in the polymerization of THF in two solvents of different polarity (CCl4 and CH3NO2). Thus, even in CCl4, where a macromolecule is in a dormant state much larger than in an ionic state (124 s vs. 8.1 s), there is approximately 10 times larger number of monomer molecules introduced into the chain by ionic chain ends. This ratio is even more than 500 in CH3NO2 solvent. We have described the ionic polymerization of THF in some details, since it is the most general one (for ROP) and, moreover, the most thoroughly studied. Other polymerizations, e.g., polymerization of oxazolines or even the controlled radical polymerization, either proceed in the same way or are almost identical kinetically [34]. In Scheme 7, it is shown how these active and inactive periods oscillate in the polymerization of THF in two solvents of different polarity (CCl 4 and CH 3 NO 2 ). Thus, even in CCl 4 , where a macromolecule is in a dormant state much larger than in an ionic state (124 s vs. 8.1 s), there is approximately 10 times larger number of monomer molecules introduced into the chain by ionic chain ends. This ratio is even more than 500 in CH 3 NO 2 solvent. We have described the ionic polymerization of THF in some details, since it is the most general one (for ROP) and, moreover, the most thoroughly studied. Other polymerizations, e.g., polymerization of oxazolines or even the controlled radical polymerization, either proceed in the same way or are almost identical kinetically [34]. Polymerization of oxazolines (OX), with the interconversion between living and dormant species, has been elaborated in detail by Saegusa and Kobayashi [35,36]. More recently, kinetic analysis of these systems has been given by Dworak [37]. The alkyl halides, in order to grow as such (covalent directly to covalent), must form the fourmembered transition state, which is forbidden according to the symmetry rules. Thus, for the propagation to proceed, an interconversion of covalent-dormant species into the ionic ones is required. Ionization of covalent dormant species proceeds as for the polymerization of THF, almost exclusively intramolecularly (Scheme 8). Dworak has calculated, that intermolecular ionization with participation of monomer, in order to be competitive, would require the impossible to reach concentration of the monomer equal to ~10 3 mol/L. Scheme 8. Polymerization of oxazoline with iodomethane [35,36]. In all these systems, the interconversions are fast comparing with the rates of growth. Thus, there are several interconversions for one monomer molecule that reacted in an act of propagation [37]. Active-Dormant Interconversion in Polymerization of Cyclic Esters The first catalytic system, successfully used in the polymerization of cyclic esters, was based on metal alkoxides (e.g., aluminum isopropoxide) and the polymerization could be considered as living Scheme 7. Conditions: [THF] 0 = 8 mol¨L´1; 25˝C [26,34], where τ is time, X is the number of monomer molecules, and indexes i, c, p and d stand for ionic and covalent propagation and depropagation, respectively. Polymerization of oxazolines (OX), with the interconversion between living and dormant species, has been elaborated in detail by Saegusa and Kobayashi [35,36]. More recently, kinetic analysis of these systems has been given by Dworak [37]. The alkyl halides, in order to grow as such (covalent directly to covalent), must form the four-membered transition state, which is forbidden according to the symmetry rules. Thus, for the propagation to proceed, an interconversion of covalent-dormant species into the ionic ones is required. Ionization of covalent dormant species proceeds as for the polymerization of THF, almost exclusively intramolecularly (Scheme 8). Dworak has calculated, that intermolecular ionization with participation of monomer, in order to be competitive, would require the impossible to reach concentration of the monomer equal to~10 3 mol/L. Polymerization of oxazolines (OX), with the interconversion between living and dormant species, has been elaborated in detail by Saegusa and Kobayashi [35,36]. More recently, kinetic analysis of these systems has been given by Dworak [37]. The alkyl halides, in order to grow as such (covalent directly to covalent), must form the fourmembered transition state, which is forbidden according to the symmetry rules. Thus, for the propagation to proceed, an interconversion of covalent-dormant species into the ionic ones is required. Ionization of covalent dormant species proceeds as for the polymerization of THF, almost exclusively intramolecularly (Scheme 8). Dworak has calculated, that intermolecular ionization with participation of monomer, in order to be competitive, would require the impossible to reach concentration of the monomer equal to ~10 3 mol/L. In all these systems, the interconversions are fast comparing with the rates of growth. Thus, there are several interconversions for one monomer molecule that reacted in an act of propagation [37]. Active-Dormant Interconversion in Polymerization of Cyclic Esters The first catalytic system, successfully used in the polymerization of cyclic esters, was based on metal alkoxides (e.g., aluminum isopropoxide) and the polymerization could be considered as living Scheme 8. Polymerization of oxazoline with iodomethane [35,36]. In all these systems, the interconversions are fast comparing with the rates of growth. Thus, there are several interconversions for one monomer molecule that reacted in an act of propagation [37]. Active-Dormant Interconversion in Polymerization of Cyclic Esters The first catalytic system, successfully used in the polymerization of cyclic esters, was based on metal alkoxides (e.g., aluminum isopropoxide) and the polymerization could be considered as living and controlled. One of the first comprehensive papers discussing this issue has been published by the Liège group of Teyssié, Jerome and Dubois [38]. Aluminum isopropoxide exists in solutions as a mixture of a trimer and tetramer (Scheme 9). and controlled. One of the first comprehensive papers discussing this issue has been published by the Liège group of Teyssié, Jerome and Dubois [38]. Aluminum isopropoxide exists in solutions as a mixture of a trimer and tetramer (Scheme 9). Scheme 9. Trimer (A3) and tetramer (A4). There was a long-lasting controversy, whether all alkoxy groups initiate or only some of them. This controversy has been resolved in our institute, as we have shown in the polymerization of εcaprolactone (CL) that in non-protonic solvents and at 25 °C in the mixture of a trimer and tetramer only trimer (cf. Scheme 9) initiates. When monomer conversion is complete, the tetramer remains intact [39]. On the other hand, Kricheldorf used higher temperatures (close to 100 °C) which caused all the alkoxy groups to be active (although the proportions of trimer and tetramer were not known) [40]. This means that at these conditions "dormant tetramer" has enough time to be converted into reactive trimer before polymerization is over. The difference in reactivities of trimer and tetramer stems from the fact, that in trimer the central Al atom is pentacoordinated and in tetramer hexacoordinated. Hexacoordinated atom does not have any coordination sites left to coordinate the monomer (cf. Scheme 9). Apparently, coordination with the central atom is a necessary condition for initiation. This may account, as Szwarc pointed out while discussing this system [25] (p. 123), for the thousand-fold faster reaction of trimer [39]. In many other systems, initiators and/or growing ionic macromolecules may form aggregates and only unimers are reactive or one of the forms of aggregates. Not knowing these subtle differences and not realizing the presence of inactive-dormant isomers, one may easily incorrectly determine concentration of active centers, and, as follows, the involved rate constants of elementary reactions. In the polymerization of cyclic esters formation of dormant aggregated macromolecules has been first shown for CL and L-lactide (LA), initiated by dialkylalkoxy aluminums, for which a kinetic equation has been developed. This allows simultaneous determination of the degree of aggregation "m", equilibrium constant and rate constants of propagation [8]. In this case, the extent of aggregation can also be determined first from the dependence of the log of rate of polymerization, rp on log [P]total ([P]total = [I]0), where [P]total is the total concentration of the active and dormant speciesinstantaneously active and dormant [41]. The corresponding kinetic system is shown in Scheme 10. The specific solution of equations describing this system for m = 2 has been given by Szwarc [25] (p. 108) and the general solution for any "m" has been proposed later [8]. There was a long-lasting controversy, whether all alkoxy groups initiate or only some of them. This controversy has been resolved in our institute, as we have shown in the polymerization of ε-caprolactone (CL) that in non-protonic solvents and at 25˝C in the mixture of a trimer and tetramer only trimer (cf. Scheme 9) initiates. When monomer conversion is complete, the tetramer remains intact [39]. On the other hand, Kricheldorf used higher temperatures (close to 100˝C) which caused all the alkoxy groups to be active (although the proportions of trimer and tetramer were not known) [40]. This means that at these conditions "dormant tetramer" has enough time to be converted into reactive trimer before polymerization is over. The difference in reactivities of trimer and tetramer stems from the fact, that in trimer the central Al atom is pentacoordinated and in tetramer hexacoordinated. Hexacoordinated atom does not have any coordination sites left to coordinate the monomer (cf. Scheme 9). Apparently, coordination with the central atom is a necessary condition for initiation. This may account, as Szwarc pointed out while discussing this system [25] (p. 123), for the thousand-fold faster reaction of trimer [39]. In many other systems, initiators and/or growing ionic macromolecules may form aggregates and only unimers are reactive or one of the forms of aggregates. Not knowing these subtle differences and not realizing the presence of inactive-dormant isomers, one may easily incorrectly determine concentration of active centers, and, as follows, the involved rate constants of elementary reactions. In the polymerization of cyclic esters formation of dormant aggregated macromolecules has been first shown for CL and L-lactide (LA), initiated by dialkylalkoxy aluminums, for which a kinetic equation has been developed. This allows simultaneous determination of the degree of aggregation "m", equilibrium constant and rate constants of propagation [8]. In this case, the extent of aggregation can also be determined first from the dependence of the log of rate of polymerization, r p on log [P] total ([P] total = [I] 0 ), where [P] total is the total concentration of the active and dormant species-instantaneously active and dormant [41]. The corresponding kinetic system is shown in Scheme 10. The specific solution of equations describing this system for m = 2 has been given by Szwarc [25] (p. 108) and the general solution for any "m" has been proposed later [8]. and controlled. One of the first comprehensive papers discussing this issue has been published by the Liège group of Teyssié, Jerome and Dubois [38]. Aluminum isopropoxide exists in solutions as a mixture of a trimer and tetramer (Scheme 9). There was a long-lasting controversy, whether all alkoxy groups initiate or only some of them. This controversy has been resolved in our institute, as we have shown in the polymerization of εcaprolactone (CL) that in non-protonic solvents and at 25 °C in the mixture of a trimer and tetramer only trimer (cf. Scheme 9) initiates. When monomer conversion is complete, the tetramer remains intact [39]. On the other hand, Kricheldorf used higher temperatures (close to 100 °C) which caused all the alkoxy groups to be active (although the proportions of trimer and tetramer were not known) [40]. This means that at these conditions "dormant tetramer" has enough time to be converted into reactive trimer before polymerization is over. The difference in reactivities of trimer and tetramer stems from the fact, that in trimer the central Al atom is pentacoordinated and in tetramer hexacoordinated. Hexacoordinated atom does not have any coordination sites left to coordinate the monomer (cf. Scheme 9). Apparently, coordination with the central atom is a necessary condition for initiation. This may account, as Szwarc pointed out while discussing this system [25] (p. 123), for the thousand-fold faster reaction of trimer [39]. In many other systems, initiators and/or growing ionic macromolecules may form aggregates and only unimers are reactive or one of the forms of aggregates. Not knowing these subtle differences and not realizing the presence of inactive-dormant isomers, one may easily incorrectly determine concentration of active centers, and, as follows, the involved rate constants of elementary reactions. In the polymerization of cyclic esters formation of dormant aggregated macromolecules has been first shown for CL and L-lactide (LA), initiated by dialkylalkoxy aluminums, for which a kinetic equation has been developed. This allows simultaneous determination of the degree of aggregation "m", equilibrium constant and rate constants of propagation [8]. In this case, the extent of aggregation can also be determined first from the dependence of the log of rate of polymerization, rp on log [P]total ([P]total = [I]0), where [P*]total is the total concentration of the active and dormant speciesinstantaneously active and dormant [41]. The corresponding kinetic system is shown in Scheme 10. The specific solution of equations describing this system for m = 2 has been given by Szwarc [25] (p. 108) and the general solution for any "m" has been proposed later [8]. As the rate of the polymerization equals: The combination of these two equations leads to (full derivation is given in [8]): Thus, "m" is known, and k p and K a can be determined from Equation (2) Plots showing the dependence of polymerization rate on the initial concentration of an initiator in the polymerization of ε-caprolactone (CL) with various R 2 AlOR'alcoholates are given in Figure 2. The combination of these two equations leads to (full derivation is given in [8]): Thus, "m" is known, and kp and Ka can be determined from Equation (2) by plotting rp 1−m (the left hand side) as a function of [I]0·rp −m (the second term of the right hand side of this equation). Plots showing the dependence of polymerization rate on the initial concentration of an initiator in the polymerization of ε-caprolactone (CL) with various R2AlOR'alcoholates are given in Figure 2. Depending on the structure of active species and conditions of polymerization, propagation proceeds on the species that either retain their integrity independently of concentration (green squires) or partially aggregate into the temporarily inactive species as described by the Equation (2). The ability to aggregate and the size of the aggregates depend on the entourage of the active species. Thus, for R2AlOR' as an initiator, there is aggregation into trimer when R = C2H5, into a dimer for i-C4H9 and no aggregation when active species are solvated by amino ligands. The absence of aggregation for propagation on Al(i-OPr)3 (not shown) is particularly spectacular, since the initiator itself is highly aggregated (cf. above) and disaggregates upon propagation, when i-OPr groups on the Al atoms are converted into macromolecules. The presented method (Equation (2)) of analysis of polymerizations with aggregation of active centers that leads to dormancy allowed analyzing the already published data not only for ROP but also for some vinyl polymerizations (Figure 3). Depending on the structure of active species and conditions of polymerization, propagation proceeds on the species that either retain their integrity independently of concentration (green squires) or partially aggregate into the temporarily inactive species as described by the Equation (2). The ability to aggregate and the size of the aggregates depend on the entourage of the active species. Thus, for R 2 AlOR' as an initiator, there is aggregation into trimer when R = C 2 H 5 , into a dimer for i-C 4 H 9 and no aggregation when active species are solvated by amino ligands. The absence of aggregation for propagation on Al(i-OPr) 3 (not shown) is particularly spectacular, since the initiator itself is highly aggregated (cf. above) and disaggregates upon propagation, when i-OPr groups on the Al atoms are converted into macromolecules. The presented method (Equation (2)) of analysis of polymerizations with aggregation of active centers that leads to dormancy allowed analyzing the already published data not only for ROP but also for some vinyl polymerizations (Figure 3). [45]). Graph has been taken from our paper [8]. In Figure 4, two chosen dependences from Figure 3 are analyzed according to Equation (2). The rates of polymerization (with 1-m exponent) are plotted against the total concentration of the active centers (equal to the starting concentration of an initiator multiplied by rp −m ). The straight lines provide kp from intercepts and Ka from the slopes. It is shown in the original paper that improper values of m do not give straight lines. Figure 3. Graphs have been taken, as above, from [8]. The determined in this way values of constants have been found practically equal to the values measured by the authors of the original papers who were using numerical method of fitting. It proves the utility of the linear analytical approach and the corresponding equation that gives an access to the analytical description of the polymerization with a reversible formation of dormant aggregates. [42]). Pink: An anionic polymerization of methyl methacrylate with Li + cation, in THF as a solvent, at´65˝C (the experimental data taken from [43]). Blue: The polymerization of oxirane on´CH 2 CH 2 O´, Cs + ion pairs, in THF as a solvent, at 70˝C (the experimental data taken from [44]). Red: The polymerization of hexamethylcyclotrisiloxane (D 3 ) on´Si(CH 3 ) 2 O´, Li + ion pairs, in THF as a solvent, at 22˝C (the experimental data taken from [45]). Graph has been taken from our paper [8]. In Figure 4, two chosen dependences from Figure 3 are analyzed according to Equation (2). The rates of polymerization (with 1-m exponent) are plotted against the total concentration of the active centers (equal to the starting concentration of an initiator multiplied by r p´m ). The straight lines provide k p from intercepts and K a from the slopes. It is shown in the original paper that improper values of m do not give straight lines. [45]). Graph has been taken from our paper [8]. In Figure 4, two chosen dependences from Figure 3 are analyzed according to Equation (2). The rates of polymerization (with 1-m exponent) are plotted against the total concentration of the active centers (equal to the starting concentration of an initiator multiplied by rp −m ). The straight lines provide kp from intercepts and Ka from the slopes. It is shown in the original paper that improper values of m do not give straight lines. Figure 3. Graphs have been taken, as above, from [8]. The determined in this way values of constants have been found practically equal to the values measured by the authors of the original papers who were using numerical method of fitting. It proves the utility of the linear analytical approach and the corresponding equation that gives an access to the analytical description of the polymerization with a reversible formation of dormant aggregates. Dormancy in Sn(Oct) 2 /ROH Catalyzed/Initiated Polymerization of Cyclic Esters: L-Lactide (LA) and ε-Caprolactone (CL) Sn(Oct) 2 /ROH catalyzed/initiated polymerization of cyclic esters will be discussed separately for its importance in a commercial polymerization of LA in the melt and a wide application in research works on a cyclic esters polymerization (altogether over 1000 papers, according to the Web of Sci.). In polymerization of cyclic esters Sn(Oct) 2 as such is not active. It gains the activity only after the reaction with, e.g., alcohols, when it is converted first into mono-and finally into dialkoxy tin (Sn(OR) 2 ) (cf. Scheme 11). Sn(Oct)2/ROH catalyzed/initiated polymerization of cyclic esters will be discussed separately for its importance in a commercial polymerization of LA in the melt and a wide application in research works on a cyclic esters polymerization (altogether over 1000 papers, according to the Web of Sci.). In polymerization of cyclic esters Sn(Oct)2 as such is not active. It gains the activity only after the reaction with, e.g., alcohols, when it is converted first into mono-and finally into dialkoxy tin (Sn(OR)2) (cf. Scheme 11). Dialkoxy tin formally behaves as other metal alkoxides [38]. The alcohol added to the system acts both as an initiator and as a chain transfer agent, which reversibly forms dormant species until it becomes an end group of a macromolecule (Scheme 12). Dialkoxy tin formally behaves as other metal alkoxides [38]. The alcohol added to the system acts both as an initiator and as a chain transfer agent, which reversibly forms dormant species until it becomes an end group of a macromolecule (Scheme 12). Dormancy in Sn(Oct)2/ROH Catalyzed/Initiated Polymerization of Cyclic Esters: L-Lactide (LA) and ε-Caprolactone (CL) Sn(Oct)2/ROH catalyzed/initiated polymerization of cyclic esters will be discussed separately for its importance in a commercial polymerization of LA in the melt and a wide application in research works on a cyclic esters polymerization (altogether over 1000 papers, according to the Web of Sci.). In polymerization of cyclic esters Sn(Oct)2 as such is not active. It gains the activity only after the reaction with, e.g., alcohols, when it is converted first into mono-and finally into dialkoxy tin (Sn(OR)2) (cf. Scheme 11). Dialkoxy tin formally behaves as other metal alkoxides [38]. The alcohol added to the system acts both as an initiator and as a chain transfer agent, which reversibly forms dormant species until it becomes an end group of a macromolecule (Scheme 12). Figure 5. Approximately tenfold excess of BuOH over Sn(Oct) 2 is needed to convert all Sn(Oct) 2 in the system into active species. When no BuOH is added, M n of the obtained polymer approaches 10 5 and apparently traces amounts of H 2 O or other proton donors present in the system serve as a coinitiator. According to Figure 5, at first, the rate of the polymerization increases with increasing [n-BuOH] (the active species are formed according to Scheme 11) and then it reaches a plateau when the concentration of active sites becomes invariant. Since the number of active species is constant and P n is equal to the [monomer] 0 /[BuOH] 0 ratio at complete conversion, the system is both living and controlled. Scheme 12. Chain transfer and creation of dormant species in the polymerization of ε-caprolactone with the Sn(Oct)2/ROH system. The [ROH]0/[Sn(Oct)2]0 ratio first strongly influences the rate of the polymerization until it reaches plateau. The established relationship is illustrated in Figure 5. Apparently, a similar (formally) mechanism takes place for the polymerization of LA initiated/catalyzed by a strong base (e.g., 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU)) and an alcohol. When alcohol is absent, then as it has been shown by Waymouth, macrocyclic PLA are formed [47,48]. Recently, a comprehensive study of the lactide LA polymerization with DBU/alcohol has been published [49]. This study encompasses the earlier works and shows, in agreement with previous observations, a simultaneous coexistence of an unreacted DBU, DBU H-bonded with growing macromolecules and dormant HO-ended macromolecules. Authors have shown that when sufficient excess of an alcohol over DBU is used, the process involves activated alcohol pathway, in which growing species are presented as activated -OH growing species (Scheme 13). However, since termination has been found, it is considered living and controlled only in a certain region of concentrations of monomer, DBU and an alcohol, as well as only at limited conversion and polymerization degree. Approximately tenfold excess of BuOH over Sn(Oct)2 is needed to convert all Sn(Oct)2 in the system into active species. When no BuOH is added, Mn of the obtained polymer approaches 10 5 and apparently traces amounts of H2O or other proton donors present in the system serve as a coinitiator. According to Figure 5, at first, the rate of the polymerization increases with increasing [n-BuOH] (the active species are formed according to Scheme 11) and then it reaches a plateau when the concentration of active sites becomes invariant. Since the number of active species is constant and Pn is equal to the [monomer]0/[BuOH]0 ratio at complete conversion, the system is both living and controlled. Recently, a comprehensive study of the lactide LA polymerization with DBU/alcohol has been published [49] [42]. This study encompasses the earlier works and shows, in agreement with previous observations, a simultaneous coexistence of an unreacted DBU, DBU H-bonded with growing macromolecules and dormant HO-ended macromolecules. Authors have shown that when sufficient excess of an alcohol over DBU is used, the process involves activated alcohol pathway, in which growing species are presented as activated -OH growing species (Scheme 13). However, since termination has been found, it is considered living and controlled only in a certain region of concentrations of monomer, DBU and an alcohol, as well as only at limited conversion and polymerization degree. Related way of formation of dormant macromolecules has also been postulated for other strong base + alcohol systems [48] as well as for catalyst acting simultaneously as initiator (INICAT) [50]. In the latter system, we postulated the following equilibrium with formation of dormant species as shown in Scheme 15. Related way of formation of dormant macromolecules has also been postulated for other strong base + alcohol systems [48] as well as for catalyst acting simultaneously as initiator (INICAT) [50]. In the latter system, we postulated the following equilibrium with formation of dormant species as shown in Scheme 15. Scheme 15. The proposed intramolecular mechanism of active-dormant interconversion for 3-[(4,5dihydro-1H-imidazol-2-yl)amino]-propanol INICAT system (in [50] the intermolecular activation has also been discussed). Formation of Dormant Species in Polymerizations of Vinyl Monomers In this subsection, formation of dormant species in the active inactive interconversion in vinyl polymerization will be briefly presented for comparison with ROP. The best known example of such transformation is an anionic polymerization of dienes with Li + counterion discussed in papers by Szwarc, Bywater and Fetters [25] (p. 34). The aggregation in this system gives a very good example of dormant species formation. However, discussion used to be so extensive and so well known that we will restrain ourselves from getting into any details. No less spectacular is the "Controlled Radical Polymerization: CRP" of vinyl monomers. In CRP, stable (persistent) radicals are used that are able to react with growing macroradicals, forming dormant species, although are unable to initiate polymerization or to react between themselves. The inactivating agents are in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6tetramethylpiperidinyl-1-oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The creation of dormant macromolecules in CRP of styrene with TEMPO is presented in Scheme 17. Formation of Dormant Species in Polymerizations of Vinyl Monomers In this subsection, formation of dormant species in the active inactive Related way of formation of dormant macromolecules has also been postulated for other strong base + alcohol systems [48] as well as for catalyst acting simultaneously as initiator (INICAT) [50]. In the latter system, we postulated the following equilibrium with formation of dormant species as shown in Scheme 15. Scheme 15. The proposed intramolecular mechanism of active-dormant interconversion for 3-[(4,5dihydro-1H-imidazol-2-yl)amino]-propanol INICAT system (in [50] the intermolecular activation has also been discussed). Formation of Dormant Species in Polymerizations of Vinyl Monomers In this subsection, formation of dormant species in the active inactive interconversion in vinyl polymerization will be briefly presented for comparison with ROP. The best known example of such transformation is an anionic polymerization of dienes with Li + counterion discussed in papers by Szwarc, Bywater and Fetters [25] (p. 34). The aggregation in this system gives a very good example of dormant species formation. However, discussion used to be so extensive and so well known that we will restrain ourselves from getting into any details. No less spectacular is the "Controlled Radical Polymerization: CRP" of vinyl monomers. In CRP, stable (persistent) radicals are used that are able to react with growing macroradicals, forming dormant species, although are unable to initiate polymerization or to react between themselves. The inactivating agents are in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6tetramethylpiperidinyl-1-oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The creation of dormant macromolecules in CRP of styrene with TEMPO is presented in Scheme 17. Scheme 16. TEMPO-a stable radical. interconversion in vinyl polymerization will be briefly presented for comparison with ROP. The best known example of such transformation is an anionic polymerization of dienes with Li + counterion discussed in papers by Szwarc, Bywater and Fetters [25] (p. 34). The aggregation in this system gives a very good example of dormant species formation. However, discussion used to be so extensive and so well known that we will restrain ourselves from getting into any details. No less spectacular is the "Controlled Radical Polymerization: CRP" of vinyl monomers. In CRP, stable (persistent) radicals are used that are able to react with growing macroradicals, forming dormant species, although are unable to initiate polymerization or to react between themselves. The inactivating agents are in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6-tetramethylpiperidinyl-1-oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The creation of dormant macromolecules in CRP of styrene with TEMPO is presented in Scheme 17. Even more widely used is the process known as atom transfer radical Polymerization (ATRP), discovered in 1995 by Matyjaszewski [11]. This process involves a reversible reaction of the macroradicals with Cu II salts, that provides reversible radical complex which is neither able to initiate polymerization, nor to react with itself (like TEMPO), (Scheme 18). extensive and so well known that we will restrain ourselves from getting into any details. No less spectacular is the "Controlled Radical Polymerization: CRP" of vinyl monomers. In CRP, stable (persistent) radicals are used that are able to react with growing macroradicals, forming dormant species, although are unable to initiate polymerization or to react between themselves. The inactivating agents are in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6tetramethylpiperidinyl-1-oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The creation of dormant macromolecules in CRP of styrene with TEMPO is presented in Scheme 17. stable (persistent) radicals are used that are able to react with growing macroradicals, forming dormant species, although are unable to initiate polymerization or to react between themselves. The inactivating agents are in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6tetramethylpiperidinyl-1-oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The creation of dormant macromolecules in CRP of styrene with TEMPO is presented in Scheme 17. Even more widely used is the process known as atom transfer radical Polymerization (ATRP), discovered in 1995 by Matyjaszewski [11] This process involves a reversible reaction of the macroradicals with Cu II salts, that provides reversible radical complex which is neither able to initiate polymerization, nor to react with itself (like TEMPO), (Scheme 18). The fast buildup of M n+1 ·LX (e.g., Cu II Cl2/ligand) establishes the persistent radical effect and controls termination [9,11]. However, in radical polymerizations at least some termination by combination or disproportionation of the macroradicals is inevitable. Nevertheless, conditions have been found allowing carrying the polymerization to the full monomer conversion with only a few percent of macromolecules terminated at the end of the process. To the same category belongs Reversible addition-fragmentation chain-transfer polymerization (RAFT), as described in Reference [52]. The cationic polymerization can also be a controlled process if the nucleophiles are introduced into the system converting reversibly the instantaneously active into the dormant ones (Scheme 19). This process, at certain conditions, is well controlled. It is not living, since carbocations easily undergo proton transfer (except at very low temperatures). The cationic vinyl polymerization with added nucleophiles has been comprehensively described in terms of reversible formation of dormant species in the Sigwalt's laboratory [53] (Scheme 20). Indeed, Sigwalt has measured the ratio kp/ktr in systems with and without nucleophiles and have found the same values [54]. Thus, it has been clearly demonstrated that the only propagating species are usual carbocations and the other species, formed in reaction with nucleophiles, are just nonpropagating, dormant macroions (oxonium, ammonium ions, etc.). These onium cations reversibly form back carbocations. Therefore, controlled polymerization, close to the living, results from The fast buildup of M n+1¨L X (e.g., Cu II Cl 2 /ligand) establishes the persistent radical effect and controls termination [9,11]. However, in radical polymerizations at least some termination by combination or disproportionation of the macroradicals is inevitable. Nevertheless, conditions have been found allowing carrying the polymerization to the full monomer conversion with only a few percent of macromolecules terminated at the end of the process. To the same category belongs Reversible addition-fragmentation chain-transfer polymerization (RAFT), as described in Reference [52]. The cationic polymerization can also be a controlled process if the nucleophiles are introduced into the system converting reversibly the instantaneously active into the dormant ones (Scheme 19). This process, at certain conditions, is well controlled. It is not living, since carbocations easily undergo proton transfer (except at very low temperatures). The cationic vinyl polymerization with added nucleophiles has been comprehensively described in terms of reversible formation of dormant species in the Sigwalt's laboratory [53] (Scheme 20). Indeed, Sigwalt has measured the ratio k p /k tr in systems with and without nucleophiles and have found the same values [54]. Thus, it has been clearly demonstrated that the only propagating species are usual carbocations and the other species, formed in reaction with nucleophiles, are just non-propagating, dormant macroions (oxonium, ammonium ions, etc.). These onium cations reversibly form back carbocations. Therefore, controlled polymerization, close to the living, results from formation of dormant macromolecules in the reaction of macrocations with nucleophiles. Polymerization becomes much slower and better conditions for the control of the process are provided. been found allowing carrying the polymerization to the full monomer conversion with only a few percent of macromolecules terminated at the end of the process. To the same category belongs Reversible addition-fragmentation chain-transfer polymerization (RAFT), as described in Reference [52]. The cationic polymerization can also be a controlled process if the nucleophiles are introduced into the system converting reversibly the instantaneously active into the dormant ones (Scheme 19). This process, at certain conditions, is well controlled. It is not living, since carbocations easily undergo proton transfer (except at very low temperatures). The cationic vinyl polymerization with added nucleophiles has been comprehensively described in terms of reversible formation of dormant species in the Sigwalt's laboratory [53] (Scheme 20). Indeed, Sigwalt has measured the ratio kp/ktr in systems with and without nucleophiles and have found the same values [54]. Thus, it has been clearly demonstrated that the only propagating species are usual carbocations and the other species, formed in reaction with nucleophiles, are just nonpropagating, dormant macroions (oxonium, ammonium ions, etc.). These onium cations reversibly form back carbocations. Therefore, controlled polymerization, close to the living, results from formation of dormant macromolecules in the reaction of macrocations with nucleophiles. Polymerization becomes much slower and better conditions for the control of the process are provided. In this way, the original idea, that addition of nucleophiles (ethers, esters, sulfides) forms a new kind of active (i.e., growing) species has finally been put to rest. Thus, let us quote Szwarc, who had written on this subject: "No new mechanism operates 'living' cationic polymerization. Neither does a new kind of species participate in these polymerizations" [25] p. 142). It has to be understood that onium ions are not propagating by themselves. Formation of dormant species has also been shown in styrene cationic polymerization initiated by protonic acids. Even though the cationic polymerization of styrene initiated by perchloric acid (HClO4) does not have the importance of ATRP, it has to be at least briefly mentioned, as it has triggered several further works involving dormant species. It shows additionally that the same system, depending on conditions (e.g., temperature), is living, living/dormant or just with termination. During the cationic polymerization of styrene initiated with HClO4, carried out at −97 °C, conversion of styrene stops at a certain conversion, that depends on the [styrene]0/[HClO4]0 ratio [55] [48]. One molecule of acid produces at this temperature one macromolecule. This result is explained by the collapse of the living ion pairs and formation of the unreactive polystyrylperchloric ester (it cannot e.g., ionize back, transfer a proton and form any species able to restart polymerization at this temperature). Choosing proper [styrene]0/[HClO4]0 ratio polymerization may go to completion, showing then typical features of living/controlled process. Thus, in terms of our pictograms (as above for THF), there is one period of growth and, after collapse of counterions, the system is not active anymore. This process is a termination reaction (Scheme 20). Scheme 20. The polymerization of styrene with HClO4 to partial conversion only. The perchloric ester at this temperature is not dormant, but just the termination product. If, however, the ratio [styrene]0/[HClO4]0 is properly chosen, then, polymerization may go to completion. At higher temperatures (e.g., −70 °C), the conversion of macroion into macroester is reversible and polymerization can be carried out to completion-the formed ester is a dormant species. Nevertheless, at still higher temperatures (e.g., at r.t.) the proton transfer takes place and up to 100 unsaturated macromolecules are formed from one acid molecule. A similar system, namely with HOSO2CF3 as an initiator, has been discussed in details by Vairon, who has determined the involved lifetimes in the stop flow experiments [56]. At some conditions, there is a living polymerization with dormant species, at higher temperatures termination takes place. Conclusions The major common feature of the presented processes, both in ROP and in some vinyl polymerizations, is the fast interconversion between active and dormant species. Then, only a fraction of all macromolecules can propagate instantaneously. The total lifetime of an average macromolecule is much higher with an introduction of dormant species, as it is a sum of the time of activity and dormancy. The periods of activity and dormancy are characterized by the ka/kd (rate constants of activation/deactivation) ratio that can be altered by experimentalist by choosing proper components of the system. An important result of the slowing down is the ability of all macromolecules to start polymerization at the same time leading to polymers of low dispersity if the rate of exchange growing dormant is fast in comparison with the rate of propagation. In this way, polymerizations Scheme 20. The polymerization of styrene with HClO 4 to partial conversion only. In this way, the original idea, that addition of nucleophiles (ethers, esters, sulfides) forms a new kind of active (i.e., growing) species has finally been put to rest. Thus, let us quote Szwarc, who had written on this subject: "No new mechanism operates "living" cationic polymerization. Neither does a new kind of species participate in these polymerizations" [25] (p. 142). It has to be understood that onium ions are not propagating by themselves. Formation of dormant species has also been shown in styrene cationic polymerization initiated by protonic acids. Even though the cationic polymerization of styrene initiated by perchloric acid (HClO 4 ) does not have the importance of ATRP, it has to be at least briefly mentioned, as it has triggered several further works involving dormant species. It shows additionally that the same system, depending on conditions (e.g., temperature), is living, living/dormant or just with termination. During the cationic polymerization of styrene initiated with HClO 4 , carried out at´97˝C, conversion of styrene stops at a certain conversion, that depends on the [styrene] 0 /[HClO 4 ] 0 ratio [55]. One molecule of acid produces at this temperature one macromolecule. This result is explained by the collapse of the living ion pairs and formation of the unreactive polystyrylperchloric ester (it cannot e.g., ionize back, transfer a proton and form any species able to restart polymerization at this temperature). Choosing proper [styrene] 0 /[HClO 4 ] 0 ratio polymerization may go to completion, showing then typical features of living/controlled process. Thus, in terms of our pictograms (as above for THF), there is one period of growth and, after collapse of counterions, the system is not active anymore. This process is a termination reaction (Scheme 20). The perchloric ester at this temperature is not dormant, but just the termination product. If, however, the ratio [styrene] 0 /[HClO 4 ] 0 is properly chosen, then, polymerization may go to completion. At higher temperatures (e.g.,´70˝C), the conversion of macroion into macroester is reversible and polymerization can be carried out to completion-the formed ester is a dormant species. Nevertheless, at still higher temperatures (e.g., at r.t.) the proton transfer takes place and up to 100 unsaturated macromolecules are formed from one acid molecule. A similar system, namely with HOSO 2 CF 3 as an initiator, has been discussed in details by Vairon, who has determined the involved lifetimes in the stop flow experiments [56]. At some conditions, there is a living polymerization with dormant species, at higher temperatures termination takes place. Conclusions The major common feature of the presented processes, both in ROP and in some vinyl polymerizations, is the fast interconversion between active and dormant species. Then, only a fraction of all macromolecules can propagate instantaneously. The total lifetime of an average macromolecule is much higher with an introduction of dormant species, as it is a sum of the time of activity and dormancy. The periods of activity and dormancy are characterized by the k a /k d (rate constants of activation/deactivation) ratio that can be altered by experimentalist by choosing proper components of the system. An important result of the slowing down is the ability of all macromolecules to start polymerization at the same time leading to polymers of low dispersity if the rate of exchange growing sed intramolecular mechanism of active-dormant interconversion for 3-[(4,5-2-yl)amino]-propanol INICAT system (in [50] the intermolecular activation has t Species in Polymerizations of Vinyl Monomers ormation of dormant species in the active inactive interconversion ill be briefly presented for comparison with ROP. mple of such transformation is an anionic polymerization of dienes with Li + papers by Szwarc, Bywater and Fetters [25] (p. 34). The aggregation in this example of dormant species formation. However, discussion used to be so own that we will restrain ourselves from getting into any details. s the "Controlled Radical Polymerization: CRP" of vinyl monomers. In CRP, ls are used that are able to react with growing macroradicals, forming h are unable to initiate polymerization or to react between themselves. The in the so called "nitroxide moderated polymerization" (mostly 2,2,6,6--oxide (TEMPO)) itself and its numerous derivatives (Scheme 16) [51]. The romolecules in CRP of styrene with TEMPO is presented in Scheme 17. More examples of the formation of the dormant macromolecules in ROP and in vinyl polymerizations (e.g., in so-called "group transfer polymerization") could be given, however the discussed examples are the most representative ones and the described phenomena also apply to other cases.	2018-01-01T12:23:17.963Z	2017-11-25T00:00:00.000	{ "year": 2017, "sha1": "b446cb0acf662c706811d30b1b2b6dcd50c5ef7c", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4360/9/12/646/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b446cb0acf662c706811d30b1b2b6dcd50c5ef7c", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
257913016	pes2o/s2orc	v3-fos-license	Immigrant assimilation in health care utilisation in Spain Abundant evidence has tracked the labour market and health assimilation of immigrants, including static analyses of differences in how foreign-born and native-born residents consume health care services. However, we know much less about how migrants’ patterns of healthcare usage evolve with time of residence, especially in countries providing universal or quasi-universal coverage. We investigate this process in Spain by combining all the available waves of the local health survey, which allows us to separately identify period, cohort, and assimilation effects. We find robust evidence of migrant assimilation in health care use, specifically in visits to general practitioners and emergency care and among foreign-born women. The differential effects of ageing on health care use between foreign-born and native-born populations contributes to the convergence of utilisation patterns in most health services after 15 years in Spain. Substantial heterogeneity by the time of arrival and by region of origin both suggest that studies modelling future welfare state finances would benefit from a more thorough assessment of migration. Supplementary Information The online version contains supplementary material available at 10.1007/s10198-023-01622-6. Introduction Migrants are common in societies of many different development levels (Interna tional Organization for Migration [IOM], 2019).Migrants' overall success largely depends on their assimilation into the host society, including in how they access and use social services.Although immigration exerts overall beneficial effects on destination countries (International Monetary Fund [IMF], 2020, Chapter 4), its impact on the sustainability of the welfare state is a subject of debate.These effects most likely depend on migrants' characteristics, the specific features of wel fare states, and the time horizon used to measure service use (Barrett & McCarthy, 2008;Christl et al., 2022;Giulietti, 2014;Hansen et al., 2017;Nannestad, 2007).In turn, as long as migration increases the ethnic heterogeneity of societies, it can also shape natives' attitudes towards resource redistribution and the welfare state itself (Alesina et al., 2021a;Alesina et al., 2021b;Dahlberg et al., 2012;Facchini et al., 2016). This paper explores the patterns of migrants' assimilation into health care in Spain.In particularly, we evaluate how length of residence affects migrants' use of this service, considered the cornerstone of the Spanish welfare state.We also explore the extent of convergence in health care utilization patterns between native and migrant populations, in terms of both assimilation and differential ageing processes. To pursue these goals, we employ different econometric models to exploit four Spanish health surveys (conducted from 2010 to 2020).These surveys contain information on respondents' time of residence in the country and include more than 80,000 natives and nearly 8,000 foreign-born individuals.This setup allows us to identify cohort, period, and assimilation effects and to carry out separate analysis by gender.Our findings suggest relevant migrant cohort effects in many services, i.e., foreign-born populations use some types of health care less than natives in their first years in the country.These results are consistent with both the healthy migrant effect and the persistence of information problems and language barriers. Assimilation-in this context, the increase in health care utilization with the number of years in the host country-is only relevant for visits to general practi tioners (GPs) by female migrants.We do not observe large differences by region of origin, although the statistical power of our analyses substantially decreases when examining different groups of migrants separately.Though the effect is modest, we argue that this outcome might be due to partial health assimilation and lim ited socio-economic progress, given that the use of health care services in Spain is higher among individuals with high educational attainment. Even if migrants use less health care at arrival and their assimilation is quite limited, the age patterns of their utilisation are different than among locals.As a result, after 20 years in Spain, migrants tend to exhibit similar levels of effective recourse to health services as the native population, with a few exceptions.Partic ularly, migrant women visit GPs more often than natives after 10 years and some cohorts of foreign-born females make higher use of emergency care. Overall, our results suggest that the differences between comparable native and migrant populations in terms of health care utilisation are relatively minor when the latter arrive to the country, and that after some time, their behaviour becomes considerably similar.Nevertheless, we identify some services where health consumption seems higher among foreign-born women.We argue that this result might indicate a modest extra cost in terms of social spending due to migration.Further simulations assessing the costs and benefits of migration in Spain might profit from these findings. To our knowledge, this study is the first to explore immigrant assimilation based on health care use in Spain.Whereas several papers provide evidence on how foreign-born populations exhibit better health than locals on arrival, how this gap tends to close with time of residence in the country (Rivera et al., 2013(Rivera et al., , 2015)), and how foreign-born populations exhibit similar or lower health service utilization (see, e.g., the surveys of Llop-Gironés et al. [2014], Norredam et al. [2009] and Sarría-Santamera et al. [2016], among many others), no previous work addresses this question linked to the dynamics of health care use. Whereas the literature on immigrant health (Antecol & Bedard, 2015;Giuntella & Stella, 2016;Hamilton et al., 2011) and specially labour market assimilation (Abramitzky et al., 2020;Bodvarsson & Van der Berg, 2013;Lalonde & Topel, 1997) is abundant, few studies consider how patterns of immigrant health care use evolve with time of residence in the host country.Studies focused on the U.S. indicate no difference in take-up rates of Medicaid means-tested benefits between natives and migrants, whose participation rises with the time spend in the country (Borjas & Hilton, 1996), as well as higher initial health expenditures among Latino migrants than locals, with certain evidence of convergence for Latino migrants who get American citizenship (Vargas Bustamante & Chen, 2011). It is also worth summarising the findings from countries providing universal access to health care.Even though, overall, these studies suggest a lower use of non-emergency health care services by migrants (vs.natives) on arrival and a certain catch-up process, this literature is not unanimous.For instance, migrant males in Canada less frequently contact a doctor on arrival, but their levels of health care usage rise to match natives after 6-8 years in the country (McDonald & Kennedy, 2004).According to Wadsworth (2013), the differences in health care use between migrants and natives in the United Kingdom and Germany are not large.In the U.K., foreigners make a little more use of GP services (but not hospitals) than natives.Their change in usage with time spent in the country does not follow a systematic pattern.In the case of Germany, if anything, migrants exhibit lower rates of utilisation of health care (both general practitioners and hospital services), but their rates seem to converge with those of natives with time of residence in the country.Finally, migrants' usage of primary care emergency services in Norway exhibits substantial variation over groups and is higher than natives' usage (perhaps at the expense of less effective access to other types of health care), and it decreases with length of residence in the country (Småland Goth & Godager, 2012). 1he remainder of our paper unfolds as follows.The second section provides some theoretical and institutional background for the analysis, while the third and fourth sections describe the database and methods employed in the analysis, respectively.Section 5 presents the empirical results and discusses their implica tions.Finally, in Section 6 we offer some concluding remarks and pathways from future research. Background Like most developed countries, Spain provides virtually universal health care cover age to every resident in the country.National authorities extended this entitlement in 2000 to undocumented migrants, with the sole requirement being registration in a local population census (with no legal consequences).Nevertheless, in practical terms, information problems or fear of retaliation due to their irregular status, jointly with the hesitancy of some regional authorities (who are responsible for health care delivery) to provide foreigners without legal residence in Spain with health cards, could hamper actual access to the National Health System (NHS) among some segments of migrants. In September 2012, in the middle of the Great Recession, the Spanish govern ment restricted the access of undocumented foreigners to primary care (with the exceptions of minors, pregnant women, and anyone who needed emergency care).The reform still allowed the affected groups to purchase insurance for a monthly fee of 60 e (individuals below 65) and 157 e (individuals above 65).Some regional governments also promptly passed legislation to protect the affected populations and provide more beneficial insurance conditions, close to those before the reform.These restrictions might have resulted in less actual access to these services and worse health outcomes (Jiménez-Rubio & Vall-Castelló, 2020;Juanmarti Mestres et al., 2021). The change in Spain's central government in 2018, after a motion of no confid ence, led to the restoration of the situation prior to the limitations on migrants' health care access.In practice, similar obstacles apparently persist because of legal loopholes (Villarreal, 2019).Bruquetas-Callejo and Perna (2020) even argue that migrants' entitlement to health care in Spain has been more of a political talking point than a subject with substantial differences between the two main political parties. Previous literature on the theoretical reasons for expecting assimilation (or non-assimilation) in health care is scant.A first reason that increased use of health services may correspond with years living in the host country, particularly relevant in contexts without universal coverage, is the higher probability of im proving health care access with longer residence in a region (Antecol & Bedard, 2015;McDonald & Kennedy, 2004;Vargas Bustamante & Chen, 2011).A second pertinent argument has to do with the existence of the "healthy migrant effect" and a process of negative assimilation in health, documented by recent studies focused on Spain (Rivera et al., 2013(Rivera et al., , 2015)).Similarly, during their first years in the country, migrants might face cultural and even linguistic barriers (Pena Díaz, 2016), whose relevance should decrease with their time spent in the country.A re lated argument refers to the role of information and knowledge on the functioning of health care systems, again likely to increase with time of residence in the host state (Devillanova, 2008).This issue could have both a negative effect on visits to GPs on arrival and a positive one on the use of emergency care (Småland Goth & Berg, 2010;Småland Goth et al., 2010).The existence of assimilation in this area is mostly an empirical question, since there are other factors that might act in the opposite direction.For instance, keeping in mind previous studies on the impact of income on health care demand, a non-linear but overall negative trend (Antón & Muñoz de Bustillo, 2010), assimilation of migrants in this domain might mitigate the the increase in health care use-see, among many others, the survey of Antecol and Bedard (2015). Data Our analysis makes use of the National Health Survey (NHS), waves 2011-2012and 2017 (Spanish Statistical Office [SSO], 2022), and the European Health Interview Survey (EHIS), waves 2014 and 2020 (Ministry of Health, 2022), administered by the SSO.These databases are the first health surveys in Spain that include precise information on the timing of immigrants' arrival in the country.The EHIS began much later than the NHS (whose first wave corresponds to 1987), but the EHIS is designed to be fully comparable to the NHS.Consequently, local authorities have discontinued the NHS, which they carried out roughly each three years until 2014.Both sources are representative of the resident population in the country aged at least 15 years old, at the regional level.Each wave includes approximately 24,000 households and follows a three-stage stratified sampling design, since it only selects one adult person from each household-households are randomly chosen from each census section-for an interview.Apart from basic socio-demographic characteristics, the questionnaires in both types of surveys cover detailed and comparable self-reported information on health status and health care utilisation.The main differences between the two sources is that the earlier questionnaire includes additional items on quality of life (e.g., information on social support) and additionally interviews a minor living each household.Hereafter, we refer to both questionnaires as national health surveys (NHSs). For the purpose of this investigation, we pool the samples of adults in the four waves mentioned above.We identify migrant status by looking at the country of birth rather than citizenship, because naturalization processes in Spain differ widely by state of origin (e.g., they are much shorter for people from some Latin American and Caribbean countries).Regarding health care use, we focus on the following items related to health services use: number of visits to general practi tioners (GPs) in the last four weeks, number of visits to specialist doctors in the last four weeks, number of hospitalisations in the last 12 months, and number of times the person used emergency care in the last 12 months. The resulting sample, after dropping the observations with missing values on any of the variables included in the analysis (1.1% of cases), comprises 80,122 nat ive and 7,807 migrant adults.Using survey weights, the latter group represents 13.8% of the sample.Thanks to ad hoc agreements with the Spanish institu tions responsible for granting access to the data (the SSO and the Ministry of Health, respectively), we are able to distinguish among foreign-born individuals from different regions of origin.The most relevant groups in demographic terms are migrants from Latin America and the Caribbean (43.4% of all the foreign-born adult population), those from European countries other than the European Union 15 (EU15) countries (19.7%), those from Africa (17.5%), and those from EU15 countries (13.6%).In contrast to other countries like Sweden or Germany, where refugees represent an important part of of the foreign population, the motivations for immigration to Spain are overwhelmingly economic, with the exception of EU15 migrants, who are mainly attracted by the benevolent climate and the lower cost of living than their countries of origin (Spanish Statistical Office, 2022). 2e show the main summary statistics of the sample in Table 1.It includes both the variables used in our analyses of health care use assimilation and those considered for exploring the channels through which such a process takes place (several health outcomes like self-perceived health, overweight status, and mental health problems). Methods In order to disentangle the effect of foreigners' length of residence on their patterns of health care use, we adopt the empirical strategy utilized by Antecol andBedard (2006, 2015) and Giuntella (2016) for analysing health assimilation.Specifically, we estimate equations of the following form: where Y i denotes a health care variable relevant to person i, X i a vector of socio demographic control variables (region, degree of urbanisation, and a cubic of age, fully interacted with migrant status, education, and marital and activity status.),A i a vector of dummy variables indicating how long an immigrant has lived in Spain (set equal to 0 for locals and excluding a category that serves as reference), C i a vector of dummy variables identifying the arrival cohort (which takes the value 0 for natives), T i a vector of dummy variables capturing the survey year, and ε i a random disturbance.We estimate equation 1 by Ordinary Least Squares (OLS) in our baseline ana lysis.We are not interested in prediction but in the marginal effects of the dummies due to cohort and assimilation variables.In this respect, OLS estimates are con sistent under weaker assumptions than others obtained from count data models (Angrist & Pischke, 2008).In any case, in Subsection 5.4, devoted to robustness checks, we present the results obtained using count data models. As there are relevant differences in health and health care use by gender, we estimate the equation of interest separately for men and women.As in the case of health status (Antecol & Bedard, 2015;Giuntella & Lonsky, 2022), ethnicity might also play a role in health services utilisation.Whereas most of the popu lation born in Spain is white, there is a considerable heterogeneity in the ethnic composition of migrant adults.This is due to the relevance of migration from Latin America, the Caribbean, and Africa.Previous research has identified relevant dif ferences in the patterns of migrant health care use by state of origin (Llop-Gironés et al., 2014).For this reason, we additionally re-estimate our model comparing locals with migrants from the EU15, the rest of Europe, Latin America, and the Caribbean and Africa.Unfortunately, considering these groups separately largely reduces the available samples, which makes the estimates quite imprecise. Pooling several cross-sections and both migrants and natives allows us to sep arately identify cohort, assimilation, and period effects.We consider three arrival cohorts based on Spain's recent economic and social conditions: before 1996 (when massive immigration began), 1996-2007 (a period of strong economic growth before the financial crisis), and 2008-2020 (just after the start of the Great Recession). In order to study assimilation, we take into account three intervals of length of stay in Spain: 0-4, 5-9, and 10 or more years.In order to identify the model, we omit the first category of time of residence.The coefficients due to arrival cohort indicate the differences in health care utilisation between migrants and natives at 0-4 years since migration.Those associated with the two dummies of length of stay in the country (5-9 and 10 or more years) capture the change in health care use for migrants with time spent in Spain.Combining the coefficients of the interaction between age and migrant status, the arrival cohort, and the time since arrival allow us to calculate how the migrant-native gap in health care use evolves over time.We can identify the period effect thanks to the inclusion of natives in the sample. In principle, the coefficients of the binary variables due to migrant cohorts would indicate the gap in health care demand between locals and migrants on arrival at 0 years old.Therefore, to make the interpretation of the results easier, we centre age at 15 years old (the lowest age at which we can observe individuals in our database), so those parameters capture the difference between foreign-born individuals at 0-4 years since migration and native population at that age. We explicitly refrain from introducing variables controlling for health status in the left-hand side of equation, as those sort of variables are jointly determined with utilisation of health care services, and both are part of the process of immigrants' assimilation to their host countries.In order to explore the potential role of these factors in shaping the use patterns of health services, we further estimate the role of immigrant assimilation in a set of health outcomes (self-reported health status, overweight status, and prevalence of mental health problems). Selection of return migration represents a potential threat to identification.For instance, if foreign-born populations who go back to their country of origin have worse health (and higher demand for health care) than stayers, the estimated coefficients would be inconsistent (downward biased).Regrettably, although return migration became a very relevant phenomenon during the Great Recession and its aftermath (Izquierdo et al., 2016;Larramona, 2013), there is little available evidence on the relationship between this phenomenon and health status for Spain.A survey of studies by Antecol and Bedard (2015), focused on other countries, suggests mixed results, so the experience of other societies does not provide a clear guide here.If, as shown by Abramitzky et al. (2014) in the case of the labour market, negative self-selection of return migration were the norm, our estimates of assimilation would be a lower bound for the actual effect of the number of years spent by migrants in Spain. Main results We display the main results of the econometric analysis in Table 2, which shows the estimates due to visits to GPs and specialists, and 3, which refers to hospital admissions and emergency care.Regarding the number of visits to GPs, we observe no difference in the frequency of use between locals and migrants on arrival in the case of males.Nevertheless, we observe that two cohorts of foreign-born women make a less intense use of health services that their native counterparts.The length of residence in Spain does not affect the utilisation of this service in the case of foreign-born men, but it increases after 10 years in Spain by 0.102 visits among migrant women.The size of this assimilation effect is not negligible: it represents nearly a quarter of the average number of visits to GPs.With regards to contacts with specialist doctors, assimilation is absent among both sexes, Nevertheless, it is worth mentioning that, on arrival, the female migrant cohort arriving between 2008 and 2020 exhibited a lower rate of utilisation of this service than natives.This effect (0.057 visits less) constitutes approximately one-third of the average number of contacts with specialists in Spain. Table 3 shows the results for hospitalisations and emergency care.They reveal no difference between male migrants on arrival and locals for the former variable, but all the cohorts of foreign-born women exhibit substantially higher hospital admissions than comparable natives.3Also, on arrival, two cohorts of migrant women exhibit a lower use emergency services than female natives.We do not observe any cohort or assimilation effects. Heterogeneity by origin In this subsection, we explore how the results on assimilation to health care differ by country of origin.Specifically, we look at foreign-born populations in the four most relevant groups of migrants: EU15, other European countries, Latin America, and the Caribbean and Africa. 4Because of sample size limitations, in many cases the estimated coefficients are not statistically different from zero, though they are not statistically different from our main results either.Therefore, we reproduce those results in the supplementary appendix and we comment on the main salient findings here.First, regarding EU15-born individuals (Table A.1 and A.2), the most interest ing finding is the positive effect of assimilation on female health care use, which does not only apply to GPs but also to specialists.Second, the results for other European migrants (Table A.3 and A.4) are completely in line with the ones presen ted in Section 5.1.Third, in the case of foreign-born populations from Latin Amer ica and the Caribbean (Table A.5 and A.6), we found that time spent in Spain has a positive effect on women's visits to GPs and specialists, but a negative impact on hospitalisations.Last, with regards to Africans (Table A.7 and A.8), the length of residence does not seem to affect health care use, but the estimated coefficients are not statistically different from those reported in the total sample. Health care use after 10 years in Spain As the number of years spent in Spain might impact the use of health care, and the utilisation patterns for these services might be different for migrants and natives, it is possible that the implications of migration for aggregate health spending vary over time.In this respect, our analysis suggests that on arrival, migrants are not an unusual burden in terms of health spending, since their rates of utilisation are no higher than their native counterparts.The only exception is hospitalisations in the case of women.In this subsection, based on those estimates, we assess whether this impact changes after the foreign-born respondents have spent 10 years in Spain. Figure 1 displays the differences in health care use between migrants and locals by type of service, sex, and arrival cohort.The results indicate that, after a decade in Spain, foreign-born males do not use health care differently than natives, with the exception of specialist doctors and two arrival cohorts whose rates of utilisation are lower than natives' rates.Concerning migrant women, their number of visits to GPs is higher than natives' visits, and the same applies to the level of their use of emergency care, for migrants who arrived between 1996 and 2007. In the appendix , we present the results of separate analyses for four different groups of migrants by region of origin (EU15, other European countries, Latin America, and the Caribbean and Africa).This exercise reveals substantial heterogeneity.For instance, some cohorts of EU15 males exhibit lower rates of use of health care services than natives, in terms of visits to GPs, special ists, and emergency care, 10 years after arrival.Differences in the levels of health care utilisation between locals and foreign-born populations from other European countries are null, with the exception of the male cohort that arrived before 1996, which has more visits to specialists than comparable natives.Individuals from Latin America and the Caribbean drive the main results: after 10 years in Spain, the number of GP visits among females is higher than native women, and the same applies to two female migrant cohorts in terms of emergency care.Last, the only gaps between African migrants and locals are a higher use of primary care by the 1996-2007 female cohort, a slightly lower number of visits to specialists by males arriving before 1996, and a lower number of hospitalisations among the latest migrant cohort compared to their native counterparts. These results illustrate that the impact of migration on health spending can vary depending on the time horizon considered and the origin of the foreign-born population.A quick back-of-the-envelope calculation, based on the total number of uses of public health services (Ministerio de Sanidad, 2022) and the cost estimated by some regional health authorities for each service (Resolución STL/353/2013, 2013), exemplifies this point.After 10 years of living in Spain, migrants who arrived between 1996 and 2007 required roughly 6% higher health care spending than natives.Similarly, African individuals from the 2007-2020 arrival cohort show a lower level of consumption of these services than locals. Another interesting outcome that emerges from this picture is that the joint ef fect of assimilation and differential ageing tends to neutralise the initial differential use of health services by some foreign-born cohorts.In other words, we observe a process of convergence in terms of health care utilisation between migrants and locals, which in certain cases even results in higher rates of utilisation by migrants. Note: The graph shows point estimates and 90%-level confidence intervals.We assume that migrants enter the country 10 years ago or earlier. Source: Authors' analysis from results in Table 2 and 3. Channels of assimilation Although the assimilation described above is somewhat limited, it is interesting to dig deeper into the potential mechanisms driving this process, even in a speculative fashion.The first possible channel is health assimilation.In this respect, using a similar econometric specification, we explore the effect of time of residence in Spain on the probability of reporting good health, overweight status, or mental health problems (Table 4).Our results suggest a pattern of negative health assimilation. In the case of females, the probability of being overweight increases by almost 8 percentage points 10 years after arrival, which is compatible with our findings revealing a higher number of visits to GPs.The higher use of health care services among migrant women is not at odds with the findings of previous literature on Spanish immigration.Whereas the employ ment rates and earnings of foreign-born, working-age populations tend to increase with time of residence in Spain (Amuedo-Dorantes & de la Rica, 2007; Izquierdo et al., 2010), their occupational assimilation is incomplete (Fernández-Macías et al., 2015;Rodríguez-Planas & Nollenberger, 2016;Simón et al., 2014).Bearing in mind that health care use seems to decrease with occupational attainment in Spain (Lostao et al., 2011), our results-indicating a higher number of visits to GPs by female migrants who have lived longer in Spain-would align with these labour market developments. In a related argument, job quality, even leaving aside pay, is substantially worse for Spanish immigrants than for locals (Díaz-Serrano, 2013;Fernández & Ortega, 2007;Gálvez-Iniesta, 2022;Gamero Burón, 2010).Recent studies document that poor working conditions might have a detrimental effect on health similar to that of unemployment (Chandola & Zhang, 2017;Wang et al., 2022).Obviously, our assessment of health assimilation is imperfect (conditioned by the survey), so job quality might be a plausible mechanism for our findings. A last potential channel has to do with acculturation.A relevant number of studies highlight the relevance of language and culture in migrants' health care access (Fassaert et al., 2009;Ndumbi et al., 2018;Pena Díaz, 2016;Småland Goth et al., 2010;Sorensen et al., 2019;Thomas, 2016).Our results are in line with this literature in that the foreign-born population segment experiencing the most intense assimilation processes are Latin Americans and Caribbeans, followed by Europeans, while the number of years in Spain does not seem to affect the rates of utilisation among Africans very much.Africans are arguably the most culturally distant migrant group from Spanish locals.Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects and controls for age (introduced though a third-degree polynomial fully interacted with migrant status), degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Robustness checks In this section, we comment on the results of several robustness checks that test the sensitivity of our results to different methodological choices.First, we re-estimate all our models using a Poisson regression model (Table A.9 and A.10 in the online appendix).Like OLS, this approach yields consistent estimates without requiring any further function of the error term (Blackburn, 2014;Wooldridge, 2010). 5eassuringly, the results are qualitative and qualitatively similar to our baseline estimations (Table A.11 and A.12. 6Our second robustness exercise consists of performing our estimations while including only those individuals who solely have public health insurance, as a way of isolating our results from the eventual distortions due to the different normative changes in the last decade.Again, our results hold, even though the degree of precision diminishes because of the smaller sample. 7The last sensitivity check explores whether our results vary when we limit our analysis to individuals aged less than 65 years old (Table A .13 and A.14).This methodological choice, which might help to ameliorate the eventual bias due to return migration, does not seem to have any influence on our results beyond reducing the native sample in a non-trivial way. Conclusion Immigration's relationship with the welfare state demands substantial attention from both policy makers and society as a whole.The access and use of health care by foreign-born populations represents a matter of relevance because of their effect on public finances, but also in terms of ensuring an adequate integration of immigrants in the host country.The results of the assessment of both issues might differ on migrants' arrival and after a longer term of residence. This research contributes to the literature by providing detailed evidence on the process of assimilation in health care utilisation by immigrants in Spain.Our findings suggest, first, that some population segments of foreign-born populations, on arrival, use part of these services less than comparable natives, which is com patible with the healthy migrant effect.Furthermore, such use increases with the time living in Spain, but only in the case of female migrants and visits to GPs.Although this suggests that the evidence of assimilation is not very strong, it is not negligible either: public health spending represented more than 30% of total expenditures in this area in 2020 (Ministerio de Sanidad, 2022; Resolución STL/353/2013, 2013).As a result of this limited assimilation and the different impact of age on the rates of health care utilisation, the patterns of consumption for these services by migrants converge with the natives' patterns and even surpass them for some female migrant cohorts and services.Note that the impact of the time spent in the host country (assimilation) and the differential effect of age on the variable of interest represent distinct phenomena.We can separate them in our analysis, thanks to the use of several waves of the database and the consideration of both natives and migrants in the main specification. Our results suggest that, even if the gap between migrants and natives in health care use is narrow, assimilation plays a non-negligible role.The existence of substantial heterogeneity by migrant group and time passed since arrival might influence the estimates of migrants' impact on public finances.As a consequence, we believe that researchers aiming to judge welfare state sustainability and those that cover migration could benefit from a more detailed modelling of the patterns of access to social services by foreign-born populations.Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Supplementary appendix Table A.5. Age, immigrant arrival cohort and assimilation effects (OLS) in vis its to GP and specialist (foreign-born population from Latin America and the Caribbean) No Note: The graph shows point estimates and 90%-level confidence intervals.We assume that migrants enter the country 10 years ago or earlier. Source: Authors' analysis from results in Table A.1 and A.2. Note: The graph shows point estimates and 90%-level confidence intervals.We assume that migrants enter the country 10 years ago or earlier. Source: Authors' analysis from results in Table A.5 and A.6.Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.R 2 is the squared coefficient of correlation between the actual and the fitted values, as suggested by Zheng and Agresti (2000).Source: Authors' analysis from national health surveys.Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.R 2 is the squared coefficient of correlation between the actual and the fitted values, as suggested by Zheng and Agresti (2000).Source: Authors' analysis from national health surveys.Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Figure 1 . Figure 1.Differences in health care use between 35-year-old migrants after 10 years in Spain by arrival cohort Figure A. 1 . Figure A.1.Differences in health care use between 35-year-old migrants after 10 years in Spain by arrival cohort (foreign-born population from EU15 countries) Figure A. 2 . Figure A.2. Differences in health care use between between 35-year-old migrants after 10 years in Spain by arrival cohort (foreign-born population from European countries other than EU15) Figure A. 3 . Figure A.3.Differences in health care use between 35-year-old migrants after 10 years in Spainby arrival cohort (foreign-born population from Latin America and the Caribbean) Figure A. 4 . Figure A.4. Differences in health care use between 35-year-old migrants after 10 years in Spain by arrival cohort (foreign-born population from Africa) Table 2 . Age, immigrant arrival cohort and assimilation effects (OLS) in visits to GP and specialist * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table 3 . Age, immigrant arrival cohort and assimilation effects (OLS) in hospital stays and visits to emergency care * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table 4 . Immigrant arrival cohort and assimilation effects (OLS) in health outcomes Table A . 1. Age, immigrant arrival cohort and assimilation effects (OLS) in visits to GP and specialist (foreign-born population from EU15 countries) Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A . 10. Robustness checks (I): Immigrant arrival cohort and assimilation effects (Poisson-regression estimates) in hospital stays and visits to emergency care Table A Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A . 12. Robustness checks (II): Immigrant arrival cohort and assimilation effects (Poisson-regression estimates) in hospital stays and visits to emergency care (population with just NHS coverage)Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A . 13. Robustness checks (III): Immigrant arrival cohort and assimilation effects (OLS) in visits to GP and specialist (population aged less than 64 years old)Notes: * * * significant at 1% level; * * significant at 5% level; * significant at 10% level.All specifications include an intercept, year and region fixed effects, degree of urbanisation, education and marital and activity status.Heterokedasticity-robust standard errors in parentheses.Source: Authors' analysis from national health surveys. Table A . 14. Robustness checks (III): Immigrant arrival cohort and assimilation effects (Poisson-regression estimates) in hospital stays and visits to emergency care (population aged less than 64 years old)	2023-04-04T01:16:18.760Z	2023-04-02T00:00:00.000	{ "year": 2023, "sha1": "5d88c7aefeb967602fe4f7df0742c856d77c521d", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10198-023-01622-6.pdf", "oa_status": "HYBRID", "pdf_src": "ArXiv", "pdf_hash": "5d88c7aefeb967602fe4f7df0742c856d77c521d", "s2fieldsofstudy": [ "Medicine", "Economics", "Political Science" ], "extfieldsofstudy": [ "Economics" ] }
2759156	pes2o/s2orc	v3-fos-license	Relation of Chlorophyll Fluorescence Sensitive Reflectance Ratios to Carbon Flux Measurements of Montanne Grassland and Norway Spruce Forest Ecosystems in the Temperate Zone We explored ability of reflectance vegetation indexes (VIs) related to chlorophyll fluorescence emission (R 686/R 630, R 740/R 800) and de-epoxidation state of xanthophyll cycle pigments (PRI, calculated as (R 531 − R 570)/(R 531 − R 570)) to track changes in the CO2 assimilation rate and Light Use Efficiency (LUE) in montane grassland and Norway spruce forest ecosystems, both at leaf and also canopy level. VIs were measured at two research plots using a ground-based high spatial/spectral resolution imaging spectroscopy technique. No significant relationship between VIs and leaf light-saturated CO2 assimilation (A MAX) was detected in instantaneous measurements of grassland under steady-state irradiance conditions. Once the temporal dimension and daily irradiance variation were included into the experimental setup, statistically significant changes in VIs related to tested physiological parameters were revealed. ΔPRI and Δ(R 686/R 630) of grassland plant leaves under dark-to-full sunlight transition in the scale of minutes were significantly related to A MAX (R 2 = 0.51). In the daily course, the variation of VIs measured in one-hour intervals correlated well with the variation of Gross Primary Production (GPP), Net Ecosystem Exchange (NEE), and LUE estimated via the eddy-covariance flux tower. Statistical results were weaker in the case of the grassland ecosystem, with the strongest statistical relation of the index R 686/R 630 with NEE and GPP. Introduction The consequences of the ongoing global climate change [1] might turn terrestrial ecosystems over large areas from carbon sink to carbon source [2][3][4]; therefore, an accurate assessment of the global carbon balance is of increasing importance. Remote sensing (RS) provides the only global and cost effective tool to monitor spatiotemporal properties of the CO 2 assimilation in various plant ecosystems [5][6][7][8][9]. One of the traditional approaches to quantify RS information is the transformation of reflectance spectra into vegetation indexes (VIs). VIs are mathematical transformations of reflectance at specifically selected spectral bands that maximize sensitivity to target biophysical variables and minimize confounding environmental factors (e.g., [10]). Since the start of the satellite era-several VIs have been developed for remote quantification of leaf chlorophyll content (Chl (a+b) ) [11], leaf area index [12], and/or other biophysical variables [13] that are important for the assessment of the health status and functioning of terrestrial ecosystems. However, most of these 2 The Scientific World Journal derived variables and VIs are rather insensitive to daily dynamical changes in ecosystem carbon fluxes (however, see [14]). Recent advances in RS provided the possibility to estimate physiological parameters related to the dynamic processes of CO 2 fixation by photosynthesis [15][16][17], namely, to (i) xanthophyll cycle pigments and (ii) chlorophyll fluorescence (Chl-F). Such approach of direct estimation of the light use efficiency (LUE) has a potential to contribute to a significant reduction of existing large uncertainties in the estimation of the carbon balance [16]. The xanthophyll cycle pigments protect the photosynthetic apparatus from overexcitation [18]. Under high light conditions, plants activate a photoprotective pathway via deepoxidative reactions of xanthophyll pigments, resulting in zeaxanthin as the final product (e.g., [19]). This process enables the dissipation of excessive energy in the form of heat [20] and is associated, along with other processes, with the reduction in photochemical efficiency of Photosystem II (PSII) [18]. These rapid and light intensity-dependent changes were firstly detected remotely as changes in absorbance around 515 nm [21] and later as changes in reflectance around 530 nm [22]. Subsequently, Gamon et al. [23] proposed Photochemical Reflectance Index (also called Plant Physiological Reflectance Index, PRI, calculated as (R 531 − R 570 )/(R 531 + R 570 )), where R XXX is a reflectance intensity at subscripted wavelength. This index has been related to changes in the zeaxanthin pigment content [23][24][25], LUE (defined as the amount of assimilated carbon per unit of incoming light) measured at leaf [25] and later also at canopy [26][27][28][29][30] and even at satellite levels [7,31,32]. Several studies linked PRI measured by eddy-covariance flux towers directly to carbon sink capacity in grassland [33] and forest ecosystems [34]. PRI index has been also shown to correlate with the foliar pigment content of Chl (a+b) , carotenoids (Car x+c ), Car/Chl ratio and Car/Chl ratio [35,36] and various Chl-F parameters, especially under the drought stress conditions [37][38][39][40]. The Chl-F emission is directly linked to the first steps of photosynthesis, to photochemistry, and it is widely used in photosynthesis research. Chl-F is produced in plant photosynthetic tissues after the absorption of light energy as one of the two deexcitation pathways competing with photochemistry and heat loss [41][42][43]. The coupling between the photochemistry and Chl-F is the strongest in PSII. Thus, the competition between Chl-F and photosynthesis makes the Chl-F an ideal noninvasive reporter of the photosynthetic activity in plant tissue. The Chl-F emission reaches its maximum intensity at 690 and 740 nm [44]. Also changes in the zeaxanthin concentration are related to Chl-F, particularly to Nonphotochemical Quenching (NPQ) [45]. Chl-F expresses dynamic changes when a previously dark adapted healthy plant is exposed to actinic light [33]. After the illumination Chl-F increases to a high peak value, it then decreases to a steady state level (F S ) [46]. Under saturating light, the peak Chl-F equals to the maximum level (F M ). The relative Chl-F decrease (R Fd ) from the dark-to-light adapted state, quantified as R Fd = (F M − F S )/F S , was shown to be linearly correlated with the light-saturated photosynthetic rate [47]. However, the only sun-induced Chl-F signal that can be tracked via passive RS sensors is "steady-state" chlorophyll fluorescence (Chl-F S ) [48][49][50]. Results of several studies suggested that solar-induced Chl-F S [51][52][53][54][55] is linked to CO 2 assimilation. Consequently, several VIs, particularly, the reflectance ratios R 686 /R 630 , R 690 /R 630 , and R 740 /R 800 have been proposed to quantify Chl-F S [37,50], where the first wavelength corresponds to the maximum intensity of Chl-F S emission, whereas the second wavelength is unaffected by Chl-F S thus serving as a normalization factor. The difference ratio R 760.59 − R 759.5 utilizes the Chl-F infilling effect [56]. The Frauenhofer Line Discriminator principle [57] is another method used in RS for Chl-F S signal extraction from reflectance measurements [34]. Nevertheless, most of the studies using the retrieval of Chl-F S from hyperspectral reflectance were carried out either in laboratory conditions or with nonimaging sensors. Only a limited number of studies attempted to map the spatial distribution of carbon fluxes which were implemented using ground-based [58,59] airborne [60,61] or spaceborne [7,31] spectroscopy sensors. In this study, we explored the ability of reflectance VIs related to the F S signal, such as R 686 /R 630 , R 740 /R 800 and PRI, to estimate the CO 2 assimilation at canopy level in grassland and forest ecosystems. First, we evaluated whether the heterogeneity of VIs of a measured grassland plot can reveal a heterogeneity in light-saturated CO 2 assimilation rates (A MAX ) under steady state conditions. Second, we followed VI changes under a change of irradiance intensity in the scale of minutes and hours and searched for correlations between VI variations and variations of physiological variables, particularly LUE, gLUE (gross light use efficiency; GPP/APAR), net ecosystem exchange (NEE) and gross primary production (GPP), measured using the eddy-covariance systems at the scale of grassland and forest ecosystems. The aim of this study was to explore potentials and limitations of scaling the carbon cycle-related physiological processes, observable via RS methods, from leaf to canopy level. This way the feasibility of larger scale RS observation of the vegetation processes at ecosystem level can be advanced. To our knowledge, this is the first study using the imaging spectroscopy to investigate plant canopy properties in relation to eddy-covariance data in the diurnal course [17]. Study Site: Montane Grassland and Forest Ecosystems. The montane grassland and forest research plots are located at the experimental study site Bílý Kříž (Moravian-Silesian Beskydy Mts., the Czech Republic, 18.54 • E, 49.49 • N, 855 m and 908 m a.s.l., resp.), which is part of the CARBOEUROPE flux tower network (http://www.carboeurope.org/). This site is characterized by cool (annual mean temperature of 5.5 • C) and humid (annual mean relative air humidity of about 80%) climate with high annual precipitation (ca. 1000-1400 mm). The geological bedrock is formed by Mesozoic Godula sandstone (flysch type) with ferric podzols. The Scientific World Journal 3 The most abundant species occurring at the natural grassland plot (association Nardo-Callunetea, class Nardo-Agrostion tenuis) that were investigated are Festuca rubra agg. (L.), Hieracium sp., Plantago sp., Nardus stricta (L.), and Jacea pseudophrygia (C. A. Meyer). Additional details on the experimental grassland and physiological properties of the dominant plant species are presented in Urban et al. [62]. The investigated forest stand was planted in 1981 with 4year old seedlings of Picea abies (L.) Karst. (99%) and Abies alba (Mill). (1%) on a slope of 11-16 • with south-southwest exposition over an area of 0.062 km 2 [63]. At the time of the experimental measurements (2005), the stand density was approximately 2 600 trees/ha (hemisurface leaf area index of about 11 m 2 m −2 ), with mean (± standard deviation) tree height of 8.5 m (±1 mm) and stem diameter at 1.3 m of 10.1 m ± (1 mm). Imaging Spectroscopy Measurements and Data Processing. The nadir viewing canopy reflectance was obtained under clear sky sunny conditions with a visible near-infrared hyperspectral Airborne Imaging Spectrometer for Applications (AISA Eagle, Specim, Oulu, FI). In total 260 spectral bands between 400-940 nm were collected with a full width at half maximum (FWHM) of 2.2 nm, and with a ground pixel resolution of 1 cm. The AISA sensor was mounted to the tower at a height of 20 meters (i.e., approximately 10 m above the top of the canopy). The acquired area of about 50 m 2 was located within a footprint of an eddy-covariance tower system. In the case of grassland, the AISA sensor was attached to a ladder at the height of 4 meters above the top of the canopy, sensing in a total area of about 4 m 2 (located completely within a footprint of an eddy-covariance system), with a ground pixel resolution of about 0.3 cm. Figure 1. shows the representative part of the Norway spruce forest, which is homogeneous even-aged monoculture, thus the assumption here is that the smaller footprint of reflectance measurements is possible to be compared with the larger footprint of eddy-covariance data. Subsequent airborne measurement covering the whole footprint area has confirmed this assumption for both forest as well as grassland ecosystem (data not shown). The acquired AISA images were transformed into radiance values using the laboratory-derived calibration files, and converted into at-sensor reflectance images (Figure 1(a)) by means of the empirical line method, using five near-Lambertian calibration panels of known flat reflectance response. An automatic supervised Maximum Likelihood classification [64] was used to distinguish and to mask shaded pixels. To ensure that only photosynthetically active pixels were used in the subsequent statistical analysis, an appropriate threshold of the green normalized difference VI (green NDVI = (R 554 − R 677 )/(R 554 + R 677 )) was applied to separate the green sunlit grass leaves from dry litter. Finally, three narrow-band VIs (PRI, R 686 /R 630 , R 740 /R 800 ) related to photosynthetic processes were derived per pixel from the AISA Eagle reflectance scans ( Figure 1 Table 1). Selection of these VIs is based on our previous work with AISA Eagle, where detailed diurnal hyperspectral measurements were coupled with Chl-F measurements (see [48] for full description). No other optical VIs were analyzed in this study. Leaf Gas Exchange Measurements of Grassland Species. The in situ CO 2 assimilation rate of intact leaves of Festuca rubra agg., Hieracium sp., Jacea pseudophrygia, Plantago lanceolata, Tarraxacum spp., and Veronica chamaedrys (L.) was measured at saturating light intensities (Photosynthetic Photons Flux Density (PPFD) ≈ 1300 ± 10 μmol (photons) m 2 s −1 , [63]) with the open infrared gasometrical analyzer Li-6400 (Li-Cor, USA). All environmental conditions inside the assimilation chamber remained unchanged and referred to the field conditions: leaf temperature between 34-37 • C, relative humidity between 35-45%. All leaves, still being attached to the plant, were measured in their natural environment and marked. This way, they were later identified on the AISA images, and all corresponding green pixels were averaged in order to minimize the angular anisotropy of the vegetation bidirectional reflectance distribution function (BRDF). 2 and H 2 O, as well as latent and sensible heat exchange between the grass stand and atmosphere, were measured using closed-path eddy-covariance systems Edisol (University of Edinburgh), containing the infrared gas analyzer (Li-6262, Li-Cor, Lincoln, USA), sonic anemometer Gill R 2 (Gill Instruments, UK), and Edisol software package. In the case of the forest research stand, the InSituFlux (InSituFlux, Sweden), consisting of the infrared gas analyzer (LI-7000 Li-Cor, Lincoln, USA), anemometer (Gill R3, Gill Instruments Ltd., Lymington, UK) and EcoFlux software, was used. The eddy-covariance systems provide time series of half an hour integral measurements of CO 2 and H 2 O fluxes. The footprint of the system covers a circular area with a radius of ca 500 m [65]. The postprocessing of eddy-covariance data was carried out based on the methodology paper Aubinet et al. [66], accommodating several modifications according to the most recent CarboEurope and FLUXNET protocols. The Quality Control (QC) Software performed the spike removal and quality check of the raw signals. After gap filling the GPP and NEE, values were modeled according to Urban et al. [63]. LUE and gLUE were calculated as follows: LUE = NEE/PPFD and gLUE = GPP/PPFD, where PPFD stands for Photosynthetic Photons Flux Density. For LUE and gLUE calculations we used PPFD values instead of commonly used absorbed PPFD, since the amount of transmitted light through the young dense forest stand is negligible (<2%; data not shown). Laboratory Analysis of Foliar Pigments. Plant leaves of grassland (n = 24) and current one-year and two-year old needles of Norway spruce trees (Picea abies (L.) Karst., 100 mg, n = 6) were sampled after the spectral measurements during the morning, midday, and afternoon (i.e., at 9:00, 12:00 and 16:00 of local time). Foliar pigments of samples, frozen into laboratory in liquid nitrogen for transportation, were extracted in 80% acetone with a small amount of 4 The Scientific World Journal R 740.0 /R 800.0 F S , early stress [37] (a) RGB 1 0 MgCO 3 . After centrifugation at 480 g for 3 min. the supernatant was used for spectrophotometric (UV/VIS 550, Unicam, Leeds, UK) estimation of chlorophyll a and b per area, according to Lichtenthaler [67], and also for High-Performance Liquid Chromatography (HPLC) quantification (TSP Analytical, USA) of individual carotenoids, as described inŠtroch et al. [68]. The deepoxidation state of the xanthophyll cycle pigments (DEPS) [69] was calculated using conversion factors published by Farber [70] as DEPS = (Z + A)/(V + A + Z) [71], where Z is zeaxanthin, A is antheraxanthin, and V is the violaxanthin content. Setup of Field Experiments. The variation of VIs was measured under three irradiance regimes: (i) instantaneous steady-state measurements under full sun irradiation, (ii) variation of VIs within dark-to-light transition in scale of minutes, and (iii) daily variation of VIs measured in onehour intervals. Within the frame of the "steady-state" experiment (i) we mapped VIs of selected experimental grassland plots (an area of about 4 m 2 ) at midday under full sunlight. 24 leaves of 6 plant species were labeled (each leaf was located on a separate plant), and their maximal CO 2 assimilation rate (A MAX ) was measured under saturating midday irradiance (PPFD ≈ 1300 ± 10 μmol (photons) m −2 s −1 ). These leaves were identified on the AISA image, captured simultaneously with CO 2 assimilation rate measurements. The computed VIs of 10-20 sunlit pixels of each leaf were averaged and correlated to the A MAX measurements. The experiment was carried out on a clear-sky day on 18th of July 2007 at 13:00 local time. In the "shade removal" experiment (ii), run in a time scale of minutes, we tested whether the VIs could estimate the CO 2 assimilation rate of labeled grass leaves exposed to saturating sun irradiation (A MAX ) not in the static steady state, but in a dynamic irradiation environment inducing the plant photoprotective reactions and consequent onset of photosynthesis. The experimental plot was divided into two parts (twice 0.5 × 4 m). The first part, containing 13 labeled sample leaves, was covered for 30 min using a black nontransparent blanket in order to dark-adapt the grass canopy prior to the imaging spectroscopy measurement. Our method of darkening enabled a lateral air convection, which prevented the physiological changes being induced by higher than ambient air temperature. The second control plot was exposed to a natural irradiation regime. Two AISA Eagle images of both plots were obtained at the 5th and 90th seconds after blanket removal. Subsequently, we computed the dynamic change of the VIs (ΔVIs) during the dark-tolight transition per leaf sample (average of 10-20 pixels) as subtraction VI DARK − VI LIGHT , where VI DARK and VI LIGHT are values obtained from the scan taken ∼5 and ∼90 seconds after removal of the blanket, respectively. These ΔVIs were The Scientific World Journal 5 statistically related to A MAX measurements of the sampled leaves. Finally, the "daily course" experiment (iii), conducted at a time scale of hours, was to correlate the daily variations of the VIs' mean values of both grassland and forest ecosystems with a daily variation of physiological variables measured in half-hour intervals via eddy-covariance flux systems. Statistical Analysis. The best-fitting models were computed to evaluate the relationship between the VIs and the physiological variables of carbon assimilation. The amount of variability of a dependent variable explained by the independent variable within the established regression model was expressed by the coefficient of determination (R 2 ). The significance of the relationships between the variables was tested using the analysis of variance (ANOVA) at two significance levels: P < 0.1 and P < 0.05, respectively. All statistical tests were performed with Statistica 7.0 software (StatSoft Inc., USA). Figure 2 present the relationships between A MAX of the 24 leaf sample and the relative change of their VIs (ΔVIs) extracted from the AISA images during the dynamic transition from the darkto-light adapted state of grassland plants. Here, significant exponential relationships (P < 0.05) were found between A MAX and Δ(R 686 /R 630 ) (R 2 = 0.51, y = 0.0484e 0.1647 x) (Figure 1(a)), and ΔPRI (R 2 = 0.51, y = 0.015e 0.0621 x) (Figure 1(b)). Δ(R 740 /R 800 ) (Figure 2(c)) showed no significant correlation with A MAX . These results indicate that, after the inclusion of temporal dynamic of the irradiance variability in the experimental setup, the relative change in VIs (PRI and R 686 / R 630 ) positively correlates with the carbon assimilation rate. "Daily Course" Experiment. Differences in the xanthophyll cycle pigments' daily variation and concentration of chlorophylls between grassland and forest stand are shown in Figure 3. Figure 3(a) displays the daily courses of xanthophyll cycle pigments measured in the leaves of grassland plants and shoots of spruce trees. The diurnal behavior is typical for sunny days, that is, the amount of de-epoxidated xanthophyll pigments (i.e., the amount of Z and A) was highest during midday. The chlorophyll content (Chl (a+b) per unit leaf area) was relatively during the day in both grassland as well as in forest ecosystems (Figure 3(b)). The forest canopy contained a higher relative amount of xanthophyll pigments in the deepoxidated state (Figure 3(a)), and also a higher absolute amount of total chlorophylls (Figure 3(b)) compared to the grassland ecosystem. In this experiment, we have only presented the results on the variation of PRI and R 686 /R 630 due to their ability to follow plant dynamic changes as had been proved in the previous "shade-removal" experiment. The daily courses and also the relationships between the daily variations of PRI and R 686 /R 630 , as well as the NEE and LUE measurements of grassland and spruce forest are given in Figures 4 and 5, respectively. In grassland, NEE gradually decreased during the day (Figure 4a), while LUE showed the typical daily course with lowest values around midday (Figure 4b). A different yet consistent pattern was observed for two consecutive days in the forest ecosystem. NEE slightly increased in the morning (till 11:00). The highest values around midday were followed by a sharp decrease in the afternoon (after 14:00) ( Figure 5(a)). The daily pattern of LUE was similar for both days, showing constant values during the day and a sharp decrease after 15:00 ( Figure 5(b)). Both NEE and LUE were higher in the forest compared to the grassland ecosystem. The daily course of the R 686 /R 630 ratio in grassland was quite stable, with the highest values at 10:30 and 11:30 ( Figure 4c). In the forest R 686 /R 630 increased in the morning hours, reached the highest values during the midday hours, and then rapidly declined ( Figure 5(c)). The daily patterns of PRI variations of grassland and forest measurements were similar; however, the time when PRI reached its daily minimum was shifted. The grassland PRI decreased between 10:00 to 12:30 (Figure 4(d)), reached its lowest value at the time of the highest irradiation intensity, and increased again at 13:30 to its morning values. In the forest ecosystem PRI decreased to the lowest value at 11:00 and then steeply increased, reaching maximum values from 13:30 to 15:30. In the late afternoon (15:30-17:00) it decreased again ( Figure 5(d)). In grassland we were not able to record these late afternoon measurements due to the mountainous terrain and the shading of the measuring plot in the late afternoon. When comparing the absolute values, PRI for forest was higher than for grassland, which corresponds with a higher xanthophyll cycle pigment content in the de-epoxidated state of spruce needles and with a higher Chl (a+b) content. The statistically significant relationships (at P < 0.05) between the daily variations in R 686 /R 630 and NEE (Table 2) were found in both ecosystems. Moreover, a strong relationship was observed also between R 686 /R 630 and LUE in the forest ecosystem (Table 2). PRI showed a significant relationship (at P < 0.1) only with LUE in grassland ( Table 2). The results of the regression analysis between the daily variations of physiological variables derived from eddy-cova-riance measurements and four VIs are summarized in Table 2. The ratio R 686 /R 630 showed a significant relationship with a high R 2 for both GPP and NEE in grassland, and also in the forest. Moreover, R 686 /R 630 was highly related to gLUE and LUE in the forest (Table 2), and to gLUE in grassland ( Table 2). The ratio R 740 /R 800 showed a relatively high dependency on all physiological variables examined for forest but none for grassland ecosystems (Table 2). Discussion Our main target was to determine if VIs can indicate dynamic physiological processes related to CO 2 assimilation rate. Under steady-state conditions, typical for RS data aquisition, we were unable to find statistically significant relationships between VIs and A MAX , indicating that a spatial scaling from the leaf up to canopy level cannot be pursued in this fashion. Grace et al. [16] pointed out that VIs related to Chl-F have the potential to provide information on the diurnal, and also on seasonal changes in photosynthesis (see also [46]). Once the temporal component with its dynamic irradiation regime had been included in our dark-to-light transition experimental setup, the variability observed in VIs Table 2: Summary of coefficients of determination (R 2 ) found for the best-fit between vegetation indices (VIs) and physiological parameters derived from eddy-covariance measurements in grassland and forest ecosystems. Gross primary production (GPP [μmol (CO 2 ) m −2 s −1 ] = NEE + R); net ecosystem exchange of CO 2 (NEE [μmol (CO 2 ) m −2 s −1 ]); gross light use efficiency (gLUE = GPP/PPFD [g C MJ −1 ]); radiation use efficiency (LUE = NEE/PPFD [g C MJ −1 ]). Values of eddy-covariance derived parameters are half-hour averages and values of VIs are averages of more than 400 000 pixels (i.e., average of one image scan acquired in half to one hour intervals), R is reflectance at subscripted wavelength and PRI is calculated as (R 532 − R 570 )/(R 532 + R 570 ). * Denotes P < 0.1 and * * for P < 0.05, as determined by ANOVA analysis. increased, probably as a result of induced changes in the electron transport rate and the onset of the xanthophyll deepoxidation processes. Consequently, also the statistically significant positive relationships between Δ(R 686 /R 630 ) and ΔPRI, and A MAX have confirmed the ability of VIs to follow the light-saturated rate of photosynthesis in the investigated species. The reflectance change at wavelengths around 690 nm was previously shown to correspond with the Chl-F emission from PSII [49,72]. Therefore, the relative change observed in Δ(R 686 /R 630 ) is comparable to the trends of the "vitality index" R Fd , significantly correlated with A MAX of outdoor grown plant species [47,73] in earlier studies. Guo and Trotter [35] published that ΔPRI for plants with contrasting photosynthetic capacities (expressed in their study as PRI at low light intensity subtracted from PRI at saturating light intensity) was negatively related to A MAX . In contrast to this, yet similar to the studies by Demmig-Adams and Adams [74], we observed a significant positive relationship, showing that a larger relative change in Z pigment content is associated with lower A MAX in both sunlit and shaded leaves. We also observed that a larger ΔPRI was associated with a higher chlorophyll index ratio (R 750 /R 710 ) [75] (see Supplementary Material available online at doi: 10.1100/2012/705872), which might explain its significant relationship with A MAX . A number of studies [25,29,30,76] investigated the possibility to use PRI derived at canopy level as a proxy for LUE in Monteith's equation [77] estimating the vegetation GPP. For instance, Rahman et al. [7] successfully used PRI derived from the Moderate Resolution Imaging Spectroradiometer (MODIS) satellite sensor for the estimation of LUE and NPP in boreal forest. However, our results showed that even if the negative effects of shade fraction and nongreen vegetation are excluded, PRI might still not be a good indicator of canopy level photosynthetic processes. Our PRI measurements were significantly related only to LUE of natural grassland and yet PRI was able to explain only 42% of LUE variability. Our result suggests that PRI relationship with LUE should be interpreted with caution, especially in ecosystems with diverse species composition, where species specific relationships might complicate the overall result. The use of PRI at larger scales originated from leaf level studies showing that the PRI variation of leaves is driven by changes in the de-epoxidation state of xanthophyll cycle pigments in the antennae that reduce the amount of light used in leaf CO 2 assimilation, and this way protect the leaf from excessive radiation [78,79]. A similar trend was observed in our flux tower LUE measurements of grassland, which slightly decreased during midday. In contrast to this, the midday depression in LUE was not visible in the forest data. This might be explained by the fact that coniferous canopy contains a large proportion of shaded compared to sun exposed needles, which might be driving the LUE diurnal course. Apparently, VIs extracted from forest AISA images corresponded mainly to the sunlit shoots on top of the canopy. Our results also stressed a necessity to exclude from VI analysis the confounding effects coming from shade fraction and nongreen vegetation. All these, and other structural factors of forest canopy, can disturb a potential relationship between VIs and the eddy-covariance observations. The lack of correlation at the canopy level might, however, be also weakened by the fact that the relative changes in the pool of xanthophylls represent only a fraction of total carotenoid pigment content [80] and that light induced changes in the leaf also include some other processes, such as changes in pH of photosynthetic membrane [80] and conformational changes in chloroplasts [81]. Porcar-Castell et al. [82] provided further evidence that pool of total carotenoids is closely linked to monoterpene emission capacity, which might further complicate the interpretation of spectral response at 531 nm. Inoue and Peñuelas [83] found out that exponential relationship between LUE and PRI was strongly affected by the soil water sontent. Ollinger [84] recently summarized and highlighted structural factors, which are strongly affecting canopy level relationships between VIs and plant biophysical properties. Apart from plant physiological and structural limitations, canopy level PRI is also affected by viewing and illumination geometry [30]. Hernándes-Clemente et al. [85] found that structural sensitivity of PRI can be reduced by selecting the reference wavelength at 512 nm, instead of 570 nm. However, the experimental results of our study found a significant relationship between canopy VIs and CO 2 assimilation-related plant physiological variables when considering their daily cycle variations. Obtained R 2 coefficients between PRI and LUE are lower than the ones reported in the recent metaanalysis [17]. This might be caused by a narrower dynamic range of PRI and LUE measured during our experiment. The narrow-band reflectance ratios of Chl-F wavelengths (R 686 /R 630 , R 740 /R 800 ) performed better than PRI, and stronger relations were observed for forest than for grassland canopy. This might be partially explained by lower forest water stress, which increased the leaf stomatal conductance resulting in a stronger Chl-F emission. Although most of the studies investigated fluorescence VIs in relation to various Chl-F-related parameters [40,49,50], several studies reported a positive relationship of photosynthesis-related variables with reflectance ratios related to Chl-F. The laboratory experiment of Dobrowski et al. [37] resulted in a positive relationship between the reflectance ratio R 690 /R 600 and CO 2 assimilation (R 2 = 0.36). Furthermore, the reflectance ratio R 695 /R 805 measured in a 5-year-old canopy of pine seedlings was significantly related to A MAX [86] during the diurnal course. In general, our results showed higher R 2 values of the reflectance ratios to LUE than to gLUE, which can be explained by the respiration component included in the "gross" measurements. The reflectance ratio R 686 /R 630 , tested in this study, was found to be a statistically significant proxy of GPP, NEE, and LUE in grassland and also of gLUE in forest ecosystems. Thus, we can hypothesize that R 686 /R 630 might be considered a potential RS indicator of the ecosystem vegetation productivity variables. Conclusions The first experimental results conducted at the leaf level revealed no statistically significant relation of "process-related" narrow-band VIs (PRI; R 686 /R 630 ; R 740 /R 800 ) to the maximum CO 2 assimilation rate measured instantaneously under the steady-state irradiation conditions. Significant correlations were only found once the relationships between VIs 10 The Scientific World Journal and physiological variables were investigated at a temporal scale (minutes and hours), taking into account the changes in irradiance intensity. When taking into account the 1.5minute duration of the dark-to-light transition, Δ(R 686 /R 630 ) and ΔPRI of natural grassland, we were able to establish significant regressions between VIs and the carbon assimilation measured under saturating light conditions. Finally, in the daily course of several hours, the simple reflectance ratio R 686 /R 630 , of the VIs investigated, showed the highest R 2 value to the measured eddy-covariance ecosystem variables with better statistical relations found in the case of spruce forest than in grassland canopy. Considering that we observed a strong statistical relation to GPP and NEE, we conclude that the R 686 /R 630 ratio may potentially act as a suitable RS indicator of vegetation productivity at the scale of the whole canopy. In general, the results of this study provide an insight relevant to the further development of leaf-canopy upscaling remote sensing approaches as well as basis for more accurate estimation of carbon assimilation and the productivity in natural canopies. However, considering spatial and temporal limitations of the presented study, many problems are yet to be resolved before a remote sensing application at larger spatial scales.	2016-05-04T20:20:58.661Z	2012-06-04T00:00:00.000	{ "year": 2012, "sha1": "82fc1ef933d7894b193409ef303144e7d6257315", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/tswj/2012/705872.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ab93900fd8efea5b8b5f382e70721bd294829cba", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Environmental Science", "Medicine" ] }
248254231	pes2o/s2orc	v3-fos-license	agronomy An Optimized Protocol for Micropropagation and Acclimatization of Strawberry ( Fragaria × ananassa Duch.) Variety ‘Aroma’ : In strawberry micropropagation, several challenges must be overcome to obtain quality plants and achieve high survival rate to ex vitro acclimatization. In this study, therefore, a set of protocols were evaluated to optimize explant (meristem) disinfection, in vitro growth (multiplication and rooting), and ex vitro acclimatization of strawberry. The results showed that explants treated with 1.0% NaClO for 5 min had a lower percentage of contamination, and achieved a higher percentage of viability, height, and number of leaves. In vitro growth was favored by the use of 1 mg L − 1 zeatin, since it allowed greater seedling growth (number of shoots, seedling height, number of leaves, number of roots and root length), and a higher pre-acclimation rate (100%). In the acclimatization phase, plants grown in a substrate composed of compost + peat combined with 4 g of humic acid achieved better response in morphological and physiological variables. In fact, the results of this study could be used to cultivate strawberry plants of the ‘Aroma’ variety with high commercial quality. Introduction Strawberry is a fruit that has high nutritional value and is a substantial source of folate, vitamin C, fiber, potassium, flavonoids, anthocyanin, phytochemicals, antioxidants [1,2], and bioactive compounds that reduce the risk of cardiovascular incidents and thrombosis [3,4]. In addition, it provides anti-cancer benefits and helps prevent age-related memory loss [5,6]. However, despite its multiple benefits and high agricultural potential, there are limitations for its cultivation due to the presence of diseases caused by viruses such as strawberry mottle virus (SMoV), Fragaria chiloensis cryptic virus (FClCV), strawberry necrotic shock virus (SNSV), and strawberry mild yellow edge virus (SMYEV) [7], and those caused by fungi such as Botrytis cinerea, Phytophthora, Pythium, Rhizoctonia, Colletotrichum spp., Verticillium dahliae, Mycosphaerella fragariae, Ramularia tulasnei, Phomopis obscurans or by the bacterium Xanthomomas fragariae [8]. Many of these diseases can be transmitted in the propagation process, which is traditionally done through stolons [9]. An interesting alternative to overcome this problem is the use of biotechnological tools that allow mass production of plants with excellent phytosanitary and genetic quality [10]. Meristematic tissue culture is a technique widely used to obtain pathogen-free and homogeneous plants over a short period of time (fungi, bacteria, and viruses) [11]. For successful micropropagation, it is essential to regulate and manage contamination during meristem establishment [12]. Likewise, it is equally important to identify the growth regulator and concentration that allow for successful establishment, multiplication, and rooting in vitro. Finally, one of the critical points in the tissue culture process is to achieve ex vitro acclimatization of micropropagated plants; therefore, it is crucial to evaluate various substrates and/or their combinations to achieve high survival rates and adequate plant growth and development [13][14][15]. Plants from in vitro culture have been reported to show high rooting and shoot generation rates [16] but, most importantly, they are free from diseases [17]. Furthermore, Nehra et al. [18] reported that in vitro propagated plants of 'Redcoat' produced significantly more inflorescences and flowers per crown compared to stolon-propagated plants, and consequently derive a higher number of fruits. However, tissue culture-propagated plants exhibit genotype-dependent behavior, suggesting the need for detailed evaluation of each strawberry cultivar [18]. In this context, the present study aimed to optimize the factors that influence the micropropagation of strawberry variety 'Aroma', and to evaluate the development of the plants during the ex vitro acclimatization process. Materials and Methods The research was carried out at the Laboratory of Plant Physiology and Biotechnology of the Universidad Nacional Toribio Rodríguez de Mendoza. As a source of explant, strawberry plants of the 'Aroma' variety grown at the experimental station located on the university campus were selected. The plants were propagated in 1.0 L containers with a sterile substrate composed of agricultural soil, peat, and river sand (ratio 2:1:1; pH 6.38). These received a preventive phytosanitary treatment with a solution of Phyton (2.5 mL L −1 ) and Chlorpyrifos (1.5 mL L −1 ), in addition to biostimulation and nutritional balance with Enziprom (5 mL L −1 ) and Basacote ® Plus 6M (4 g plant −1 ), respectively. The phytosanitary treatment was carried out at 5-day intervals for 8 weeks. Culture Medium and Growth Condition In this study, Murashige and Skoog (MS) base medium [19], supplemented with 22.5 g L −1 sucrose, 0.15 g L −1 ascorbic acid, 0.10 g L −1 myo-inositol, 1.0 mL L −1 Plant Preservation Mixture, and 6 g L −1 Agar was used. The pH was adjusted to 5.8 and then autoclaved at 121 • C and 1.5 bar for 20 min. Explants were grown at 25 ± 1 • C and a photoperiod of 16 h light provided by white fluorescent tubes (3000 lux). Disinfecting Plant Material Stolons free of disease symptoms and nutritional deficiencies were harvested. Leaves and roots were removed, gently rinsed under running water, and placed in ascorbic acid solution (150 mg L −1 ) for 20 min. The crowns were then immersed in a soapy solution for 15 min and then placed in fungal solution (Carbonyl 1 g L −1 ) for 15 min. At the end of each disinfection process, three rinses with sterile distilled water were performed. Experiment 1: In Vitro Establishment Under aseptic conditions in a laminar flow chamber, explants were sterilized by immersion through independent treatments with sodium hypochlorite (0.5 and 1% NaOCl, for 3 or 5 min), hydrogen peroxide (0.5 and 1% H 2 O 2 , for 3 or 5 min), or mercuric chloride (0.1% HgCl, for 1.5 or 3 min). At the end of each treatment, three rinses with sterile distilled water were performed. The experiment consisted of 10 treatments and 10 replicates. Each repetition corresponded to one experimental unit (explant). Sterile meristems (0.3 mm) were extracted and seeded in test tubes with 15 mL of base culture medium (Figure 1a). After 6 weeks, the percentage of phenolization, contamination, viability, seedling height, and number of leaves were recorded. Then, following the experimental design and treatments of the multiplication-rooting stage, the seedlings were separated and sown in polyethylene trays with a substrate composed of a mixture of sphagnum peat + perlite in a 1:2 ratio (special mixture, Klasmann Substrates Select, Geeste, Germany) with pH 6.0 ( Figure 1c). The trays were placed in transparent containers with a thin film of water, then covered with parafilm (gradually removed) and maintained at a temperature of 25 • C, 80% relative humidity, and light intensity of 3000 lux. After 16 days, the percentage of pre-acclimation (survival) was recorded. Experiment 3: Acclimatization The most vigorous and uniform hardened seedlings ( Figure 1d) were transferred to a shaded nursery (70% shade) and transplanted into 5" × 8" nursery bags containing four types of substrates (Table 1): compost (coffee pulp), burned rice husk, coconut fiber, and perlite, which were mixed with peat in a 2:1 ratio. After 7 days of acclimatization, humic acid was added (0 or 4 g EKOTRON ® 70 GR), depending on treatment. The average temperature and relative humidity during the acclimatization period were 16.4 • C and 77.9%, respectively. The experiment was conducted under a 4 × 2 factorial design (substrate types × humic acid dose), with six replicates. Each replicate corresponded to one experimental unit (plant). After 35 days post-transplanting, the following parameters were evaluated. Determination of Plant Growth Parameters Plant height (longest shoot), number of leaves, number of shoots or crowns, root length, number of roots, aerial biomass (fresh and dry matter), root biomass (fresh and dry matter), leaf area, and root volume were recorded. To record dry matter, plants were dried in an oven at 60 • C until a constant weight was achieved. Leaf area of all leaves/plant was determined using a regression equation: where y = leaf area cm 2 , x = length per leaf width, and coefficient of determination R 2 = 0.99 [20]. Experimental Design and Data Analysis All experiments were conducted under a complete randomized design (CRD). Data were subjected to analysis of variance and significant means were tested by Tukey's test (p ≤ 0.05) using InfoStat statistical software version 2018. Table 2 shows that the lowest percentage of contamination (10%) was obtained when the explants were treated with 1.0% NaClO for 5 min. In addition, it was also the treatment that allowed the highest percentage of viability (60%), height (5.7 cm), and number of leaves (3.7) per established seedling to be recorded. On the other hand, the highest percentages of contamination (80%) were recorded in the treatments with H 2 O 2 1.0% for 3 and 5 min, NaClO 0.5% for 5 min and H 2 O 2 0.5% for 3 min, the latter being also the treatment with no viability rate (Table 2). Experiment 3: Acclimatization The morphological performance of the seedlings during the acclimatization process is shown in Table 4. The greatest plant height (15.45 ± 0.91 cm), number of crowns (7.33 ± 0.82), number of roots (26.25 ± 0.76), and leaf area (516.17 ± 145.42 cm 2 ) were recorded in plants grown on substrate 1 + 4 g humic acid. In substrates 1 and 3 (both with 4 g humic acid), the highest number of leaves was recorded, with values of 15.17 ± 2.14 and 15.17 ± 1.33, respectively. Regarding root length, substrate 3 + 4 g of humic acid yielded better root growth, achieving roots up to 32.21 ± 2.43 cm in length (Table 4). Table 5 shows that the highest biomass, in terms of fresh weight (foliar: 29.46 ± 10.40 g; root: 12.94 ± 1.84 g) and dry weight (foliar: 5.40 ± 1.67 g; root: 1.31 ± 0.17 g) was achieved in plants grown on substrate 1 + 4 g of humic acid. Regarding root volume, it was observed that the highest value of this variable was recorded in substrate 3 (14.04 ± 4.08 mL) and substrate 1 (13.45 ± 2.19 mL), both with 4 g of humic acid, followed by substrate 3 (13.03 ± 1.69 mL) with no humic acid. The physiological characteristics of the plants during the acclimatization process are shown in Table 6. Plants grown on substrate 1 + 4 g humic acid presented higher chlorophyll index and stomatal conductance, reaching values of 40.69 ± 2.57 SPAD and 434.47 ± 14.20 mmol/m 2 /s, respectively. By comparison, plants grown on substrate 4 without humic acid presented lower water potential (−16.74 ± 1.71 Bar). Micropropagation Stage Although strawberries are generally propagated by stolons, this method does not necessarily guarantee high-quality plants due to the risk of spreading diseases [9]. Therefore, in vitro meristem culture is an ideal technique to obtain healthy and homogeneous plants [11,21,22]. However, to achieve a high rate of success, it is absolutely necessary to optimize protocols, thus minimizing material losses from tissue oxidation and contamination, or low response to in vitro growth conditions [23]. In the present study, meristems treated with 1% NaClO for 5 min achieved higher viability (60%) and lower contamination (10%). Similar results were reported by Munir et al. [24], where disinfection of strawberry meristems ('Oso Grande' and 'Toro' varieties) with 0.5% NaOCl for 10 to 15 min allowed recording a survival rate of 75% and contamination lower than 15%. It is worth mentioning that the addition of antioxidants plays an important role in improving the response during in vitro establishment as it reduces phenolization of explants [11,25]. During multiplication, it was observed that the use of zeatin (1 mg L −1 ) largely benefited the development of shoots, leaves, and roots. Similar effects have been described in the micropropagation of crops such as blueberry, where the addition of zeatin (1 mg L −1 ) favored the development of vigorous plants with an increased number of shoots and leaves [26]. In contrast, the results of this study show that the addition of AG 3 (0.5 mg/L) or thiadizuron (1 and 1.5 mg L −1 ) clearly resulted in lower shoot induction. The low efficiency shown by these growth regulators (AG 3 and thiadizuron) may be related to the fact that they need to act synergistically with other phytohormones to enhance their effects [15,27]. However, Zakaria et al. [28], during multiplication of three strawberry cultivars (Festival, Sweet Charly, and Florida) with 2 mg L −1 thiadizuron, achieved a shoot regeneration percentage above 70%, suggesting that the response is also related to the regeneration potential of each variety and to the concentration of phytohormones. Even though all explants installed during the multiplication stage formed roots, the use of zeatin (1 mg L −1 ) clearly promoted better root system development. In this regard, Domínguez and Donayre [29] state that in the pre-acclimation stage, it is essential that seedlings should exhibit high vigor and a good root system, so as to ensure their survival. This, in fact, was confirmed in this study, since the seedlings that showed the best morphological behavior were highly adaptable during the transition to ex vitro conditions, with 100% survival (pre-acclimation). Similar results were described by Valencia-Juárez et al. [30], achieving 100% survival of strawberry seedlings of 'Nikté' variety. Jofre-Garfias et al. [31] also recorded up to 90% survival in strawberry of 'Buenavista' variety. Although the best results were obtained with the use of zeatin, its high cost can be a limiting factor for implementing commercial micropropagation protocols in companies and nurseries. That is why, in recent years, coconut water has been used in micropropagation protocols for crops such as olive [32], hazelnut [33], and Raja Bulu banana [34], in certain cases as a substitute element for zeatin [32]. In this study, the use of coconut water produced encouraging results for plant height (5.41 ± 1.00 cm), number of leaves (13.00 ± 3.38), and survival (between 84.45 to 88.89%), but had no significant effects on shoot formation or root development. In plant tissue culture, coconut water is a source of beneficial nutritional and hormonal substances, but it is not always sufficient to promote successful micropropagation, so combining it with cytokinins can improve results [33]. In any case, considering the high prices of zeatin, the use of coconut water is justified from an economic point of view. Acclimatization Stage During acclimatization, plants must have a well-developed root system [35], enabling the plant to have properly assimilated water and nutrients, in addition to being well fixed to the substrate [36]. Accordingly, findings of this study indicate that plants grown in the 1 + 4 g humic acid substrate exhibited, in general, better morphological development. This result, together with the characteristics of the substrate, may be related to the application of humic acid, which in turn promotes root and vegetative growth by improving cation exchange capacity and increasing the number of favorable microorganisms in the soil, thereby improving the adaptive ability of plants [37][38][39]. Importantly, the substrate needs to exhibit good porosity and filtration [40]. Regarding the chlorophyll index, plants grown on substrate 1 + 4 g humic acid had favorable values (40.69 SPAD) for their growth and development, as reported, for example, in peach cultivation, where normal values vary in the range of 39-56 SPAD [41]. Similar values have also been recorded in hydroponic strawberry cultivation with 38.5 to 49.5 SPAD for young and old leaves, in different substrate (50% coco peat + 50% perlite) [42]. Nitrogen is a required element during the growth stage to ensure structural and osmotic functions [43]; therefore, the measurement of the chlorophyll index is useful as an indicator of nitrogen content. Conversely, unsuitable substrate can lead to water stress, resulting in changes in the photosynthetic system and lower chlorophyll levels [44]. Hence, suitable mixtures are vital to ensure good plant development, using easily accessible and low-cost materials [45]. Stomatic conductance is related to gas exchange in leaves, where high readings denote good water supply [46]. Substrate 1 + 4 g humic acid enables a better physiological response of strawberry plants, whereas plants grown on substrate 4 with or without humic acid recorded low levels of stomatal conductance, which may lead to reduced transpiration rate and photosynthetic carbon assimilation [47,48]. This demonstrates, together with water potential, a negative metabolic regulation of developmental processes in plants linked to water stress [49], which was more favored in substrate 4. In this way, changes in stomatal conductance are useful to determine and regulate water loss in plants, which are crucial to establish adequate irrigations [50]. By comparison, the highest level of water potential was recorded in substrate 1 + 4 g of humic acid, which was beneficial for the plants; on the contrary, the plants growing in substrate 4 without humic acid exhibited low values, which affected the growth and development processes [51], which may be due to the delay in cell divisions that occurs at the foliar and root level [52]. Moreover, water potential is a useful indicator to check the water status and its measurement indicates water demand of strawberry plants [53,54]. Accumulated biomass levels were even lower in plants grown on substrate 4. It has been reported that variations in water potential and stomatal conductance led to reductions in plant biomass, because water deficit seriously affects photosynthetic activity [55]. This is demonstrated by Cordoba-Novoa et al. [56], where low water potential (−22.10 Bar) together with low conductance led to a reduction in water in leaves and low biomass accumulation. Furthermore, substrate 4, being more porous (72.73%) than the others, retained less water and generated a greater water deficit, leading to less leaf development in the plants [57], making it unsuitable for acclimatizing strawberry plants of 'Aroma' variety. Conclusions The results achieved in this study describe an efficient protocol for in vitro propagation and ex vitro acclimatization of strawberry variety 'Aroma'. Indeed, treatment with NaClO (1%) for 5 min showed high efficiency in reducing contamination and increasing meristem viability. Favorably, zeatin at a concentration of 1 mg L −1 was shown to promote growth, sprouting, rooting, and survival of new seedlings. During ex vitro acclimatization, the use of compost + peat-based substrate (substrate 1) plus humic acid yielded more vigorous plants with better morphological and physiological characteristics.	2022-04-20T15:21:34.019Z	2022-04-17T00:00:00.000	{ "year": 2022, "sha1": "54ab3d068f53b8b080a3a20b05181db81997fa22", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4395/12/4/968/pdf?version=1650186514", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "c678c0cd7ad5003c505e09d46430a4b420d550d9", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "extfieldsofstudy": [] }
255810954	pes2o/s2orc	v3-fos-license	Horizontal gene transfer in an acid mine drainage microbial community Horizontal gene transfer (HGT) has been widely identified in complete prokaryotic genomes. However, the roles of HGT among members of a microbial community and in evolution remain largely unknown. With the emergence of metagenomics, it is nontrivial to investigate such horizontal flow of genetic materials among members in a microbial community from the natural environment. Because of the lack of suitable methods for metagenomics gene transfer detection, microorganisms from a low-complexity community acid mine drainage (AMD) with near-complete genomes were used to detect possible gene transfer events and suggest the biological significance. Using the annotation of coding regions by the current tools, a phylogenetic approach, and an approximately unbiased test, we found that HGTs in AMD organisms are not rare, and we predicted 119 putative transferred genes. Among them, 14 HGT events were determined to be transfer events among the AMD members. Further analysis of the 14 transferred genes revealed that the HGT events affected the functional evolution of archaea or bacteria in AMD, and it probably shaped the community structure, such as the dominance of G-plasma in archaea in AMD through HGT. Our study provides a novel insight into HGT events among microorganisms in natural communities. The interconnectedness between HGT and community evolution is essential to understand microbial community formation and development. Background Horizontal gene transfer (HGT), also known as lateral gene transfer, is defined as the movement of genetic material between phylogenetically unrelated organisms by mechanisms other than parent-to-progeny inheritance. Usually, this horizontal flow of genetic material utilizes three routes: conjugation, transduction, or transformation. Currently, it is widely believed that HGT plays an important role in prokaryotic evolution. Through the acquisition of new genes and functions, the recipient organism can accelerate evolution and adapt to new ecological niches [1,2]. With the availability of a large number of complete genome sequences, many possible transferred genes were identified in prokaryotes [3,4]. For example, 755 of 4288 genes were recognized as horizontally transferred into Escherichia coli since it diverged from the Salmonella lineage about 100 million years ago [5]. Also, the hyperthermophilic bacteria Thermotoga maritima is believed to have undergone high rates of genetic exchange with archaeal species sharing its extreme habitat [6]. Overall, it is estimated that 1.6-32.6 % of the genes in microbial genomes have been acquired by HGT [7]. Although HGT has been widely studied in isolated microorganisms, the understanding of HGT events in natural prokaryotic communities is superficial because of the complexity of unculturable characteristics of most microbes on the earth [8,9]. In fact, many microorganisms live as communities and interact with each other. They exhibit complex social relationships and co-evolve to continuously adapt to the specific environment. As an important driving force of evolution, HGT is thought to significantly influence the dynamics of microbial communities [10,11]. Therefore, studies of HGT events in a mixture of microbial genomes living in natural communities are worthwhile and are now expected to be realized through the development of metagenomics [12,13]. With metagenomic and metafunctional genomics data, the developments in microbial communities can be explored at the genome scale using the novel approaches of metagenomics, and it is possible to enhance our understanding of the principles of multi-species consortia and biocomplexity [14]. A recent study reported that the percentage of possible HGT events is close to that of complete genomes in several representative samples, including whale fall, Sargasso sea, farm soil, and human gut [11]. Also, some metagenomic sequencing projects mentioned the possible gene transfer events among various microbial genomes in a specific environment [15][16][17]. Many viewpoints, which were formed by studying individual prokaryotic genomes, suggested that HGT events contributed to the evolution and adaptation of species [18]. These points can be proposed in microbial communities reflected in metagenome data. For example, HGT events in metagenomes could be driven by factors of both environment and community composition, could accelerate evolution and adaptation to environments, and could happen to some species more than others [11]. However, these points are largely in a state of conjecture because of the challenge of the complexity of sequenced genome collections; therefore, there is an urgent need to confirm and study these HGT in detail. As mentioned above, the main challenge of analyzing HGT in metagenomes is the complexity of metagenomic sequences, which cannot be circumvented because most of the current sequenced data were recovered directly from environmental samples. To be more specific, an awkward situation, i.e., large numbers of mixed short DNA reads or contigs belonging to many different species (we know little about the population structure for some environmental samples and how many species the communities comprise), made it difficult to analyze genomic sequences and detect HGT in metagenomic sequences using existing computational methods. Fortunately, with the rapid development of metagenome projects, people can now use a few well-studied metagenomic datasets that avoid the above troubles. One example is the dataset of metagenomic sequences in the acid mine drainage (AMD) constructed from studies by the Banfield group [15]. As a typical metagenome, the biofilm growing on the surface of flowing AMD belongs to a low-complexity and relatively self-contained ecosystem [19]. With the advance of highthroughput sequencing strategies, genomes of microorganisms in the AMD community were directly sequenced from the environment. The researchers definitely identified 11 microorganisms with complete or near complete genomic sequences. More importantly, each contig can be exactly traced to its source species. Furthermore, microorganisms in AMD have demonstrated the possibility of gene transfer, and some evidence has been observed. For example, some possible phage genes and integrases with a broad host range were found, and gene transfer through transduction is thus possible among these AMD organisms [15]. With these properties of data, one may apply the computational methods to metagenomic sequence analysis just like it is applied to individual genomes. In this paper, we used the AMD metagenomic sequences as a model to investigate the putative HGT events that occurred among members within this natural community of microorganisms. We first annotated the protein-coding regions in the metagenomic sequences using the current computational tools. With a phylogenetic approach based on accurate gene reannotation, we detected a set of gene families with a total of 119 genes that showed phylogenetic tree incongruities, implying that they are probably horizontally transferred among genomes in AMD and the nine previously isolated organisms. Among them, 14 HGT events were determined as transfer events among the eight AMD members. The 14 transfer events happened in both directions of gene movement between bacteria and archaea. Further functional and pathway analysis of transferred genes revealed that these HGT events affected the functional evolution of archaea or bacteria in AMD, and they probably shaped the community structure of the AMD ecosystem. With several representative cases via computational analysis of metagenomes in this paper, our exploratory study also presented a supporting view of the role of HGT within community members in the functional distribution and evolution among community members. HGT identification and overview of HGTs among AMD microbial genomes We first reported our identification of the genes that were horizontally transferred among the genomes including eight organisms in AMD and nine previously isolated organisms. Using the method of strict reciprocal best basic local alignment search tool (BLAST) hit [20,21] based on the accurate gene reannotation by the current computational tools [22] or annotation from the National Center for Biotechnology Information (NCBI) [23], we obtained 251 orthologous gene families that were present in more than seven genomes in which at least one genome was from AMD, while 185 of them showed phylogenetic tree incongruities using an approximately unbiased test (AU test) [24]. Furthermore, 66 of the 185 phylogenetic trees were excluded because the differences in the branch length were not the same as those of the organism tree, which were probably caused by different evolution rates and could not represent HGT events. Therefore, 119 genes [Additional file 1] with significant evolution incongruence were identified in the study. Of these 119 genes, most of them demonstrate a function related to metabolism, especially amino acid transport and metabolism (Fig. 1). In fact, the metabolic network of E. coli has a lot of changes and growth to acquire various external nutrients in the past 100 million years caused by HGT instead of gene duplication [25]. In this case, over 66 % of transferred metabolism-related genes can build a metabolically flexible lifestyle to facilitate the environmental adaptation in this extreme acidic and metal-rich environment [26]. Therefore, it is reasonable to hypothesize that the 119 genes indicate possible horizontal transfer events among genomes in AMD and the nine previously isolated organisms. We further analyzed the transfer events with a clear evolutionary path in which only AMD organisms were involved. To this end, after a rigorous manual selection from the 119 possible transfer events, 14 genes were determined to be HGT among the eight AMD organisms (Table 1). Furthermore, possibly involved genomes among the eight AMD organisms, the possible donor and recipient organisms, and functions of the 14 genes were identified and listed in Table 1. It is interesting that genetic exchanges of these genes occur between bacteria and archaea in both directions in this community. We noted that two transfer events from bacteria to archaea, which included 11 of the 14 genes, are crucial to the development of the AMD community. This makes a lot of sense because two iron-oxidizing chemoautotrophic bacteria, Leptospirillum ferrodiazotrophum and Leptospirillum rubarum, dominate this relatively self-contained ecosystem [19]. With respect to the transfer events in this direction, functions of the involved genes affect the AMD microbial community. In the next two subsections, the details of the putative HGT events in this direction are reported. Herein it is noteworthy that the transfer events in the other direction, i.e., from archaea to bacteria, also suggested the significance of transferred genes to the recipients. For instance, two genes, MesJ and Mdh, were horizontally transferred from archaea to bacteria L. ferrodiazotrophum and L. rubarum. The MesJ protein is a wellknown cell-cycle protein that is directly responsible for lysidine formation and thus is essential for decoding AUA codons in vivo [27]. Mdh, which was proved to be horizontally transferred in a previous study [28], plays a key role in the cell during growth on methanol. The CcmA gene, which was transferred from archaea to the bacterium L. ferrodiazotrophum, is the first gene of an eight protein-encoded operon involved in cytochrome c maturation and heme delivery [29]. The evolution of cytochrome c domains has been reported to involve gene transfer events [30]. CcmA was identified as the essential gene of the cytochrome c ATPase; therefore, CcmA is likely to be transferred to assist the electron transfer process. In this study, we focused on the 14 horizontally transferred genes that could be traced in the AMD evolutionary process; other genes also have the possibility of horizontal transfer among the AMD community members. HGT events are widely involved in microbial genomes in natural communities; however, the computational identification of HGT in genomic sequences remains very difficult in practice [11]. We could have identified more HGT events among the AMD metagenomes. However, to more thoroughly investigate the impacts of HGTs, we selected the genes that were most likely transferred and made a large contribution to the AMD community. A second notable aspect of this study is that we aimed to examine HGT events between members within the microbial community. To be sure, novel genes of a recipient genome were recognized as being transferred by a donor species either from an external environment or internal community. However, as a relatively self-contained ecosystem in an extreme environment, the AMD microorganisms can demonstrate the social behaviors of both cooperation and competition. In this light, we argued that the movement of genetic materials among organisms within the community should be essential to their performance as a whole. Finally, it is certain that the gene transfer events studied in this work were determined by the strict standard of HGT identification adopted here, which also implies that these putative HGT events were identified amongst distantly related species. HGT might be easier between closely related species for which the barriers to the transfer more easily can be surmounted [11]. However, because of the environmental challenges, some gene transfer events would greatly benefit some species or the entire community, leading to a better adaptation to the environment, even with the effort for the exchange of genetic material between distantly related species. In other words, the HGT events reported in this manuscript should be regarded as being nontrivial and essential to the AMD community because of the strong selective pressure and the harsh environment in AMD. Acquisition of antibiotic resistance in G-plasma via HGT associated with ribosomal proteins Among the 14 horizontally transferred genes, nine were transferred from bacteria to the archaeon G-plasma. Moreover, they encoded ribosomal proteins and the related subunits. Following, we report our detection and analysis of the consequence of these transferred genes on G-plasma and the AMD microbial community. Ribosomal proteins are widely distributed in prokaryotes, and they are usually involved in information processing (e.g., replication, transcription, and translation) or central metabolism. Genes for ribosomal proteins are generally considered to be housekeeping genes and relatively recalcitrant to HGT; because of this, their sequences are routinely used as phylogenetic markers [31]. However, ribosomal genes have shown a remarkable possibility of being transferred in many prokaryotic genomes [3]. For example, a phylogenetic study on ribosomal protein S14 revealed an unexpected tree topology that was explained by horizontal transfer [32]. Also, ribosomal proteins L32 and L33 have phylogenetic incongruities that resulted from gene transfer [33]. In the current study, one of the evident horizontal transfer events was that of ribosomal protein genes. In L. ferrodiazotrophum, we found a contig (gi: 251772484) containing 10 ribosomal genes: rpsJ, rplD, rpsC, rplP, rplX, rplE, rplR, rpsE, rpsK, and rpsD. Of these genes, rpsJ, rplD, rpsC, and rplP are usually clustered as operon S10, while rplX, rplE, rplR, and rpsE are usually clustered as operon spc. All of these ribosomal genes are in the positive strand of the contig [Additional file 2: Figure S1], and their protein name and function are listed in Table 2. Looking into the phylogenetic tree of these ribosomal genes, most of them (except RpsK) show obvious disagreement with the species tree, strongly indicating possible horizontal transfer events. To be specific, the ribosomal genes of G-plasma often have a close relationship with those of L. ferrodiazotrophum and L. rubarum instead of other plasma species, and they are placed in a cluster of bacteria rather than archaea in the gene tree. The same pattern holds for the ribosomal genes of both the spc operon (rpsE in Fig. 2, others in Additional file 2: Figures S2-S4) and S10 operon [Additional file 2: Figures S5-S8]. Therefore, ribosomal genes of G-plasma show a high possibility of being transferred from bacteria. Considering the close relationship and the same habitat as Leptospirillum, it makes sense that L. ferrodiazotrophum and L. rubarum are the donor of these ribosomal genes for the archaeon G-plasma. Notably, these genes were identified as transferred clusters, including two known operons, from bacteria to archaea as shown in Additional file 2: Figure S1. This feature is clearly consistent with the theory of a selfish operon, in which operons are viewed as mobile genetic entities that are constantly disseminated via HGT, although their retention could be favored by the advantage of the co-regulation of functionally linked genes [34]. As a result, functional information for these transferred genes may be inferred by comparing them with the related or analogous genes. An earlier study focusing on the horizontal transfer of rps14, which is also a member of the spc operon, led to the conclusion that antibiotic resistance can be conferred because rps14 is known to be involved in antibiotic resistance through the binding of puromycin besides its major role in the assembly of ribosomal 30S subunits [32,35]. In the current study, although rps14 does not appear in both G-plasma and the two Leptospirillum bacteria, we noted that rpsE, which is in both G-plasma and Leptospirillum, has a function that is similar to that of rps14, i.e., antibiotic resistance. Therefore, we may safely conclude that G-plasma can acquire this function through horizontally transferred spc operon genes. In addition, the S10 operon has two genes, rplP and rplD, that are involved in antibiotic resistance. These observations led to the explanation that G-plasma acquired these genes for antibiotic resistance. As predicted by the selfish operon model [34], these transferred gene clusters should allow cells in the AMD environment to demonstrate the metabolic benefits of antibiotic resistance; moreover, they can enhance the fitness of G-plasma as the recipient species. Another question of interest is how the transferred genes associated with antibiotic resistance influence the microbial community in AMD. We may just as well learn this point from their role in L. ferrodiazotrophum and L. rubarum, the two most dominant organisms in AMD. The amount of antibiotic resistance proteins in L. rubarum was correlated with the growth stage of biofilms [36]. In early growth stage samples, L. rubarum dominates the community, and its repressors of antibiotic resistance genes are abundant. With the growth of biofilms, increasing types of microorganisms compete for limited resources (e.g., nitrogen, oxygen, and phosphate), forcing L. rubarum to reinforce its competitiveness and self-protection. A previous study demonstrated that antibiotic resistance proteins from L. rubarum were more abundant during latestage growth [36]. Clearly, antibiotic resistance is closely related to the population of individual species in the natural microbial community. It is interesting to note that Gplasma is the dominant archaeon in AMD. Based on proteomic data across 28 microbial community samples, G-plasma constituted about 9.0 ± 4.9 % of the community [37]. Deduced by analogy, G-plasma and other archaea should also encounter a similar situation, and the archaeon with a competing advantage will survive easier than archaea without the advantage. As shown by the population distribution, G-plasma is no doubt a strong competitor. The horizontal acquisition of antibiotic resistance genes is a possible advantage that protects and enhances its competitiveness, making G-plasma the largest group among AMD archaea. As a result, both these genes and their horizontally transferred relatives that provide antibiotic resistance influence the growth of species and shape the population structure of the AMD microbial community. Taken together, the transferred gene clusters of ribosomal genes are associated with antibiotic resistance, leading to the fitness improvement of the archaeon G-plasma and the community structure in AMD. It should be pointed out that the requirement of antibiotic resistance, as the selective pressure, may force microorganisms to overcome the possible difficulties of horizontal transfer because these horizontally acquired antibiotic resistance genes are of great importance in the life of G-plasma in the toxic environment of the AMD, and they may be vital in making G-plasma the largest group of AMD archaea. Possible impact of gadBC operon transfer on acid resistance in Ferroplasma In this subsection, we discuss the putative horizontal transfer of the gadB gene of the gadBC operon from bacteria to two archaea of the Ferroplasma genus. The gadBC operon corresponds to the function of acid resistance. Acid resistance is perceived as an essential property of microorganisms living in AMD. The glutamate decarboxylase (GAD) system, which is common in bacteria and some eukaryotic genomes, has been extensively studied for its major role in acid resistance in organisms such as in E. coli, Shigella flexneri, and Listeria monocytogenes [38][39][40][41]. An essential role of biochemical pathways is to yield cell-cell messengers [41]. The GAD system is usually composed of three genes, gadA, gadB, and gadC. The gadA and gadB genes usually have high sequence similarity and encode two biochemically indistinguishable glutamate decarboxylases, and the gadC gene encodes a glutamate/GABA antiporter. The gadB and gadC genes are organized into a functionally important operon, gadBC [42]. By producing alkaline γ-aminobutyrate and utilizing an intracellular proton, functions that involve the gadBC operon, cells can adapt to low pH [43]. Through investigation of the phylogenetic tree of gadB, we found that there was an obvious incompatibility with the organism tree, and gadB from Ferroplasma fer1 and Ferroplasma fer2 were misplaced within the cluster of bacteria; the closest relatives were genes from L. ferrodiazotrophum and L. rubarum (Fig. 3). This incompatibility indicates a possible transfer event. Moreover, transposase genes were found in the neighborhood of the gadBC operon in both F. fer1 and F. fer2, providing additional evidence for the HGT events [44]. Because of the low conservation and poor annotation quality of the gadC gene in public databases, it is difficult to estimate the true representation of gadB in most microbial genomes even with examples of its potential role in a few well-known organisms such as E. coli and S. flexneri. Although the gadC gene in both F. fer1 and F. fer2 is located close to that of L. ferrodiazotrophum and L. rubarum in the phylogenetic tree, we did not analyze its confused and incongruous evolution in the current study. However, because there was only one transcription start site for the gadBC operon regardless of the inducing condition [45] and no obvious promoter could be found upstream of gadC, we cannot exclude the possibility of the same transfer event including the gadC gene, leading to the transfer of both the gadB and gadC gene. Furthermore, the prediction of 13 transmembrane passes with TMHMM 2.0 [46] inferred the integral function of gadC in Ferroplasma. An ancestor of Ferroplasma originally acquired the gadB gene by horizontal transfer (transferred as the gadBC operon), and L. ferrodiazotrophum and L. rubarum might be the donors because of their close relationship and presence in the same habitat in which they overcame the acid environment to use more resources. On the basis of the function of the GAD system, it is reasonable to suggest that the acquisition of the gadBC operon allowed F. fer1 and F. fer2 to better resist the extreme acid environment. Although there are other mechanisms to resist a low-pH environment [47], the horizontally transferred gadBC operon plays an important role in the efficient utilization of a proton by F. fer1 and F. fer2 to resist the extreme acidic environment. Combined with the putative transfer events of the spc operon and S10 operon from bacteria to G-plasma, there seems to be a tendency toward the movement of genes with operon structures from bacteria to archaea. In general, this agrees with the selfish operon hypothesis [34]. However, the two cases described in this report indicate that most transferred genes involved in the same operon have a closely functional relationship. Moreover, they play an essential role in the requirements of antibiotic or acid resistance in recipient archaea. This differs from the viewpoint that the majority of genes in transferred operons are nonessential genes with related functions [34,48]. In an environment that seems so harsh, organisms have to retain their beneficial genetic resources to survive the external stresses of the environment. Discussion As an important driving force of the evolution of microorganisms, HGT has been extensively studied in prokaryotes. However, the theories of HGT of natural microorganisms in extreme environments remain open; currently, these theories seem to be largely in a state of conjecture because of the challenges associated with the complexity of sequenced metagenomes. In this paper, we used AMD metagenomic sequences as a model metagenome, which avoided the difficulties of HGT identification in metagenomic data from natural environmental samples. The strategy designed to identify HGT events is based on a strict standard in the current study. Because phylogenetic reconstruction was assumed to cause inherent noise and possible artifacts, we used an AU test, which is regarded as an appropriate approach to evaluate the phylogenetic topology to yield a discriminating result and dramatically reduce the noise of phylogenies. Herein, we attempted to focus on a set of putative HGT events that occurred among members within the AMD microbial community. As we have seen with our computational identification strategy, apparent HGT events occurred among the microorganisms in AMD. Our investigation thus provided insight into understanding a potential role of HGT in the environmental microbial communities. Although there are possible limitations of computational identification of HGT events, the bioinformatics strategy avoids the experimental difficulties and the challenges associated with the complexity of multiple species in a natural niche. Furthermore, because the methodology of computational identification has been widely used in genomics research, studies based on these methods give researchers important clues to explore HGT events and genomic evolution [49][50][51]. Through gene transfer, functional traits were introduced into the recipient microorganisms, and those that overcame selective pressure are presented by heredity. For example, antibiotic resistance, transferred via the HGT of ribosomal genes in AMD, allowed microorganisms to grow in the presence of certain poisonous compounds. Also, with the acquisition of acid resistance, such as that via HGT of the gadBC operon, cells could remove intracellular protons and adapt to the acidic environment. Therefore, these transferred genes may greatly contribute to the metabolic and physiological requirements of the extreme environment and provide microorganisms with innovative functional solutions to adverse environmental problems. Aside from the impact on the recipients, the influence of HGT also influenced the structure of the entire community or environment. As mentioned above, transfer events of ribosomal genes enhanced the competence of G-plasma and greatly influenced the community structure in AMD. Previous studies demonstrated that gene transfer may promote cooperation among microorganisms in some situations and was correlated with biofilm formation [52]. It should also be noted that the extreme environment and community could influence the possibility of HGT. For example, the acid solution prevents gene transfer by the uptake of naked DNA in AMD. Consequently, gene transfers were definitely influenced by both the natural environment and community members. In summary, our findings, which are based on bioinformatics analysis, revealed that gene transfer events potentially contribute functional innovation to the recipients, as well as further shape the dynamic evolution and population structure of the special microbial community. Furthermore, from an evolutionary standpoint, it is reasonable to discuss HGT in the context of the community instead of single microorganisms to understand the social relationships and dynamics of community members in a natural environment. It has been pointed out that communities are ideal candidates for genetic exchange among microorganisms [53]. In comparison, single microorganism had low competitive competence; therefore, living as consortia could benefit many members in the community through events such as the acquisition of new genes by HGT. Especially in biofilms, which are microbial cells in densely packed communities, the possibility of HGT is very high [52]. Eventually, HGT in a community will accelerate single microorganism evolution and adaptation to the environment, and the subsequent evolution of social relationships will also be influenced. Therefore, trends of gene transfer and community dynamics are highly correlated. Research on the interconnectedness between HGT and community evolution will expand our view of the impacts on the recipient, community, and environment. At present, the clustering property of transferred genetic materials remains uncertain and is therefore an interesting problem for HGT studies. In the current study, our two detailed cases clearly exemplified this issue: they were all connected with operon structure, especially the large gene clusters in the ribosomal gene transfer. As we mentioned above, the selfish operon model argued that operon organization allowed efficient horizontal transfer of genes that were otherwise susceptible to loss by genetic drift [34]. Moreover, recent research showed that the amount of genetic material that can be moved horizontally may include small gene fragments, entire operons, superoperons that encode complex biochemical pathways, and whole chromosomes [54]. Thus, it is not surprising that our two cases were identified as transfer events at the operon level. However, compared with transfer events of a single gene, to what extent did HGT events involve gene clusters? If genes were transferred by cluster structure, what intrinsic factor caused it? These questions are largely unsolved mysteries of genomic evolution. The current study provides nontrivial support to extend the research to natural community microorganisms. A further problem is how these transferred genes were accepted and how they retained their function in the recipient genome. Whether these newly acquired genes can survive in the recipient genome depends on the evolution of their sequence characteristics, transcription, and expression mechanisms. To this end, we analyzed the genes associated with ribosomal proteins that were transferred from bacteria to G-plasma. We examined the sequence pattern of regions upstream of the transferred ribosomal genes in the recipient genome. As a result, the translational and transcriptional signals in the upstream region were much like those of G-plasma instead of those of the donor bacteria. This is compatible with the fact that phylogenetic methods were used to detect ancient transfer events. It is uncertain whether this appearance was caused by the convergent evolution of the originally transferred signals, or if these genes used the signals from the recipient genome. A recent study demonstrated that operons were often acquired with their regulators to facilitate the evolution of the transferred genes [55]. This is in accord with the speculation that gadB and gadC of the gadBC operon might have been transferred together and used one transcription signal that was located upstream of gadB. In this case, the convergent evolution of transferred genes with their original translation regulatory regions seems likely. However, it is clear that further investigation will be required to reach a full understanding of this problem. Conclusions Using the AMD metagenomic data as a model metagenome, our exploratory study provided unique insight into HGT events among microorganisms in natural communities. It is important to discuss all possible contributions of HGT among community members to an individual species, the entire microbial ecosystem including its dynamic evolution, and the related environment. In this regard, our analyses partly address these outstanding questions and shed light on functional metagenomics and social evolution in microbes from a metagenomic perspective. This study will stimulate future investigation of these topics propelled by an interdisciplinary exchange between microbiologists, evolutionary biologists, and bioinformaticians. Obtaining sequences and gene annotation Complete or near-complete sequences of 11 organisms were constructed from three samples in AMD [15,56] and downloaded from NCBI [23] (http://www.ncbi.nlm.nih.gov/). Three ARMAN organisms were not considered in this study because of their small genome size and gene number. Recently, several tools to predict coding regions in metagenomic sequences were developed [20,21,52]. The eight remaining organisms (including two bacteria and six archaea), were analyzed using MetaGeneAnnotator [57] to identify genes and MetaTISA [22] to locate Translation Initiation Sites (TISs). Besides the AMD microorganisms, nine previously isolated organisms (five bacteria and four archaea) were added to the phylogenetic analysis to obtain improved resolution. Protein sequences of these nine isolated organisms were downloaded from NCBI to allow orthologous gene family identification. Information about these organisms is listed in Table 3. Phylogenetic tree construction To construct the gene family phylogenetic tree, the strict reciprocal best BLAST hit method [20,21] with E-value cutoff 10 −5 , identity >25 %, and alignment length >60 % was used to identify orthologous gene families. These gene families were aligned with ClustalW 2.0 [58] using default parameters. For each gene family, a maximum likelihood tree was built using the proml program in the PHYLIP package [59]. Each dataset was replicated 100 Fig. 4 Phylogenetic tree of the 16S rRNA from genomes in this study. Microorganisms from acid mine drainage are shown in bold times, and the last consensus tree was decided by majority rule with Consense in the PHYLIP package [59]. The organism tree was constructed using 16S rRNA as a phylogenetic marker (Fig. 4). The corresponding 16S rRNA sequences of AMD organisms were predicted using RNAmmer [60], and those of other genomes were downloaded from RDP database (Ribosomal Database Project) [61]. Accordingly, a maximum likelihood tree of these organisms was built with MEGA 5.0 [62].	2023-01-15T14:07:38.079Z	2015-07-04T00:00:00.000	{ "year": 2015, "sha1": "67099682a16a57ed991a33b3686fb26da5e71f8b", "oa_license": "CCBY", "oa_url": "https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-015-1720-0", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "67099682a16a57ed991a33b3686fb26da5e71f8b", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [] }
234795438	pes2o/s2orc	v3-fos-license	Targeting SNARE-Mediated Vesicle Transport to Block Invadopodium-Based Cancer Cell Invasion During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells’ invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types. involved in cancer cell invasion is necessary to advance the development of anti-invasion drugs, as agents to mitigate metastatic progression of cancer and increase survival of patients. Invadopodia are sub-cellular, membrane-associated structures that mediate cancer cell invasion and facilitate metastasis. These cancer-specific protrusions function to degrade the extracellular matrix (ECM), allowing cancerous cells to invade through these barriers, breach tissue compartments, intravasate into blood and lymphatic systems, extravasate and subsequently colonize secondary tissue sites (3)(4)(5). The primary tumors of many metastatic cancers display increased expression of key proteins involved in invadopodia formation (e.g. Tks5, EGFR), compared to non-metastatic cancers (6)(7)(8). Furthermore, the invasive and metastatic potential of primary tumors often correlates with their ability to form invadopodia (9). Invadopodia thus represent attractive targets for the inhibition of cancer cell invasion as part of the metastatic cascade. Invadopodia contain an F-actin core, which is enriched in actin modeling proteins such as Arp2/3, N-WASP, Tks5, and cortactin. Formation of invadopodia is also dependent on the localization and activation of epidermal growth factor receptor (EGFR) and integrins, which elicit intracellular signaling cascades that recruit and activate signaling molecules such as FAK and Src kinase (10,11). Together, these proteins make up the core structure of invadopodia, integrate signaling pathways to induce localized actin polymerization within invadopodia pre-cursors, and support the formation of membrane protrusions. The microtubule network is also crucial to invadopodia formation and maturation (12,13). Microtubules have been shown to have important roles in the regulation of MT1-MMP activity at invadopodia (14). As well, microtubules and microtubuleregulating proteins have been implicated in the modulation of focal adhesion dynamics and cell-ECM interaction at invadopodia (15,16). Maturation of invadopodia corresponds with the delivery of matrix metalloproteinases MT1-MMP, MMP-2, and MMP-9, which gives these distinct structures their degradative phenotype by initiating ECM remodeling (17,18). The delivery and localization of all invadopodia-associated proteins is essential for invadopodia formation and function, and is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE-MEDIATED VESICLE TRAFFIC AND INVADOPODIUM FORMATION SNAREs are mediators of vesicle-based trafficking in cells and are central to both constitutive and regulated trafficking pathways. SNAREs form complexes between vesicle and target membranes, leading to fusion of the membranes and allowing delivery of vesicle contents to target compartment. In this manner, SNAREs contribute to the biosynthetic secretary pathway, endocytic recycling pathways, and regulated membrane traffic such as neurosecretion or insulin release. While much is known about SNARE-mediated membrane trafficking in some systems, our understanding of the membrane traffic in the context of cancer cells has significantly increased in recent years. SNARE-mediated trafficking of invadopodial proteins, to and from the plasma membrane, contributes to the remodeling of membranes and the localized enrichment of signaling components, adhesion receptors, and ECM degrading enzymes at sites where invadopodia form (Figure 1). Several studies point toward the role of specific SNAREs in trafficking invadopodium-associated proteins to promote cellular invasion and migration of malignant cancer cells (19)(20)(21)(22). The localization and activation of EGFR and b1 integrin to sites of cell-ECM attachment are important for invadopodia formation and function. Membrane trafficking pathways, involving SNAREs SNAP23 and Syntaxin13, have been shown to contribute to invadopodium formation through the delivery of EGFR and b1 integrin to the cell membrane (21). This trafficking pathway also delivers Src kinase, in association with EGFR and b1 integrin, to these sites (21). b1 integrin signaling stimulates SNARE complex formation, involving SNAP23 and syntaxin13, promoting the association of Src with EGFR, leading to receptor phosphorylation and activation (21). The association of Src, EGFR, and b1 integrin downstream of b1 integrin activation then promotes invadopodia formation and cellular invasion (23). Expression of SNAP23 constructs with cytoplasmic deletions and syntaxin13 dominant-negative mutants were shown to perturb invadopodia formation and cell invasion of ECM in vitro (21). Secretion of MMPs correlates with the metastatic potential of cancers (6,17,18), and evidence suggests that MT1-MMP is a key protease that drives cancer cell invasion. It is clear that vesicle-mediated delivery of MT1-MMP promotes invadopodia maturation, and this is important for ECM remodeling and cell invasion (20,22). For example, using several different cell culture models, SNARE complexes containing SNAP23, VAMP3 and syntaxin13 (20), or SNAP23, syntaxin4, and VAMP7, have been shown to contribute to invadopodia formation, by mediating the trafficking of MT1-MMP (22). These studies have demonstrated that both expression and function of the SNAREs were required for invadopodia formation and MT1-MMP localization to invadopodia (22). VAMP3 and syntaxin13 were also found to be involved in the secretion of MMP2 and MMP9 during ECM remodeling in invasive cancer cells (20). Collectively, these studies reveal that several, possibly overlapping, SNAREmediated membrane trafficking pathways contribute to invadopodium formation and function, and how these pathways are interconnected and coordinated is an area of active investigation. REGULATION OF SNARE FUNCTION AS A THERAPEUTIC TARGET The regulated assembly of SNARE complexes is necessary for the delivery of invadopodial proteins to the surface of cancer cells during cell invasion. Therapeutic targeting of SNARE proteins is therefore a potential approach for the inhibition of invadopodiabased invasion and subsequent metastatic spread of malignant cells; however, the diverse roles that SNARE proteins play in crucial physiological functions suggests that targeting SNAREs themselves may lead to detrimental side effects. While targeting a potential anti-SNARE therapy specifically to cancer cells is a theoretical possibility, a more attractive strategy might be to target regulators of SNARE function and complex formation to more specifically disrupt membrane trafficking pathways that are supporting invadopodium formation and invasive activity. SNARE complex formation displays high specificity during membrane trafficking, providing fidelity in trafficking pathways (24), and this is achieved in part by post-translational regulation of SNARE activity. While much is known about the regulation of SNARE complex formation in some contexts, how SNAREs are regulated in invasive cancer cells is just emerging. One important mechanism for regulation of SNAREs is phosphorylation, and this has been well described in other systems (25,26). In cancer cells, trafficking of MT1-MMP, involving SNAP23 and syntaxin4, has been shown to be modulated by phosphorylation of syntaxin4, with its dephosphorylation correlating with increased interaction with SNAP23 and increased invadopodium formation (22). The kinase(s) and phosphatase(s) responsible for phosphorylation and dephosphorylation of syntaxin4 in this context remain to be identified. MUNC18C SNAREs that have been implicated in cancer cell invasion have also been shown to be regulated by accessory proteins, including the Sec1/Munc18 (SM) family. SM family proteins are key regulators of SNARE-mediated membrane fusion, and they function by interacting with members of the syntaxin family of SNARE proteins (27). In one model, the binding of an SM protein to its cognate syntaxin is believed to modulate the syntaxin's conformation to a primed open state (28). The "open" conformation of the syntaxin facilitates its association with other SNARE proteins necessary for the formation of a fusogenic SNARE complex. Munc18c is a known partner of syntaxin4 (29), a SNARE involved in the delivery of MT1-MMPand EGFR-containing vesicles to invadopodial membranes (30). Munc18c has been reported to promote the formation of a syntaxin4-VAMP7-SNAP23 complex in MDA-MB-231 cells during invadopodia formation (22). A potential method to inhibit the delivery of invadopodial proteins to sites of invadopodia formation was studied, whereby Munc18c binding to endogenous syntaxin4 was perturbed. Exogenous expression of the 29 amino acid N-terminus of syntaxin4 (Stx4-N-term), containing the site that binds Munc18c, impaired the association of endogenous Munc18c and syntaxin4, possibly by competitively inhibiting syntaxin4-Munc18c binding (31). Cells expressing Stx4-N-term demonstrated decreased levels of syntaxin4-containing SNARE complexes, lower cell surface GELSOLIN AND SUPERVILLIN Other Syntaxin4-regulatory molecules have been identified, including those in the gelsolin/villin superfamily (32). Gelsolin is a multifunctional actin-binding protein, which and can regulate the cytoskeleton by capping and severing F-actin filaments (33). Interestingly, gelsolin has also been found to play a role in the regulation of insulin exocytosis in pancreatic islet b-cells (34). Syntaxin4 mediates insulin granule docking at the plasma membrane of b-cells, forming a complex with SNAP25 and VAMP2 (35). Gelsolin was found to interact directly with the HA domain (amino acids 39-70) of syntaxin4 under resting conditions, suppressing SNARE complex formation (32). Upon glucose stimulation, gelsolin releases from Syntaxin4, allowing for the formation of cognate SNARE complexes necessary for insulin exocytosis. b cells overexpressing the HA domain of syntaxin4 were observed to secrete insulin in the absence of glucose, underscoring the importance of gelsolin in regulating insulin granule release. Gelsolin and supervillin (another member of the gelsolin/ villin superfamily) have been shown to localize to invadopodia where they regulate actin dynamics (36)(37)(38). Knockdown of both proteins in COS-7 and MDA-MB-231 cells was found to negatively affect MT1-MMP-dependent matrix degradation at invadopodia, as well as cellular invasion (37). Downregulation of gelsolin has also been shown to play a role in regulating the invasion and motility of MDA-MB-231 and PC-3 cells (39). Given gelsolin's established role as a Syntaxin4-binding protein, it is plausible that members of the gelsolin/villin superfamily may be regulating SNARE complex formation to influence the delivery of cargo to the invadopodial membrane. Further research should be directed towards determining if gelsolin and supervillin associate with SNAREs during invadopodiumbased cell invasion, and how perturbing their expression or function might influence invadopodial dynamics. CDC42 The vesicle SNARE VAMP2 also has a potentially regulated role in invadopodia biogenesis. A complex of VAMP2-Syntaxin1A-SNAP25 plays a well understood role in insulin exocytosis in pancreatic b cells (40). Cdc42 can directly interact with VAMP2 in CHO-K1 cells, and this interaction promotes the formation of a complex with Syntaxin1A (40). Expression of a VAMP2 Nterminal peptide, corresponding to the binding site of cdc42, resulted in decreased insulin secretion in cells stimulated with glucose, demonstrating functional significance of VAMP2 A B (42), and syntaxin13 (21) are SNAREs whose expression has been shown to be upregulated in cancerous cells or have been identified to influence cellular invasion directly. Further investigation into proteins that associate with these SNAREs should be pursued, as these would represent potential druggable targets for impeding invadopodium-driven metastatic invasion. SNARE-dependent trafficking of proteins to invadopodia holds potential as a point of therapeutic intervention in metastatic progression. An effective approach to interfere with SNARE-dependent invadopodium formation and function is to target SNARE interactions with regulatory proteins that have been shown to be involved in invadopodia function ( Figure 2). Such an approach has already been successful in vitro in MDA-MB-231 cells (31). These results provide a promising avenue for the development of anti-metastatic agents targeting SNARE regulatory molecules. Specific interactions between SNAREs and Munc18c, gelsolin, supervillin, as well as other unidentified SNARE regulatory proteins, represent potential targets to combat metastasis in patients with invadopodiaforming cancer subtypes. AUTHOR CONTRIBUTIONS GG and OG drafted the manuscript and generated the figures. MC edited and revised the manuscript and the figure. All authors contributed to conceptualization. All authors contributed to the article and approved the submitted version.	2021-05-21T13:31:41.548Z	2021-05-21T00:00:00.000	{ "year": 2021, "sha1": "bcef1db19a5b5d40ffd1fd5335f615281cfcf00a", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fonc.2021.679955/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bcef1db19a5b5d40ffd1fd5335f615281cfcf00a", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
54671271	pes2o/s2orc	v3-fos-license	Fluorescent Properties of Hymecromone and Fluorimetric Analysis of Hymecromone in Compound Dantong Capsule Fluorescence spectra of hymecromone (4MU) aqueous solutions are investigated at different pHs. Two fluorescent species of 4MU, neutral molecular form and anion form, are considered to be the main fluorescent forms. Quantum yields of the two forms are measured to be 0.74 at pH 5.98 and 0.95 at pH 9.75, respectively.The ionization constant of 7-hydroxyl proton of 4MU is determined to be pK a = 7.85 ± 0.03 by a pH-fluorescence method. Addition of methanol into 4MU aqueous solution leads to a blue shift of maximum emission wavelength from 445 nm to 380 nm, and a decrease in fluorescence intensity. 3D fluorescence spectra of Chinese patent drug Compound Dantong Capsule (CDC) and its four component herbal drugs are also investigated. Based on their fluorescent properties, a novel fluorimetric method is proposed for the selective determination of 4MU in CDC without preseparation. The new method is suitable for the routine quality evaluation of CDC. The chemical composition of CDC includes 4MU (also named Dantong) and four kinds of Chinese herbal medicines, including Herba Isodonis Lophanthoidis (Xihuangcao), Herba Artemisiae Scopariae (Yinchen), Herba Andrographis (Chuanxinlian), and Rhei Radix et Rhizoma (Dahuang). The content of 4MU is one of the main quality indexes of CDC [21]. Earlier, the spectrophotometric method was applied for the determination of 4MU in CDC according to the absorbance of 4MU at 372 nm [19]. Later, since the interference of coexistent components, high performance liquid chromatographic methods (HPLC) were developed for the determination of 4MU in CDC [21][22][23]. So far, no fluorimetric method was reported for the determination of 4MU in CDC and other medicinal samples. Fluorimetry has been recognized as one of the most useful analytical methods owing to its high sensitivity, good selectivity, simplicity, speediness, and low cost. It can be suitable for analysis of some complex samples such as traditional Chinese medicine containing a fluorescent component, especially in the case of a routine analysis. Three-dimensional fluorescence spectra (3D fluorescent fingerprint) have been used in our lab for qualitative identification of Chinese herbal medicines [24]. Moreover, a number of fluorimetric methods have been developed for quantitative determination of active components in medicinal materials, such as paeonol in Cynanchi Paniculati Radix (Xuchangqing) [25], camptothecin in common Camptotheca fruit (Xishuguo) [26], and arctiin in Arctii Fructus (Niubangzi) [27]. In this study, the fluorescent properties of 4MU are investigated, and its quantum yield and 7-hydroxyl proton ionization constant are measured. The fluorescent properties of CDC and its four consisting Chinese herbal medicines are also studied. Based on the spectral differences between 4MU and other components in CDC, a fluorimetric method is proposed for the determination of 4MU in CDC sample without preseparation. Experimental 2.1. Apparatus. Fluorescence measurements were performed on a Hitachi (Tokyo, Japan) F-7000 spectrofluorimeter equipped with a xenon lamp, 1 cm quartz cell, and a UV-29 filter placed into the emission light path to remove secondary spectrum. The excitation and emission slits (band pass) 5 nm/5 nm were used throughout the work. Absorption spectra were recorded using a Shimadzu (Kyoto, Japan) UV-2501PC recording spectrophotometer with 1 cm quartz cell. An Orion (Beverly, USA) 868 pH/ISE meter was used for pH measurement. and diluted to appropriate concentration with 0.05 mol L −1 H 2 SO 4 when it was used. Britton-Robinson buffer solution was a mixture of phosphoric acid, boric acid, and acetic acid (each 0.02 mol L −1 ) and adjusted to an appropriate pH by addition of 0.1 mol L −1 NaOH solution. All the buffer chemicals were of analytical grade. The water used throughout the study was doubly-deionized and verified to be free from fluorescence. General Procedure for Spectral Measurement. A series of 10 mL volumetric flasks was added appropriate amount of 4MU or CDC and buffer solutions. The mixtures were diluted to the mark with water and mixed well. Fluorescence or absorption spectra were measured at room temperature. Meanwhile, Raman scattering of water at excitation wavelength of 350 nm was measured (expressed as R, for calibration of 3D fluorescence spectra) [24]. Determination of Ionization Constant. A number of solutions containing the same concentration of weak acid HB and different pH were prepared. Fluorescence intensity and pH of each solution were measured. Then the ionization constant p was calculated according to the following equation [28]: where HB and B were the fluorescence intensity of HB and its conjugate base B; they can be measured at sufficient acidic pH where all HB species exist in the neutral molecular form and at sufficient alkaline pH where all HB species exist in the anion form, respectively. Measurement of Fluorescence Quantum Yield. Quinine bisulphate was used as a reference (quantum yield 0.55 at excitation wavelength of 313 nm) in measuring quantum yield of 4MU. For the measurement, quinine bisulphate and 4MU solutions were prepared in proper concentration so that the absorbance ( ) of the two solutions was similar and not larger than 0.05. Absorption and fluorescence spectra were recorded, and then quantum yields were calculated according to the following equation [28,29]: where and were the fluorescence quantum yield of unknown and the reference, and were the integral fluorescence intensity of unknown and reference solutions, and and were the absorbance of unknown and reference solutions at their excitation wavelengths, respectively. 2.6. Sample Preparation. Dissolve 9.21 mg CDC powder in 100 mL methanol and dilute to 0.921 g mL −1 with methanol as sample solution. Fluorescence Properties of 4MU 3.1.1. 3D Fluorescence Spectra of 4MU. 3D (three-dimensional) fluorescence spectra of solvent blank and 4MU aqueous solutions at pH 5.98 and at pH 9.75 were measured as shown in Figure 1. The spectra indicated that the solvent used in this study was basically no fluorescence. 4MU can produce strong fluorescence in near neutral condition with maximum excitation wavelength ( ex ) of 320 nm and maximum emission wavelength ( em ) of 445 nm, while in weak alkaline condition, the fluorescence intensity enhanced, ex red shifted from 320 nm to 360 nm, but em remained constant. Figure 1 revealed that pH has an important influence on fluorescence. Effect of pH on Fluorescence. As shown in Figure 2, effect of pH on fluorescence excitation and emission spectra of 4MU at various pHs were studied. In acidic conditions (pH 1.97-6.72), along with the decrease in pH, fluorescence intensity declined, ex centered at 320 nm, and em red shifted slightly from 445 nm to 455 nm. In the range of pH 7.12-10.43, along with the increase in pH, fluorescence emission at 445 nm enhanced, but the excitation band centered at 320 nm declined, meanwhile a new excitation band centered at 360 nm emerged and an iso-fluorescence point [30] formed at 330 nm. In the range of pH 10.80-13.38, along with the increase in pH, fluorescence emission at 445 nm gradually quenched while ex and em remained constant. The relationship between pH and fluorescence intensity was summarized in Figure 2(d). The variation of fluorescence spectra implies that the solution equilibrium or molecular structure of 4MU changed along with the change in pH. According to Figure 2, we suppose that the proton dissociation and hydrolysis process of 4MU is as shown in Figure 3. The structure of 4MU contains a benzene ring (linked with 7-OH) and a lactonic ring (include lactone bond and 2-C=O). In strong acid conditions, the 2-carbonyl oxygen protonated [28,30] to form a cationic species (type I), leading to a decrease in fluorescence intensity and a red shift in emission wavelength. In near neutral conditions (pH 4.0-7.0), 4MU exist mainly as molecular form (type II) which have strong fluorescence with ex of 320 nm and em of 445 nm. In weak alkaline conditions (pH 9.0-12.0), 7-hydroxyl proton dissociated and 4MU exist mainly as anion form (type III) which have stronger fluorescence with ex of 360 nm and em of 445 nm. In strong alkaline conditions (pH > 12.0), the hydrolysis of lactone bond take place [28,31], leading to a fluorescence quenching. Ionization Constant of 7-Hydroxyl Proton. With the above discussion, ionization constant of 7-OH proton can be determined in suitable conditions. Using the pH-fluorescence method described in Section 2.4, the ionization constant of 7-hydroxyl proton was determined to be p = 7.85 ± 0.03 (Table 1). In addition, according to the relationship between fluorescence intensity and pH, we can graphically estimate the ionization constant. In Figure 2(d), the abscissa of the intersection of curve (a) and curve (b) is about 7.85, which is just the p . The ionization constant determined above is close to the ionization constant of umbelliferone; p = 7.61 ± 0.03 [28]. The little difference between the two constants reflects the impact of 4-methyl on the acidity of 7-hydroxyl proton. declined, while a new emission band centered at 380 nm emerged. When the percentage of methanol reached 100%, the em blue shifted to 380 nm; however, the fluorescence intensity was less than that in aqueous solutions. Therefore, we choose water as the appropriate solvent in establishing a fluorimetric method in the following study, and control the methanol percentage in aqueous solution no more than 20% to obtain sensitive and stable fluorescence. Measurement of Fluorescence Quantum Yield of 4MU. Using the method described in Section 2.5, quantum yields of 4MU in near neutral (pH 5.98) and weak alkaline (pH 9.75) conditions were measured, as shown in Table 2. In near neutral solution, 4MU exist as molecular form; its quantum yield at excitation wavelength 320 nm was measured to be 0.74. In weak alkaline solution, 4MU exist as anion form; its quantum yield at excitation wavelength 360 nm was measured to be 0.95. These results indicate that 4MU is an excellent fluorophore. A fluorimetric method for determination of 4MU in Chinese patent drug should be very sensitive either in near neutral or in weak alkaline conditions. 3D Fluorescence Spectra of Four Kinds of Chinese Herbal Medicines in CDC. The key point for developing a fluorimetric method is to understand the fluorescent properties of the analyte and other components in the sample, then find a way to avoid interference of the coexistent components on the fluorescence of the analyte. For these reason, 3D fluorescence spectra of four kinds of Chinese herbal materials in CDC were measured and shown in Figure 5. From Figure 5 we showed that all the four kinds of Chinese herbal medicines contain fluorescent components. The interference of these components on the fluorescence of 4MU seems to be a serious problem. However, we noted that the concentrations of these herbal medicines in Figure 5 (400 g mL −1 ∼ 100 g mL −1 ) were much higher than the concentration of 4MU in Figure 1 (18.5 ng mL −1 ). So, whether these herbal medicines in CDC interfere with the fluorescence of 4MU should be considered further from the view point of concentration. Figure 6 shows the 3D fluorescence spectra of CDC dilute solutions at pH 5.85 and at pH 9.22. Comparing Figure 6 to Figure 1, we figured out that the two pictures are basically the same. The fluorescence peaks presented in Figure 5 are not emerged in Figure 6. This result indicated that the fluorescence of those four herbal medicines in CDC is too weak to be seen, either because of their low concentration or due to their poor luminous ability. So, we can conclude that the coexistent components in CDC do not interfere with the fluorescence of 4MU. The content of 4MU in CDC can be determined simply by a fluorimetric method. From Figures 6 and 2, we also know that the fluorimetric method may be performed at near neutral or at weak alkaline pH. For simplicity, we prefer the method to be performed at near neutral pH so that the fluorescence intensity of CDC water solution can be measured directly without using a buffer solution. Determination of 4MU in CDC by a Standard Curve Method. A series of standard aqueous solutions containing different amounts of 4MU, from 1.55 to 31.0 ng mL −1 , were prepared. Fluorescence spectra, as shown in Figure 7(a), and fluorescence intensity at measuring wavelength of ex / em = 320 nm/445 nm of each solution were measured. A linear calibration curve of fluorescence intensity against concentration was plotted, as shown in Figure 7(b). The regression equation obtained was = 5.5 + 104.1 (ng mL −1 ), with correlation coefficient = 0.9998 ( = 13). A number of solutions containing different amounts of CDC were prepared and their fluorescence intensities at wavelength of ex / em = 320 nm/445 nm were recorded. Using the calibration curve, the 4MU content in CDC sample was determined to be 33.35 ± 0.10%, as shown in Table 3. Determination of 4MU in CDC by a Standard Addition Method. As shown in Figure 8, a standard addition method was employed to verify the result obtained above. The regression equation obtained was = 636.9 + 105.3 (ng mL −1 ), with correlation coefficient R = 0.9998 ( = 5). The content of 4MU in CDC sample was calculated to be 33.3%, which was coincident with the result obtained by standard curve method. According to the CDC drug label, the content of 4MU in CDC is 100 mg per grain (0.3 g). It is evident that the analytical result of this method agrees with the drug label. Conclusion Hymecromone (4MU) is an excellent fluorophore. Acidity and solvent have important influence on its fluorescence. In near neutral aqueous solutions, 4MU exist mainly as neutral molecular form which can be produced strong fluorescence at 445 nm. In weak alkaline solutions, 4MU exist mainly as anion form which can be produced stronger fluorescence at 445 nm. In methanol solution, the fluorescence peak at 445 nm blue shifted to 380 nm and the fluorescence intensity declined in a certain extent. There is a good linear relationship between fluorescent intensity and 4MU concentration. The 3D fluorescence spectra of Chinese patent drug CDC dilute solutions are very similar to the 3D fluorescence spectra of 4MU, indicating that the main fluorescent component in Conflict of Interests The authors declare that they have no conflict of interests.	2018-12-08T20:13:43.939Z	2013-12-04T00:00:00.000	{ "year": 2013, "sha1": "4fdce51aba200ad8ba67c33d1c808eea00ff7fd7", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/jspec/2013/147128.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "4fdce51aba200ad8ba67c33d1c808eea00ff7fd7", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Chemistry" ] }
233543561	pes2o/s2orc	v3-fos-license	Analytic integrability of certain resonant saddle We provide suﬃcient conditions of analytic integrability for a family of planar differential system with a p : − q resonant saddle at the origin. The conditions are then shown to be necessary in several ﬁnite dimensional families where the calculations can be performed explicitly. It is conjectured that the conditions are in fact complete for all families. Though the form of the equations is quite simple, their study merits further attention as they exhibit an interesting dichotomy between ﬁnite and arbitrary dimensional components of the center variety. Introduction and main results The classical center problem at the origin for real planar differential systems ˙ x = −y + P (x, y ) , where P, Q are analytic functions without constant and linear terms, can be generalized to study the integrability problem of a p : −q resonant singular point at the origin of systems ˙ x = p x + F 1 (x, y ) , ˙ y = −q y + F 2 (x, y ) , (1) with p, q ∈ N and where F i (0 , 0) = ∂ x F i (0 , 0) = ∂ y F i (0 , 0) = 0 for i = 1 , 2 .In the case that F 1 and F 2 are specific polynomials and for some particular resonance ratios, the integrability problem of system (1) has been studied by several authors [1,2,8,10,11,[13][14][15]17,21] .However few results are known for arbitrary resonance ratios p : −q .The order of the resonant saddle is recently studied in Dong et al. [3] , Dong and Yang [4] , Dong et al. [5] for polynomials of arbitrary degree. In this paper we aim to give a characterization of the analytic integrable resonant saddles at the origin of the complex differential system in C 2 , ˙ x = p x, ˙ y = −qy + f (y ) x, 0 = p, q ∈ N , (2) where f (y ) = i ≥1 a i y i is an analytic function without constant term.The resonance 1 : −1 of system (2) was studied in Giné and Valls [12] .Some specific polynomial systems (2) with p : −1 resonance have been studied in Fer čec and Giné [7] .If system (1) has a analytic integrable saddle at the origin we say that the origin is a generalized center.The main theorem is the following obtained studying the necessary conditions for low values of p, q and the degree of f . Theorem 1. System (2) has an analytic integrable saddle at the origin if one of the following conditions holds: (1) a i = 0 for i ≤ n with n = 1 + p/q ; (2) a i = 0 for i ≥ n with n = 1 + p/q ; ( 3 k ) a i = 0 for i = 1 and i = k + 1 and p does not divide k . In order to investigate the universality of these conditions, we have taken a system of reasonably large degree ( f of degree 6) and for a range of p and q have obtained the necessary and sufficient conditions to have an analytic integrable saddle for system (2) .The calculations grow rapidly in complexity for higher degrees of f or for large values of p and q .However, we have been able to verify the following after explicit computations.Theorem 2. If f is a polynomial of degree at most 6, then conditions (1)(2)(3) of Theorem 1 are the necessary and sufficient conditions for a generalized center for all p, q ≥ 1 with p + q ≤ 7 .Proof.Theorem 1 establishes the sufficiency of the conditions.A number of necessary conditions were then computed using two approaches.The first is detailed in the next section.The second was a more traditional approach seeking a first integral of the form x q y p + . . . .Finally, a Groebner Basis computation was carried out to simplify the computed expressions to the ones given in Theorem 1 . These results strongly suggest the following conjecture.Conjecture 3. If f is a polynomial, conditions (1)(2)(3) of Theorem 1 are the necessary and sufficient conditions for a generalized center for all p, q ≥ 1 . Although the system (2) is of a very simple form, the completeness of the conditions in Theorem 1 is not at all obvious.Furthermore, the nature of the conditions is quite unusual-exhibiting an interesting dichotomy between finite codimension components (cases 1 and 3 k ) of the center variety and arbitrary (resp.infinite) dimensional components (case 2) in the polynomial (resp.analytic) case. Remark. A further investigation of this system would be of much interest-especially to understand to what extent the conjecture holds when f is an analytic function.Furthermore, if the analytic case is indeed more general, is it possible to write the additional center conditions in an explicit form? Method to find the necessary conditions The necessary conditions are usually obtained proposing a power series as a formal first integral of system (2) .However we will see that this is not the case in this work.Any p : −q resonant saddle can be written, doing a rotation and a change of time if necessary, as system (1) .If we propose a formal first integral for system (1) , the leading term h 0 (x, y ) of this formal first integral must a first integral of the linear part of the system (1) .Then we look for a formal first integral of the form H(x, y ) = x q y p + . . .where the dots denotes the higher order terms.Hence the derivative of H along the trajectories of system (1) give us ˙ where v i are polynomials in the coefficients of system (1) called resonant saddle quantities of system (1) at the origin.We have formal integrability at the origin when all of the v i are zero and in this case the system has a formal integrable resonant saddle at the origin, see [9,20,22] .Moreover the formal integrability around an isolated singular point implies local analytic integrability around it, see for instance [16] .Other methods to study integrability of a resonant saddle and the recursive formulas to compute resonant saddle quantities were also investigated in Wang and Huang [17] , Wang et al. [18] , Wang and Liu [19] .However we are not going to construct the formal first integral around the origin of system (2) .We are going to use the blow-up method described in Fer čec and Giné [6,7] .The method is based on applying the blow-up (x, y ) → (x, z) = (x, y/x ) at the origin of system (1) .Doing this blow-up the origin is replaced by the line x = 0 , which contains two singular points that correspond to the separatrices at the origin of system (1) .We call these new singular points p 1 and p 2 which are (p + q ) : −p and (p + q ) : −q resonant saddles, respectively.In [9] the following results are proved that we will use to derive the method for computing the necessary integrability conditions of system (1) around the origin.Theorem 4. The p : −q resonant singular point at the origin of system (1) is analytically integrable if and only if one of the points p 1 or p 2 are analytically integrable. Proposition 5.The first nonzero necessary condition for integrability of the p : −q resonant singular point at the origin of system (1) is the same that the first nonzero necessary integrability condition of the points p 1 or p 2 . Hence, we apply the blow-up (x, y ) → (x, z) = (x, y/x ) , to system (1) and in the variables (x, z) we get a system of the form ˙ where F (0 , 0) = 0 .The most powerful of the method is that now we can propose the power series as a formal first integral of system (4) , where f i (z) are functions of z.However we will see that in the case that we have an integrable resonant saddle at the origin of system (4) these f i (z) will be polynomials of degree at most i .Now we compute the derivative of ˜ H along the trajectories of system (4) , i.e., and we get a system of equations defined by the coefficient of each power of x .The constant term gives the differential equation Taking into account that to have formal integrability f 1 (z) must be a polynomial then we have that c 1 = 0 which implies that The power x 1 has as a coefficient the differential equa- , which implies, either (2 p) / (p + q ) ∈ N or c 2 is zero and f 2 (z) too.From the fact that p, q ∈ N then exist f k 0 (z) such that (k 0 p) / (p + q ) ∈ N or (k 0 q ) / (p + q ) ∈ N (taking the other saddle point p 2 ).From here, for the next powers of x we have the following ordinary recursive differential equation where g k (z) is a polynomial in z which depends on the previous polynomials f i for i = k 0 , . . ., k − 1 .The solution of the differential Eq. ( 6) takes the form where c k is an arbitrary constant.In order to have formal integrability, f k (z) must be a polynomial.The integral that appears in expression ( 7) can give a logarithmic term and this term is the term that prevents formal integrability of system (4) and consequently by Theorem (4) of the original system (1) .The logarithmic term appears, in fact, by the power s −1 with nonzero coefficient in the integrant of the integral of (7) .The first nonzero coefficient of the power s −1 coincides with the first nonzero resonant saddle quantity defined in (3) .However if the resonant saddle quantity is zero then coefficient of the power s −1 is also zero and the method always gives a polynomial attending to the computation of f k in (7) .Therefore the blow-up method always gives polynomials and sometimes also logarithmic terms.In fact this is true if we do not use the blow-up method presented here.More specifically, system (1) can be formal integrable around the origin but if we propose a formal first integral in the original variables (x, y ) the recursive procedure can do not give always polynomials for the functions f i , see for instance [6] and references therein.Usually, induction method works to prove that, for any arbitrary i, if certain f i (z) does not have the logarithmic term then next function f i +1 (z) does not have them either.However, sometimes this is not the case.Nevertheless, in Fer čec and Giné [6] , it is shown that if we add certain property P that must satisfy any previous f i , for i = k 0 , . . ., k − 1 , then f k does not have a logarithmic term.This method was applied to system (2) to find the necessary conditions that give the proof of Theorem 2 . Proof of Theorem 1 Since ˙ x = px, the integrability and linearizability conditions of (2) are the same. Case (1) Under the assumptions of statement (1) of Theorem 1 , system (2) takes the form where n = 1 + p/q .Hence the condition implies that f (y ) = y n +1 h (y ) for some analytic function h (y ) .Choosing the new variable Y = y n x, the system (2) takes the form Since n = 1 + p/q , we have n − p/q > 0 .Hence the system (9) has a node and can have no resonant terms because as it has two analytic separatrices.Consequently there exist a change of variables which linearizes the node.In particular, there is a Hence, from Y = y n x, we find that W = yφ 1 /n (y, y n x ) satisfies ˙ W = −qW and (x, W ) becomes a linearizing change of coordinates. If m > 0 then we perform the change of coordinates X = x 1 /m and Y = yx 1 /m , bringing the system (10) where 11) has a linearizable node at the origin because there are two analytic separatrices and the ratio of eigenvalues is ) > 0 , for m = p/q − 1 . Hence, there is an analytic function φ(X, Y ) with φ(0 , 0) = 1 so that, taking Z Clearly φ(w k x 1 /m , w k yx 1 /m ) will also have the same property for w a primitive m th root of unity.And so, replacing φ by The solution to this linear differential equation is g = e −ak/p x qk/p kb p e ak/p x −qk/p dx.(13) If p does not divide qk then the exponential can be expanded as a power series and the integral can be evaluated term by term to express g as a power series in x .It is straight forward to show that this series must be convergent.On the other hand, if p divides qk, then there will be terms in log (x ) in the series for g arising from terms of the form x −1 in the integrand of (13) .Thus, assuming p and q are coprime, a sufficient condition for the existence of g, and hence , is that p does not divide k .It is easy to construct a Darboux first integral from the Darboux factors x, y, e x and .In fact the cofactors are k x = p, k y = −q + x (a + by k ) , k e x = px and k = kbxy k and a Darboux first integral will be given by H = x q y p e ax (1 + y k g(x )) p/k .Furthermore, we can use these factors to construct an explicit linearizing change of coordinates.Indeed, if H = x q y p ψ (x, y ) is the first integral then X = x and Y = yψ 1 /p linearizes the system. Conclusions For system (2) we have computed the necessary conditions to have formal integrability using the new method developed in Fer čec and Giné [6,7] for certain degrees of the function f (x ) .The main result ( Theorem 1 ) gives the sufficiency of such conditions using different methods for each case, as the blow-up method to a node, as well as the recursive method used in the last case. The are several methods to find the necessary conditions but there is no global method to find the sufficiency and in this work we propose some new techniques to find the sufficiency which are given in the proof of Theorem 1 . A conjecture is given for the analytic case of f (x ) .Furthermore, if the analytic case is indeed more general, it is open if such additional center conditions can be written in an explicit form. Declaration of Competing Interest Authors declare that they have no conflict of interest. Bernoulli equation.In this case we seek a Darboux factor of the form = 1 + y k g(x ) , for some analytic function g(x ) .Then has cofactor k = kbxy k if and only if pxg , if necessary, we can assume that φ is analytic in x and y .Finally, if we take W = yφ(x 1 /n , yx 1 /n ) we see that ˙ W = −qW and (x, W ) is a linearizing change of coordinates.	2021-05-04T22:04:48.034Z	2021-05-01T00:00:00.000	{ "year": 2021, "sha1": "478cd816b1e6146b140dc3fc628cfbf14ea91c54", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1016/j.chaos.2021.110821", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "df657114386fa4bc4a2726b8ccc5f00f10396751", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Physics" ] }
255852640	pes2o/s2orc	v3-fos-license	Investigation of Telomerase/Telomeres system in Bone Marrow Mesenchymal Stem Cells derived from IPF and RA-UIP Idiopathic Pulmonary Fibrosis and Rheumatoid Arthritis associated usual interstitial pneumonia seem to have the same poor outcome as there is not an effective treatment. The aim of the study is to explore the reparative ability of bone marrow mesenchymal stem cells by evaluating the system telomerase/telomeres and propose a novel therapeutic approach. BM-MSCs were studied in 6 IPF patients, 7 patients with RA-UIP and 6 healthy controls. We evaluated the telomere length as well as the mRNA expression of both components of telomerase (human telomerase reverse transcriptase, h-TERT and RNA template complementary to the telomeric loss DNA, h-TERC). We found that BM-MSCs from IPF, RA-UIP cases do not present smaller telomere length than the controls (p = 0.170). There was no significant difference regarding the expression of both h-TERT and h-TERC genes between patients and healthy controls (p = 0.107 and p = 0.634 respectively). We demonstrated same telomere length and telomerase expression in BM-MSCs of both IPF and RA-UIP which could explain similarities in pathogenesis and prognosis. Maintenance of telomere length in these cells could have future implication in cell replacement treatment with stem cells of these devastating lung disorders. Introduction Idiopathic pulmonary fibrosis (IPF) is the most devastating form of fibrosing lung diseases with a median survival of 3 years and a prognosis which is worst than that of many cancers [1,2]. The underlying pathologic pattern is that of usual interstitial pneumonia (UIP). Although recent studies have suggested that IPF is the result of repeated injuries in different sites of the lung epithelium followed by aberrant wound healing with inadequate repair of the epithelial damage, pathogenesis of IPF still remains poorly understood. Consequently and despite recent advances [3][4][5], there is no effective treatment currently available which can lengthen patient's survival other than lung transplantation. Thus, the need for more effective treatment becomes imperative. Mesenchymal stem-cells (MSCs) are one of the most intriguing novel therapeutic approaches in the field of chronic diseases [6][7][8][9][10][11][12] because of the ability to repair injured tissues. They possess high proliferative capacity and ability to differentiate in adipocytes, condrocytes, osteocytes, endothelial, epithelial and neuronal cells depending on the culture conditions [13]. Moreover, there are data suggesting that bone marrow (BM)-MSCs have the ability to differentiate and function as airway and parenchymal lung cells [14]. It has been shown in animal model of bleomycin (BLM)-induced fibrosis that (BM)-MSCs express several chemokine receptors such as CXCR4 which ligand, CXCL12, is induced in murine lungs [15][16][17] suggesting that bone marrow stem cells could be recruited and mobilized to the injured lung through a CXCR4 dependent mechanism. Recently we have shown that CXCR4 is overexpressed in BM-MSCs of patients with IPF [18] suggesting that the abovementioned scenario could also be applied to humans. Telomeres are repeated DNA sequences acting as protective caps for chromosomes. Telomere shortening is one of the molecular mechanisms underlying ageing and critically short telomeres trigger chromosome senescence and lead to cell death [19]. Telomerase is a specialized polymerase that adds telomere repeats to chromosomes compensating the telomere shortening and consists of two components: a catalytic component, telomerase reverse transcriptase (h-TERT) and an RNA component (h-TERC) [20]. The aim of our research was to investigate the reparative ability of BM-MSCs in patients with IPF by evaluating telomere expression and telomere length and propose a novel therapeutic approach for this dismal disease. In our research we have also included patients with rheumatoid arthritis (RA) as in this disease, when there is interstitial lung involvement and unlike the rest of collagen tissue disorders, the most predominant pathologic pattern is UIP and in this case prognosis is similar to IPF [21]. We aimed to prove that telomere length and telomerase expression are not different in patients group compared to healthy controls proposing the possibility of cell replacement treatment as a novel therapeutic approach. Patients We have studied prospectively 6 patients with IPF, 7 patients with RA-UIP whose characteristics are shown in Table 1. Patients were recruited from the Interstitial Lung Disease Unit (ILDU) at the Department of Thoracic Medicine of Heraklion. The control group included 6 subjects, age-matched with the patients who underwent posterior iliac crest aspirate because of suspected hematologic malignancies. We have included in our research those with negative biopsy results and thus considered as healthy subjects. These patients have been studied retrospectively and we did not have details other than age and gender in order to be matched with the patients. The diagnosis of IPF was made in 3 cases by surgical biopsy and the histologic diagnosis of Usual Interstitial Pneumonia (UIP) was obtained. In the remaining 3 cases the diagnosis was made according to the recently published ATS/ERS guidelines [1]. The diagnosis of RA-UIP was made in 2 cases by surgical biopsy and the histologic diagnosis of UIP was obtained. In the remaining 5 cases, patients had a "definite RA-UIP" pattern based on HRCT criteria, with basilar predominant reticulation, traction bronchiectasis and honeycombing, with limited ground-glass opacities [22]. Diagnosis of RA was based on clinical criteria in accordance with the international societies guidelines [23]. Ethical Committee of the University of Crete has approved the study and all participants (patients and control subjects) were informed on the scope of the study and gave their written informed consent [18,24]. BM-MSCs in vitro expansion and differentiaition BM mononuclear cells (BMMCs) obtained from posterior iliac crest aspirates were cultured in Dulbecco's Modified Eagle Medium-Low Glucose (DMEM-LG; Gibco/ Invitrogen, Paisley, Scotland)/10% fetal calf serum (FCS; Hyclone, Logan-Utah,USA)/100 IU/ml Peniciline-Streptomycin (MSC medium) and MSCs were grown and flow cytometric analysis of MSCs in each passage was performed as previously described [25,26]. Upon reaching confluency at passage 2 (P2) trypsinized MSCs were centrifuged and after performing a cell count in a Neubauer haemocytometer at least 1.000.000 cells were used for further processing. MSCs from P2 were induced for differentiation. Adipogenic differentiation was induced following 21-day culture of cells in MSC medium supplemented with 10% FCS/0.5 mM 1-methyl-3-butylisoxanthine/1 μM dexamethasone (Dex)/0.2 μM indomethacin/10 μg/ml insulin and adipogenesis was assessed by Oil Red O staining. Osteogenic differentiation was induced following 21-day culture of cells in MSC medium supplemented with 0.1 μM Dex/0.15 mM ascorbate-2-phosphate/3 mM Real-time reverse transcriptase-polymerase chain reaction assay MSCs at P2 were homogenized in the TRIzol W reagent (Invitrogen, Carlsband, CA), total RNA was extracted and cDNA synthesized by reverse transcription (RT) with the Thermoscript™ RT kit (Invitrogen). Genes mRNA expression was measured using a real-time RT-PCR assay with SYBR-Green I. β-actin was used as the internal control, in order to normalize h-TERT and h-TERC expression levels. The mRNA-specific primers used are listed in Table 2. Telomere length measurement The relative telomere length was estimated by real-time PCR as described originally by Cawthon [27], with minor modifications. β-Globin (HBG) was used as control single-copy-gene. Primer sequences are listed in Table 3 Statistical analysis Differences in relative telomere length and m-RNA expression of h-TERC and h-TERT genes between patients group and controls have been tested with Kruskal-Wallis test and a value of p < 0.05 has been considered significant. MSCs immunophenotype and differentiation potential Immunophenotypic analysis of MSCs from all groups of patients and healthy controls at the end of P2 demonstrated that cultures constituted of a homogenous cell population positive for CD73, CD90, CD146, CD105, CD29, CD44 and negative for CD45 and CD34 surface antigens. P2 MSCs were able to differentiate towards the adipogenic, osteogenic and chondrogenic lineages in healthy individuals, as well as in all groups of patients. Telomerase expression and telomere length We found that the relative telomere length in both patients group (IPF and RA-UIP) did not differ compared to healthy controls (p = 0.17). This finding suggests that BM-MSCs obtained from patients group are able to maintain telomere length and therefore could be proposed as possible replacement treatment (Table 4 and Figure 1). Both h-TERT and h-TERC genes were expressed at the m-RNA level in all patients' group and control subjects. However, we did not observe any statistically significant difference in gene expression of h-TERT and h-TERC between study groups (p = 0.107 and p = 0.634 respectively) ( Table 5). Discussion It is well known that IPF is the result of multiple injuries in different sites of lung epithelium followed by inadequate repair characterised by the migration of resident fibroblasts, BM progenitors of fibroblasts (fibrocytes) and fibroblasts derived from a process called epithelialmesenchymal transition (EMT), formation of fibroblast and myofibroblast foci and exaggerated production of extracellular matrix (ECM). The subgroup of patients with RA associated interstitial lung disease who present with the underlying pathologic pattern of UIP has the same prognosis with IPF [22] suggesting the possibility of shared pathogenetic pathways. For both diseases there is still no effective treatment other than lung transplantation. Cell replacement treatment with MSCs is promising because of the proliferation capacity and ability of these cells to repair injured tissues. Telomeres shorten successively with each cell division and when they achieve a critical length, activate a p53-dependent mechanism that leads to apoptosis or replicative senescence [28]. Short telomeres are expected to compromise the replicative potential of progenitor cells that remain in tissues after injury. We aimed to show that telomeres from patients with IPF and RA-UIP are able to maintain telomeres length despite the high proliferation capacity and thus maintain the reparative ability. Our most important finding is that BM-MCSs from patients present relative telomere length which did not differ compared to healthy controls and thus could be used for autologous transplant. Moreover, they express, although weakly, both genes of telomerase at the same level with healthy controls suggesting a plausible mechanism for the maintenance of telomere length. The findings of the current study are not similar with previous findings of our group [29]. However, it should be stressed that these were preliminary findings of our work and patients included in that cohort were older, had more severe disease (DLCo < 30%), with few of them on ambulatory oxygen, than the patients included in the current cohort. In addition, it was previously observed that 25% of sporadic cases with IPF had shorter telomeres in peripheral leukocytes without coding mutations in telomerase [30] and we believe that this scenario could also be applied to BM-MSCs explaining the discrepancy of the findings between the two studies. Recent studies have shown that MSCs may abrogate fibrosis, but unfortunately in most of them, an animal model of BLM-induced fibrosis has been used and as it is known, this model does not represent the progressive and lethal nature of IPF. Nonetheless, MSCs seem to partially abolish lung injury in animal models of fibrosis, emphysema and inflammatory lung injury and participate in organ regeneration [31]. Interestingly, lung engraftment of MSCs administered systematically occurs at low levels in normal mice whereas it is increased in injured murine lung after exposure to BLM. MSCs adopt an epithelial-like phenotype and have a beneficial effect by reducing inflammation, collagen deposition and metalloproteinases activation within lung tissue [32]. Additionally, in another study it was observed that prominin-1/CD133(+) epithelial progenitor cells expanded from adult mouse lung and of bone marrow origin, are able to express both stem and haematopoietic cell markers and differentiate in vitro into type II epithelial cells. When intratracheally administered in mice treated Figure 1 Relative telomere length in the three study groups. with BLM, they engrafted into the lungs, differentiated into type II epithelial cells and suppressed proinflammatory and profibrotic gene expression protecting form the development of pulmonary fibrosis [33]. Alternatively to BM-MSCs, embryonic stem cells (ESCs) which present similar ability to differentiate in any cell of the body have been studied as replacement treatment. Alveolar epithelial type II cells derived from human ESCs were transplanted in the lungs of mouse treated with BLM and differentiated into type I alveolar epithelial cells abrogating the inflammatory and fibrotic response [34]. The system telomerase/telomere has been implicated in the pathogenesis of IPF. It was observed that mutations in both components of telomerase are present in 8-15% of patients with familial IPF [35,36]. On the other hand, mutations in the essential telomerase genes are also present in 1-3% of sporadic cases [30,37]. Plainly mutations lead to loss of telomerase function and to telomere shortening. Interestingly though, it was observed that in sporadic cases of IPF telomeres are short in lymphocytes, granulocytes and alveolar epithelial cells compared to age-matched controls even in absence of telomerase mutations [30,37]. Considering the fact that BM-MSCs have high mitotic activity and go through a large number of replication, one may expect that they express high levels of telomerase in order to prevent telomere shortening. Surprisingly and in accordance with our findings, it was observed that adult stem cells express low levels of telomerase and telomerase activity is low [38,39]. This may be a defensive mechanism against malignant transformation as it was shown that adult stem cells play an important role in cancer development and maintenance [40][41][42][43]. Plainly, there may be another mechanism of telomere maintenance other than telomerase. It is suggested that the alternative lengthening of telomeres pathway, a recombination based DNA replication mechanism, may maintain telomere length [44,45]. In addition, it was observed that subtelomeric hypomethylation facilitates telomere elongation in mammalian cells suggesting that such epigenetic modification of cromatin may occur also in MSCs [46]. On the other hand, even a low telomerase expression and activity are required for both replication and differentiation as it is shown that MSCs from a telomerase activity knocked-down mouse failed to differentiate into adipocytes or condrocytes and that telomerase overexpressing human MSCs have enhanced in vivo bone formation potential [47,48]. In conclusion, we have shown that BM-MSCs from patients with IPF and RA-UIP maintain the same telomere length with healthy donors suggesting the possible use of these cells in cell replacement treatment for both diseases. The same treatment has been also proposed for other chronic diseases such as amyotrophic lateral sclerosis (AML) [49] as it was found that human BM-MSCs from patients present the same telomere length with healthy donors. Plainly, caution is recommended as there are a lot of issues that need to be clarified such as whether the level of lung engraftment is sufficient for regenerative purposes, the likelihood of cell rejection, and the possibility of causing local damage i.e. favouring the development of fibrosis or lung neoplasms [50]. Definitely, further studies are needed before start using stem cells safely for pulmonary diseases.	2023-01-17T15:12:51.013Z	2012-07-02T00:00:00.000	{ "year": 2012, "sha1": "00f9ac5dd55d05cc0dc3664191e1ecfa27175fc5", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1186/1476-9255-9-27", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "00f9ac5dd55d05cc0dc3664191e1ecfa27175fc5", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [] }
231662055	pes2o/s2orc	v3-fos-license	Concept and Building Blocks of a Business Model: A Systematic Literature Review —Business model has been drawing attention from both industry and academia. It plays a crucial role in business operations and strategy. In the revolutionary and competitive environment, business model innovation is likely to lead to better business performance. However, there have been diverse interpretations on the concept and building blocks of a business model. The purpose of this paper is to provide some common understanding on the concept and its core building blocks in order to progress to the development of an innovative business model. A systematic literature review has been undertaken to gain insights into the key themes of the topic, before some recommendations for business model development are made. I. INTRODUCTION The term of -business model‖ has drawn growing interest to both industry and academics in recent years.Many studies have shown the importance of a business model for companies, and that business model innovation has close relationship with an organization's success [1]- [3].However, the concept of business model is still divided among researchers [4], [5].In addition, no generally accepted opinions about key components or building blocks of a business model exist.Different researchers tend to give varied descriptions about a business model [6]. There are some studies and systematic literature reviews on business models.For example, Osterwalder and Pigneur have classified business models into five categories [7].They are (1) separation of business model, (2) -long tail model‖, (3) multilateral platforms, (4) free as a business model, and (5) open business model.Barth et al. propose a conceptual framework for sustainable business model in argi-food industry based on a systematic literature review covers the papers published from 1990 to 2014 [4].Both of them provide insights for the business model development, but some of the cases in the literature are out of date.Furthermore, Foss and Saebi have conducted a systematic literature review on business model innovation based on the paper published from 2000 to 2015 and suggest that a conceptual clarification is still needed for this emerging field [5]. As business and global economy are developing at rapid speed in recent years, it is necessary to review the concept and recent development of business models.This paper aims to increase the understanding of business models by including the most recent paper up to 2019.This paper Manuscript received May 15, 2019; revised August 7, 2019.Bin Gao, Shaofeng Liu, Genhua Pan, and Aira Patrice R. Ong are with the University of Plymouth, UK (e-mail: tony.gao@plymouth.ac.uk). examines existing definitions of a business model and its key building blocks in order to make recommendations for business model development.The two research questions to be addressed are: RQ1: What is the essence in the concept of a business model and how has the research on business models evolved? RQ2: What are the key building blocks of a business model? II. SYSTEMATIC LITERATURE REVIEW PROCESS This section covers the research method and research process.In this paper, the Scopus database and ScienceDirect are selected for the systematic literature review because they are comprehensive sources with a strong focus on business studies, science, and medical literature.The research process is shown in Fig. 1 that consists of four stages: planning, searching, refining, and analysis.Following the systematic literature review process, we started searching the two databases by using keywords -business model‖, -business model components‖ and -business model innovation‖.Phrases implying similar meanings such as -business model building block‖ are considered.Boolean operations such as -AND‖ and -OR‖ are incorporated in the search.Initially the search returned over 2,000 results.Then we applied a list of inclusion/ exclusion criteria to refine the results, for example, to include only the sources that full-text articles are available, peer-reviewed, with high topic relevance.Repetitive results returned from the two databases are removed.Cross references are considered.Plus two papers recommended by experts in the field.In the end, 45 articles ae selected for the thematic analysis.The distribution of these 45 papers is illustrated in Fig. 2. As it can be seen from the Figure, there is a steady growth of interest in the last few years.Furthermore, the papers are from a variety of academic fields, as illustrated in Fig. 3, ranging from business, social sciences, economics, and environmental sciences etc.It provides the evidence that the business model research is an interdisciplinary research and attracts attention from heterogeneous disciplines. III. THEMATIC ANALYSIS Thematic analysis focuses on eliciting the main themes by analyzing the collection of literature.Three main themes emerged from the analysis of the 60 papers.First theme is centered around the business model definition.Second theme is about the components of a business model, or the building blocks of a business model.Third theme is sustainable business model.Sustainable business model is an emerging field as some researchers realize that business model concept should not be confined to merely economic aspects [8]- [10].Some of the sustainable models have been developed from the manufacturing industry [11]- [13].The framework of a sustainable business model includes four dimensions: life cycle thinking, multiple stakeholders, value exploitation, and the triple bottom lines (i.e.economic, social, and environmental value).There have been a small number of models associated with the healthcare industry [14].Several studies examined the relationship between business sustainability and health care stakeholders [15]- [17].There are three key stakeholders in the healthcare industry, including patients, practitioners and policymakers.Lopes et al. argue that the sustainability of a business model plays an important role in hospital management [18]. A. Concept and Definitions of a Business Model The concept of a business model could date back to the 1990s and the research evolution experienced four main stages.The first stage is the emerging stage of business model.In around 2000 with the rapid development of information technology and the internet boom, several e-business models emerged and some of them turned to be very successful [19].At this stage, the research on business model emerged, with an emphasis on the definitions and classifications.In the second stage, the components and building blocks were proposed by several studies to describe a business model in a systematic way [20].However, different researchers from different background proposed varied building blocks, and they could not reach unanimous agreements among each other.The next stage, researchers focused on the case study of business model implementation [21], [22].The importance of business model innovation is commonly agreed with practitioners and academics [23].In the fourth stage, the concept of sustainability draws increasing attention, as researchers agree on the fact that a company should undertake corporate social responsibility and pay attention to sustainable development.Some researchers fill the gap between business model design and implementation from the sustainability perspective [24].Fig. 4 illustrates the four stages of the business model evolution.Among the four stages in the Fig. 4, the first stage is of great importance because understanding the definitions of business models is an essential process for developing innovative business models.During the stage of business model definitions, there are several studies with varied definitions about business models.For example, Stewart and Zhao define business model as -a statement of how a firm will make money and sustain its profit stream over time‖ [25].Chesbrough and Rosenbloom state that business models are descriptions of how a firm or an organization does business [26].Furthermore, Chesbrough and Rosenbloom propose that a business model is a -focusing device that mediates between technology development and economic value creation‖ [27]. Morris et al. state that the business model is a concise representation of how an interrelated set of decision variables in the areas of venture strategy, architecture, and economics are addressed to create sustainable competitive advantage in defined markets [28].Richardson proposes that the business model could be seen as the conceptual and architectural implementation of a business strategy and as the foundation for the implementation of business processes [29]. Osterwalder and Pigneur define business model as the rationale of how an organization creates, delivers and captures value.Teece defines the business model as the architecture of a firm's value creation, delivery and appropriation mechanisms provides the basis for a much needed dimensionalization of the business model constructs [30].Casadesus-Masanell and Ricart suggest that business model is -a reflection of the firm's realized strategy‖ [31].Gassmann et al. summarize business model as the understanding in four dimensions: Who, What, How and the Value [32]. Joyce and Paquin develop three layers of a business model, which include the economic factors, social benefits, and environmental impact from the sustainable perspectives [8].The case of Nestle Nespresso was then reanalyzed from a holistic view of economic, environmental and social perspectives. As it can be seen from the variety of definitions, -value‖ is the most important characteristic of a business model.In addition, -strategy‖ and -sustainability‖ appear frequently, which means that a business model should reflect an organization's strategy and sustainability.Based on the above definitions, it is reasonable to draw the conclusion that business models should address -value‖. B. Building Blocks of a Business Model Amit and Zott propose three key building blocks for a business model from the transaction perspective.They include transaction contents, transaction structure, and transaction governance [33].Transaction contents include the goods, information, and capabilities that are required to exchange.They argue that transaction cost could be reduced via a properly designed business model. Additionally, Krumeich et al. divide a business model into five aspects, including value offering, value capture, value creation, cooperation, and financial model [20].Chesbrough and Rosenbloom suggest that business model should consists of six building blocks, value proposition, target market, value chain for creating and distributing the offerings, competitive strategy, cost and revenue model, and a value network [26].Moreover, Bocken et al. propose four building blocks for a business model, including value proposition, value creation, value capture, and value delivery [13].Furthermore, they also pointed out from the sustainable development perspective, that is, value proposition should include not only the economic value but the ecological benefits and social value as well.Richardson develops a business model that consists of three main building blocks: value proposition, value creation and delivery, and value capture [29].The value proposition includes the concepts of what does a company offer and who will benefit from the value offering.However, we argue that it is better to separate the value beneficiary components and value propositions, which could help clearly identify who will benefit from the business offerings. In addition to value proposition, Gassmann et al. propose the Who-what-how-why business model that describes a business model of which the first two (who and what) address its external dimensions and the second two (how and why) address its internal dimensions [32].They also propose 55 business model and case studies to illustrate how the business model works in the real world.Value Proposition, value creation and delivery, value capture, and value intention. Table I summarizes the selected building blocks from literature.As can be seen from the Table I, value and its elements have been the most frequently identified as key building blocks in the literature.In addition, -transaction‖ and -strategy‖ are also seen as important components for a business model. C. Sustainable Business Models However, traditional commercial business models usually focus on only obtaining economic revenue and are in ignorance of corporate sustainability.As sustainable development draws increasing concern in recent years, scholars realized that business model should not only focus on the economic perspective, but also should consider social Journal of Economics, Business and Management, Vol. 8, No. 2, May 2020 and environmental perspectives.Some companies would like to innovate their business model from selling products to the sale of service in order to move to a circular business model [37].Many business model innovations are involved with sustainability [38], [39].For instance, Geissdoerfer et al. design the process for business model innovation, which include concept design, detail design, and implementation [24]. Geissdoerfer et al. discuss the methods to bridge the design and implementation gap during the business model process [40].Laasch examines the differences between traditional commercial business model and sustainable business model.The value logics of sustainable business models should integrate social, environmental and economic value.Sustainable business models focus on benefitting a diverse set of stakeholders.The differences mainly are in that a traditional business model merely focuses on economic value capture, making profit for the companies, whereas a sustainable business model has a widely range of stakeholders, i.e. more value beneficiaries. IV. RECOMMENDATIONS FOR FUTURE BUSINESS MODEL DEVELOPMENT Based on the thematic analysis in the previous section, it is evident that value and its elements are highlighted in the business model definitions, building blocks and sustainability in the literature.A business model should help identify value, create value, deliver value and capture value.In this regard, we make the following recommendations for future business model development. A business model should address four key dimensions in order to create a complete value circle.(1) Value beneficiary who will benefit from the value offering?(2) Value proposition -what is the company's offering?(3) Value chainhow do we achieve the value proposition?(4)Value capture-why does a business model work commercially?The complete value circle with the four dimensions is shown in Fig. 5.This section also provides the justification of the four building blocks. A. Value Beneficiary One of the important building blocks is value beneficiaries, which usually refer to stakeholders.It describes who will benefit from the value proposition and which market segments to target at [32].Based on different markets a business targets at, a business can be classified as local business, regional business or international business.As a business gradually grows from a small start-up to a larger company, it means that the expansion of the business from local to international with greater influence and more beneficiaries.Furthermore, this building block includes dealing with customer relationships.Good relationship with customers could help improve customer loyalty.Customer relationship involves effective interactions with the target customers.A sustainable business needs to achieve continuous improvement of current communication channels and explore new relationships with potential customers. From a holistic point of view, successful business models should help companies to create value to beneficiaries, ranging from customers, suppliers, and communities. B. Value Proposition The second key building block is value proposition.Most papers have agreed on this component, which is regarded as the core component of a business model [41], [42].Value proposition describes what product and/or service offering of the company.Some successful companies offer both products and service, to increase the competitiveness.For manufacturing industry, most value proposition predominantly focuses on certain products, innovated with technology [43].While for healthcare industry, most value proposition predominantly focuses on service.Some researchers studied the business model innovation in hospital management and developed patients-oriented business models from the patients-caring point of view. C. Value Chain The third dimension is value chain.Value chain describes how a business arranges its activities and resources to achieve the value proposition.It involves the main activities that are necessary for a business to provide its offerings.Some studies also called this process value exchange [9], [44]. The value chain building block includes several subconstructs, as shown in Fig. 6.They are key resources, key activities, key partnerships, and distribution channels [32].Key resources are defined as the assets that are required to achieve the value proposition.The resources include intellectual, physical, financial, and human resources, as well as technology.Key activities refer to what a firm must do to create the value proposition.Key partners include suppliers, joint ventures, and strategy alliance.For some start-up business, joint ventures could help reduce some risks. D. Value Capture The fourth dimension is value capture.The value capture has close relationship with the value proposition as it describes how the profit is realized .i.e.why the business will survive and thrive.From economic perspective, value capture includes revenue model and cost structure [45].From social perspective, it should include social benefits and impacts.For example, in the healthcare industry, a successful robust and resilient business model should not only bring itself value captures, but also relieve the pains of patients and increase their social welfare. V. CONCLUSION Business model research has been ongoing for more than a decade.Different scholars hold different views about the concept of business model and from multidisciplinary background.The interests in business model development is growing rapidly especially since 2016.The development of a sustainable and innovative business model is of great importance in the current fierce competitions.Understanding the concept and key components of a business model is the foundation to business model innovation.Based on a systematic literature review, this article recommends four key dimensions for future business model development, namely value proposition, value beneficiary, value chain, and value capture.These four dimensions summarize the main components of a business model that are interdependent with each other.When all four dimensions are addressed, a complete value circle can be created aiming for a healthy, sustainable business.Value proposition is regarded as the key building block of a business model, as it describes what benefits a business creates.Value beneficiary refers to who will benefit from the value proposition.Value chain is closely related to the value proposition because it describes how the value can be achieved.Value capture describes why a business will survive and thrive.This article helps make progress towards better understanding of the concept of business model and contributes to future development of innovative, sustainable business models. Fig. 3 . Fig. 3. Classification of field of business model research. TABLE I : SELECTED BUILDING BLOCKS OF A BUSINESS MODEL	2020-12-24T09:12:41.355Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "c49bce3b6d0a0f28e10acefb067c7bf2e3fadf0b", "oa_license": "CCBY", "oa_url": "http://www.joebm.com/vol8/616-UA2012.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "3360413dc7a881327bb5d22a93a1c74917956f94", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Computer Science" ] }
211230505	pes2o/s2orc	v3-fos-license	Ethyl Acetate Fraction of Aqueous Extract of Lentinula edodes Inhibits Osteoclastogenesis by Suppressing NFATc1 Expression Bone tissue is continuously remodeled by the coordinated action of osteoclasts and osteoblasts. Nuclear factor-activated T cells c1 (NFATc1) is a well-known transcription factor for osteoclastogenesis and transcriptionally activated by the c-Fos and nuclear factor-kappa B (NF-κB) signaling pathways in response to receptor activation of NF-κB ligand (RANKL). Since excessive RANKL signaling causes an increase of osteoclast formation and bone resorption, inhibition of RANKL or its signaling pathway is an attractive therapeutic approach to the treatment of pathologic bone loss. In this study, we show that an ethyl acetate fraction (LEA) from the shiitake mushroom, Lentinula edodes, inhibited RANKL-induced osteoclast differentiation by blocking the NFATc1 signaling pathway. We found that the water extract and its subsequent ethyl acetate fraction of L. edodes significantly suppressed osteoclast formation. Comparative transcriptome analysis revealed that LEA specifically downregulated a set of RANKL target genes, including Nfatc1. Next, we found that LEA suppresses Nfatc1 expression mainly through the inhibition of the transactivity of p65 and NFATc1. Moreover, treatment of LEA rescued an osteoporotic phenotype in a zebrafish model of glucocorticoid-induced osteoporosis. Collectively, our findings define an undocumented role of the shiitake mushroom extract in regulating bone development. Introduction Bones are constantly degraded and regenerated throughout life. This process is known as bone remodeling, which is critical for bone shape and integrity [1]. Bone remodeling is carefully regulated by osteoclasts and osteoblasts. Disturbing these processes leads to skeletal disorders, such as osteoporosis and osteopetrosis [2,3]. Glucocorticoid-induced osteoporosis (GIO) is the most frequent form of secondary osteoporosis and increases the risk of bone fractures [4]. Glucocorticoids exert adverse effects on bone metabolism, including a decrease in the number and function of osteoblasts and an increase in the life span and differentiation of osteoclasts. Long-term glucocorticoid treatment is associated with the development of GIO. Osteoclasts are multinucleated giant cells that are responsible for bone degradation. They are formed by the fusion of mononuclear precursors of the hematopoietic stem cell lineage [5,6]. Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand LEA Suppresses Osteoclast Differentiation To examine the effect of L. edodes on osteoclast differentiation, we first prepared extracts of L. edodes using three different solvents ( Figure 1A). Then, bone marrow-derived macrophages (BMMs) as osteoclast precursors (OCP) were treated with ethyl acetate extract, ethanol extract, or water extract in the presence of M-CSF and RANKL. The formation of TRAP-positive multinucleated osteoclasts was significantly inhibited by the water extract, but not by the ethyl acetate extract or the ethanol extract ( Figure 1B and Supplementary Figure S1). The findings that the water extract had no effect on the proliferation of osteoclast precursors indicate that the water extract directly controls the differentiation ability of OCP cells ( Figure 1C). The water extract was further fractionated using three different organic solvents and subsequently determined for anti-osteoclastogenic activity. LEA exhibited the most potent anti-osteoclastogenic activity ( Figure 1A,D). LEA rarely affected OCP proliferation ( Figure 1E). LEA Modulates a Set of Osteoclast-Related Gene Expression in RANKL-Induced Osteoclastogenesis To elucidate the inhibitory mechanism of LEA on RANKL-induced osteoclast differentiation, we performed genome-wide transcriptome analysis of BMMs with or without LEA during RANKL-mediated osteoclastogenesis. Analysis of RNA-seq data revealed 4740 differentially expressed genes in any pairwise comparison among the three conditions (no RANKL (-R), RANKL (R), RANKL + LEA (R+LEA)). K-means clustering classified the genes into six gene clusters that were differentially modulated by RANKL and LEA (Figure 2A and Supplementary Table SI). Gene ontology (GO) analysis revealed that each cluster was enriched in genes related to distinct biological functions ( Figure 2B). Clusters I and VI showed the enrichment for GO terms associated with bone resorption and osteoclast differentiation. Specifically, LEA selectively downregulated cluster I, but not cluster VI (Figure 2A). Because RANKL is known to induce osteoclast-related gene expression and LEA inhibited RANKL-induced osteoclast differentiation ( Figure 1D), we focused particularly on the effect of LEA on RANKL-induced genes. As shown in Figure 2C, 1283 genes were two-fold upregulated by RANKL treatment. Upon LEA treatment, a total of 768 genes (441 upregulated and 327 downregulated genes) were differentially expressed. Interestingly, most of the downregulated genes belonged to cluster I ( Figure 2D and Supplementary Table SII). In addition, gene set enrichment analysis (GSEA) scoring plots showed significant enrichments of osteoclast development and osteoclast differentiation pathways ( Figure 2E). Examination of the leading-edge subset of these genes identified 23 osteoclast development genes and six osteoclast differentiation genes, respectively. The datasets from GSEA were further confirmed by qRT-PCR ( Figure 2F). BMMs were treated with the fractions as in (D), and cell proliferation was measured by MTT assays. Error bars represent the mean result ± SD of three independent experiments; * p < 0.05, *** p < 0.001. LEA Modulates a Set of Osteoclast-Related Gene Expression in RANKL-Induced Osteoclastogenesis To elucidate the inhibitory mechanism of LEA on RANKL-induced osteoclast differentiation, we performed genome-wide transcriptome analysis of BMMs with or without LEA during RANKLmediated osteoclastogenesis. Analysis of RNA-seq data revealed 4740 differentially expressed genes LEA Inhibits NFATc1 Expression during Osteoclastogenesis Given that c-Fos, NF-κB, and NFATc1 are key transcription factors involved in RANKL signaling for osteoclast differentiation [7], we first examined the effect of LEA on the mRNA expression of c-Fos, p65, and Nfatc1. Our RNA-seq dataset showed that LEA significantly repressed RANKL-induced Nfatc1 expression, whereas there was no effect on c-Fos and p65 expression ( Figure 3A). Consistent with the RNA-seq results, our qRT-PCR analysis confirmed that LEA selectively inhibited NFATc1 expression ( Figure 3B). We further evaluated the protein levels of those transcription factors. Similarly, LEA selectively diminished NFATc1 levels without affecting the protein levels of c-Fos and p65 ( Figure 3C). osteoclastogenesis. Error bars represent the mean result ± SD of three independent experiments; * p < 0.05, p < 0.01, * p < 0.001. LEA Inhibits NFATc1 Expression during Osteoclastogenesis Given that c-Fos, NF-κB, and NFATc1 are key transcription factors involved in RANKL signaling for osteoclast differentiation [7], we first examined the effect of LEA on the mRNA expression of c-Fos, p65, and Nfatc1. Our RNA-seq dataset showed that LEA significantly repressed RANKL-induced Nfatc1 expression, whereas there was no effect on c-Fos and p65 expression ( Figure 3A). Consistent with the RNA-seq results, our qRT-PCR analysis confirmed that LEA selectively inhibited NFATc1 expression ( Figure 3B). We further evaluated the protein levels of those transcription factors. Similarly, LEA selectively diminished NFATc1 levels without affecting the protein levels of c-Fos and p65 ( Figure 3C). Total RNA was prepared after treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of LEA (10 μg/mL), and qRT-PCR was performed using primers specific for Nfatc1, c-Fos, and p65. Results represents the means ± SD of three independent experiments. * p < 0.001 versus only RANKL treatment. (C) Whole cell lysates were prepared from M-CSF/RANKLtreated BMMs with or without LEA (10 μg/mL) for 0, 1, 2, and 3 days, and analyzed by immunoblotting with NFATc1, c-Fos and p65 antibodies. β-Actin was probed as a loading control. Total RNA was prepared after treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of LEA (10 µg/mL), and qRT-PCR was performed using primers specific for Nfatc1, c-Fos, and p65. Results represents the means ± SD of three independent experiments. * p < 0.001 versus only RANKL treatment. (C) Whole cell lysates were prepared from M-CSF/RANKL-treated BMMs with or without LEA (10 µg/mL) for 0, 1, 2, and 3 days, and analyzed by immunoblotting with NFATc1, c-Fos and p65 antibodies. β-Actin was probed as a loading control. LEA Suppresses NFATc1 Expression by Inhibiting the Transactivities of Both p65 and NFATc1 It is well documented that diverse signaling pathways such as MAPKs (ERK, JNK, and p38), Akt, and NF-κB signaling pathways modulate osteoclast differentiation [23][24][25]. To gain further insight into the mechanism by which LEA controls the expression of NFATc1, we investigated whether LEA influences RANKL-induced signaling pathways, including Akt, ERK, p38, JNK, and NF-κB. BMMs cultured with M-CSF for 24 h were pretreated with DMSO or LEA for 1 h and subsequently stimulated with RANKL for the indicated times ( Figure 4A). We observed that RANKL treatment increased the activation of Akt, ERK, NF-κB, p38, and JNK within 15 min. However, LEA treatment had no obvious changes in RANKL-induced signaling pathways ( Figure 4A). LEA Suppresses Prednisolone-Induced Osteoporosis in Zebrafish Larvae To examine the in vivo efficacy of LEA against osteoporosis, we employed zebrafish for this study since zebrafish are an ideal model system for the in vivo analysis of GIO [26,27]. Because c-Fos and NF-κB, as well as NFATc1 itself, transactivate Nfatc1 expression, we performed an Nfatc1 luciferase reporter gene assay to determine whether LEA affects their transactivities. Expression of NFATc1, c-Fos, or p65 increased Nfatc1 reporter gene transcription. LEA treatment almost completely abrogated transactivities of NFATc1 and p65, but has a modest inhibitory effect on c-Fos transactivity ( Figure 4B). LEA Suppresses Prednisolone-Induced Osteoporosis in Zebrafish Larvae To examine the in vivo efficacy of LEA against osteoporosis, we employed zebrafish for this study since zebrafish are an ideal model system for the in vivo analysis of GIO [26,27]. Zebrafish larvae at 10 days post-fertilization were treated with or without LEA in the presence of prednisolone (25 µM) for three days, then whole-mount bone staining was performed. We observed that bone mineralization was severely decreased by prednisolone. However, LEA treatment reduced prednisolone-induced osteoporosis ( Figure 5). Zebrafish larvae at 10 days post-fertilization were treated with or without LEA in the presence of prednisolone (25 μM) for three days, then whole-mount bone staining was performed. We observed that bone mineralization was severely decreased by prednisolone. However, LEA treatment reduced prednisolone-induced osteoporosis ( Figure 5). Discussion Although the shiitake mushroom possesses diverse bioactivities, such as immune modulation, antitumor, liver protection, cholesterol lowering, antiviral, and blood pressure lowering activities, there are few reports describing the effects of L. edodes on osteoblastogenesis or osteoclastogenesis [26,27]. Saif et al. demonstrated that the L. edodes water extract showed in vitro bone-inducing effects on human osteoblastic cells [22]. Another study demonstrated that water extracts of mushrooms decrease bone resorption and improve bone formation in vivo [28]. However, the effects of L. edodes extracts on osteoclastogenesis have yet to be fully elucidated. In the present study, we investigated the potential role of the shiitake mushroom in modulating the differentiation process in which osteoclast precursors are differentiated into mature osteoclast by RANKL. Using solvent fractionation methods, we discovered that the ethyl acetate fraction of aqueous extracts of L. edodes had strong antiosteoclastogenic activity. Recently, Fang et al. showed that an ethyl acetate fraction of aqueous Discussion Although the shiitake mushroom possesses diverse bioactivities, such as immune modulation, antitumor, liver protection, cholesterol lowering, antiviral, and blood pressure lowering activities, there are few reports describing the effects of L. edodes on osteoblastogenesis or osteoclastogenesis [26,27]. Saif et al. demonstrated that the L. edodes water extract showed in vitro bone-inducing effects on human osteoblastic cells [22]. Another study demonstrated that water extracts of mushrooms decrease bone resorption and improve bone formation in vivo [28]. However, the effects of L. edodes extracts on osteoclastogenesis have yet to be fully elucidated. In the present study, we investigated the potential role of the shiitake mushroom in modulating the differentiation process in which osteoclast precursors are differentiated into mature osteoclast by RANKL. Using solvent fractionation methods, we discovered that the ethyl acetate fraction of aqueous extracts of L. edodes had strong anti-osteoclastogenic activity. Recently, Fang et al. showed that an ethyl acetate fraction of aqueous methanol extracts of shiitake mushroom induced apoptosis in cancer cells by arresting cell cycle [15]. It will be interesting to examine the effects of the LEA used in this study on other biological activities. Our transcriptome analysis demonstrated that LEA negatively regulated a set of osteoclast-related gene expression. It is noteworthy that LEA significantly inhibited RANKL-induced NFATc1 expression, which is a key transcription factor for osteoclastogenesis. Additionally, GSEA analysis suggested that LEA treatment controlled expressions of Tnfrsf11a/Rank, which is a key regulator of osteoclastogenesis via RANKL-RANK signaling ( Figure 2E) [29]. DEG analysis, however, showed that Rank gene is not significant with a cutoff of FDR-adjusted p < 0.05 and 1.5-fold ( Figure 2C and Supplementary Table SII). Moreover, our qRT-PCR study clearly revealed that LEA treatment had no apparent effect on MCSF-or RANKL-mediated Rank expression (Supplementary Figure S2). These results indicate that LEA may suppress Nfatc1 expression by regulating RANK downstream rather than Rank expression. NFATc1 expression during osteoclastogenesis is tightly regulated via the NF-κB and c-Fos signaling pathways [7,30]. The NF-κB components p65 and p50 are recruited to Nfatc1 promoter upon RANKL stimulation. Calcium signal-induced activation of NFATc1 also triggers Nfatc1 expression by binding to its own promoter in cooperation with c-Fos [31]. In our study, LEA did not affect cellular signaling of NF-κB and c-Fos activation, as well as their transcription and translation. Interestingly, LEA selectively abolished NF-κB/NFATc1 transactivation for NFATc1 expression and did partially affect c-Fos transactivation. These results suggest that LEA might block p65 and NFATc1 localization at the Nfatc1 promoter. Similarly, Ha et al. observed that water extract of Uncaria sinensis inhibits RANKL-induced NF-κB transactivation without modulating the activation of MAPK and NF-κB by RANKL [32]. Additional studies are needed to understand the exact mechanism underlying the effect of LEA on NFATc1 expression. Since LEA efficiently suppressed osteoclast differentiation in this study, we anticipate that LEA will be effective for osteoporosis treatment. GIO is one of the serious side effects of glucocorticoid treatment, resulting in vertebra fractures. Recent studies revealed that glucocorticoids produce osteoporosis simultaneously by stimulating osteoclastogenesis and attenuating osteoblast formation [2,4]. Furthermore, we and others have shown that prednisolone treatment induces an osteoporosis phenotype in zebrafish larvae [26,27]. Based on our findings that LEA treatment prevented bone loss in a zebrafish GIO model, we believe that LEA prevents glucocorticoid-induced bone loss by affecting both osteoclasts and osteoblasts. Further studies are required to isolate bioactive compounds from LEA. Nevertheless, our present results indicate that LEA could have therapeutic value in treating GIO. Preparation of Water Extract and Its Fractions Using L. edodes Fresh fruiting bodies of L. edodes strain Chamaram were sliced and freeze-dried for 72 h. 150 g of the freeze-dried materials were soaked in various solvents (water, ethyl acetate, or ethanol) for 6 h and sonicated for 1 h in an ultrasonic bath (DAIHAN-Sci, Seoul, Korea). After filtration, each extract was evaporated using a rotary vacuum evaporator (RV8, Germany). The solvent fractions of crude water extract have been prepared as previously described with minor modifications [33]. The crude water extract (59.5 g) was suspended in water, and then sequentially partitioned with equal volume of hexane, ethyl acetate (EtOAc) and n-butanol (BuOH). The remaining aqueous extract was used as an aqueous fraction. Each fraction was evaporated using vacuum evaporator. The yields afforded EtOAc (1.6% w/w), BuOH (7.7% w/w) and aqueous (90.7% w/w) fractions. The hexane fraction exhibited the lowest yield of 0.01%, thus, this fraction was excluded from the next analysis. Osteoclast Differentiation and TRAP Staining Osteoclast precursor cells were prepared as previously described [34]. In brief, bone marrow cells were collected by flushing tibias and femurs from six-to eight week-old ICR male mice. Cells were cultured in α-minimum essential medium supplemented with 10% FBS and M-CSF (5 ng/mL) for 16 h. Non-adherent cells were harvested and cultured with M-CSF (30 ng/mL) for three days. Suspended cells were removed and adherent cells were used as OCP cells. Osteoclast differentiation were performed as recently described [35]. Briefly, OCP cells were cultured in 48-well plates with 30 ng/mL of M-CSF and 100 ng/mL of RANKL, in the presence or absence of LEA (10 µg/mL). On day 3, the cells were fixed with 3.7% formaldehyde in phosphate-buffered saline and stained for TRAP using an acid phosphatase leukocyte kit (Sigma-Aldrich, St. Louise, MO, USA). TRAP-positive, multinucleated cells containing three or more nuclei were counted as osteoclasts under a light microscope. Cell Viability Assay To examine the effect of L. edodes on cell proliferation, osteoclast precursor cells were treated with the extracts of the L. edodes (EtOAc, ethanol, and water) or the fractions of the L. edodes (EtOAc, BuOH, and aqueous), and MTT assays were performed after 24 or 48 h using Cell Proliferation KIT 1 (Roche Diagnostics, Mannheim, Germany). RNA-seq Total RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany). Libraries were prepared from 2 µg of total RNA using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., Palo Alto, Santa Clara, CA, USA). High-throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2500 (Illumina, Inc., San Diego, CA, USA). mRNA-Seq reads were mapped to reference mouse genome (mm10 assembly) using HISAT2 with default parameters. RNA expression was quantified using the analyzeRepeats.pl command in HOMER. DEGs were assessed with DESeq2 using getDiffExpression.pl in HOMER. The Cut-offs parameters for significantly up-or down-regulated genes were set at fold change ≥1.5 and FDR < 0.05 (Benjamin-Hochberg). To generate the heatmap of K-means clustering, we used the Morpheus web site (https://software.boradinstitute.org/morpheus/). To find GO terms enriched in DEGs, we performed Metascape tool [36]. Gene Set Enrichment Analysis were performed on the entire genes (a total of 24940 genes) using MsigDB gene sets [37]. Western Blot Analysis Whole cell lysates were prepared with lysis buffer (25 mM Tris, pH 7.9, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 5% glycerol, protease inhibitor cocktails, 1 mM sodium orthovanadate, and 2.5 mM sodium pyrophosphate). Protein concentration of cell lysates was determined by the Protein Assay Kit (iNtRON biotechnology, Inc., Seoul, Korea). Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Freiburg, Germany), and subject to Western blotting using the indicated antibodies. The antibodies used in this study were as follows: p-Akt, Akt, p-ERK and ERK antibodies from Cell Signaling Technology (Danvers, MA, USA); p-IκB, NFATc1 and p65 antibodies from Santa Cruz (Santa Cruz, CA, USA); IκB and β-Actin antibodies from Sino Biological (Beijing, China); and p-p38, p38, p-JNK, JNK, and c-Fos antibodies from Millipore (Burlington, MA, USA). Reporter Gene Assay 293T cells were plated in 12-well plates at 80% confluence and transfected with an Nfatc1-Luc reporter plasmid and expression vectors for c-Fos, p65, and Nfatc1 in the presence or absence of LEA (10 µg/mL) for 24 h. Cells were lysed in Reporter Lysis buffer (Promega, Madison, WI, USA) and assayed for luciferase activity using SpectraMax i3x (Molecular Devices, San Jose, CA, USA). Fish Maintenance and Drug Treatment Zebrafish were raised under standard maintaining conditions in a circulating water system at 28 • C, with day-night (14 h light/10 h dark) cycles. The fish were fed live brine shrimp three times a day. The male and female zebrafish with high potential to produce fertilized eggs were chosen for spawning. After removing the unfertilized eggs, embryos were kept at 28 • C, with day-night (14 h light/10 h dark) cycles. At 10 dpf, the larvae were treated with 25 µM prednisolone of LEA (10 µg/mL). At 13 dpf, the larvae were collected for whole-mount skeletal staining. All experimental protocols were approved by the Animal Care and Use Committee of the Chungbuk National University (CBNUA-1244-19-02, 8 March 2019). Whole-Mount Skeletal Staining Alizarin red staining on zebrafish larvae was performed as previously described [35]. Briefly, larvae at 13 dpf were fixed in 10% neutral buffered formalin. After bleaching pigmentation with 3% H 2 O 2 solution, the larvae were stained with 1 mg/mL alizarin red stain/1% KOH, pH 4.2 and then sequentially washed with 20% glycerol/1% KOH, 40% glycerol/1% KOH, and 60% glycerol/1% KOH. Images of stained larvae were obtained using a stereo microscope SMZ18 (Nikon, Tokyo, Japan). For quantification of bone mineral density, the areas of the first ten stained vertebrae (V1-V10) were calculated using the ImageJ densitometry program. Statistical Analysis All quantitative data were presented as mean ± SD. GraphPad Prism (GraphPad Software Inc. La Jolla, CA, USA) was used for all analyses. Statistical analysis was done using the one-way ANOVA test, followed by Tukey's Multiple Comparison Test. Accession Numbers RNA-seq datasets were deposited at NCBI Gene Expression Omnibus (GSE 142866).	2020-02-20T09:17:12.321Z	2020-02-01T00:00:00.000	{ "year": 2020, "sha1": "2013b386e2be7a505e09a935f118a52b5be35f0b", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1422-0067/21/4/1347/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d325539800486adac9c9b48c518b00f1cd89f011", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
237727097	pes2o/s2orc	v3-fos-license	Saving energy in China’s industry with a focus on electricity: a review of opportunities, potentials and environmental benefits Industry is the largest electricity consuming sector in the world. China consumes about 25% of global electricity demand, and 69% of this is used in industries. The high electricity demand in industry is responsible for 45% of CO2, 25% of SO2, 34% of NOx and 14% of PM emissions in China. This study aims to fill the knowledge gap on the potential for electricity savings in China’s industries, thereby providing important implications for the potential of reducing emissions in electricity-intensive industrial subsectors in general. Available studies are reviewed and compared to identify electricity-saving potentials. The findings show that China’s industrial energy system is shifting to higher electricity and relatively lower fossil fuel use due to accelerated end-use electrification. China’s industry can reduce electricity use by 7–24% in 2040, compared to baseline levels, and generate emission reductions of 192–1118 Mt-CO2, 385–2241 kt-SO2, 406–2362 kt-NOx and 92–534 kt-PM2.5. The iron & steel subsector has the largest contribution to the industrial electricity savings, followed by non-ferrous metals, chemicals, cement and pulp & paper. Policies that combine environmental targets, demand-side efficiency and supply-side retrofits in the power sector should be adopted. Given the different performance of policies in terms of energy savings and emission reduction, sector- and region-specific policies would be preferred. 10% by nuclear, 8% by renewables and the remaining 4% by oil (IEA, 2018). The high share of fossil fuels makes the power sector one of the largest emitters of greenhouse gases (GHGs) and air pollutants. In 2016, 41% of CO 2 emissions, 36% of SO 2 emissions, 15% of NO x emissions, 6% of PM 2.5 emissions around the world are produced by electricity generation (IEA, 2016b). It is expected that global power consumption will keep growing over the next decades due to the acceleration of industrialization and electrification in developing economies. As a major source of GHG emissions, many countries are making great efforts to decarbonize their power supply. Besides climate change, health impacts of air pollution are of great attention. An estimate by the World Health Organization shows that 4.2 million premature deaths globally are attributable to air pollution in 2016 (WHO, 2018). Improving energy efficiency in end-use sectors would therefore generate multiple benefits, by reducing both types of emissions, as well as limiting the needs for investments in new power capacities and grid extensions. Industrial processes are responsible for 42% of global electricity demand in 2016 (IEA, 2018). This makes the industry sector an attractive target for reducing emissions through the efficient use of electricity. This study focuses on electricity conservation in industrial sectors by performing an in-depth review of available literature on the most consuming subsectors and key energy-saving technologies, taking the largest electricity consumer, China, as case. China consumes 25% of global electricity demand, which is double the total consumption of the European Union, and 69% of the national electricity consumption is used by the industrial sector, in 2016 ( Fig. 1). As a large manufacturing country of energyintensive products, China also leads the electricity consumption at the subsector level (followed by the USA, Russia, India, respectively), accounting for around 50% of global electricity consumption per industrial subsector (Fig. 1). The high electricity demand increases air pollutants from the power sector in China, for example, around 27.2% of SO 2 emission, 26.9% of NO x emission and 14.8% of PM are caused by power generation (NBS and MEP 2018). Annually, more than 1 million people die prematurely in China as a result of exposure to high concentrations of PM, of which power generation is estimated to contribute to 39% of mortality (Gao et al., 2018). While the annual concentration of PM 2.5 and PM 10 decreased by 5.4% and 1.9% in 2017 (Huang et al., 2018), respectively, compared to 2016 levels, concentrations are still higher than the Chinese national level II targets (PM 2.5 of 35 μg/m 3 and PM 10 of 70 μg/m 3 ) (MEP and AQSIQ 2016), stressing the need for future emission reductions. The high electricity demand delivered by a coal-dominated power generation sector, and its contribution to poor air quality require integrated approaches to help improve air quality. The Chinese government is taking serious efforts to control emissions of air pollutants by requiring more strict emission standards for energy-intensive sectors (e.g. cement, chemical and iron & steel) (MEP & AQSIQ 2019), retiring small coal-fired power units (NDRC, 2019) and limiting energy consumption per unit product (e.g. primary aluminum and crude steel) (MIIT, 2017). Furthermore, China is also under huge pressure to reduce GHG emissions and has committed to cutting its carbon intensity by 60 to 65% by 2030 from a 2005 baseline, peaking GHG emissions by 2030. About 45% of CO 2 emissions from China's industrial sector can be attributed to electricity consumption, reaching 2690 Mt-CO 2eq in 2015 (CEADs, 2017). Promoting electricity conservation is an effective way to both reduce overall emissions of GHG and air pollutants from coal-fired power generation, offering synergies for electricity intensity, air quality (Yue et al., 2018), climate change (IRENA, 2017) and health effects from ambient air pollution (Abel et al., 2019), as well as economic benefits (U.S. EPA 2015). Although studies have looked previously at energy efficiency Zhang et al., 2020) and emission reduction (Khanna et al., 2019;Zhou et al., 2018a) in China, a comprehensive understanding of electricity use and conservation in China's industry perspective is still lacking. Most studies have focused on direct fuel use in end-use sectors, as fuel demand is still larger and contributes to direct emissions of the end-use sectors. Moreover, the studies on electricity often take a top-down view on electricity demand, without sufficient detail on the electricity end-uses and subsectors. To fill this gap, this study provides an industry-wide overview of electricitysaving potentials in China, based on available studies, and estimates associated benefits on emission reduction potentials. China can be a great case bringing guidance for countries dominated with industry and coal power (e.g. Australia, India, Mongolia, South 1 3 Africa and Poland) in saving energy, cleaning up air and mitigating climate change. Within industry, five energy-intensive subsectors (i.e. iron & steel, cement, non-ferrous metals, pulp & paper and chemical) together account for 65% of China's industrial energy consumption 1 and are analyzed in-depth in this study. These industries are also highlighted by the IEA (IEA, 2016a(IEA, , 2017, EIA (EIA, 2018) and China's development plan for energy conservation and emission reduction (State Council of China, 2017). The five energy-intensive subsectors are complemented by the textile industry, which is a non-energy-intensive industry, but a large electricity consumer. This review-based research provides important insights in the way forward for reducing electricity-derived emissions in the industry for the most polluting countries, with an in-depth focus on the big contributing subsectors and key manufacturing processes. Specifically, the main contribution of this study is answering the following four questions. (1) How much electricity can China's industry save? (2) Which industrial subsectors have the highest potentials of electricity savings and associated emission reduction? (3) Which research fields should be given more attention? (4) How to design and implement (industrial energy) policies to jointly achieve electricity, climate H u n g a r y Q a t a r N e w Z e a l a n d I r a q R o m a n i a P e r u P o r t u g a l U z b e k i s t a n S i n g a p o r e A l g e r i a B a n g l a d e s h G r e e c e C z e c h R e p u b l i c I s r a e l S w i t z e r l a n d C o l o m b i a A u s t r i a K a z a k h s t a n V e n e z u e l a C h i l e P h i l i p p i n e s F i n l a n d B e l g i u m P a k i s t a n N e t h e r l a n d s (NBS 2017). Calculated by authors. Note: the y-axis in the Bubble plot represents a logarithmic scale and clean-air goals (at national, regional and sectoral levels)? The study presents the available literature and classified these studies by industrial subsectors in Section 2 and 3 discusses the methodological differences between the studies. The main results, in terms of electricity-saving potentials and associated environmental benefits in China's industrial sectors are presented in Section 4. Finally, conclusions and policy implications are given in Section 5 and 6, respectively. Review method A systematic review method is carried out to address the questions in this study, consisting of seven steps (Fig. 2). In the first step, four questions are proposed related to the knowledge gap on understanding the role and potential of electricity savings for reducing air emissions in industry. In the second step, targeted keywords are used, combined with the snowballing technique to identify relevant publications from various databases (e.g. Google Scholar, Web of Science and Scopus). Keywords reflecting the study purpose include "electricity saving", "energy saving", "efficiency" and "China's industry". Snowballing was used to check for additional publications in the reference list of the identified papers and for studies that cited the identified papers. During the third step, the available publications are analyzed to discuss their main characteristics (in Section 2). The selected studies allowed obtaining relevant data to be analyzed. In the fourth step, the methodological differences between the studies are discussed to be able to interpret the modeling results (Section 3). In the fifth and sixth steps, the collected data on energy consumption Fig. 2 Methodological steps Energy Efficiency (2021) 14: 60 1 3 and intensity from the studies are systematically processed to estimate the electricity-saving potentials (Section 4). The framework is completed in the seventh step, highlighting the role of electricity as a key energy carrier and identifying the highest potential subsectors (Section 5). Furthermore, suggestions on joint policy design and further research are provided in the seventh step (Section 6). Literature selection Table 1 gives an overview of studies that focused on energy-saving potentials in China's industrial subsectors (i.e. iron & steel, cement, non-ferrous, pulp & paper and chemical sectors), including some of their key characteristics (such as studied period, model used and research scope). A discussion on the literature review of energy-saving potentials in China's industry is given below and is structured by subsector, 2 starting with studies that focus on industry as a whole. China's industry Limited studies were found that evaluate the future potentials of China's entire industry with detailed subsector information. The study of Ouyang and Lin (2015) analyzed the driving forces of energy-related CO 2 emissions in China's industry and suggested that energy efficiency improvement through efficient technology promotion and phasing out of inefficient production capacities was a major cause of CO 2 emission reduction in the period 1991-2010. Another study of Meng et al. (2014) analyzed the electricity-saving potentials of 20 economic sectors in China and showed that the chemical and mechanical sectors have large potentials for electricity efficiency improvement. These studies emphasize the considerable energy-saving potentials in China's industry and indicate energy efficiency improvement is key to achieving sustainable development. However, they did not quantify future energy (and electricity) demand and emissions. IEA (2016a) and EIA (2018) give an overview of energy efficiency improvement in China's industry but provide limited information on industrial electricity use. ERI (Dai et al., 2013), LBNL and THU (Zhou et al., 2018b) Zhang (2015a) and identified optimum EET options from an economic perspective. In summary, these studies indicate production structure adjustment (i.e. shift from Basic Oxygen Furnace (BOF) to Electric Arc Furnace (EAF)) and technological progress as the main forces to reduce future energy use and emissions in CISI. In addition, actions to improve energy efficiency by EETs can bring synergies for climate change, air quality and public health to be achieved cost-effectively (Wu et al., 2016). Cement industry 12 studies focus on energy efficiency improvement and emission mitigation in China's cement industry (CCI, Table 1). Xu et al. (2012) showed that 25% of energy use (2009) could be saved if best available technologies were implemented. Hasanbeigi et al. (2013a) evaluated the cumulative saving potentials (CSPs) and associated CO 2 emission reduction of 23 technologies in the period 2010 to 2030. However, CSPs provide limited transparency for policy-makers for understanding energy savings and emission reductions by year, when designing policies. To fill this gap, Wen et al. (2015a) and Li et al. (2017) simulated the future trends of energy consumption and CO 2 emissions using single-objective optimization models. Zhang et al., (2015bZhang et al., ( , 2015c 2016) modeled the multiple benefits of 37 EETs in energy savings and emission reduction (CO 2 and air pollutants) as well as health effects contributed by PM 2.5 from CCI for the period 2010-2030 at various regional levels. Yang et al., (2013aYang et al., ( , 2013b) estimated the benefits of 18 mitigation measures, monetized in $/t-CO 2 by MACCs (marginal abatement cost curves), at national and provincial level. In summary, the studies indicate that improving energy efficiency, switching to low-carbon fuels, reducing the clinkerto-cement ratio and integrating carbon capture into cement production are the main energy saving and carbon mitigation measures which support a transition to sustainability in the cement sector. The integration of carbon capture technologies and reducing of the clinker content in cement are identified to provide the largest cumulative CO 2 emission reductions in a long-term perspective. Table 1 includes 12 studies that investigate energy efficiency and carbon emission reduction in China's non-ferrous metals industry (CNFMI). Based on a top-down approach, Wang and Feng (2018), Shao (2017) and Wang and Zhao (2017) examined the energy efficiency performance of CNFMI and found that an energy efficiency improvement potential of more than 20% remains in CNFMI. Because the CNFMI sector consists of multiple products, it is difficult for bottom-up models to cover all production processes. Thus, bottom-up models are used to analyze specific subsectors. For example, Gao et al. (2009), Zhang et al. (2015d and Hao et al. (2016) predicted the GHG emissions in 2020 for China's primary aluminum industry by assuming different levels of electricity intensity (kWh/t-Al) based on national plans. Wen and Li (2014) evaluated the energy-saving potentials of 67 EETs for five main non-ferrous metals (aluminum, copper, lead, zinc and magnesium) during 2010-2020. Kermeli et al. (2015) assessed the technical and economic potentials of 22 EETs for aluminum industry at global level (including China) up to 2050. In summary, both technological progress and rising electricity prices have greatly promoted the decline in energy consumption and GHG emissions. In addition, promoting non-ferrous metals recycling through the implementation of circular economy is also of great importance to reduce energy consumption in the future. As the transition to clean power generation cannot be achieved short-term, The detailed scope of each study can be seen in the Supplementary material (Table S1- decision-makers can prioritize the use of EETs to achieve energy saving and emission reduction targets in CNFMI (Wen and Li, 2014;Zhang et al. 2015d). Table 1 and Table S2 (Supplementary material), several approaches have been adopted to analyze energy efficiency and intensity, yet few have evaluated the energy-saving potential in China's pulp & paper industry (CPPI). Lin and Zheng (2017) and Zheng and Lin (2017) studied energy efficiency using a total factor energy index, implying that CPPI could save at least 40% of its energy use. Kong et al. (2017) These studies indicate that efforts to improve energy efficiency will benefit both the economy and the environment by reducing energy costs and emissions of water pollutants and GHG. Closing small capacity and updating production technology have been identified as effective ways to improve energy efficiency in the CPPI. China's NDRC (2018) and MIIT (2012) also pointed out that dry-wet stock preparation, high-efficiency pulp washing and efficient double-disc refiner are efficient technologies that both conserve energy and mitigate emissions. In addition, 36 emerging technologies to substantially reduce energy use and GHG emissions compiled by Kong et al. (2012) should be promoted. Chemical industry Research on China's chemical industry (CCMI) is mainly limited to energy efficiency evaluation (Han et al., 2015;Long, 2014, 2015;Lin et al., 2012) and carbon emissions (Kahrl et al., 2010;Lin and Long, 2016;Liu et al., 2011;Zhou et al., 2010;Zhu et al., 2010). Only a few studies focus on future efficiency improvement potentials in CCMI and its subsectors, especially for electricity demand (Table 1). The chemical industry is a multi-product sector. Top-down approaches (Lin & Long, 2014Lin et al., 2012) used aggregated economic indicators to describe the industry as a whole. Lin and Long (2015) studied the energy efficiency improvement for the chlor-alkali industry in Shandong Province. Technological progress has proven an efficient way to improve energy efficiency. Studies on how to achieve the potentials by specific technologies are rare. Zhu et al. (2015) and Zhang et al. (2012) identified technological opportunities for energy savings and water-pollution reduction for ammonia production in the short term. Yue et al. (2018) estimated the electricity-saving potentials and associated emission reduction (GHG and air pollutants) of 60 electricity-saving measures in CCMI, covering 4 electricity-intensive chemicals. These studies reveal that efficient technology promotion and switching feedstocks (ammonia: coal-based to natural gas-based; PVC: calcium carbide-based to ethylene-based) are the main factors to reduce energy use and emissions of water and air pollutants. Increasing energy prices can drive the diffusion of EETs. Modeling factor identification Model-based scenario analysis nowadays plays a key role in informing decision-makers about future trends in the energy system. However, a slightly different hypothesis or scenario storyline can result in large differences in projected energy consumption. Therefore, it is necessary to understand studies in terms of their system boundaries, data sources, modeling approaches, key driver assumptions, assumed policy instruments and explicit technologies modeled. Firstly, as shown in Table S2, the base-year final energy and electricity use varies in the included studies as a result of different input data and system boundaries. For example, China's iron and steel sector as identified in Zhang et al. (2014) shows a lower final energy use in base-year 2010, of 16 EJ, compared to Hasanbeigi et al. (2013b) (from China's Statistics Bureau-CSB (NBS 2017)) and ERI (Dai et al., 2013), which is around 17 EJ. The processes of steel production and its specific energy consumption differ between various studies Karali et al., 2016;Ma et al., 2016;, and conflict with statistics for 2010 (ERI and CSB). The studies Zhang et al., 2017) are based on the same energy consumption per unit product (17.72 GJ/t-steel) to explore future energy efficiency improvement. Different statistical methods and system boundaries (e.g. the major process energy intensity of iron & steel in the GAINS model is lower than official data in 2010) are responsible for the divergences of base-year energy consumption among the listed studies (Zhang et al., 2014). Secondly, modeling approaches and included drivers are key factors that affect projections. Various modeling approaches are used to analyze energy-saving potentials (Table 1). These can be classified into three general types: (1) top-down models, (2) bottomup models and (3) integrated models (Karali et al., 2012). The drivers used typically vary per modeling approach (Table S3 for an overview of the key drivers used in the included studies). Top-down models (e.g. SFA, co-integration and LMDI) have a macroeconomic perspective and use socio-economic variables to predict energy use developments. Sectoral economic activities are represented through aggregated production functions. For example, the studies (Lin and Zhang, 2013;Lin et al., 2012) evaluated the electricity-saving potentials in CNFMI and CCMI by 2020 based on top-down approaches. However, topdown approaches (characterized by implicit technology representation like R&D progress) fail to capture technological details of energy conservation and are unable to incorporate different assumptions about how discrete EETs and costs will evolve in the future (Table S4) (Karali et al., 2012). In the list of Table 1, bottom-up models with specific technology representation are widely used to explore future energy demand and emissions in different sectors. However, some studies using bottom-up models (e.g. ECSC, ISEEM, NET and IWPCTP) include very limited interactions with the macro-economic system (Table S3). Integrated models (e.g. GAINS-ECSC and MFA-TE-LMDI) combine top-down and bottomup modeling approaches through hard or soft linking. However, integrated models are typically limited in the amount and details of key drivers compared to top-down or bottom-up models alone (Table S3). Detailed strengths and weaknesses of various models used in China were assessed by Zhang et al. (2015d) and Mischke et al. (2014). Additionally, power pinch method based on the concept of thermodynamics is widely used to analyze energy efficiency gap (Klemeš et al., 2018), but the applied coverages are mostly on optimizing hybrid power systems from the supply side (Rozali et al., 2014(Rozali et al., , 2019, rarely on quantitatively estimating the electricity savings in the demand side (Hackl & Harvey, 2013) for China, like industry and building. Therefore, the power pinch analysisrelated studies are out of this research scope focusing on industrial efficiency improvements in terms of electricity. While the pinch method should be given more attention to extend the application in annual strategic deployment of energy conservation, or to explore more efficient energy systems in combination with the demand side. Thirdly, although some studies use the same model, the policy instruments or type and specific EETs modeled in alternative scenarios can result in different results for energy savings. Tables S4 and S5 give an overview of policy instruments and specific technologies modeled in the different studies, respectively. Technology selection and diffusion are the main factors that affect energy savings in the studies using bottom-up approaches. For example, study collected 41 EETs for CISI, while 25 technologies are cost-effective. The results show that the technical and cost-effective energy savings are 4.63 GJ/t and 3.89 GJ/t, respectively, which are higher than the results from study (also on the basis of an ECSC model), i.e. 3.08 GJ/t and 1.93 GJ/t. The main reasons are the inclusion of more EETs for EAF-steelmaking, casting, rolling and finishing processes in study than study (Table S5). Results and discussion Based on the literature collection in Table 1, a comparative analysis of electricity savings for China's five key industrial subsectors is conducted. Moreover, the share of electricity savings in total final energy savings is assessed for each sector to understand the role electricity savings can play in reducing industrial energy use and providing an intuitive understanding of the relationship between electricity savings and 1 3 total energy savings. Opportunities to improve electricity efficiency in China's textile sector are also discussed. A detailed description of final energy consumption is presented in the Supplementary material. Finally, the synergies of electricity saving in relation to climate change and air quality are quantified (Tables 2, 3 and 4). China's industry Figure 3 shows the future electricity consumption and saving potentials in China's industry. Industrial electricity consumption grows steadily in all IEA scenarios (IEA, 2016a), with an average annual rate of 1.8% (with a range of 1.2-2.3%), which is higher than the annual growth rate of industrial final energy use of 0.5% (− 0.2-1.1%). The projected electricity use in 2020 for IEA's 450 and THU's Reference scenarios (Zhou et al., 2018b) are similar. THU predicts that electricity use will peak at 2025, with an average annual growth rate of 0.8% during 2015-2025 (range 0.5-1.2%), and then decline with 0.5% after 2025 (range 0.5-0.6%). In comparison to the baseline scenarios, industrial electricity consumption is expected to decrease by 4% in 2020 (range 2-5%), 10% in 2030 (range 6-15%), 14% in 2040 (range 7-24%) and 3% in 2050. China's industrial energy system is expected to shift to higher electricity and lower fossil fuels in the future due to accelerated end-use electrification. As shown in Fig. 4, the share of electricity consumption in industrial final energy use is expected to increase from an average of 24% in 2015 (range 20-28%) to 33% in 2040 (range 21-39%). Meanwhile, electricity savings are estimated to be 31% (with a range of 23-39%), 26% (20-29%) and 25% (11-33%) of total industrial final energy saving in 2020, 2030 and 2040, respectively. Promoting electricity conservation therefore is an effective way to reduce overall consumption in China's industry. Iron & steel industry As shown in Table 1, six studies focus on the electricity-saving potentials in CISI. Some of these studies (Hasanbeigi et al., 2013b;Ma et al., 2015a) show that with proven EETs a considerable amount of electricity can be avoided. Only two studies (Wang et al., 2007;Zhang et al., 2014) present the evolution of electricity use and efficiency improvements in the future (Fig. 5). Electricity use in CISI is expected to peak around 2020 and then decline slightly (by 0.1-0.4% per year) till to 2030. With the diffusion of advanced technologies, 1 3 electricity intensity would be able to decrease, with 10% in 2020 (range 8-12%) and 23% in 2030 (range 18-27%). The electricity-saving potential is estimated to be 16% in 2020 (range 13-17%) and 23% in 2030 (range 13-31%), compared to the baseline. Driven by the increasing proportion of EAF, electricity consumption is expected to increase from 10% of final energy use of CISI in 2010 to 13-14% in 2020 and 14-16% in 2030. There are not many long-term studies, but Chen et al. (2014) project in a baseline scenario, a share of 22% in 2050. Generally, the proportion of coal keeps declining in the final energy mix of CISI, and electricity will increasingly become a more dominant energy carrier. Efficient use of electricity can save 2-3% and 2-5% of final energy use of CISI in 2020 and 2030, which accounts for 11-12% and 10-12% of energy savings in 2020 and 2030, respectively. Five major processes (coke Unit: TWh Projected electricity use Electricity-saving potential making, sintering, iron making, steel making, and casting, rolling & finishing) are involved in steel production (see Table S1). The largest potential of electricity savings is found in the process of casting, rolling and finishing, which contributes to around 52% of the electricity savings in the steel industry. Direct rolling technology is one of the key cost-effective measures to reduce electricity demand in the finishing mills. Besides improving the casting and rolling process, considerable potentials of electricity savings are expected by promoting the penetration of new EAF systems such as direct current arc furnaces and twin shell furnaces. Figure 6 and Table 2 show the electricity-saving potential in China's cement sector. The included studies mainly focus on the short term (i.e. 2010-2030). Baseline scenarios show that electricity use in CCI will approach a peak of 211 TWh in 2020 (range 180-259 TWh) and then decline to 90 TWh by 2050. Compared to the baseline, electricity use can be reduced by 8% in 2020 (range 4-21%), 15% in 2030 (range 10-33%) and 15% in 2050 (range 9-18%). Compared to 2010, the electricity intensity is expected to be able to decrease by 27% in 2030 (range of 20-33%) and thereafter reach world best practice level (i.e. 54-60 kWh/t-cement) in 2050 (Worrell et al., 2008). Electricity accounts for around 10% (range 8-11%) of final energy use and changes not significantly during the period 2010 to 2030. Around 10% (range 6-16%) and 10% (range 6-14%) of final energy savings are contributed to electricity savings in 2020 and 2030, respectively, which account for 1% and 2% of final energy use. There are not many long-term studies, but Li et al. (2017) project that the share of electricity use would grow if a carbon tax increased from 50 $/t-CO 2 in 2020 to 215 $/t-CO 2 in 2050. The main reason is that the tax will promote energy system changes towards lower coal and higher electricity. Around 1.5% of final energy use can be saved by improving electricity efficiency in 2050. Cement manufacturing consists of four major production processes, i.e. fuel preparation, raw material preparation, clinker making, grinding and finishing. Around 50% of the electricitysaving potential can be released through improving the electricity use efficiency in the processes of raw material preparation and cement grinding. It is worth noting that general measures, such as highefficiency motors and energy management systems, have huge potentials to reduce electricity demand. These measures represent around 20% of total electricity savings in the cement sector. Meanwhile, Non-ferrous metals industry Research for the CNFMI is mainly concentrated on two aspects, i.e. GHG emissions (Gao et al., 2009;Zhang et al. 2015d;Hao et al., 2016) and historical energy efficiency investigations (Shao, 2017;Wang and Feng, 2018;Wang and Zhao, 2017). Studies on predicting the energy conservation trajectory in the whole non-ferrous metals industry are rare. As shown in Table 1, most of these studies focus on aluminum production because it accounts for 74% and 75% of total energy consumption and electricity consumption in the whole non-ferrous metals industry, respectively (CNMIA, 2017). Future electricity demand in CNFMI will keep growing with the expected increasing demand for nonferrous metals. The increasing electricity demand would result in large indirect emissions of GHG and air pollutants. Figure 7 shows that future electricity savings in the non-ferrous metals sector are considerable in 2020. For the overall non-ferrous metal industry, projected electricity use could be reduced by 41% in 2020 (with a range of 31-52%), corresponding to around 132 TWh (range 99-164 TWh). For aluminum production, electricity use is expected to be reduced by 8% in 2020 (range 6-10%). Both technological progress and increasing use of secondary metals greatly promote the decline in electricity consumption and GHG emissions. However, increases in the share of secondary production of non-ferrous metals are limited by the availability of scrap (IEA, 2017). Thus, the adoption of cost-effective technologies should be prioritized to improve electricity efficiency and reduce emissions by CNFMI. As the dominant energy carrier, the share of electricity in final energy use of CNFMI increased from 38% in 2000 to 66% in 2016 (NBS 2017). The increased electricity use comes mainly from aluminum production. Electricity consumed by aluminum production accounts for 49% of total final energy use of CNFMI. In 2020, around 4% (range 3-5%) of final energy use in CNFMI can be avoided by improving electricity efficiency in aluminum production. Electricity savings in aluminum production can contribute to 58% (range 56-60%) of energy savings of CNFMI in 2020. Saving electricity in aluminum production thus can significantly reduce overall energy consumption in CNFMI. Actions to improve the alumina refining process are expected to unleash around 10% of total electricity-saving potentials in China's aluminum sector. The most cost-effective way to reduce the electricity intensity of primary aluminum is to retrofit and upgrade outdated electrolytic cells, which can contribute more than 80% of the total electricity savings in the aluminum sector. Therefore, the government should prioritize the penetration of high-efficiency electrolyzers, such as new reduction cells with low temperature and low voltage, and TiB2/C composite cathodes with high wettability and impermeability. Figure 8 shows the electricity-saving potentials in CPPI in 2010. Four percent of the electricity use in CPPI can be avoided though implementing 3 process electricity-saving technologies (i.e. vacuum system optimization, high-efficiency double-disc refiners, refiner improvements) and 2 general measures (i.e. adjustable-speed drivers and energy-efficient lighting). It is worth noting that Germany as one of the most efficient paper producers still has 16% electricity-saving potentials though promoting 11 electricity-saving technologies . As the world's largest paper producer, the electricity savings (4%) in China's paper sector thus may be underestimated due to the few technologies included in the study (Kong et al., 2017). The shortcoming makes it difficult for policy-makers to fully understand the electricity savings and emission reduction potentials, and to make reasonable and cost-effective choices. Similar to other sectors, the energy system of CPPI is shifting towards higher electricity and lower coal use. Final energy consumption has peaked in 2009 and then declined with an average annual rate of 2% during 2009-2016 (Supplementary material). However, electricity use continued to grow, with an average annual rate of 5% in the period 2009 to 2016. The share of electricity demand thereby increased from 18% in 2000 to 29% in 2016. According to Kong et al. (2017), electricity savings (2 TWh) of CPPI can contribute to 3-4% of energy savings, representing around 1% of final energy use in 2010 by this sector. However, due to the limited studies and especially scope of included electricity conservation measures, these results are expected to be an underestimation. Two major production processes (i.e. pulping and papermaking) are included in paper manufacturing. Electricity savings in the pulping process account for around 20% of total electricity savings in CPPI. The papermaking process has the highest potential for reducing electricity use (accounting for nearly 60% of total electricity savings). Besides these two major processes, approximately 20% of the electricity-saving potentials can be accessed by cross-cutting Unit: TWh Electricity use Electricity saving potential 1 3 measures (e.g. adjustable-speed drives and antiscaling technology for cooling water systems). With the increasing share of wood-based pulping (10% in 2010 to 13% in 2017 (CTAPI, 2018)), more advanced electricity-saving technologies can be introduced to CPPI, like high-efficiency grinding, enzymatic pretreatment and chemical modification of fibers . It is necessary to build a cost-effective technology roadmap to facilitate the electricity-saving potentials and investments for CPPI in the future. Chemical industry As shown in Fig. 9, only two studies Yue et al., 2018) focus on future electricity-saving potentials in the CCMI. Top-down approaches show a large electricity saving potential for the overall chemical industry in 2020, of around 40% (ranging from 29 to 50%). Bottom-up approaches (Yue et al., 2018), on the other hand, estimate a lower possible reduction of electricity use by 16% in 2025 (range 15-18%), 25% in 2030 (range 22-28%) and 35% in 2035 (range 31-39%), compared to business as usual. As one of the main energy carriers in CCMI, electricity consumption in 2016 doubled in comparison to 2005, with an average annual growth rate of 8%, which is higher than the growth rate of final energy use (5%). The share of electricity in the final energy mix increased from 12% in 2005 to 15% in 2016. With accelerating electrification in CCMI, electricity consumption is expected to further increase (IEA, 2013;Yue et al., 2018). Efficient use of electricity by adopting 60 EETs can save 6% in 2030 (range 5-7%) and 9% in 2035 (range 8-10%) of final energy use of CCMI. Investments in energy efficiency in the chemical industry should focus especially on calcium carbide manufacturing (in particular the calcium carbide furnace), which is expected to deliver around 50% of the electricity savings among three key chemical subsectors (i.e. ammonia, chlor-alkali and calcium carbide). The chlor-alkali sector produces two major electricity-intensive chemicals, i.e. caustic soda and PVC. Efficient ion-exchange membrane electrolyzer (e.g. oxygen depolarized cathodes membrane electrolyzer, and zero electrode-distance membrane electrolyzer) and dry-process acetylene are key to reducing electricity costs in caustic soda and PVC production, respectively. Ammonia manufacturing includes four major processes, i.e. gas generation, shift conversion, gas purification and ammonia synthesis. China's ammonia production is dominated by coal feedstocks, which account for 86% of total ammonia capacity. The process of gas purification thus has the highest potential for conserving electricity (representing around 30% of total electricity savings in China's ammonia sector), which can be achieved by implementing high-efficiency desulfurization and decarbonization technologies (e.g. pressure swing adsorption for removing CO 2 ). Replacing and upgrading low efficiency ammonia synthesis towers can further release around 27% of the electricity-saving potential. Considering the electricity performance of each process, policy-makers and managers are suggested to scale up efficiency by targeting key areas within an industrial sector. This presents an option to access significant reductions in a rapid way with high cost performance. Textile industry The textile industry in China is not considered an energy-intensive sector. However, CTI consists of a large number of factories which together consume significant amounts of energy. CTI consumes 2% and 4% of China's industrial final energy and electricity consumption, respectively. Final energy use of CTI increased dramatically in the period 2000-2016, with an average annual growth rate of 5%. Within the energy consumption in CTI, electricity is one of the dominant energy sources, with an increasing share from 23% in 2000 to 45% in 2016 (NBS 2017). However, existing studies mainly focus on carbon emissions (Huang et al., 2017;Moubarak, 2013, 2014b) and have limited information on electricity savings in CTI. An international comparison of energy efficiency shows the large gap between China (31 MJ/$) and USA (11 MJ/$) in the textile industry (Peng et al., 2015a, b). Lin and Zhao (2016b) found the CTI sector has a 20-33% available potential to improve energy efficiency. Various energy efficiency opportunities (including available and emerging EETs) in the textile industry have been summarized by LBNL, many of which are cost-effective Price, 2012, 2015). As a dominant energy carrier, efficient use of electricity can effectively reduce the overall energy consumption and indirect emissions in the sector. Electricity-saving potentials of available technologies for major textile processes (i.e. spinning, weaving and wetting processing) are shown in Fig. 10. Most of the technologies are applied to the electric motor-driven systems (i.e. pumps, fans and compressed air). Thus, promoting efficient motor systems is key to realizing electricity efficiency improvement in CTI (Lin and Zhao, 2016a, b). Energy consumption in China's textile industry will continue to increase, while the growth rate is expected to slow down due to energy efficiency improvements. Various measures (e.g. industrial scale adjustment and technology upgrading) reduce energy use in CTI, of which technology upgrading is the most effective way (Lin and Zhao, 2016a;. It is worth noting that the energy efficiency of the entire motor-driven systems in the textile industry leaves much room for improvement. Summary of electricity savings and associated emissions reduction In this study we aim to include total industrial electricity demand, including purchased power from the grid as well as self-generated power (e.g. by combined heat and power (CHP) plants). For most literatures, we found that the energy demand data is consistent with this definition; however, not all studies are clear in this regard. In terms of savings potential as share, there is little impact to be expected since this is based on the percentual saving potentials of measures that are applied to total electricity demand. The total development of industrial electricity demand may however be underestimated to a certain extent because of this. Tables 2 and 3 summarize the saving potentials for final energy and electricity use in China's industry and for the five industrial subsectors. The electricity-saving potentials in China's industry show an increasing trend in the period 2020 and 2040, from 4 to 14%. Within total industry, electricity savings in the CISI sector have the largest contribution, followed by CNFMI, CCMI, CCI and CPPI 3 in 2020. The non-ferrous metal sector has the largest 1 3 electricity-saving potential with 25%, followed by chemicals (24%), iron & steel (16%), cement (8%) and pulp & paper (4%) in 2020. In 2030, the electricity-saving potentials of CISI and CCI increase to 23% and 15%, respectively. By 2030, the chemical sector (25%) still has greater potentials than the iron & steel and cement sector. In general, CNFMI and CCMI have more opportunities to improve electricity efficiency, while the saving contribution to the whole industry is less than that of the iron & steel sector. Based on the data collected in Table 2, co-benefits of electricity savings in terms of emission reduction of GHGs (CO 2 , CH 4 and N 2 O) and air pollutants (SO 2 , NO x and PM 2.5 ) in China's industry are calculated (Fig. 11). The emissions of GHGs and air pollutants can potentially reduce by 153 Mt-CO 2eq and 699 kt in 2020, and by 1123 Mt-CO 2eq and 5137 kt in 2040, respectively. The maximum reduction of CO 2 , SO 2 and NO x in 2040 equals 42%, 63% and 68% in comparison to 2015 industrial indirect emissions, respectively. The emission benefits from electricity savings are important to China for achieving climate and clean-air goals (e.g. reducing 60-65% of CO 2 intensity by 2030 compared that of 2005). Discussion of impact modeling factors on results Sectoral energy/electricity consumption and savings vary quite strongly in the included studies. In order to understand these differences, key factors that impact results are summarized below. These factors are partly based on the results from Sect. 3 that analyzes model approaches and assumptions, recorded in Tables S1, S3, S4 and S5. In addition, a comparison of energy demand levels in baseline scenarios and energy intensity trends, which is included in the Supplementary material, is used. • System boundary is identified as an important factor that especially has great influence on projections in sectors with multiple products, like non-ferrous metals and the chemical industry. As shown in Fig. S5, final baseline energy use of CNFMI projected by bottom-up approaches developed by Wen and Li (2014) T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 Electricity-saving potential Technology Fig. 10 Electricity-saving potential per technology applied in major textile processes. Note: T1-T25 refer to the technology summarized by Hasanbeigi and Price (2012): No. 3, 7, 12, 16, 34, 58, 97, 117, 121, 127, 132, 133, 134, 135, 138, 139, 140, 141, 142, 143, 146, 153, 154, 157 and 158, respectively; the black vertical lines represent ranged potential value from low to high; the blue and red markers mean average value and non-ranged value, respectively Energy Efficiency (2021) value from low to high; the blue and red markers mean average value and non-ranged value, respectively 1 3 differences in the scope of included products (the study of Wen and Li (2014) includes other materials besides aluminum production (e.g. lead, copper and zinc)). Another example is the chemical industry, where a wide range in potentials occurs, from 6 to 20% in 2020, as shown in Table 2. A main difference in sector coverage concerns which products are included and should therefore always be taken into account when comparing energy-saving potentials. Detailed system boundaries for each study are shown in Table S1. • The modeling approach is another key factor resulting in significant divergences among projections. Most included top-down approaches focus on modeling short-term horizons (up to 2020), while bottom-up and integrated approaches focus more on the medium and long term (Table 1). Modeling projections by top-down approaches often give bigger energy demand result than other modeling approaches. For example, the studies (Lin & Wang, 2014b; using a co-integration model show a much higher energy demand in 2020 for CISI (33 EJ) and CCI (9 EJ) than average levels projected by bottom-up modeling (14 EJ for CISI and 6 EJ for CCI, Supplementary material). This could be related to the higher level of technology detail in bottom-up approaches and assumed lower energy intensity developments. Moreover, there is a larger range in alternative scenario results in terms of energy savings. A key difference among model structures is the drivers used for energy modeling as shown in Table S3 (detailed discussion in Sect. 3) and is related to the next point on socio-economic indicators. • The assumed development of Social and economic indicators (e.g. GDP (Edelenbosch et al., 2017), population and urbanization ) strongly impacts production functions. The modeled output of industrial products in turn directly affects the development of energy demand and thus energy savings for a given sector (Fig. S12). A large level of assumed production (e.g. the found range is 364 to 881 Mt-steel by 2020) usually results in a high energy use in the baseline (ranging from 8 to 17 EJ by 2020). This also impacts the alternative scenarios, together with varying assumptions regarding implemented energy efficiency technol-ogies and policy measures, as indicated in the next point. • Assumed efficiency improvement room and technology characterization largely determine the modeling potentials of energy savings. Differing collected levels of energy-saving technology impact bottom-up approaches with rich technology representations (as described in Sect. 3 and Table S5). Technology cost is a main barrier for measures when considering necessary investment. High levels of assumed policy instruments (e.g. carbon tax, technology subsidies) can reduce the relative cost of technologies and thus increase the technology implementation level Zhang et al., 2018). This level is also referred to as the technology diffusion rate and has a direct impact on the saving potential. For example, scenarios modeled by Liu et al. (2017), who included 23 technologies with all nearly 100% implementation rates in 2030 for CCI, show a higher potential energy savings (relative savings of 20%) than other studies (e.g. 9-16% projected by Zhang et al. (2015b)), which included more technologies, but with a lower diffusion rate. Key differences in included policy measures found are the technology representation and the level of economy regulatory incentives. • Data sources also affect the modeling results to a certain extent. For example, the energy demand for CISI projected by Wang et al. (2007) and LBNL are lower than for other studies, probably because the energy service demands are based on historical data up to 2005. Different data sources and statistical methods used have different qualities and timeliness. The impact of data sources is difficult to quantify, but for long-term trends the impact on sectoral energy use and intensity seems to be limited (Supplementary material). The absolute development of energy or electricity use in the scenarios and therefore absolute savings are difficult to compare among studies, e.g. due to different activity levels assumed (e.g. more or less iron and steel production). Therefore, the focus was on percentual changes of the alternative scenario compared to the baseline scenario, with the only difference being the implementation of energy efficiency measures, while activity data in the scenarios remain the same. There may however already be some energy efficiency Energy Efficiency (2021) 14: 60 Page 21 of 28 60 improvement included in the baseline scenario, such as is to be expected in real life (e.g. the energy intensity in GJ/t-steel in most included studies is lower in the end year compared to the base year, see Supplementary material). The potentials are then an underestimation of the energy efficiency improvement compared to base year energy efficiency levels. On the other hand, for quite some studies the base year is 2010 or before, and it is to be expected that the energy intensity has improved in the current situation. However, the actual decrease in intensity, since the base year, may be higher or lower than assumed in the baseline scenario. As another factor, in most studies new and emerging technologies are not included in the energy efficiency potentials, meaning that more potential would be available in the future when these technologies become commercial. Therefore, the potentials should be seen as broad indications and would likely be expanded by new and emerging technologies. Conclusions Industry is the largest energy using sector in China, resulting in high emissions of GHGs and air pollutants, while large potentials for improving energy efficiency exist. With accelerating end-use electrification, coalintensive electricity has become a dominant energy source in China's industry. This systematic review evaluates energy-saving potentials in China's industry, while especially focusing on electricity savings in five industrial electricity users (representing more than 50% of total industrial electricity consumption) that are generally targeted by the IEA and the Chinese government. Results show varying levels of electricity-saving potentials, depending on sector, modeling approach (e.g. top-down, bottom-up) and scope (included products). Based on the results, synergies of electricity saving and climate change and air quality are quantified. Countries (e.g. India, Poland, Czechia and Australia) structured with heavy industry and coal-based power would get large benefits through saving electricity in end-use sectors. Main conclusions are as follows: ( (2) China's industrial energy system is shifting to higher power and lower fossil fuels. The share of electricity in final energy use is expected to further increase to 33% (21-39%) by 2040. Electricity-saving potentials, in China's industry, are expected to amount to 4% (2-5%), 10% (6-15%) and 14% (7-24%) of baseline electricity use in 2020, 2030 and 2040, respectively. The electricity savings contribute to 31% (23-39%), 26% (20-29%) and 25% (11-33%) of total industrial final energy saving in 2020, 2030 and 2040, respectively. (3) Electricity-saving potentials (%) and contributions (TWh) vary for different industrial subsectors. The iron & steel sector (93 TWh) contributes to the largest electricity savings, followed by non-ferrous metals, chemical, cement and pulp & paper sector. The nonferrous (25%) and chemical sector (24%) have the largest potentials to save electricity in 2020. In addition, motor-driven systems in the textile industry leave much room for improving energy efficiency. (4) Increasing energy efficiency in industry can generate synergies to mitigate climate change and improve air quality by reducing emissions of GHGs and air pollutants. In 2040, the potential co-benefits achieved with saving electricity in industry can be reduced emissions of 192-1118 Mt-CO 2 , 1995-11,603 t-CH 4 , 2851-16,584 t-N 2 O, 385-2241 kt-SO 2 , 406-2362 kt-NO x and 92-534 kt-PM 2.5 . Many options exist to reduce emissions from power generation (e.g. more efficient technology, fuel substitution and end-of-pipe measures), but end-use energy efficiency promotion is often cost-effective and can achieve multiple national energy and environmental goals. Policy implications and directions for research A trend towards higher electricity and lower fossil fuels is observed in China's industrial energy system. The 1 3 efficient use of electricity can contribute to around 30% of total industrial final energy savings in China. This study shows that promoting electricity savings in specific end-use sectors is a win-win strategy for demandside management and supply-side structural reform. The decrease of electricity demand could reduce emissions of GHGs and air pollutants while limiting the need for construction of new capacity (Peng et al., 2018). Based on the reviewed analysis and discussion, this study highlights the following policy implications (from national, sectoral and regional levels, respectively) and directions for future research. (1) As for national government, it is strongly suggested to further implement energy conservation and emission reduction through a joint policy of enhancing environment quality and energy efficiency on both demand and supply sides. Electricity savings provide a large potential reduction of air pollutant and GHG emission and need to be included in air quality and climate policy. In addition, policies integrating demand-side savings into power sector planning would help to design a pathway to phase out small and old coalfired units (e.g. < 300 MWh and > 20 years) with super-polluting (Tong et al., 2018). To economically achieve the sustainable development goals, a cost-effective technology roadmap for industry should be designed, including details on the energy-intensive subsectors, to maximize benefits of electricity savings, emission reduction and improving public health. These benefits outweigh investments. The findings suggest that energy efficiency technology will help policies for climate and air quality to be more cost-effective. (2) Promoting electricity conservation in different end-use sectors is an important policy measure. Within industry, policies should prioritize the iron & steel industry with the largest electricity-saving contribution (around 93 TWh in 2020), followed by non-ferrous metals (80 TWh), chemicals (69 TWh) and cement (15 TWh). When implementing sectoral efficiency policies, it would be more effective and economical if decision-makers are able to allocate limited resources to key areas. For example, the efforts for the non-ferrous metals should focus on, e.g. aluminum electrolysis (which consume more than 70% of electricity use in CNFMI) and motor-driven systems for textiles. It is worth noting that increasing waste material recovery and recycling for non-ferrous metals and pulp & paper also helps to reduce electricity use but is limited by the available resources. Learning from experiences in the European Union, standardized recycling systems of waste resources (for example, managing the waste material classification and investing in new recycling capacity) should be established to increase the recycling rates of waste materials. Furthermore, new policy of value-added-tax rebates should be formulated to incentive the enthusiasm of enterprises to participate in waste resource recovering and recycling. (3) The impacts of regional grid distribution to address air pollution (Yang et al., 2013a;Yue et al., 2018) and climate change (Peng et al., 2018;Wang and Zhao, 2017) should not be ignored. This study suggests that regional decision-makers need to not only understand the co-benefits of emission reduction and public health, but also different levels of power supply (and consumption) and air quality related to the possible relocation of electricity-intensive industry. Strict regional standards for large electricity consumers (e.g. aluminum and chlor-alkali sectors) should be carried out to restrict new capacity and phase out inefficient capacity in regions with higher emissions of power generation (e.g. Northeast and North grids). Meanwhile, regions in grids with lower emissions of power generation (e.g. Northwest grids) may consider expanding industrial capacity. Regional development policies should include these priorities to effectively address air quality improvements. (4) Regional governments should understand gridspecific synergies related to the regional allocation of abatement targets. Synergies between electricity savings and emission reduction vary per region due to the different levels of power use and specific emission intensities. To achieve national reduction goals effectively, this study suggests that different responsibilities should be assumed by different regions. Therefore, specific policies should be designed to set regional targets. Regions in grids with high potentials of electricity savings and emission reduction should bear the largest responsibility; the mini-mum burden should be allocated to regions in grids with lower multiple benefits. (5) Future research should focus on studying electricity-saving trajectories in the non-ferrous metals, textile and pulp & paper sectors, for which few studies are available. Furthermore, expanding the analysis to other electricity consuming sectors and to provincial/regional level will help to fully understand the contribution of electricity saving to environment and public health, as well as the impacts on grid distribution. Moreover, determining savings helps to identify options to reduce and optimize investments in new power plants and grid expansion, resulting in additional economic benefits. Quantifying the linkages between the supply and demand sides would warrant further research to optimally assess the impacts on power system. (6) Model-based scenario analysis is expected to continue to play a key role in assessing energy systems. A spread in modeling results for different policy scenarios (e.g. energy efficiency, pollutant control and tax measures) is unavoidable due to differing assumptions for urbanization and industrial electrification (especially in developing countries like China and India). Therefore, it is necessary to further improve modeling structures, upgrade key drivers (e.g. GDP, population and urbanization) and technology representations (e.g. specific technology) to design more effective policies.	2021-09-09T20:48:04.577Z	2021-07-28T00:00:00.000	{ "year": 2021, "sha1": "6180d3f6545c8eb0723e79ab730de22da1758e44", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s12053-021-09979-4.pdf", "oa_status": "HYBRID", "pdf_src": "SpringerNature", "pdf_hash": "5688fc064a8a8e6c03d818e0130d2ed786d9e2e9", "s2fieldsofstudy": [ "Environmental Science", "Engineering" ], "extfieldsofstudy": [ "Business" ] }
10053956	pes2o/s2orc	v3-fos-license	Community attitudes to the appropriation of mobile phones for monitoring and managing depression, anxiety, and stress. BACKGROUND The benefits of self-monitoring on symptom severity, coping, and quality of life have been amply demonstrated. However, paper and pencil self-monitoring can be cumbersome and subject to biases associated with retrospective recall, while computer-based monitoring can be inconvenient in that it relies on users being at their computer at scheduled monitoring times. As a result, nonadherence in self-monitoring is common. Mobile phones offer an alternative. Their take-up has reached saturation point in most developed countries and is increasing in developing countries; they are carried on the person, they are usually turned on, and functionality is continually improving. Currently, however, public conceptions of mobile phones focus on their use as tools for communication and social identity. Community attitudes toward using mobile phones for mental health monitoring and self-management are not known. OBJECTIVE The objective was to explore community attitudes toward the appropriation of mobile phones for mental health monitoring and management. METHODS We held community consultations in Australia consisting of an online survey (n = 525), focus group discussions (n = 47), and interviews (n = 20). RESULTS Respondents used their mobile phones daily and predominantly for communication purposes. Of those who completed the online survey, the majority (399/525 or 76%) reported that they would be interested in using their mobile phone for mental health monitoring and self-management if the service were free. Of the 455 participants who owned a mobile phone or PDA, there were no significant differences between those who expressed interest in the use of mobile phones for this purpose and those who did not by gender (χ2(1), = 0.98, P = .32, phi = .05), age group (χ2(4), = 1.95, P = .75, phi = .06), employment status (χ2(2), = 2.74, P = .25, phi = .08) or marital status (χ2(4), = 4.62, P = .33, phi = .10). However, the presence of current symptoms of depression, anxiety, or stress affected interest in such a program in that those with symptoms were more interested (χ(2) (1), = 16.67, P < .001, phi = .19). Reasons given for interest in using a mobile phone program were that it would be convenient, counteract isolation, and help identify triggers to mood states. Reasons given for lack of interest included not liking to use a mobile phone or technology, concerns that it would be too intrusive or that privacy would be lacking, and not seeing the need. Design features considered to be key by participants were enhanced privacy and security functions including user name and password, ease of use, the provision of reminders, and the availability of clear feedback. CONCLUSIONS Community attitudes toward the appropriation of mobile phones for the monitoring and self-management of depression, anxiety, and stress appear to be positive as long as privacy and security provisions are assured, the program is intuitive and easy to use, and the feedback is clear. Introduction Reducing the burden of mental disease requires a combination of effective prevention, early intervention, treatment, and self-management, and a critical aspect of these functions is for individuals to monitor their mental health. Self-monitoring brings about actual improvements in mood and behavior and enhances individuals' compliance with treatments [1,2]. Historically, paper diaries have been the primary mode of monitoring, but patients can find them cumbersome. Noncompliance is also common, as demonstrated by Stone et al [3] who compared patients' actual and reported compliance with diary keeping. By embedding a photosensor into the binder containing the paper diary forms that detected light and recorded when the binder was opened and closed, the researchers ascertained that actual compliance was 11%, whereas participant-reported compliance was 90%. In contrast, compliance with an electronic diary was 94% [3]. However, computer-based monitoring also has inherent limitations in that it relies on users being at their computer at scheduled times, which can be inconvenient, and, as a result, nonadherence after short periods of time is also common with this delivery channel. Retrospective recall of symptoms, mood, or behavior can also be unreliable [3]. To be maximally effective, individual self-monitoring needs to take place regularly and in real time to reduce recall bias and increase accuracy. Mobile phones offer a solution. Their take-up has reached saturation point in most developed countries and is increasing in developing countries; they are carried on the person and they are usually turned on. A further advantage is that mobile phones allow the gathering of frequent instantaneous reports of mood and behavior while people go about their everyday business. Termed Ecological Momentary Assessment, or EMA, mobile phone monitoring has been shown to more accurately represent the true natural history of transitory states than dispersed measurements and may decrease user burden [4]. With mobile phones, users can be prompted to respond, and these "just-in-time" prompts can be scheduled for key times. Mobile phone monitoring is also potentially more convenient than paper-or computer-based recording. Mobile phones have been used for monitoring within behavioral health applications such as alcohol consumption [5] and gambling [6]. They have also been used to deliver simple interventions, for example, to manage migraines [7], enhance physical activity [8], cease smoking [9], and control weight [10]. However, the use of mobile phones in mental health monitoring and management is in its infancy. Furthermore, community attitudes toward the use of mobile phones as a mental health tool are unknown. Until recently, mobile phones were primarily viewed as tools of communication and social identity, but increasingly they are evolving into a personal multipurpose tool for their owners, with routine functions now including reminders for medical appointments, timekeeping, and note taking. While preliminary research has been undertaken exploring community attitudes toward the use of mobile phones for health monitoring, such as for asthma [11], attitudes to the appropriation of mobile phones for mental health monitoring and management have not yet been investigated. Derived from the field of marketing, the term "appropriation" refers to the processes that take place when new uses are invented for existing technologies and when these uses develop into routine practices and spread within a user community [12]. The aim of this study was to investigate community attitudes toward the appropriation of mobile phones for mental health monitoring and management as groundwork for the development of a digital tool for self-monitoring and self-management of depression, anxiety, and stress. Methods A mixed method approach was used consisting of an online survey, focus group discussions, and interviews. The target population was Australian adults over 18 years of age with or without depression, anxiety, or stress. Participation was voluntary and anonymous in all 3 study components. Development and Administration of Study Tools Questions for the online survey, focus group discussions, and interviews were generated, reviewed, and amended by the research group in consultation with the members of the community program team at the Black Dog Institute, a mood disorders unit in Australia. The final set of questions explored the following issues: • current usage of mobile phones • attitudes toward using a program on mobile phones or the Internet for monitoring and managing depression, anxiety, or stress • possible ways in which participants might use such a program • any key features that such a program should have • demographic information and mental health history of participants The survey was pilot tested using QuestionPro [13]. The survey's usability and functionality were assessed and improvements made prior to it being posted on the website of the Black Dog Institute [14]. The website provides information and tools about mood disorders for the public, health professionals, and workplaces. A broad cross section of the public uses the site, including those with mood disorders and those without (eg family, carers, friends, students, and interested individuals). The site is consistently in the top 5 Google ranks for mood disorders in Australia. The survey was voluntary and was open to any site visitor. Initial contact with potential participants was made on the Internet. The survey consisted of 46 questions over 12 Web pages. Some pages had more items than others, although the maximum number of items per page did not exceed 7. Forty-four of the 46 items were mandatory, each was highlighted, and it was not possible for respondents to proceed to the next page until they had completed the mandatory items. Because there was no back button, respondents were unable to review or change their answers. To prevent multiple entries, a cookie was placed on the participant's computer. There was no Internet protocol (IP) check or log file analysis. As this was an open survey, no log-in or registration was required. The data were collected over a 3-month period from May to August 2009. Focus group participants and interviewees were recruited through community organizations, a variety of companies, and the Black Dog Institute community programs. Similar questions to those in the online survey were used in the focus group discussions and the interviews to facilitate later triangulation of results. To protect participants' confidentiality within the interviews and focus groups, demographic information and mental health history were completed anonymously by a paper and pencil questionnaire that participants sealed in an envelope and gave to the researcher. An experienced moderator conducted each of the focus groups with 1 or 2 observers present who took notes. The sessions were audio taped for later transcription and analysis. Informed Consent Process Individuals who clicked on the link to the online survey were provided with a statement about the study that included its purpose, the length of time to complete the survey (approximately 15 minutes), how the data were being stored (initially on the secure QuestionPro server then in password-protected files in the university's secure server for 7 years), and the name of the chief investigator (author JP), before being invited to give their informed consent online and to access the survey. The survey was anonymous. However, those who wished to enter a draw to win an iPod Nano for completing the survey were invited to separately provide their name, phone number, and email address. This information was stored in a secure password-protected file on the QuestionPro server and then transferred to the university's secure server for long-term storage. The informed consent process for the focus group discussions and interviews was similar. Individuals who expressed interest in being interviewed or participating in the focus group discussions were provided with a written outline of the study which included its purpose, the length of time the interview/focus group would take (approximately one hour), how the data were being stored (audiotaped for later transcription and storage for 7 years in password-protected files in the university's secure server) and the name of the chief investigator (author JP). They were then invited to give their informed consent to participate in the interview or focus group. No identifying information was collected. The study was approved by the University of New South Wales Human Research Ethics Committee. Advertising and Recruitment The online survey, focus group discussions, and interviews were advertised simultaneously through Facebook, the websites of the University of New South Wales and Black Dog Institute, and the intranets of a variety of companies and consumer organizations. The advertisement for the online survey can be found in Multimedia Appendix 1. The advertisements for the interviews and focus groups had similar wording to the advertisement in Multimedia Appendix 1 with the exception that participants were reimbursed A$50 for their time and travel expenses instead of being given the opportunity to enter into the draw for the iPod Nano. Participants From May to August 2009, 655 unique visitors accessed the online survey of whom 48 were ineligible because they were either under 18 years old (n=13) or did not live in Australia (n=35). Of the 607 eligible respondents, 525 (86.5%) completed the survey. In all, 6 focus group discussions involving 47 participants (70% female) were conducted from June to August 2009; 4 groups were held in urban areas and 2 in rural towns in New South Wales, Australia. Of the urban groups, 2 specifically targeted young people aged 18 to 28 years. A further 20 people were involved in the interviews, all of whom lived in Sydney, Australia. Table 1 provides demographic and mental health data for the participants involved in the 3 related studies. Analysis Data from each component of the study were analyzed separately and then triangulated. Triangulation involves the exploration of a research question by using multiple data gathering methods in order to get a better understanding of the subject matter, to cross-check the research findings, and to increase their validity [15]. In our studies, we also used the information from the focus group discussions and interviews to shed light on and illustrate the findings of the online survey. Descriptive analyses and tests of group differences within the survey data were conducted using PASW Statistics Version 18 (SPSS Inc, Chicago, IL, USA). Within the interviews and focus group transcripts, salient themes and principles were identified using the "thematic analysis" technique, a qualitative method for identifying, analyzing, and reporting patterns of meaning within data [16]. Data are organized and described in rich detail within a theoretical framework. In this study, an existentialist or realist framework was used whereby the experiences, meanings, and the reality of participants were identified and reported as expressed, in contrast to other frameworks which focus on, for example, the manner in which participants' meanings are "constructed" within the broader context of society [16]. Current Mobile Phone Behavior The majority of survey respondents (455/525 or 86.7%) owned a mobile phone or personal digital assistant (PDA) of whom 83.3% (379/455) reported using it at least daily. Making and receiving calls and sending and receiving short message service (SMS) messages were the predominant functions used (see Figure 1). Other functions commonly used included the Internet (for downloading songs and videos, listening to music, accessing email and Facebook, and listening to the radio: see Figure 2), camera, alarms, memos and reminders, calendar and appointments, clock, games and calculator. Nearly half (255/525 or 48.6%) indicated that they did not use their mobile phone to access the Internet. Reasons included the cost (150/525 or 28.5%), lack of need because they have Internet access on a computer (169/525 or 32%), their mobile phone is not Internet-enabled (66/525 or 12.6%), they haven't had the need (67/525 or 12.7%), or a variety of other reasons, such as not knowing how to access the Internet via the mobile phone, finding it too difficult or complicated, or because the phone is poor quality with a small screen. Focus group and interview findings converged with those of the online survey, with the exception of accessing the Internet. All focus group participants owned a mobile phone and the majority (39/47 or 83%) said they used it every day, predominantly for social reasons, but 72% (34/47) said they also used their mobile phone for work. However, only 18/47 (38%) accessed the Internet on their mobile phone and this was primarily for email, Facebook, Twitter, music, directions, games, Google, and Internet browsing. Reasons participants gave for not using the Internet on their mobile phone included the cost, not knowing how to use it, having no need to use it, or the Internet was not available on their mobile phone. Responses from interviewees were similar. While all owned a mobile phone or PDA, usage varied widely from a few times a week to more than 50 times a day. However, similar to the survey respondents and the focus group participants, the majority (19 of the 20 interviewees) reported using their phone at least once a day. The different uses to which their mobile phones were put were also similar: all participants reported making phone calls, 18/20 (90%) used text messaging, and a minority used their mobile phone for checking emails, taking photos, and other functions such as a clock, a calculator, a calendar, or an alarm or to set reminders. However, 13/20 (65%) did not use the Internet on their phone, and a similar proportion reported that they did not know how to download a program or application on their phone. The reasons participants gave for not using the Internet on their mobile phone were that they had Internet access at home or work, the cost was too high, or their phone didn't allow Internet access. Interest in Using a Mobile Phone Program for Monitoring and Self-management The majority (399/525 or 76%) of survey respondents indicated that they would be definitely be interested (245/525) or likely (154/525) to be interested in using a program on their mobile phone to monitor and manage their mood, anxiety, or health. Among the 455 respondents who owned a mobile phone or PDA, there were no significant differences between those who were interested in using their mobile phones in this way and those who were not by gender (χ2 1 , = 0.98, P = .32, phi = .05), age group (χ2 4 , = 1.95, P = .75, phi = .06), employment status (χ2 2 , = 2.74, P = .25, phi = .08) or marital status (χ2 4 , = 4.62, P = .33, phi = .10). However, the presence of symptoms of depression, anxiety, or stress was associated with reported increased interest in using such a program (χ 2 1 , = 16.67, P < .001, phi = .19). Specifically, standardized residuals indicated that among participants who stated that they were not interested in using the program, there were fewer with current mental health symptoms than expected (and an overrepresentation of participants with no symptoms; P < .05 and P < .01, respectively). Of the 9.9% (45/455) of respondents who reported current symptoms of depression, anxiety, or stress and were not interested in the program, 68.9% (31/45) indicated that they did not think using a mobile phone program to track moods could help people to manage their depression, anxiety, or stress. These results were supported by information from the focus group discussions. The majority (33/47 or 70%) of focus group participants also said they were interested in the notion of using their mobile phone to track their mood, anxiety, or health. Reasons given included speed, convenience, ease of access, the importance of being able to monitor and reflect on mood changes during the day, the opportunity to improve self-awareness, self-management, and well-being, access to support when it was not possible to get to a doctor, the view that it would be less confronting than face-to-face consultation, and the possibility of helping isolated people feel connected. Comments included: A mobile phone application would be a highly convenient, portable, and discreet way of tackling one's condition. Everyone uses the Internet and mobile phones. It could help those who are isolated and have mental health issues. You have your phone with you most of the time, so you would be able to record moods more accurately. Reasons given by those who were not interested in using their mobile phone to monitor their mental health included not liking to use their phone or technology, concerns that it would be too intrusive or privacy would be lacking, and not seeing the benefit of tracking mood and behavior. For example: If the technology is too difficult, then it only adds to the stress and a sense of failure. It seems impersonal and too generalized to be able to capture the emotions of each individual. Mode of Using the Program Of the 399 survey respondents who indicated they were interested in using a monitoring and management program delivered via their mobile phone, 93.7% (374) indicated that they would want a username and password to log on. The length of time per session for which they would be prepared to use such a program ranged from 1 to 90 minutes (median = 5 minutes). The mean number of mood dimensions that participants were interested in tracking at any one time was 5.6 (SD 3.2). Most of these respondents (329/399 or 82%) thought that they would use the program at least daily, some suggesting multiple times per day (196/399 or 49.1%). However, there were significant differences in the expected mode of use according to whether respondents reported current symptoms or not. The participants who were without current symptoms (96 out of 399) indicated that they would use such a program less often (χ2 1 , = 4.52, P = .03, phi = .11) and for shorter periods (mean 7.28 minutes, SD 6.03) than the 303 respondents with current symptoms (mean 10.7 minutes, SD 11.28; t 301 = 3.82, P < .001 [two-tailed]). Information presented in the interviews illustrated the online survey findings. For example, comments from the 13 (of 20) interviewees who reported that a program to monitor depression, anxiety, or stress would definitely be helpful included: It is text savvy and good for the younger generation. Yes, it would be great, another avenue to use and to help people feel that they are not alone. It could be a great motivational tool for people to look after themselves better. Maybe [it would be good] if someone cannot afford psychological treatments or for people who like to keep their feelings to themselves. In addition, 5 interviewees felt that such a program might be helpful, but with caveats. For example, one asked, "If someone such as a doctor is already helping to manage one's moods, why would you use the Internet?" Another 2 interviewees thought that face-to-face contact with a professional would be more beneficial. In response to the question of whether they would use the program themselves if there were no cost, 17 answered positively, and the majority said that they would use it at least once a day. I would be very interested [in using the program] because it's a new tool and not intrusive, and it would be handy. I would be interested, not for my moods but for my health. Other reasons given included "my phone is always with me." Of the interviewees who reported that they would not use a mobile phone mental health program, the reasons given included that they use their mobile phone only for basic functions like phone calls and they did not see how monitoring moods or behavior would help them if they were depressed, stressed, or anxious. Key Functions and Features Required In answer to questions about the key functions required in such a program, 78.7% (314/399) of the survey respondents interested in using the program said they would find it helpful to receive SMS reminders to track their moods; 93% (371/399) nominated that they would want to receive feedback about the information they had entered into such a program; and 89% (355/399) were interested in receiving self-help suggestions (Figure 3). Comparison of respondents with (303/399) and without (96/399) current depression, anxiety, or stress showed that significantly more of those with symptoms indicated that they would find it beneficial to receive SMS reminders to track their moods and behaviors (χ2 1 , = 9.98, P = .002, phi = .165). However, there was no significant difference between those with and without current symptoms in whether they saw feedback on monitoring information as a requirement (χ2 1 , = .01, P = .91, phi = -.02) or whether they would want to receive self-help suggestions (χ2 1 , = .51, P = .47, phi = -.04). Detailed discussion took place in the focus groups about what constituted necessary features of a mobile phone mental health program. Privacy was highlighted as an issue of significant importance and the majority said that a secure log-in comprising username and password should be a mandatory feature. The need for the program to be simple to use and "foolproof" was also emphasized. Usage should be quick (maximum 10 minutes) and easy; one participant suggested that the benchmark for its ease and privacy of use was whether it could be used on public transport. SMS reminders were seen as helpful as long as users had the option of varying their delivery so that they did not become intrusive. Feedback was deemed to be very important, and data presented in graphical form was the most popular format suggested. Functionality allowing day-to-day and week-to-week comparison was also seen as important. Participants suggested that entering data on the mobile phone and receiving it via computer would be an acceptable method that would also resolve some issues of privacy. Allowing users flexibility of choice regarding how much information is to be fed back and over what time frame was also seen as key. Interviewees reported similar attitudes. They highlighted the importance of privacy and security of information, and the majority said they would want a username and password to access the program. A majority also indicated that they would want to receive short message service (SMS) reminders to track their moods/behaviors. The minority who did not want reminders indicated that they would find them annoying and bothersome. If given the choice, 86.2% (344/399) of survey respondents interested in using a mobile phone mental health program indicated that they would allow their doctor to access or receive information about their moods and behavior from the program. Interviewees were similarly minded, saying that "it would be good to have a human on the end to make sense of the data I enter," and "yes, it would be a helpful, precise, and efficient way of tracking what's happening." Of the 91 survey respondents who were against the idea, the major reason given was that it would constitute a breach of privacy for them, while others explained that they don't have a doctor whom they see regularly. There was an even split between those with current symptoms (48/91 or 52.7%) and those without. One interviewee said he did not see the point because "if I am seeing a doctor for my health or mental illness, I would already be telling him what is going on." Discussion This study explored, for the first time, community attitudes toward the appropriation of mobile phones for mental health monitoring and self-help. Triangulation of results from the 3 study components suggests that, overall, participants were positive about the idea of conceptualizing mobile phones as a mental health tool but the acceptance was conditional upon a number of key features being included. These included the need for the program to be simple and straightforward to use and the need for its security and privacy to be guaranteed, especially for information sent to the mobile phone. A user name and password were considered to be mandatory. Text message reminders were seen as helpful as long as they were not intrusive, and feedback graphs were deemed to be important. However, there were differences in expected mode of use between participants with current depression, anxiety, or stress and those without. Respondents with current symptoms indicated that they would be prepared to use a mobile phone program more often and for longer periods, and they were also significantly more likely to want to receive SMS reminders to track their moods and behaviors. Nevertheless, both groups indicated that they would want feedback on monitoring information and to receive self-help suggestions from such a mobile phone program. Thus, while there appears to be a community willingness to accept a broadening of the conceptualization of mobile phones to embrace functions associated with improving mental health, there are caveats to the appropriation of mobile phones for the new functions. The implications for clinicians and eHealth providers are clear. Most mental health programs, whether delivered face-to-face, by telephone, book, or computer, rely on their clients monitoring their symptoms or activities, either as an integral component of the service or as a complement to it. The information is useful for individuals to help them gain control of their condition and for service providers to review the effectiveness of their service. However, to successfully facilitate self-monitoring and self-management via mobile phones, clinicians and eHealth developers must place additional importance on ensuring that the mobile phone programs are secure, private, and easy to use. A further implication arising from the data concerned the apparent lack of understanding about the rationale for and benefits of mood tracking among some respondents. Nearly 10% of the sample, the majority of who reported current depression, anxiety, or stress, indicated that they did not see how a mobile phone program for monitoring moods would help people manage their depression, anxiety, and stress. It is not known if they were expressing doubt about monitoring per se or monitoring specifically on a mobile phone. If the former, then considering that the recent National Survey of Mental Health and Wellbeing in Australia [17] found that 65% of people with mental health conditions do not access services, our results suggest that a health promotion campaign outlining the benefits of self-monitoring for mental health may be helpful. Limitations Having been recruited primarily through the websites of the University of New South Wales and the Black Dog Institute, the intranets of a variety of companies and consumer organizations, and via Facebook, the convenience sample for the online survey is likely to be unrepresentative of the broad population. Visitors to the site were self-selected, and because Internet access is not equal among all socioeconomic and demographic groups, biased estimates on variables related to socioeconomic status may have resulted [18]. Nevertheless, we felt it was justified to recruit from these online sources because our research questions pertained to the use of electronic technologies. Post hoc inspection of the survey sample indicates that ownership and usage of mobile phones was representative of the Australian population. However, there was a stronger representation of survey respondents with current or lifetime depression, anxiety, or stress compared with the Australian population. Another limitation was that our studies took place only in Australia and, although the prevalence of mental health problems in Australia is similar to that in other developed countries and mobile phone penetration is as high, the results may not generalize to populations in other developed countries. The Future In line with the results of our research, we are now developing a mobile phone monitoring and self-help program at the Black Dog Institute. The "myCompass" system will provide users with a tool to monitor and manage depression, anxiety, and/or stress via the Internet on their mobile phone or computer. With the assistance of optional screening questions, users can receive tailored suggestions about mood and behavior dimensions they might find helpful to monitor, or they can select from a menu of monitoring dimensions themselves. They can also choose the time of day they want to monitor and whether they would like to receive regular SMS or email messages to prompt them. Both real time and retrospective assessment of moods, events, and behavior will be available, and users will be able to receive graphical feedback of their data with situational information if desired. They may also choose to receive brief self-management modules (involving interactive cognitive-behavioral strategies), motivational messages, stories, information, and tips to help them to manage depression, anxiety, and stress. Alternatively, the system will offer a selection of strategies derived from algorithms based on user data. The program is designed as a stand-alone tool for the public that is secure and easy to use. Once the digital program is fully developed and pilot tested, a large-scale randomized controlled trial is planned to measure outcomes arising from use of the program. Conclusion Mobile phones are the way of the future. Ownership has reached saturation point in most developed countries, and in developing countries it is increasing exponentially. The simplification and rapid development of digital technology together with the way in which mobile phones are carried on the person and switched on and positive community attitudes make the mobile phone a useful vehicle for enhancing access to evidence-based monitoring and self-help for people with mild to moderate high-prevalence mental health conditions.	2017-04-20T14:42:08.143Z	2010-12-19T00:00:00.000	{ "year": 2010, "sha1": "b00744dbd0ebd8f6f73e1683aa20f89c9dcf977c", "oa_license": "CCBY", "oa_url": "https://doi.org/10.2196/jmir.1475", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "b8f4b65a1ed992f8a6fc908bd2458cbb17c32482", "s2fieldsofstudy": [ "Psychology", "Sociology" ], "extfieldsofstudy": [ "Psychology", "Medicine" ] }
204527627	pes2o/s2orc	v3-fos-license	Morality in Phenomenological Structuralism This work explores the nature of morality within Paul C Mocombe’s structurationist theory of phenomenological structuralism. The author posits that morality or moral standards are associated with the linguistic and ideological desires power and power positions of those who control the resources and mode of production of a material resource framework via their language, ideology, ideological apparatuses, and communicative discourse i.e., social class language game. Its moral practices and statements constitute a part of the superverse/multiverse as phenomenal properties of subatomic particles once disaggregated as lived experience. This does not mean that morality is universal; instead, it is contingent upon the material resource framework and the evolutionary stage of consciousness development as constituted in the framework. Introduction This work explores the nature of morality within Paul C Mocombe's structurationist theory of phenomenological structuralism. The author posits that morality or moral standards are associated with the linguistic and ideological desires power and power positions of those who control the resources and mode of production of a material resource framework via their language, ideology, ideological apparatuses, and communicative discourse i.e., social class language game. Its moral practices and statements constitute a part of the superverse/ multiverse as phenomenal properties of subatomic particles once disaggregated as lived experience. This does not mean that morality is universal; instead, it is contingent upon the material resource framework and the evolutionary stage of consciousness development as constituted in the framework [1][2][3][4][5][6][7][8][9][10]. Theory and Methods Paul [26][27][28][29] structurationist sociology, phenomenological structuralism, which attempts to resolve the structure/agency problematic of the social sciences, builds on the ORCH-OR theory and panpsychism of Hameroff [10], while holding on to the multiverse hypothesis of quantum mechanics and Haitian ontology/epistemology, which the authors reject because it is not a more down-to-earth view point Hameroff [10]. For Mocombe [26][27][28][29] quantum superposition, entanglement, and evidence in Haitian vodou of spirit possession, which represent ancestors from a parallel world, Vilokan, of the earth's of which we ought to pattern our behaviors and structures, are grounding proofs for the acceptance of the multiple worlds hypothesis of quantum mechanics. Within the latter hypothesis, the understanding is that each possibility in a superposition evolves to form its own universe, resulting in an infinite multitude of coexisting 'parallel' worlds. The stream of consciousness of the observer is supposed somehow to 'split', so that there is one in each of the worlds-at least in those worlds for which the observer remains alive and conscious. Each instance of the observer's consciousness experiences a separate independent world and is not directly aware of any of the other worlds Hameroff [10]. It is within this multiple world's hypothesis that Mocombe constitutes the notion of consciousness in the universe according to his theory of phenomenological structuralism. For Mocombe, consciousness is a fifth force of nature, a quantum material substance/energy, psychion, the phenomenal property of which is recycled/entangled/superimposed throughout the multiverse and becomes embodied via the microtubules of brains. It is manifested in simultaneous, entangled, superimposed, and interconnecting evolutionary material resource frameworks as embodied praxis or practical consciousness, which in-turn becomes the phenomenal properties of material subatomic particle energy, psychion consciousness that is recycled/entangled/superimposed throughout the multiverses [30][31][32][33][34][35][36]. Discussion and Conclusion Hence, as outlined above, phenomenological structuralism posits consciousness to be the by-product or evolution of subatomic particles unfolding with increasing levels of abstraction within an evolutionary material resource framework enframed by the mode of production, language, ideology, ideological apparatuses, and communicative discourse of bodies who control the material resource framework recursively reorganizing and reproducing the ideals of the latter factors as their practical consciousness across entangled/superimposed multiple worlds. They are only conscious of the subjects and objects of only one world at a time, however. Thus, in phenomenological structuralism the understanding is that the structure of reality determines language via its generative grammar and how we ought to live in the multi worlds where consciousness is found. However, the language, and its usage, i.e., social class language game, of those who control the material resource frameworks of the worlds conceal that relationship via their mode of production, ideologies, ideological apparatuses, and communicative discourse, which is evolutionary. In other words, like the Wittgenstein Ian position of the Tractatus, Mocombe's theory of phenomenological structuralism assumes that there is a uniform grammatical structure to language determined by the logical-empirical structure of entangled and superimposed quantum and physical realities. The grammatical structure of linguistic utterances attempts to capture the subjects and objects of those realities and how we ought to live with the subjects and objects in them. In being-in-the-world s with others and objects, this logical-grammatical structure, however, is concealed by the evolution and developmental knowledge, and its usage practical activity, of those who control the material resource framework of the world's via the stage of development of their language, ideology, ideological apparatuses, social relations of production, and communicative discourse. Be that as it may, the latter comes to constitute an evolutionary social class language whose linguistic systemicity and usage comes to determine our conception of reality, and the classes, categories, and forms of life we belong to and interact in and with, which, depending on its stage of development and relation to the True nature of reality as such, is either accepted or constantly deferred by those in its speech community who are marginalized or not represented in its evolutionarily developed linguistic systemicity. The latter process, language usage, under the guise language game, language as a tool, is what Wittgenstein captures in his second treatise on language as developed in the philosophical investigations. That is, the classes and categories created by the dominant social class language game of a material resource framework, in their efforts to capture the subjects, objects, and activities of the logical-grammatical structure of reality and how we ought to live within it, constitute reified classes, categories, and forms of life, language games, whose meanings and praxes as defined by the dominant social class language game are either accepted or deferred by those classified in them. The latter may inturn seek to reify their form of life that they are marginalized for, or categorized in, as a distinct alternative practical consciousness to that of the dominant order thereby undermining the attempted universality of the logical-grammatical structure of the dominant order for notions of diversity, intersectionality, etc. So, in Mocombe's theory of phenomenological structuralism, Wittgenstein's two theories of language and meaning must be read as one philosophy as opposed to two, one supported by analytical philosophy and the other by postmodernism/post-structuralism. We have a plethora of language games classes, forms of life, and categories in the world, which structures our language, because of the ability to defer meaning in ego-centered communicative discourse and the developmental stage of the human mind and body vis-à-vis the actual True structure of reality. The language of science, like its predecessor religion, attempts to capture the logical-empirical structure of quantum and physical reality, and how we ought to live within it, amidst the utterances and practical consciousnesses of the masses given their abilities to defer meaning in ego-centered communicative discourse and the classes, categories, and forms of life they are classed in/with by the dominant social class language game as they attempt to capture the overall nature of reality via language. ICP.MS.ID.000513. 1(3).2019 Hence within the theory and methodology of phenomenological structuralism, there is, contrary to David Hume's bundle of perception hypothesis, a human essence, which is tied to the embodiment and structuring structure of the phenomenal properties of superimposed, entangled, embodied, and recycled subatomic particles, the processes of which are unbeknownst to us as of the writing of this work, as they are recursively reorganized and reproduced via the superverse and its multiverses. Just the same, universalism and truth are also tied to the science and physics of the remaining processes of phenomenological structuralism. Subatomic/chemical particles with phenomenal properties constitute objects and subjects that are external and internal to the perceiving human actor who know them the objects and subjects as both external and internal phenomenon endowed with, and mediated by, linguistic and ideological meanings, stemming from the evolutionary modes of production, of other human actors who presupposed their aggregated existence. The essence, universalism, and truth of an object and subject lies in the phenomenal properties of their subatomic and chemical particles once demystified and demythologized, from linguistic and ideological meanings and understandings associated with the evolutionary mode of human production, by the techniques of phenomenology and the scientific process. Be that as it may, for phenomenological structuralism, in keeping somewhat with the empiricist logic of Bertrand Russell, outside of human desires there are no moral standards. Morality or moral standards are associated with the linguistic and ideological desires power and power positions of those who control the resources and mode of production of a material resource framework via their language, ideology, ideological apparatuses, and communicative discourse i.e., social class language game. Its moral practices and statements constitute a part of the superverse/multiverse as phenomenal properties of subatomic particles once disaggregated as lived experience. This does not mean that morality is universal; instead, it is contingent upon the material resource framework and the evolutionary stage of consciousness as constituted in the framework. In that sense, assuming the phenomenal properties of subatomic particles get recycled/superimposed/entangled between the superverse and its multiverses as I am positing here, morality is an epiphenomenon of lived-experience and becomes an emergent property of the superverse and its multiverses, which constitute the laws platonic forms or concepts such as beauty, justice, egalitarianism, etc. of Haitian metaphysics that human reason, which are the recycled subatomic neuronal/chemical particles of the superverse and multiverse operating through DNA and its aggregation as the brain and mind perception, can reflect upon to constitute their beingin-the-world practical consciousness in relation to the language, ideology, etc., i.e., social class language game, of those who precedes individual existence. Each multi world has its own practical activity or consciousness, based on the material structure of its resource framework, by which we ought to recursively organize and reproduce our being in them. However, the evolutionary nature of both our being-in-the-material resource framework and the material resource framework itself is limited by the stage of development of our embodied practical consciousness in them. In this sense, morality in Mocombe's theory of phenomenological structuralism is both naturalistic and emotive.	2019-09-19T09:05:09.339Z	2019-06-11T00:00:00.000	{ "year": 2019, "sha1": "f5a82f2c86f42d067eb6ad2cd074766192322be5", "oa_license": "CCBY", "oa_url": "http://crimsonpublishers.com/nrs/pdf/NRS.000508.pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "14a459362ff8de72ca41fa4eaf4c30d7c954c076", "s2fieldsofstudy": [ "Philosophy" ], "extfieldsofstudy": [ "Philosophy" ] }
51688637	pes2o/s2orc	v3-fos-license	Relativity of quantum states in entanglement swapping: Violation of Bell's inequality with no entanglement The entanglement swapping protocol is analyzed in a relativistic setting, where shortly after the entanglement swapping is performed, a Bell violation measurement is performed. From an observer in the laboratory frame, a Bell violation is observed due to entanglement swapping taking place, but in a moving frame the order of the measurements is reversed, and a Bell violation is observed even though no entanglement is present. Although the measurement results are identical, the wavefunctions for the two frames are different--- one is entangled and the other is not. Furthermore, for boosts in a perpendicular direction, in the presence of decoherence, we show that a maximum Bell violation can occur across non-simultaneous points in time. This is a signature of entanglement that is spread across both space and time, showing both the non-local and non-simultaneous feature of entanglement. I. INTRODUCTION The nature of entanglement has always been a point of intrigue since the early days of quantum mechanics [1]. In the last few decades, advances in experimental techniques have been able to test directly the spooky action at a distance by demonstrating its effects at increasingly larger distances. Adapting terrestrial free-space methods [2,3], entanglement distribution and quantum teleportation to distances over 1000 km have now been performed [4,5], using space-based technology. Another fundamental test is to measure the bounds to the speed of influence due to entanglement [6,7]. In such experiments it is advantageous to have widely separated and near-simultaneous measurements to ensure that the two events are outside of the light cone of influence of each other. This allows one to close the locality loophole [8][9][10], where conspiring parties may mimic results that are attributed to entanglement. Combining special relativity with quantum mechanics leads to peculiar results see e.g. [11,12]), such as from the fact that simultaneity is relative according to the * tim.byrnes@nyu.edu observer's frame. Suarez and Scarani noticed that, for near-simultaneous measurements of an entangled pair, it is possible to reverse the order of measurements by moving to a suitable frame [13]. This inspired several experiments [14][15][16][17] and recently it was shown that this paradox can be resolved by taking into account the uncertainty in the time of measurement [18]. These relativistic contexts have rekindled debate on the foundations of quantum mechanics. It is a strange fact of modern physics that non-relativistic quantum mechanicswhich is not constructed with relativity in mind at allstill gives consistent results with special relativity, such as the impossibility of superluminal communication due to the no-cloning theorem. Investigations of relativistic effects beyond the no-signaling principle have been made [19,20], focusing on quantum causality and possible applications of event-order swapping to quantum circuits. In this paper, we examine the entanglement swapping protocol [21] in a relativistic setting, where, after entanglement has been swapped, a Bell test is used to verify the presence of the swapped entanglement. We consider two scenarios in particular, which highlight two peculiar relativistic quantum effects. In Scenario 1, we contrast the interpretations of an experiment of two observers, which observe the Bell test and the entanglement swapping to take place in different orders (Fig. 1). In the moving frame (Rob), we show that it is possible to observe a Bell violation, despite the fact that entanglement swapping has not occurred at all. In fact, Rob observes a Bell violation between two particles that has no entanglement whatsoever between them, directly or indirectly, or at any point in time. This paradoxical effect occurs because of the quantum correlations, which -even in the absence of entanglement-determine the random measurement outcomes of the verification to give a Bell violation. In Scenario 2 depicted in Fig. 2, we consider Rob to be moving in a perpendicular direction, and he is in control of the Bell test. In this case, when there is some decoherence present, we find that there is an optimal time offset for him to make the two measurements to obtain the maximum Bell violation. In this case the results of the Bell test point to correlations resulting from entanglement that are spread in both space and time. A similar experiment to Scenario 1 was considered by Peres [22] in the context of delayed-choice entanglement swapping, and has been experimentally demonstrated since [23,24]. Variations of the theme have been investigated, where entanglement can be verified between particles that have never co-existed [25]. The difference of our scenario to these past works is that the order of the measurements depends upon the reference frame, due to relativity of simultaneity. In particular, in Ref. [26] the verification measurements and the Bell measurement are time-like separated. This procedure is complementary to, but differs from, our procedure where there are space-like separations between the Bell and verification measurements. Other works have investigated special relativistic effects on entanglement swapping [27,28] and on teleportation [29][30][31][32], but none have considered a setup where it is possible to switch the time ordering of measurements. In relation to Scenario 2, several concepts relating to entanglement in time, and temporal Bell inequalities have been investigated [33][34][35][36]. These differ from our scenario, since the nature of entanglement that they study is across time-like intervals and between the same system at different points in time. In our case we consider entanglement in a more conventional sense, with a space-like orientation and involving two particles. Specifically, the consecutive measurements on two different systems show stronger-than-classical correlations if the time difference between the two measurements matches the time it takes light to traverse the distance between the two systems as measured in a stationary frame. We note that throughout this paper we neglect the relativistic effects that occur locally on the quantum state, and assume that qubit measurements can be performed faithfully. As has been discussed in several other works [37][38][39][40], depending upon the encoding of the logical qubit states, the local quantum state may be subject to Lorentz transformations. For example, the polarization of a photon is not a Lorentz invariant quantity, and appears differently in different frames. Since this is dependent upon the particular physical implementation, and can in principle be accounted for if the relative velocities are known, . Two reference frames (a) the laboratory (Lara) with coordinates (t, x, y, z); (b) an observer Rob with coordinates (t ′ , x ′ , y ′ , z ′ ), who is moving in the negative x-direction are shown. Initially, four qubits are prepared, labeled by A, B, C, D, where pairs AC and BD are entangled (indicated by the wiggly lines). A Bell measurement and a verification measurement, consisting of two single-qubit measurements, are then made. The order of the measurements depends upon the observer's frame. In order to verify the Bell violation, the results of the Bell measurement must be classically transmitted to the verification measurements (or vice versa). Classical communication is denoted by thick solid lines, the dashed lines are to guide the eye. we disregard this effect and consider only the combined effect of quantum measurements with relativity of simultaneity. II. SCENARIO 1: ENTANGLEMENT SWAPPING A. Laboratory frame (Lara) We now analyze the entanglement swapping protocol described in Fig. 1(a) in detail. In this frame, the protocol appears as conventional entanglement swapping, where first a Bell measurement is made to swap the entanglement, then is followed by a verification measurement. Here, a Bell measurement is a measurement which projects qubits C and D onto one of the four Bell states, while a verification measurement are made individually on A and B, projecting them onto a product state. For concreteness, we consider the situation where all the measurements are stationary in Lara's frame. Qubits A and B are at the same x-coordinate, and are separated from qubits C and D by a suitably large distance. Initially, the qubits are prepared in the state where the four Bell states are defined as for i, j ∈ {0, 1}. These Bell states are equivalent to the traditional Bell states with \|Ψ 00 = \|Φ + , \|Ψ 01 = \|Φ − , \|Ψ 10 = \|Ψ + , and \|Ψ 11 = \|Ψ − . After the Bell measurement is made the state collapses to one of the four outcomes with probability p ij = 1/4 Soon afterwards (such that the events are space-like separated), a verification measurement is performed on qubits A and B to perform a Bell test. After the result of the Bell measurement has been classically transmitted, the appropriate CHSH quantity [41,42] is computed. The four states labeled by i, j are subject to their respective Bell tests and the result is averaged [43,44]. Note that performing the Bell test separately on each of the four outcomes is necessary, otherwise the outcome would be a completely mixed state. For instance, the CHSH quantity for \|Ψ 00 is evaluated where the operators for qubits A and B arê for n, m ∈ {0, 1} respectively. This a group of four measurements -only one of which is performed for a particular run of the experiment. The results of the verification measurement are used to compute the CHSH quantity for a general state \|ψ ij , which can be written (see Appendix) Each state observes a positive maximal Bell violation and the average is also a maximal Bell violation. We note that none of the verification data that is taken is thrown away -in this sense no post-selection is performed. The data is however conditionally processed according to the appropriate CHSH quantity (6) for the the Bell state \|Ψ ij in question. B. Rob's frame with x-boosts Now consider the moving frame of Rob, who is moving in the −x direction. Due to the Bell measurement and the verification measurements being space-like separated, Rob observes that the latter occurs before the former [ Fig. 1(b)]. Starting from (1), the verification measurement yields Substituting this into (6) of course gives S = 0, which does not violate the Bell inequality, as expected for a product state between A and B. The state collapses in the basis of the verifying measurements (5). For measurement settings n, m ∈ {0, 1} and measurement outcomes l A , l B ∈ {0, 1}, the state after the measurement is which occur with probabilities Here the states are the eigenstates of (5) The Bell measurement then projects the CD qubits onto the Bell states (2). If the Bell measurement returns \|Ψ ij , then the obtained state is which occurs with probability for the outcome (i, j, l A , l B ). Now as before, we evaluate the CHSH correlation (6) for each of the i, j outcomes. Then the average value conditioned on the outcome (i, j) is Now, putting the expectation value in the same form as (6), we obtain Substituting this into (6), this yields a Bell violation S ij = 2 √ 2 for all i, j. Averaging over all i, j of course again gives a Bell violation, giving the same result as Lara's frame. C. Relativity of entanglement We have seen that, ultimately, the results of the Bell test agree in both frames. At one level, this is not surprising since it is a relativistic principle that measurement outcomes must agree in all frames. Furthermore, the only role of the boosted frame is to reverse the order of the measurements. Since the two types of measurements commute (they are on separate qubits, AB and CD), the fact that the same outcomes are obtained is always guaranteed [22,[45][46][47]. What is unusual is that the interpretation of the experiment is completely different in the two frames. Between the two measurements, the quantum state is different, due to the differing order of the measurements. In Lara's frame, the intermediate state is (3), which is an entangled state between qubits AB. This appears as entanglement swapping from the initial configuration to between AB and CD. Using a conditional Bell test which depends upon the result of the Bell measurement, a Bell violation is observed. Thus the origin of the Bell violation can be straightforwardly attributed to the presence of entanglement between AB. On the other hand, the intermediate state in Rob's frame is (8), which has no entanglement at all. In fact, in Rob's frame there is never any entanglement between AB from the beginning to the end of the sequence. The Bell violation appears to be more akin to manipulation of random data such as to violate a Bell inequality, since it is achieved by communication of appropriate side information by the Bell measurement on CD and the outcomes at AB are completely random by (9). To see this point, let us compare Rob's point of view to a parallel thought experiment where there are only qubits at A and B. The same verification measurements are performed on each qubit, and a conditional Bell test is performed. In this case, first prepare a completely mixed state at AB: where I A , I B are 2 × 2 identity matrices on A, B. The measurement basis n, m of the verification are randomly chosen in the same way as (5). Since the initial state is a completely mixed state, one obtains the outcomes with probability p The demon then communicates the result to the Bell tester. We note the demon does not have to perform a Bell measurement to produce this probability distribution, he may simply follow the formula in (17). The Bell tester then evaluates the corresponding CHSH inequality (6). Following identical algebra to that following (13), one finds that the Bell inequality is violated. Obviously in this scenario, the Bell inequality is violated not because there is entanglement. Here the demon has information about potentially non-local information of the measurement outcomes at A and B, and the use of the formula (17) simply mimics the probabilities of the Bell measurement that would be performed in Rob's frame. The only difference between Fig. 1(b) and the demon example is that entanglement is used to produce the probabilities (12). The role of entanglement in Rob's frame is to produce the duplicate results at CD as given in Eq. (8). The entanglement replaces the non-local powers of the demon, which bridges the distance between A and B. The question is then whether it is appropriate to interpret Fig. 1(b) as verification of entanglement between A and B. In our opinion, this situation seems better described as "entanglement-assisted fictitious violation of a Bell inequality", rather than genuine entanglement detection. The puzzling aspect of this is that the only difference is a change of observer, and the experiment itself is identical. Thus if the order of the measurements were physically being reversed, one might be able to explain the discrepancy by claiming that they are fundamentally different experiments. However, since they are different only due to a change of observer, it appears more important in this case to understand what the correct interpretation is. One might argue that there is in fact no entanglement in Lara's frame. In Lara's frame, the Bell states at AB emerge completely randomly, and are statistically equivalent to a completely mixed state. In such an interpretation, the Bell violation then should occur due to a similar procedure as the demon -the random verification measurements of the state (15) are manipulated into a fictitious Bell violation. However, this would require the outcomes of the verification measurements l A , l B being suitably correlated to the Bell measurement outcomes i, j, which cannot be produced by a completely mixed state. It therefore suggests that the procedure as given in Sec. II A is necessary to produce the necessary correlations. From these observations it appears that the existence of the entanglement of the wavefunction is not universally agreed upon by different observers [39]. That is, much like the notion of relativity of space and time itself, the entanglement of the wavefunction is also a relative quantity that is observer-dependent. The fact that relativistic effects can effect entanglement has been observed in several works [37][38][39][40]. However these cases are somewhat different in that there exists a transformation law that can convert quantities between frames. In this case, due to the quantum measurements, there is no such transformation that can convert the quantum states. This Fig. 1(a), according to Rob's frame who is moving in the +y direction relative to Lara's frame. The Bell measurement for Lara acts on qubits C and D simultaneously, which corresponds to a non-simultaneous Bell measurement with time difference ∆t ′ for Rob. The time difference between the verification measurements are adjustable and have a time difference of ∆t ′ in Rob's frame. (b) Bell violations for measurement times t ′ M as marked in units of the depolarizing rate η, for qubit separations such that ηγτ ′ = 1. point was also observed in other contexts [48,49], where relativistic effects were combined with quantum measurements. III. SCENARIO 2: BELL CORRELATIONS IN SPACE-TIME The same entanglement swapping setup can illustrate another effect, by considering boosts in other directions. Now consider that Rob is moving in the +y direction, and he is in control of the verification measurement such that there is a time offset ∆t ′ between them. The Bell measurement is performed in the laboratory frame (Lara) as before, and the two verification measurements occur at a time t ′ M ± ∆t ′ /2, where t ′ M is the midpoint between the two times. Dashed quantities refer to Rob's frame and undashed for Lara's throughout. We also consider that some decoherence in the form of a depolarizing channel is present. This could be, for example, from storing the qubits in an imperfect quantum memory. Working in Lara's frame, we may calculate the outcome of the Bell violation test in the following way. The state starts in (3) immediately after the Bell measurement. The depolarizing channel acts for a time t M −∆t/2 until qubit A is measured. After the measurement of qubit A, the the depolarizing channel again acts on the state for a time ∆t until qubit B is measured. The measurements and Bell test are performed as before, and the average CHSH inequality can be evaluated (see Appendix) to give where we have written the final result in terms of Rob's variables according to the transformation where γ = (1 − β 2 ) −1/2 , β = v/c, v is Rob's velocity relative to Lara's frame, and L ′ is the distance between the qubits A and B. Here τ ′ = βL ′ /c is the time offset in Rob's frame between the measurements such that in Lara's frame they are simultaneous. The calculation can be performed directly from the point of view of Rob, but due to the principle that all observers should obtain the same results, the same results are obtained in the same way as before. Interestingly, we note that Rob can actually use (19) in order to determine his own relative velocity, by maximizing his observed Bell violation. The results are illustrated in Fig. 2(b). If there is no decoherence (η = 0) as can be deduced from (19), one recovers the maximal Bell violations of situation in Fig. 1. In the presence of decoherence, we see that in Rob's frame ∆t ′ is optimal for Rob's verification measurement when ∆t ′ = τ ′ . This equality occurs at the particular time when the Bell verification measurement is performed simultaneously in Lara's frame. This result is natural from the point of view of Lara's frame, as any other ∆t ′ would correspond to measuring the CHSH correlations at different times. From the point of view of Rob, due to a different notion of simultaneity, he must deliberately offset his times in order to get the maximum Bell violation. The time that he must offset his time is precisely such that in Lara's frame, the measurements are carried out simultaneously. In the limit of very strong decoherence, the Bell violations would only occur in a very narrow window of measurements centered around the time ∆t ′ = τ ′ . Rob would then conclude that due to the optimal time offset for his measurements, there are Bell correlations both non-locally as well as non-simultaneously. This has similarities with previous works examining bounds on entanglement in time [33][34][35][36], illustrating that nonlocal quantum correlations can be engineered by measurements separated in time. As we have shown here, these correlations have influence not only in a non-local fashion, but more generally across space-time. IV. SUMMARY AND CONCLUSIONS In this paper we have presented two quantum thought experiments, which highlight the peculiar nature of entanglement when relativity is involved. In the first scenario, we considered transformations such that the order of the Bell measurement and the verifications were reversed. Depending upon the observer, the interpretations of the same experiment are radically different. In one frame, entanglement swapping is completed deterministically and entanglement between qubits A and B is accordingly verified. In the other frame, random outcomes of the verification measurement are manipulated into a Bell violation with classical feed-forward and no entanglement between qubits A and B is present at any time. The different interpretations mean that different observers disagree on whether the wavefunction is entangled or not. This example points to the possibility that quantum entanglement being a relative quantity, much like other concepts such as simultaneity in special relativity. In the second scenario, the phenomenon of entanglement across space-time can be observed, where the maximum Bell violation occurs for measurements at different times in a suitable reference frame. In this case the effect occurs because the Bell correlations are best observed when the verification measurements are made in the same reference frame as the entangled state itself. By preparing the state via entanglement swapping, the state is projected in the reference frame of the Bell measurement. It is now widely recognized that entanglement is a resource for performing useful quantum information tasks, such as quantum computing, quantum teleportation, and quantum cryptography. But what is a physical resource? A key feature of a physical resource is a principle of invariance such that it can be quantified in a basisindependent (i.e. observer-independent) way. In this paper, we have shown that quantum entanglement of a two-particle state may be an observer-dependent quantity. How can something that disappears for one observer -and then reappears for another observer -possibly be a physical resource? Entanglement is a mathematical statement about the separability of two quantum states. A mathematical statement cannot be a physical resource, any more than, say, the Pythagorean theorem. This suggests that the physical resources are in fact the non-local Bell violating correlations that entanglement seems to encode in one frame, but not in another, an idea which was suggested independently in other works [50][51][52]. It is these non-local correlations -which are present in both frames -that are the true physical resources. We derive the generalized expression for the CHSH correlations given by (6). The well-known expression for the state \|Ψ 00 = (\|0 \|0 + \|1 \|1 )/ √ 2 reads To obtain the CHSH correlations for the other Bell states, we introduce the operator Introducing factors of unity before and after the operators, the corresponding correlation reads The transformation only acts on theB m operator and can be evaluated as Substituting this into (A4) we obtain which is Eq. (6). Appendix B: Derivation of Eq. (19) We consider that initially the state is prepared in the state given (1). At t = 0, the Bell measurement in Lara's frame collapses the state to The depolarizing channel subsequently acts on this state until Rob performs the verification measurement. The time evolution of the state before the first verification measurement is given by Let us first assume that ∆t > 0. At the time t M −∆t/2 in Lara's frame, Rob performs a measurement on the state ρ AB (t) for the qubit at A. We note that sinceÂ n and B m act on different Hilbert spaces,Â n andB m commute. As such, the order of CHSH verification measurement is irrelevant. The state immediately after the first verification measurement becomes σ (n) is the measurement operator on qubit A. This state again evolves in time due to the depolarizing channel as The second form of the equation shows that the state is equivalent to simply evolving the state to a time t M + ∆t/2 under the depolarizing channel, and then performing a measurement. If the order of the measurements were reversed (i.e. ∆t < 0) we would follow the same procedure and find that the final state is the measurement at the later time t M − ∆t/2. Hence in general the procedure is equivalent to taking the expectation value with respect to the state ρ AB (t M + \|∆t\|/2). The above reasoning can be used to evaluate the expectation value as in (6). For a given i, j outcome we have S ij = (−1) j n,m=0 Tr Â nBm+i+j ρ AB (t M + \|∆t\|/2) Taking an average over all outcomes, we obtain To relate time t in Lara's frame to the time t ′ in Rob's frame, we use the standard relativistic transformation where β = v c , v is Rob's velocity relative to Lara's frame, c is the speed of light, and γ = (1 − β 2 ) −1/2 . The time difference ∆t between the measurement of A and B in Lara's frame transforms as where Suppose that the average time t M of measurement in Lara's frame is t M = (t A + t B )/2 where t i , (i = A, B) is the time to make the measurement on particles at A and B. Then, t M would transform as We then have so that Substituting (B11) and (B15) into (B9) gives (19).	2018-06-06T20:06:20.000Z	2018-06-06T00:00:00.000	{ "year": 2018, "sha1": "611ebf172d1ffa0e54b7699a009e19ab096ca2bd", "oa_license": null, "oa_url": "https://www.sciencedirect.com/science/article/am/pii/S0375960120301006", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "d3d6e728c3cd4672880c82a9f7ef8a2219391810", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
119151326	pes2o/s2orc	v3-fos-license	Finding Dynamics for Fractals The famous Laplace's Demon is not only of strict physical determinism, but also related to the power of differential equations. When deterministically extended structures are taken into consideration, it is admissible that fractals are dense both in the nature and in the dynamics. In particular, this is true because fractal structures are closely related to chaos. This implies that dynamics have to be an instrument of the extension. Oppositely, one can animate the arguments for the Demon if dynamics will be investigated with fractals. To make advances in the direction, first of all, one should consider fractals as states of dynamics. In other words, instead of single points and finite/infinite dimensional vectors, fractals should be points of trajectories as well as trajectories themselves. If one realizes this approach, fractals will be proved to be dense in the universe, since modeling the real world is based on differential equations and their developments. Our main goal is to initiate the involvement of fractals as states of dynamical systems, and in the first step we answer the simple question"How can fractals be mapped?". In the present study Julia and Mandelbrot sets are considered as initial points for the trajectories of the dynamics. INTRODUCTION French mathematicians P. Fatou and G. Julia invented a special iteration in the complex plane (1,2) such that new geometrical objects with unusual properties can be built. One of the famous is the Julia set. Later B. Mandelbrot suggested to call them fractals (3). Famous fractals appeared as self-similar objects were introduced by Georg Cantor in 1883, and Koch snowflake was discovered in 1904. Fractals are in the forefront of researches in many areas of science as well as for interdisciplinary investigations (24,26). One cannot say that motion is a strange concept for fractals. Dynamics are beside the fractals immediately as they are constructed by iterations. It is mentioned in the book (6) that it is inadequate to talk about fractals while ignoring the dynamical processes which created them. That is, iterations are in the basis of any fractal, but we still cannot say that differential equations are widely interrelated to fractals, for instance, as much as manifolds (16). Our present study is intended to open a gate for an inflow of methods of differential equations, discrete equations and any other methods of dynamics research to the realm of fractals. This will help not only to investigate fractals but also to make their diversity richer and ready for intensive applications. Formally speaking, in our investigation we join dynamics of iterations, which can be called inner dynamics, with outer dynamics of differential and discrete equations. We are strongly confident that what we have suggested can be in the basement of fractals through dynamical systems, differential equations, optimization theory, chaos, control, and other fields of mathematics related to the dynamics investigation. The concept of fractals has already many applications, however, the range would be significantly enlarged if all the power of differential equations will be utilized for the structures. This is why our suggestions are crucial for fractals considered in biology, physics, city planning, economy, image recognition, artificial neural networks, brain activity study, chemistry, and all engineering disciplines (19)(20)(21)(22)(23)25), i.e. in every place, where the geometrical objects in physical and/or abstract sense may appear (2). Significant results on the relation of fractals with differential equations were obtained by Kigami (14) and Strichartz (15). In the books (14,15), fractals were considered as domains of partial differential equations, but not as building blocks of trajectories. In this sense one can take advantage of the results of (14,15) in the next development of our proposals. The same is true for the studies concerned with deep analyses of fractals growth performed in (17,18). The most important fractals for our present study are Julia sets which were discovered in 1917-1918 by Julia and Fatou, both of whom independently studied the iteration of rational functions in the complex plane (5). They established the fundamental results on the dynamics of complex functions published in their papers (1,2). In 1979, Mandelbrot visualized Julia sets. Although they are constructed depending on the dynamics of simple complex polynomials, the Mandelbrot set has a complicated boundary, and the Julia sets associated with points close to the boundary usually have amazing and beautiful structures. Julia and Mandelbrot fractals are very great achievements for set theory, topology, functions theory, chaos, and real world problems. Therefore, studies in the area has to be at the frontiers of modern sciences and applications. Additionally, the development of researches on the basis of fractals is of significant importance to any possible direction starting from the basis of set theory. In general, a fractal is defined as a set that displays self similarity and repeats the same patterns at every scale (8). Mandelbrot (4) defined fractals as sets for which the Hausdorff dimension is either fractional or strictly exceeds the topological dimension. Based on this, self-similarity can occur in fractals, but, in general, a fractal need not be self-similar. The easiest way to indicate that a set has fractional dimension is through self-similarity (11). Besides the iteration of rational maps, there are various ways to construct fractal shapes. The well known self-similar fractals like Sierpinski gasket and Koch curve are constructed by means of a simple recursive process which consists in iteratively removing shrinking symmetrical parts from an initial shape. These types of geometrical fractals can also be produced by an iterated function system (9, 10), which is defined as collections of affine transformations. In our paper, we make a possible study of mapping fractals, which is simple from one side since it relates to classical functions, but from another side it is a developed one since we apply the mapping function in a new manner, which nevertheless still relates to the original ideas of Julia. There are two sides of the fractal research related to the present paper. The first one is the Fatou-Julia Iteration (FJI) and the second one is the proposal by Mandelbrot to consider dimension as a criterion for fractals. In our study, both factors are crucial as we apply the FJI for the construction of the sets and the dimension factor to confirm that the built sets are fractals. In previous studies the iteration and the dimension factors were somehow separated, since self-similarity provided by the iteration has been self-sufficient to recognize fractals, but in our research the similarity is not true in general. We have to emphasize that there is a third player on the scene, the modern state of computers' power. Their roles are important for the realization of our idea exceptionally for continuous dynamics. One can say that the instrument is at least of the same importance for application of our idea to fractals as for realization of Fatou-Julia iteration in Mandelbrot and Julia sets. Nevertheless, we expect that the present study will significantly increase the usage of computers for fractal analysis. Moreover, beside differential equations, our suggestions will effect the software development for fractals investigation and applications (29,30). Studying the problem, we have found that fractal-like appearances can be observed in ancient natural philosophy. Let us consider the Achilles and tortoise in the Zeno's Paradox (13), (see Fig. 1(a)). In the paradox, Achilles is observed at the initial moment t 0 = 0 with the distance d 0 from the tortoise. Suppose that Achilles runs at a constant speed, two times faster than the tortoise, then he would reach the previous position of the tortoise at moments t 1 , .. from the tortoise, respectively. Now contemplate Fig. 1(b), where the heights of the red lines are proportional to the distance of Achilles from the tortoise at the fixed moments, and denote the diagram by S 0 . The set S 0 demonstrates the entire dynamics for t ≥ t 0 . Fix i ≥ 0, and let S i be the similar diagram which consists of all the lines for the moments which are not smaller than t i . Let us consider the collection of the states {S i }, i ≥ 0. One can assume that there exists a map B such that the equations which symbolize a dynamics, are valid. It is easily seen that S 0 is self-similar to each of its parts S i , i > 0. Nevertheless, the Hausdorff dimension of the set S 0 is equal to one. For this reason, we call S i , i ≥ 0, pseudo-fractals, due to the similarity. The trajectory {S i }, i ≥ 0, is also a pseudo-fractal. The sketch of the trajectory is seen in Fig. 1(c). Our present study is an extension of the ancient paradigm, since we will investigate dynamics having all points of a trajectory as well as the trajectory itself fractals. FATOU-JULIA ITERATIONS The core of the present study is built through FJI and the quadratic map P : C × C → C defined by where C denotes the set of complex numbers. Consider the equation z n+1 = P (z n , c), n ≥ 0. The points z 0 ∈ C are included in a fractal F depending on the boundedness of the sequence z n , and we say that the fractal F is constructed by FJI. The so-called filled-in Julia set, K c , is constructed by including only the points z 0 ∈ C such that the sequence z n is bounded (32). Moreover, in the simulation, those points z 0 ∈ C where {z n } is divergent are colored in a different way, correspondingly to the rate of divergence. The term Julia set J c , usually denotes the boundary of the filled Julia set, i.e., J c = ∂K c . The Mandelbrot set, M, is also generated by a Fato-Julia iteration. In this case, we consider the equation z n+1 = P (z n , c), and include in the set M the points c ∈ C such that {z n (c)}, z 0 (c) = 0, is bounded. Here again, the points c ∈ C corresponding to divergent sequences z n are plotted in various colors depending on the rate of the divergence. HOW TO MAP FRACTALS To describe our way for mapping of fractals, let us consider a fractal set F ⊆ A ⊂ C, constructed by the following FJI, where F : A → A is not necessarily a rational map. We suggest that the original fractal F can be transformed "recursively" into a new fractal set. For that purpose, we modify the FJI, and consider iterations to be of the form or explicitly, where f is a one-to-one map on A. Next, we examine the convergence of the sequence {z n } for each z 0 ∈ f (A). Denote by F f the set which contains only the points z 0 corresponding to the bounded sequences. Moreover, other points can be plotted in different colors depending on the rate of the divergence of {z n }. To distinct the iterations (4) from the Fatou-Julia iterations let us call the first ones Fractals Mapping Iterations (FMI). It is clear that FJI is a particular FMI, when the function is the identity map. The mapping of fractals is a difficult problem which depends on infinitely long iteration processes, and has to be accompanied with sufficient conditions to ensure that the image is again fractal. The next theorem is the main instrument for the detection of fractal mappings. Accordingly, we call it Fractal Mapping Theorem (FMT). Theorem 1. If f is a bi-Lipschitz function, i.e. there exist numbers l 1 , l 2 > 0 such that Proof. Fix an arbitrary w ∈ F f . There exists a bounded sequence {w k } such that w 0 = w and f −1 (w k+1 ) = F (f −1 (w k ). Let us denote z k = f −1 (w k ). Our purpose is to show that {z k } is a bounded sequence. Indeed Hence, the boundedness of {w k } implies the same property for {z k }, and therefore, we have z 0 = f −1 (w) ∈ F. Now, assume that w ∈ f (F). There is z ∈ F such that f (z) = w and a bounded sequence {z k } such that z 0 = z and z k+1 = F (z k ). Consider, w 0 = w and w k = f (z k ), k ≥ 0. It is clear that the sequence {w k } satisfies the iteration (3) and moreover The following two simple propositions are required. Lemma 1. (7) If f is a bi-Lipschitz function, then where dim H denotes the Hausdorff dimension. It is clear that a bi-Lipschitz function is a homeomorphism. Shishikura (12) proved that the Hausdorff dimension of the boundary of the Mandelbrot set is 2. Moreover, he showed that the Hausdorff dimension of the Julia set corresponding to c ∈ ∂M is also 2. It implies from the above discussions that if f is a bi-Lipschitz function and F is either a Julia set or the boundary of the Mandelbrot set, then their images F f are fractals. In what follows, we will mainly use functions, which are bi-Lipschitzian except possibly in neighborhoods of single points. Now, we apply FMI to a Julia set J , and the iteration will be in the form with Fig. 2(e). DISCRETE DYNAMICS Discrete fractal dynamics means simply iterations of mappings introduced in the last section. Let us consider a discerete dynamics with a bi-Lipschitz iteration function f and a Julia set J 0 = J as an initial fractal for the dynamics. The trajectory is obtained by the FMI such that J k+1 = f (J k ), k = 0, 1, 2, 3, . . .. The last equation is a fractal propagation algorithm. CONTINUOUS DYNAMICS To demonstrate a continuous dynamics A t with real parameter t and fractals, we use the differential equation such that A t z = φ(t, z), where φ(t, z) denotes the solution of (9) with φ(0, z) = z. Thus, we will construct dynamics of sets A t F, where a fractal F is the initial value. To be in the course of the previous sections, we define a map f (z) = A t z and the equation Thus the FMI (4) in this case will be in the form In what follows, we assume that the map A t is bi-Lipschitzian. This is true, for instance, if the function g in (9) is Lipschitzian. Then the set A t F for each fixed t is a fractal determined by the FMI, and we can say about continuous fractal dynamics. As an example we consider the differential equation dz/dt = −z, 0 ≤ t ≤ 1, with the flow A t z = ze −t . It represents a contraction mapping when it is applied to the iteration (10), whereas the unstable dynamical system A t z = ze t corresponding to the differential equation dz/dt = z represents an expansion mapping. Now, we will focus on the autonomous system of dif-ferential equations The solution of the last system in polar coordinates with initial conditions ρ(0) = ρ 0 and ϕ(0) = ϕ 0 is given by Thus, the map can be constructed by where x(t) = ρ(t) cos(ϕ(t)), In Fig. 5(a), the fractal trajectory of system (11) is seen with the Julia set as the initial fractal. Parts (b)-(g) of the same figure represent various points of the trajectory. Next, let us consider the non-autonomous differential equation and the map which is determined by the solutions of Eq. 13. The map is not of a dynamical system since there is no group property for non-autonomous equations, in general. This is why, Eq. 10 cannot be used for fractal mapping along the solutions of the differential equation (13). However, for the moments of time 2πn, n = 1, 2, . . . , which are multiples of the period, the group property is valid, and therefore iterations by Eq. 10 determine a fractal dynamics at the discrete moments. In the future, finding conditions to construct fractals by non-autonomous systems might be an interesting theoretical and application problem. We have applied the map with a = 0.01 and the Julia set corresponding to c = −0.175 − 0.655i as the initial fractal. The results of the simulation are seen in Fig. 6. Since the moment t = π 2 is not a multiple of the period, the section in part (b) of the figure does not seem to be a fractal, but in part (c), the section is a Julia set. CONCLUSION Despite the intensive research of fractals lasts more than 35 years (3), there are still no results on mapping of the sets, and our paper is the first one to consider the problem. To say about mathematical challenges connected to our suggestions, let us start with topological equivalence of fractals and consequently, normal forms. Differential and discrete equations will be analyzed with new methods of fractal dynamics joined with dimension analysis. Next, the theory for dynamical systems which is defined as iterated maps can be developed. Therefore, mapping of fractals will be beneficial for new re-searches in hyperbolic dynamics, strange attractors, and ergodic theory (33,34). The developed approach will enrich the methods for the discovery and construction of fractals in the real world and industry such as nanofiber engineering, 3D printing, biotechnologies, and genetics (25,27,28,31).	2018-03-20T15:01:38.000Z	2018-03-20T00:00:00.000	{ "year": 2018, "sha1": "9eee7c281f806f4241517ca0a3bd5497fd67b517", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "111f032dd92e6b4d4183b1806af8836b46a1d9f7", "s2fieldsofstudy": [ "Mathematics", "Physics" ], "extfieldsofstudy": [ "Mathematics" ] }
236951040	pes2o/s2orc	v3-fos-license	Decarbonising the Portland and Other Cements—Via Simultaneous Feedstock Recycling and Carbon Conversions Sans External Catalysts The current overarching global environmental crisis relates to high carbon footprint in cement production, waste plastic accumulation, and growing future energy demands. A simultaneous solution to the above crises was examined in this work. The present study focused on decarbonizing the calcination process of the cement making using waste plastics and biowastes as the reactants or the feedstock, to reduce the carbon footprint and to simultaneously convert it into clean energy, which were never reported before. Other studies reported the use of waste plastics and biowastes as fuel in cement kilns, applicable to the entire cement making process. Calcination of calcium carbonate and magnesium carbonate is the most emission intensive process in cement making in Portland cements and Novacem-like cements. In the Novacem process, which is based on magnesium oxide and magnesium carbonates systems, the carbon dioxide generated is recycled to carbonate magnesium silicates at elevated temperatures and pressures. The present study examined the Novacem-like cement system but in the presence of waste plastics and biomass during the calcination. The carbon dioxide and the methane produced during calcination were converted into syngas or hydrogen in Novacem-like cements. It was established that carbon dioxide and methane emissions were reduced by approximately 99% when plastics and biowastes were added as additives or feedstock during the calcination, which were converted into syngas and/or hydrogen. The reaction intermediates of calcination reactions (calcium carbonate–calcium oxide or magnesium carbonate–magnesium oxide systems) can facilitate the endothermic carbon conversion reactions to syngas or hydrogen acting as non-soot forming catalysts. The conventional catalysts used in carbon conversion reactions are expensive and susceptible to carbon fouling. Two criteria were established in this study: first, to reduce the carbon dioxide/methane emissions during calcination; second, to simultaneously convert the carbon dioxide and methane to hydrogen. Reduction and conversion of carbon dioxide and methane emissions were facilitated by co-gasification of plastics and bio-wastes. Introduction Current environmental challenges relate to meeting global CO 2 emission targets, managing tons of plastics waste, and meeting the future energy needs. Cement production presents a major opportunity for addressing concerns related to waste plastics and biowastes as energy sources and chemical feedstock. This work identified and explored issues associated with this opportunity. These include: Review of emission specifications and energy requirements for lime and clinker production Comparative tonnage of present day energy sources vs. coal Available plastics/energy sources Feed-stock recycling of tires in cement production Emission and toxicity concerns Reactions of Engineers and contractors did not embrace these alternate cements due to high costs [11], and they prefer strong materials and strict building standards [9,12,13]. Geopolymer cement (USD 161.00) costs nearly thrice as much as the Portland cement (USD 51.00) [14,15]. High costs and lack of field testing prohibit the use of these new cements. Simultaneous Decarbonization, Wastes Management, and Clean Energy Production in Portland Cements The present study offers a novel step-change process to decarbonize the cements during the calcination via co-gasification of biomass and waste plastics, to cut down on emissions and energy requirement. In this section, energy requirement and source of emissions in cement making, roles of waste plastics and biowastes in reducing these emissions, and energy requirements are discussed. Emissions from Calcination of Carbonates, the Raw Materials Used in Cements CaCO 3 , MgCO 3 , and dolomites are the raw materials used in cement making [16,17]. Calcination of calcite (limestone, CaCO 3 ) requires higher energy than the calcination of dolomite and magnesite (MgCO 3 ). The incipient evolutions of CO 2 for magnesites, dolomites, and calcites occur at 640 • C, 730 • C, and 906 • C, respectively. In total, 1.092 kg CO 2 is released per kg magnesia (MgO) and 0.477 kg CO 2 per kg dolomite. The energy demand for MgO production ranges between 5 and 12 GJ/t MgO [18]. A cement clinker is made by calcining a homogeneous mixture of limestone (CaCO 3 ) and clay or sand (silica and alumina source) in a rotary kiln at~1450 • C (reaction 1) [9]. 3CaCO 3 + SiO 2 → CaSiO 5 + 3CO 2 (1) Subsequently, Portland cement is produced by grinding the clinker with~5% of gypsum (calcium sulphate). There are two sources of CO 2 emissions in Portland cement: (1) burning the coal as fuel and (2) calcination of limestone to lime. The focus of the present study was to reduce CO 2 produced during the calcination of limestone to lime (reaction 2) as well as during the calcination of magnesite to MgO, a Novacem-like cement system. About 65% of CO 2 emissions are due to the calcination of raw materials, mainly from reaction 2. Energy consumption for lime (CaO) production is 4.25 GJ/t of quicklime [19]. The remaining 35% is due to fuel combustion. The amount of CO 2 released is 1 kg/kg cement during calcination. Almost equal amounts of CO 2 are released from heating up the required amount of coal. Coal consumption is 0.2 t/t cement. To produce 1 kg of clinker, 1.16 kg of limestone is required [20], of which CaO content is 0.65 kg/kg clinker. Emissions from 1 t clinker production are calculated as: 1 t × 65% × 0.79 = 0.51 t CO 2 from CaCO 3 calcination [21]. CaCO 3 → CaO + CO 2 (2) Role of Waste Plastics in Reducing the Emissions in Cement Processing About~104 Mt of waste plastics are projected to enter our environments by 2030 [22]. Wrong waste management practices of end-of-life (EOL) plastics pose huge environmental challenges. Plastics production and incineration will account for 56 Gg.t of carbon emissions between now and 2050 [23,24]. Halogenated and PVC plastics release dioxins, polychlorinated biphenyls, HBr, and furans into the environment. Harsh HCl gas from PVC can damage treatment plants and incinerators. Demand for silicones in electrical, electronics, medical, and other industries led to their increased land filling and the loss of valuable resources [25,26]. Toxic odors and severe temperatures constrain silicones repurposing. Net emissions factors for plastics for different materials management options are given in Table 2 [27,28]. The driving forces of plastics recycling schemes are energy recovery and cutting emissions, penalties, energy consumption, non-renewable resources, and manufacturing costs [29,30]. Energy recovery from waste plastics depends on their calorific values (kJ/kg): coke~25,000-30,000, PE~44,800, PP~42,700, PS~41,900, PET~23,200, PVC~1800, and epoxy (resin)~32,000 [31,32]. More than 90% of the plastics produced (300 Mt/y) are not recycled [33]. Waste Plastics as Fuel A cement plant with 1 Mt capacity can consume between 10,000 and 30,000 Mt of plastics as fuel per year. About 50,000 t of waste plastics can be treated as fuel with 3000 to 4000 t of lime in a shaft kiln to generate syngas, which can support high temperature processes, such as glass foundries and iron and steel production replacing the fossil fuels [38,39]. Waste Plastics as Chemical Feedstock Waste plastics are used as fuel in the cement industry but not as chemical feedstock (as raw material) to reduce the CO 2 emissions thus far. Feedstock recycling of plastics is a sustainable solution to manage the plastic wastes such as mixed and halogenated plastic wastes and silicone wastes not suitable for recycling. Chemical feedstock recycling processes can extract valuable resources, e.g., C, H, Cl, and Si, from waste plastics, silicones, and biomass without considerable pre-treatment or depolymerization. During chemical feed stock recycling, plastics decompose without burning, producing chemically useful materials, and can convert to syngas at cement making temperatures [34,40]. Syngas is a renewable fuel with similar properties to natural gas that contains H 2 and CO and has many applications, as seen in Figure 1 [41]. It is a precursor for liquid fuel production via Fischer-Tropsch process and a main source of H 2 in the refineries [42]. Waste plastics as chemical feedstock in iron and steel industry reduce CO 2 emissions, acting as reductants and as the source of syngas [29][30][31]43]. Up to 30% reduction in CO 2 emissions is demonstrated in iron ore reduction using waste plastics as feedstock to partially replace coke as the reductant. Blast furnace and coke ovens treat waste plastics as chemical feedstock to produce syngases. The advantage of feedstock recycling in blast furnace approximates to 50 GJ/t of mixed plastics [44]. Biomass/Biowastes Biomass/biowastes are generally considered carbon neutral because the CO 2 emitted to the atmosphere during combustion is absorbed while growing the replacement biomass. However, emissions accrue during farming, harvesting, processing, and delivering the fuel. A "carbon neutral" emissions factor for biomass is 0.04 kgCO 2 e/kWh (net CO 2 e emissions assuming carbon sequestration) and 0.39 kgCO 2 e/kWh when all emissions accrued at the point of consumption are considered [45]. The biomass has the following composition: cellulose 42%, lignin 29%, and hemicellulose 7% [46]. At high pyrolysis temperatures, biomass exhibits increased amounts of H 2 and CO and decreased amounts of CO 2 in the gases [47]. Carbon neutral natural rubber components in tires contribute to lower CO 2 emissions [48]. Decarbonizing Cement via Chemical Feedstock Recycling of Wastes This study focused on decarbonizing the calcination phase (reaction 2) of Portland cement and Novacem-like cements and converting the CO 2 generated during the calcination to hydrogen and/or syngas. This was achieved by calcining the mixture of plastic wastes and biowastes and the carbonates (calcium carbonate or magnesium carbonate). The author's previous studies formed the basis for the proposed decarbonization in Portland and Novacem-like cement processing. The author's earlier studies similar to the Novacem process detail the low temperature and the high pressure carbonation (−13 • C and 6 bar) of silicate rich magnesites dumped as wastes and calcination reactions of MgCO 3 to MgO producing CO 2 [49][50][51][52][53]. Novacem production involves carbonation of magnesium silicates under elevated temperature and pressure (180 • C/150 bar). The carbonates produced are heated up to 700 • C to produce MgO, where the CO 2 generated is recycled back to carbonate the silicates [11]. Author's earlier studies were extended by the author to incorporate plastics and biowaste during the calcination of MgCO 3 to MgO, resulting in a great reduction in the carbon footprint and simultaneous production of hydrogen [54]. Author's research on plastic degradation and use of plastics and forestry wastes in materials processing was the inspiration to extend the application of organic waste material to cement processing [29,30,[55][56][57][58]. The author's work on non-soot forming the catalytic ability of calcine intermediates, e.g., the MgCO 3 -MgO system in carbon conversion reactions, the dry reforming reaction to produce syngas and/or enriched hydrogen [59,60], underpins the application of waste organic materials in reducing the emissions in cement making. The present study adopted the strategies discussed in the earlier works of the author, i.e., the use of organic wastes to decarbonize the calcination reactions of CaCO 3 to CaO (reaction 2) of Portland cements and calcination reactions of MgCO 3 to MgO of Novacemlike cements ("green" alternatives to Portland cement), which, to date, remain high carbon footprint processes. Similar chemistries between MgCO 3 -MgO and CaCO 3 -CaO systems enabled the author to extrapolate the results from one system to the other, accounting for slight differences in calcination temperatures and the amounts of CO 2 released during the calcination reactions. The catalytic ability of these systems is exploited in carbon conversion reactions (dry reforming reactions) to produce syngas and hydrogen during the calcination phase. The authors' study on replacing silica and coke with silicone wastes in ferrosilicone production formed the basis for recommending the use of silicone wastes in place of expensive silica in cement clinker production (reaction 1) [61]. Objectives This study sought to resolve the high carbon footprint associated with Portland and Novacem-like cements and unsuitable plastic waste management strategies simultaneously. The CaCO 3 -CaO system of Portland cement and the MgCO 3 -MgO system of Novacemlike cements are reported with the overarching aim to minimize emissions, energy, and pollution. As the calcination phase (reaction 2) is the major emitter of global CO 2 , this study aimed to minimize the emission during this phase by introducing waste plastics and biowastes as chemical feedstock. This work did not focus on the use of plastic and bio wastes as fuel sources. Specific objectives included establishing the criteria for suppressing the CO 2 (mainly from calcination of carbonates and gasification of wastes) and CH 4 emissions (from co gasification of wastes) during the calcination and increasing the H 2 generation during the calcination. Experimental The experiments involved the study of calcination reactions of CaCO 3 (reaction 2) and MgCO 3 , responsible for the most emissions in cement making, in the presence of plastics and/or biomass. These experiments were designed to establish the criteria for emissions reduction and emission conversions to hydrogen, the source of green energy. The study included monitoring the off-gas composition from calcination experiments. Pinus radiata was vacuum dried at 80 • C for 2 h and packed to a density of 400 kg m -3 in a furnace. The proximate and the ultimate analyses details of the Pinus radiata and the plastics are given in Table 3 [62,63]. Calcination experiments were carried out in a laboratory setup with furnaces [53,54,61]. Isothermal calcination of CaCO 3 samples was carried out at 1250 • C and 1450 • C in an electricity operated horizontal tube furnace in an inert (argon) atmosphere at a flow rate of 1.0 L/min. The MgCO 3 samples were subjected to isothermal calcination at 1000 • C in an IR image gold furnace and an arrangement of internals for heating. MgCO 3 samples (20 to 50 mg) packed inside the silica tube were introduced at 1000 • C in the middle of a graphite heating element. Helium at~50 mL/min was maintained. Total sulfur/% 0.08 Off-Gas Compositions A gas chromatographic (GC) analyzer (SRI8610C Chromatograph Multiple Gas #3 GC) configuration equipped with a thermal conductivity conductor (TCD) and a continuous IR gas analyzer were used to measure off-gases, CH 4, and CO 2 periodically for CaCO 3 and CaCO 3 + resin studies. The amounts of H 2 could not be monitored during the calcination studies due to the limitation in the IR used. The volatiles from the MgCO 3 +resin +biomass system were measured with MTI Activon M200 series micro gas chromatograph (GC) instrument. The thermal conductivity detectors with a 5A molecular sieve column at 60 • C was used to measure H 2 and CO. A Poraplot U column at 40 • C was used to measure CO 2 , CO, CH 4 , C 2 H 4, and C 2 H 6 . The evolution rate was determined as the wt.% of initial Wt. of sample/min. X-ray Diffraction (XRD) XRD with a copper Kα source operated at 45 kV and 40 mA and scanned at a step size of 0.026 • and a scan rate of 1 • /min and X'pert High score software were used for phase identification of calcined MgCO 3 with epoxy resin at 1200 • C. Figure 2 illustrates the results from isothermal calcination reactions of CaCO 3 with and without the plastic resin (resin). Figure 2 shows calcination of CaCO 3 (2.36 g) at 1450 • C without the resin (test 1), calcination of CaCO 3 (2.36 g) with the resin (2.37g) at 1450 • C (test 2), and calcination of CaCO 3 (2.36 g) with the resin (2.06 g) at 1250 • C (test 3). Test 2 showed almost no traces of CO 2 but only CH 4 , while test 3 showed reduced amounts of CO 2 and almost equal amounts of CH 4 . These figures show the effects of the resin and the temperatures in suppressing the CO 2 emissions to almost zero at high temperatures during the calcination. In these tests (tests 3 and 4), the resin quantity was kept almost equal or slightly less than the CaCO 3 at 2.36 g. To summarize, calcination of CaCO 3 in test 2 showed more CH 4 and negligible amounts of CO 2 at 1450 • C; test 3 showed almost equal amounts of CO 2 and CH 4 at 1250 • C, demonstrating the effect of temperatures. Additionally, test 2 had slightly higher resin content than in test 3. Both the high temperature and the higher resin content could be responsible for suppressing the CO 2 significantly. Hydrogen content was not measured during these tests. Biomass effect was not studied during calcination of CaCO 3 . A summary of test details and results of calcination reactions of MgCO 3 ·xH2O and CaCO 3 with various ratios of biomass and plastics and at different temperatures is shown in Table 4, including the experimentally observed (y%) and the expected values for the gas evolution. The expected gas composition was calculated based on the mass% of different components (carbonates, biomass, and resin) present in each sample [54,60]. Cumulative gas compositions determined by GC are shown in Table 5. Resin amount about three quarters that used in test 7; approximately equal amounts of resin, MgCO 3 , and biomass 7 Calcination of MgCO 3 Test 8, Table 4, 1000 • C Note: In MgCO 3 tests, the amounts of resin + biomass was greater than MgCO 3 . All tests were at 1000 • C. High temperatures and high plastic (resin) content favored suppression of CO 2 above 95%, as seen from the test results of tests 5, 6, 7, and 8 (Tables 4 and 5), whereas biomass contributed to less suppression, e.g., up to 82% reduction in CO 2 but up to 230% increase in hydrogen. A higher resin content than the biomass (test 7) during the calcination resulted in CO 2 reduction up to 99%, and in CH 4 , there was a reduction up to 97% accompanied by 360% increase in H 2 compared to the expected value. Test 7 could be an ideal scenario to produce H 2 enriched gas. Resin content approximately equal to or less than the biomass during the calcination resulted in 76% reduction in CO 2 and~63% reduction in CH 4 but 4684% increase in H 2 compared to the expected value (Test 8). Discussion Note: biomass was not used in CaCO 3 tests. During calcination of MgCO 3 ·xH 2 O, the effect of temperature was not studied. Another interesting observation was the absence or the negligible amounts of CO, contrary to what was expected. Low Carbon Portland Cement and Novacem-Like Cement The calcination phase of the cement production is the most emission intensive process. Attempts to reduce CO 2 emissions during the using waste plastics as the chemical feedstock were never reported before. Cement kilns use shredded waste plastics as fuel but not as chemical feedstock. The current study demonstrated the CO 2 reductions during the calcination reactions of calcium carbonate (Portland cements) and magnesium carbonate (Novacem-like cements) in the presence of resin and/or biomass (Figures 2 and 4-7), indicating the feasibility of using cements/clinkers production as waste plastic conversion facilities. Owing to their similar chemistries, the results from MgCO 3 studies can be extrapolated to CaCO 3 (taking into consideration the higher calcination temperature of CaCO 3 (906 • C), which is close to the iron ore reduction temperatures. The role of plastics and biomass as feedstock in greatly reducing the carbon footprint of calcination reactions in cement making as well as the conversion of carbon from calcination reaction to syngas/hydrogen are explained in the following sections. Chemical Feed Stock Recycling of Plastics Plastics pyrolysis shows two phases, solid carbon and gas, namely CH 4 and H 2 , which are thermodynamically stable at 1100 • C [64]. CH 4 and the solid carbon further undergo catalytic transformation to syngases. The following reactions characterize the chemical feed stock recycling of waste plastics: Plastics decomposition (pyrolysis) results in reaction 3: Pyrolysis product from reaction 3 undergoes methane cracking (greater than 557 • C) (reaction 4): C n H m (g) → nC (s) + H 2 (g); CH 4 = C (s) + 2H 2 ; ∆H = 75.6 kJ/mol (4) Syngas production is governed by the following reactions: The Boudouard reaction (reaction 5,~701 • C): Water gas shift reaction (reaction 6): Water gas reaction or char reforming (reaction 7, greater than 700 • C): C + H 2 O → H 2 (g) + CO; ∆H = 131 kJ/mol (7) Dry reforming reaction (~700 • C in presence of catalysts) (reaction 8): Methane reforming reaction (reaction 9) CH 4 + H 2 O → 3H 2 + CO; ∆H = 206 kJ/mol Reactions 7 (water gas reaction), 8 (dry reforming reaction), and 9 (methane reforming reaction) result in various ratios of syngas. These reactions are endothermic and high temperature reactions requiring catalytic support. The main benefit of CO 2 reforming methane (reaction 8, where CO 2 acts as the oxidizing agent) is, when H 2 /CO is~1, it is suitable for synthesizing oxygenated chemicals, e.g., methanol, acetic acid, aldehydes, ethanol, a wide variety of alcohols, olefins, and gasoline [65]. Oxygenates facilitate easy and safe storage and transport of energy. Methanol mixed with dimethyl ether (DME) is an excellent fuel for diesel engines with a high cetane number and beneficial combustion characteristics. The energy input for the CH 4 dry forming reaction (reaction 8) is 20% higher than the steam reforming (or the methane reforming) reaction 9, resulting in syngas of varying H 2 /CO molar ratios. The drawback of the methane reforming (reaction 9) is that the H 2 /CO ratio 3:1 is greater than that required for the Fischer-Tropsch process. Chemical Feedstock Recycling of Biomass Biomass undergoes similar reactions as waste plastics during pyrolysis (refer to Section 5.2). Gases generated during the pyrolysis of biomass are CO, H 2 , CH 4 , and CO 2 ; other products of pyrolysis include H 2 O and char depending upon the ambience [66]. Steam gasification/reduction chemical processes of biomass often occur at temperatures above 700 • C governed by: methane cracking (reaction 4), Boudouard (reaction 5), water gas shift (reaction 6), char reforming (reaction 7), dry reforming reaction (reaction 8), and methane reforming (reaction 9). During the gasification of biomass in an inert environment at 900 • C, cellulose contributes to CO, hemicellulose promotes CO 2 generation, while lignin aids H 2 and CH 4 generation. Reduction in CO 2 Emissions during Calcination Calcination of inorganic carbonates in reducing atmosphere (reactions 10 and 11) serves to capture or utilize CO 2 , the chemical H 2 storage system for CH 4 , and the fuels from syngas [66][67][68][69][70]. Plastics and the biomass provide the reductive atmosphere to reduce the CO 2 emissions during calcination. H 2 produced in the reductive calcination can be a means to produce CH 4 or CO/syngas from the CO 2 emitted [71]. The methane cracking reaction (reaction 4) reduces the CO 2 generated during calcination of MgCO 3 and CaCO 3 in the presence of plastic/biomass. Reduced CO 2 emissions (reaction 10 and 12) in the presence of a reductive atmosphere of H 2 and N 2 mixtures was reported [71]. The H 2 and the C, the products of reaction 4, react with MgCO 3 (reactions 10 and 12) and CaCO 3 (reactions 11 and 13), resulting in reduced CO 2 emissions (reactions 10 and 11). MgCO 3 + C → MgO + 2CO (12) Calcination of magnesite in a reductive hydrogen atmosphere results in decreased CH 4 and increased CO content. Amounts of CH 4 formed in reactions 10 and 11 depend upon MgCO 3 content, i.e., the amount decreases as MgCO 3 content decreases. The CO increases as the MgO content increases. MgO calcined reductively catalyzes the reverse water gas shift (reaction 14), leading to CO generation. This results in reduced CO 2 emissions below 820 • C, which means H 2 increases above 820 • C [71][72][73]. However, reaction 14 was reported to occur above 1000 • C during iron oxides reduction [74]. The reduction in CO 2 emission is greater than the reduction in CH 4 if no carbon deposition occurs during the dry reforming reaction (reaction 8) [42,75]. Methane Conversions Resin to carbonates ratio during calcination governs the CO 2 /CH 4 ratios. When resin/CaCO 3 is equal to or less than one, CH 4 /CO 2 emission is high (Figure 2). When resin/MgCO 3 ratio is high, both CH 4 and CO 2 emissions are reduced by 94% (test 6, Figure 5). The presence of biomass during MgCO 3 calcination results in higher CH 4 /CO 2 , while the CO 2 is reduced up to 82% (Figure 4). Increase in CH 4 can be attributed to reaction 3. In total, 100% of the CO 2 from MgCO 3 calcination can be transformed to CH 4 in the presence of H 2 and the catalysts Co/Ca/CoO (reaction 15) [76]. CH 4 conversion (reduction in CH 4 ) at high temp0eratures is ascribed to reactions 4, 8, and 9, leading to H 2 generation (tests 5, 7, and 8). Reduction in CH 4 (reaction 8) depends on CO 2 /CH 4 ratios as well as the temperatures. A high CO 2 /CH 4 ratio (reaction 8) results in high conversion of CH 4 , demonstrating the positive effect of CO 2 as a soft oxidant at temperatures greater than 700 • C. The dry reforming reaction (reaction 8) requires a cheap and pure source of CH 4 and CO 2 . Pure CO 2 is released during cement production from calcination of MgCO 3 or CaCO 3 , and the CO 2 from pyrolysis of biomass and the plastics ensure CO 2 is greater than CH 4 (reaction 2) (Tables 4 and 5). It should be noted that more CO 2 is released from MgCO 3 (52%) compared to the CaCO 3 (44%) stoichiometrically during calcination. Under the experimental conditions, MgCO 3 calcination can result in sudden copious amounts of CO 2 (calcination temperature~700 • C) compared to that from CaCO 3 (calcination temperaturẽ 900 • C). Increasing the amount of CH 4 (from biomass and plastics) can increase H 2 generation from reaction 4. Figures 6 and 7 show increased H 2 and greatly reduced CO 2 during the calcination of MgCO 3 in the presence of plastics and biomass. It is anticipated CaCO 3 follows a similar trend owing to its similar chemistry to MgCO 3 . This is attributed to reactions 4, 7, 8, and 9 directly contributing to increased H 2 and syngas (H 2 and CO). Co-Pyro-Gasification of Waste Plastics and Biomass vs. Individual Gasification of Wastes Hydrogen enriched syngas production is attributed to several factors. Co-pyrogasification of plastics and biomass blends increases the quality and the composition of syngas (H 2 /CO ratio) [66,77]. The present study showed the biomass and plastics blend enhanced the hydrogen generation while reducing CO 2 and CH 4 emissions. Using only plastics greatly reduced the CO 2 emissions with negligible gen-eration of hydrogen; the biomass use only decreased CO 2 emissions to an extent but fa-cilitated the generation of both H 2 and CH 4 (Figures 6 and 7, Tables 4 and 5). Co-pyro gasification of plastic wastes and biomass converts wastes predominantly to gas rather than to char and tar [77]. Increasing the CO 2 promotes a high yield of syngas [73]. CaCO 3 and MgCO 3 , plas-tic wastes and biomass, were the main sources of excess CO 2 in the present study. When steam is present, the water gas shift reaction (reaction 6) shows reduction in CO and increase in H 2 yields [72]. It should be noted that, in the present study, CO was not observed. If the water gas shift reaction 6 is not present, soot formation through me-thane cracking can occur (reaction 4). Temperature Effects In this stud, calcination of CaCO 3 at 1450 • C (test 2) showed almost no CO 2 content compared to calcination at 1250 • C (test 3) and increased methane in the absence of any external catalysts (Figure 2). The reductive H 2 atmosphere can lower the calcination temperature by more than 150 • C compared to a non-reducing atmosphere [71]. The sudden spike in CH 4 seen in Figure 2 may be attributed to reaction 11 from increased H 2 . Increasing the gasification temperature of the biomass usually promotes syngas production while concurrently inhibiting the biochar production [78]. A slight decline in the syngas at temperature above 800 • C is ascribed to the reverse water gas shift reaction (reaction 14). Conditions for high H 2 yield are discussed below. High calcination temperatures increase H 2 and CO contents, simultaneously decreasing the CO 2 content by facilitating the hydrocarbons cracking (reaction 4) [47]. Reaction 4 favors higher H 2 generation at temperatures above 900 • C [73]. CH 4 formation is favored at low temperature and elevated pressure. There is a decrease in CO content at temperatures above 800 • C. High H 2 yield is attributed to low amounts of CH 4 in reaction 8 and high temperature when CO 2 /CH 4 is high [60]. Char Formation Char formation has immediate relevance to the endothermic carbon reforming reactions (reactions 8 and 9), which require catalysts. These catalysts also catalyze soot formation (reaction 4) [42] resulting in catalytic fouling, affecting the stability of the catalysts and increasing the costs of the dry reforming process (reaction 8), which hinders its commercialization. Reducing the char formation is important in carbon reforming reactions (reactions 8 and 9). In the present study, decreasing amounts of CH 4 and CO 2 and hydrogen generation during the calcination of MgCO 3 confirmed the occurrence of carbon reforming reactions (reactions 8 and 9) without the aid of external catalysts. The XRD trace of the calcined MgCO 3 at 1250 • C in the presence of plastics showed no carbon formation (Figure 8). This was attributed to the high temperatures (cement making temperatures) and the high amounts of pure CO 2 generated from the calcination reactions (reaction 2) as well as from the co-pyro gasification of biomass and plastics. Lignin present in biomass contributes to high amounts of char [79]. To suppress biochar production, it is necessary to increase the temperature and the heating rate, which can promote the syngas production [78]. Thermodynamic calculations indicate the required temperature to be 1035 • C for 50% CO 2 conversion in reaction 8 without the catalyst. High CH 4 and CO 2 conversions at temperatures 700 • C require catalytic systems such as metal oxides, monometallic and bimetallic catalysts, and supported metal catalysts [80][81][82]. Steam reforming reactions (reactions 9 and 16) are favored at temperatures above 900 • C and 15-30 atm using nickel-based catalysts. However, carbon fouling of the catalysts is a serious issue. Steam can eliminate the carbon formed as quickly as its formed. Alkali compounds improve the water gas reaction or the char reforming reaction (reaction 7) at temperatures above 700 • C [83,84]. Though reaction 5, the Boudouard reaction, can be a source of char formation, it does not occur above 700 • C [73]. When CO 2 /CH 4 is high and temperature is above 700 • C, the coke deposition is diminished due to the oxidation reaction of CO 2 with the surface carbon (reaction 5) [84]. Conversion of CO 2 and CH 4 is determined by the ratio of CO 2 /CH 4 and the carbon or the soot formation [42]. Presenting CO 2 to the catalytic dry reforming process (reaction 8) reduces the soot deposition. The CO 2 from the calcination reactions of carbonates during cement making ensures CO 2 is greater than CH 4 , thus reducing the carbon formation (Tables 4 and 5). In the present study, more than 70% CO 2 conversions were achieved without an external catalyst. MgCO3-MgO and CaCO3-CaO Catalytic Systems Generated In Situ The hydrogen generation in calcination reactions is governed by the steam (reactions 9 and 14) and the dry reforming reactions (reaction 8). The efficacy of these reactions relies on external catalytic systems, e.g., nickel-based catalysts. A 90% CO 2 conversion was achieved for an MgO promoted catalytic system [75] promoting a partial reduction of CO 2 . In the present study, high conversions up to 99% were realized for both CH 4 and CO 2 , accompanied by H 2 generation without the use of external catalysts (Table 4). This was attributed to the MgCO 3 -MgO and the CaCO 3 -CaO systems acting as catalysts generated in situ. Freshly prepared MgO and CaO on their own or in combination act as catalysts for the carbon conversion reactions [85]. The catalytic ability of CaO is better than MgO during the biomass conversions [86]. The characteristics that make MgCO 3 -MgO or CaCO 3 -CaO desirable catalysts for carbon reforming reactions include: Lewis basicity, mesoporosity, high reactivity and stability, small crystal size, high specific surface, high adsorption, and reduced carbon formation, promoting both steam forming and dry forming of CH 4 . Lewis bases considerably improve CO 2 reforming of the CH 4 reaction 8, resulting in values higher than the equilibrium values of H 2 . Freshly formed MgO from basic MgCO 3 has a high specific surface, mesoporosity, low bulk density, low crystallite size, and nitrogen adsorption up to 100 cm 3 /g, making it catalytically active [50][51][52]87,88]. MgO calcined reductively catalyzes the reverse water gas shift reaction, leading to decreases in CO 2 (reaction 14). The catalytic effect of CaO increases syngas production from mixed plastic wastes and from the halogenated plastics and the PVC fractions. Lime serves as a passage for fuel and gas and simultaneously binds halogen and other harmful pollutants [38,89]. CaO's catalytic action prevents formation of dioxins and furan and tar containing cleavage products and oil at temperatures greater than 900 • C, hence facilitating the use of halogenated plastic waste streams. CaCO 3-CaO suppresses the release of toxins such as C 6 H 6 and HBr [90]. It was demonstrated that Portland cement making can effectively be treated as a plastic/biowaste and carbon conversion facility without the use of any costly external catalysts. Syngas Production-Proposed Mechanism It is proposed that one of the major reactions taking place during calcination in the presence of waste plastics and/or biomass is the reaction between CH 4 from pyrolysis (reaction 3) and MgCO 3 or CaCO 3 to produce MgO or CaO and syngas (reactions 17 and 18). As calcination of carbonates progresses, the CO 2 released reacts with CH 4 , resulting in increased amounts of H 2 and CO (as MgO content increases, the amount of CH 4 decreases, and the amounts of H 2 and CO increase). Hence, it was concluded the MgCO 3 -MgO system or the CaCO 3 -CaO systems generated in situ effectively catalyzed the dry reforming reaction (reaction 8) without coke deposition (Figure 8). However, CO (Figures 4-7) was not detected in the present study. It is possible that high temperatures, composition of reactants, and CO 2 /CH 4 could effectively suppress CO emissions. It was proposed that the catalytic actions of MgCO 3 -MgO and CaCO 3 -CaO systems not only catalyzed reactions 8 and 9 to produce H 2 /CO but also catalyzed the subsequent conversion of syngas to hydrogen and other, smaller hydrocarbon molecules, which could be building blocks to other useful fuels and chemicals. Composition of the reactants (e.g., MgCO 3 , resin, biomass) controlled the product gas distribution, e.g., as in selective production of H 2 (test 7), or the mixed distribution of CO 2 :CH 4 :H 2 in the product mixture in test 8. Applications Potential applications of the present study include extending similar strategies to more problematic materials, such as using halogenated waste materials in Sorel cements and Alinite clinker, and using silicones to replace sand, a costly commodity in Portland cement clinkers, and feedstock recycling of tires as sources of both plastics and biomass in cement making to combat high carbon footprint. Decarbonising Sorel Cements and Alinite Clinker Using Halogenated Waste Plastics Developing environmentally safe processes to handle halogenated plastic wastes is vital due to stringent environmental regulations. CaCO 3 and MgCO 3 inhibit the release of toxins such as C 6 H 6 , HBr, and dioxins, enhancing the pyrolysis process [90,91]. Hence, cement making is an ideal platform to repurpose halogenated plastic wastes. Silicones for Eco-Efficient Clinker Production Concrete and cement clinker production use a significant amount of sand, the world's second most consumed natural resource [95]. Silicones can replace high pure silica and coke in ferrosilicon production at cement making temperatures [61]. Silicone polymers possess organic and inorganic moieties with valuable resources such as silica, methane, carbon, and hydrogen. The organic moiety of silicones can reduce the emissions, while the inorganic moiety contributes silica. If co-pyro-gasified in the presence of biomass, the carbon emitted can be converted to hydrogen. SiO 2 from silicones can better replace the sand in cement clinkers in addition to offering similar emission and energy benefits derived from the waste plastics. Use of virgin silicones, siloxanes, and silanes in energy enabling technologies and as energy and materials results in energy savings and greenhouse gas (GHG) emission reductions. The CO 2 emission cuts realized in Japan, North America, and Europe using virgin silicone products amount to~54 Mt/y [96]. A pathway for the direct production of clinker (calcium silicates) from calcite and waste silicones to eliminate the use of silica is shown in reaction 21. This reaction demonstrates reduction in CO 2 and energy consumption and simultaneous production of syngas and H 2 . Waste or virgin silicones can replace silica in reaction 1. CaCO 3 calcined in the presence of silicones (polydimethylsiloxane (C 2 H 6 OSi) (Figure 9)) at cement making temperatures can directly produce clinker (CaSiO 5 ) and syngas with a great reduction in CO 2 emissions (reaction 21) while simultaneously facilitating silicone waste management. CaCO 3 + (C 2 H 6 OSi)n → CaSiO 5 + CO + H 2 (21) Figure 9. Structure of a linear silicone polymer (polydimethylsiloxane). Tires as Source of Both Plastic and Biomass The present study indicates the potential use of tires as a chemical feedstock to gain the benefits of emission and energy as well as to use the rubber ash generated in situ during the pyrolysis/gasification as sand replacement in the cement system. Scientists are working on ways to replace sand in concrete with other materials, e.g., rubber tire ash. Rubber tires that comprise both synthetic rubbers (plastics) and natural rubbers (carbon neutral biomass) can be the ideal candidates to reduce the GHG emissions (CO 2 and CH 4 ) and to generate H 2 in cement making, as proposed in the present study. It should be noted that ash content from biomass and waste plastics is negligible and is unlikely to alter the material properties (ash content from plastics and wood-LDPE, HDPE, PP, and PVC-less than 0.05%; wood 0.45%; rubber tires 5.7%; and coke/coal 18.4%) [97]. Other Industrial Applications This study has relevance not only to cement industries but also to iron and steel industries (where CaO and MgO are used as fluxes) in regard to dead burned magnesia production, carbothermic reduction of magnesium, carbon conversions, waste valorization, and emission and energy reduction while supporting the hydrogen economy and the generation of precursors for new materials. Use of plastics and biowastes can result in considerable reductions by about 200 • C in reaction temperatures (~1600 • C) during dead burned magnesia (DBM), fused magnesia (FM) production, and carbothermic reduction of Mg. Dead burned magnesia (DBM) currently makes up the largest portion of produced magnesia intermediate products, and there is growing demand and market share for FM [98]. Conclusions IEA's Sustainable Development Scenario (SDS) aim is to stay below 1.5 • C global warming, by adopting carbon mitigation strategies in cement sector. The findings of present study on decarbonsing the cement production using waste streams as chemical feedstock, and to simultaneously convert the CO 2 produced during cement production to clean energy, are most relevant to the IEA's SDS aim. The cement sector as a potential waste plastics/rubber tires treatment facility to simultaneously meet the emission targets, convert the GHG emissions to hydrogen, and maximize the recovery of resources present in waste materials, e.g., Si, H, CH 4 , and C, was discussed. The study focused on developing Novacem-like low carbon cements and decarbonizing Portland cements. Use of waste plastics and biomass as chemical feedstock (co-pyrogasification) to reduce the carbon footprint in the calcination step of cement making was demonstrated, which was never reported before. It should be noted the use of wastes as fuel in cement making was not considered in this study. Therefore, emission and energy benefits reported in this study were in addition to the benefits from using the wastes as fuel. Up to 99% reduction in GHG in Portland cement and Novacem-like cements production was established in this study. The effects of temperature, the ratio of the plastics: biomass: carbonates in controlling GHG emissions, H 2 production, and catalytic ability, and carbon fouling of the calcine intermediates are examined. High temperatures and high plastic content favored suppression of CO 2 more than 95%, whereas biomass contributed to less suppression, i.e., up to 82% reduction in CO 2 but up to 230% increase in hydrogen. A higher resin content than the biomass during calcination resulted in CO 2 reduction up to~99% and CH 4 reduction up to~97%, accompanied by 360% increase in H 2 compared to the expected value. A higher biomass content than the resin during calcination resulted in 76% reduction in CO 2 and 63% reduction in CH 4 but 4684% increase in H 2 compared to the expected value. When CO 2 /CH 4 were high and the temperature was above 700 • C, the coke deposition was diminished, thus preventing the carbon fouling of the catalytic calcine intermediates. Increasing the gasification temperature of the biomass also suppressed biochar formation. It was concluded that the catalytic actions of MgCO 3 -MgO and CaCO 3 -CaO systems not only catalyzed reactions 8 and 9, the carbon conversion reactions to produce H 2 /CO, but could catalyze subsequent conversion of syngas to hydrogen and other smaller hydrocarbon molecules as well. Use of mixed plastics, including halogenated plastics, silicones, and biomass from the waste inventory as chemical feedstock in cement making was examined. CaCO 3 minimizes the negative impacts of dioxins and toxic emissions from halogenated waste plastics during feedstock recycling and syngas production. The strategies presented in the present study can be applied to Alinite clinkers and Sorel cements production using halogenated plastic wastes with similar emissions and energy benefits. Recommendations for direct clinker production from silicone/silicone wastes (as sand replacement), solid residues from tires (ash), and silicones (silica) from rubber tires or silicone polymers used as the feedstock can offer emission and energy benefits. They can replace sand in direct production of a cement clinker (CaSiO 5 ).	2021-08-09T05:26:16.974Z	2021-07-27T00:00:00.000	{ "year": 2021, "sha1": "a946fd88740e9685c27641d839b33f94d9278dab", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4360/13/15/2462/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a946fd88740e9685c27641d839b33f94d9278dab", "s2fieldsofstudy": [ "Environmental Science", "Chemistry", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
125346248	pes2o/s2orc	v3-fos-license	Supersymmetric soliton solution in (1+1)-dimensional Ultracold Quantum Gases We obtain by dimensional reduction a $(1+1)$ supersymmetric system introduced in the description of ultracold quantum gases. The correct supercharges are identified and their algebra is constructed. Finally novel static self-dual solutions emerge, satisfying a Liouville type differential equation. Introduction Since its birth in the early 1970's in the context of high energy physics and mathematical physics, supersymmetry has found a growing number of applications strongly influencing many areas both in experimental and theoretical physics. Originally proposed as a graded extension of the Poincaré algebra [1], it was soon recognized that it can also be considered in the systems that exhibit Galilean invariance and this lead to the construction of a graded super-Galilean algebra [2] in d = 3 + 1 space time dimensions. Constructing the supersymmetric extension of the Galilean invariant d = 2 + 1 Jackiw-Pi model [3], Leblanc et al [4] discovered the existence of 2 graded superalgebras and related this to the possibility of finding BP S equations in the bosonic sector. After that, there were several developments related to Galilean supersymmetry in diverse contexts [5]. In (1 + 1) dimension Galilean supersymmetry was considered to study ultra cold quantum gases [6,7,8]. Ultracold quantum gases not only are interesting by the physics that they describe, but also are a useful tool in the modeling of others branches in physics [9]. One interesting example of this modeling was considered in Ref. [6,7]. There, the authors propose that by combining a vortex line in a one-dimensional optical lattice with a fermionic gas bound to the vortex core, it is possible to tune the laser parameters such that a nonrelativistic supersymmetric string is created. This could allow to test experimentally several aspects of superstring and supersymmetry theory. From the theoretical point of view, the model that describe that proposal presents a supersymmetric structure. Despite the theory has interactions, the authors only found the generators of the super-Galilei algebra for the free theory. This fact constrast with a basic feature of supersymmetry theory, where the full Hamiltonian is generated by the supercharge algebra. Later, in ref. [8] was shown the existence of supersymmetry charge whose algebra generate the Hamiltonian with quartic interactions. Nevertheless a supersymmetry transformation associated to a charge be able to generate the full Hamiltonian of the theory was not found. In this paper we will show the existence of a supersymmetry related to a charge generating the full Hamiltonian of the theory presents on Ref. [6,7]. Also we will construct the complete supersymmetry algebra. In order to discus this aspects we will present the (1 + 1) model study in Ref. [7] as the dimensional reduction of a Maxwell-Chern-Simons model proposed by Manton[10]. This has no influence in the derivation of the correct supersymmetry generators but leads us to the presences of interesting solitonic equations in the system. For the bosonic sector these equations reduce to a Liouville type differential equation. We analyze this case and construc the solucion for this type of Liouville equation. The Model Let us start by considering some features of the model proposed by Manton. This model is governed by a (2+1)-dimensional action consisting on a mixture from the standard Landau-Ginzburg and the Chern-Simons model, where the matter is represented by a complex scalar field φ(x), Here γ, κ and λ are real constants, D µ = ∂ µ + iA µ (µ = 0, 1, 2) is the covariant derivative and B = ∂ 1 A 2 − ∂ 1 A 2 the magnetic field. The term γA 0 is related to the possibility of a condensate in the ground state [11] and J T i is a constant transport current. It was show by Manton in Ref. [10] that this theory presents Galilean invariance with the requirement that the transport current transforms as J T i → J T i + γv i under a boost. With this consideration, we can choose a frame where J T i = 0. The field equations in this frame takes the form where E i = ∂ i A 0 − ∂ 0 A i is the electric field and J i is the supercurrent defined by The first equation of this system is the non-linear Schrödinger equation.The second is the Ampère's law in two dimensions.The last equation is the Chern-Simons version of the Gauss law, which here takes a different form from that presents in the Jackiw-Pi model [3]. The energy of the system for static field configurations reads as For finiteness we require that the energy vanishes asymptotically. This fixes the asymptotic behavior of the fields where α is common phase angle. With these conditions the magnetic flux reads where N is a topological invariant which takes only integer values. Following to Hassaïne et al. [12], we can rewrite the expression (4) as where we have used the Gauss law of the equations (2) and the identity \|D i φ\| 2 = \|(D 1 ±iD 2 )φ\| 2 ∓B\|φ\| 2 ±ǫ ij ∂ i J j . For λ = ∓ γ 2κm + γ 2 2κ 2 the potential terms cancel, and we see that the energy is bounded below by a multiple of the magnitude of the magnetic flux (for positive flux we choose the lower signs, and for negative flux we choose the upper signs): So this bound is saturated by fields obeying the self-duality equations Motivated by these results and the previous works on the (1+1)-dimensional supersymmetry in ultracold quantum gases [7,6,8] we are interested here on the dimensional reduction of the supersymmetric extension of the model (1). Such extension can be carried out by considering the inclusion of non-relativistic (down-spinor) fermion ψ [4]: where the coupling constants are given by and we have include a Pauli term for the fermion corresponding to a down-spinor. This action is invariant under the following supersymmetry transformation if the coupling constants satisfy Where η 1 appearing in (12) is a complex Grassmann variable. In order to analyze the lineal problem [8,13], it is natural to consider a dimensional reduction of the action (10) by suppressing dependence on the second spacial coordinate, renaming A y as B. Then, the action (10) becomes Where we have introduced the matter densities, The Gauss law constraint for this action is Note that this constraint has an additional constant term from that appearing on Ref. ( [8,13]). The equation can be solved as Using these expressions for the magnetic field and its derivative, the action (14) takes the form Here the action contains new coupling constants defined as Following the method proposed in Ref. [13], the gauge field A x may be eliminate from the action (18) via a gauge transformation. Indeed, after transforming the matter fields as with the action can be written simply as The last term appearing in this action is a constant of motion, where N = dxρ(x), and use has been made of the identity Thus, dropping this term, the action can be written as This is the model study in Ref. [7,6], in the context of description of ultracold gases dynamics. The action (25) is similar to the action derived by dimensional reduction of the Jackiw-Pi model [8]. The difference is that our model also include the chemical potential terms. Let us now consider the possible supersymmetry transformations that leave unchange the action (25). One obvious supersymmetry of the system (25) takes place when bosons and fermions are interchanged according to and the coupling constants are related by It is interesting to note that the transformation (26) is also a supersymmetry of the model study in Ref. [8]. The fact that is less evident is the existence of a second supersymmetry. In discussing this supersymmetry, note that the the action (25) is invariant under the following transformation and Notice that combining equations (19) and (29) we obtain Eq. (11). As the transformation (26), the expression (28) is also a supersymmetry of model explored in Ref. [8]. Another interesting fact is that the condition for coupling constants imposed in (29) is a particular case of the equation (27). This implies that the following combination of the precedent two supersymmetries is also a supersymmetry of the accion (25) if the condition (29) is kept. The supersymmetry algebra In this section we shall study the algebra of the generators associate to the transformations (26) and (31). In the representation of the supersymmetry algebra the generator associated to the supersymmetry (26) can easily be fond to be In order to write the supersymmetry algebra we define the Poisson brackets for the functions of the matter field as where the subscripts r and l refer to right and left derivatives and in particular we have Using the definition of Poisson bracket it is easy to get Following the Ref. [7] we can define a second generator which generate the free part of the Hamiltonian Nevertheless, as we mentioned in the introduction, the previous works does not propose a supercharge be able to generate the full Hamiltonian of the model explored in Ref. [7], that is the Hamiltonian derived from the action (25) In addition we know, from the Ref. [8], that the supersymmetry (28) is generated by the supercharge which generate a Hamiltonian only with potential terms quartic in the fields. Based in this idea and in the fact that the transformation (31) is a combination of the supersymmetry (26) and (28), it seems natural to define a supercharge Q 2 , such that it is a linear combination of Q 1 and Q (1) 2 We will show that this charge generate the Hamiltonian (38) . Using this charge we can calculate where The first of the brackets was calculated in Ref. [8] and its result is which can be reduced, after using the definition of the B 1 and eliminating the constant term d 2 xB 2 1 ρ, to The second bracket can be developed as follows It can be easily checked that Thus In similar form we have for the third bracket From (47) and (48) we get The last bracket gives Then the full bracket takes the form The algebra is completed by the following bracket The first of this brackets can be calculated to give Where we have used that The second bracket is In order to show that this expression may be identified with the linear momentum of the system we use the Noether's theorem. Let {θ c } = {φ, φ † , ψ, ψ † } the set of the fields of the our system, where c runs from 1 to 4. The theorem establish that if under a variation of the fields δθ c , the variation of the Lagrangian density is a surface term, δL = ∂ µ X µ , then exist a conserved current associated with such variation of the fields. The Noether current, assuming the summation convention over the index c, is We are interested on the zero component of this current associated to the transformations where Note that, from the Noether theorem, the only restriction for X µ is that ∂ µ X µ be a surface term. Using (57), the linear momentum is The energy-momentum-tensor, then may be defined as whereδ µ ν = 1 if µ = ν andδ µ ν = 0 if µ = ν. Finally it is easy to check that the remaining brackets are zero The soliton solution Consider now the derivation of the self-dual equations. As discussed in Ref. [14] the field B plays an important role in the derivation of self-dual equations. Indeed the expression (17) of B involves the existence of a novel soliton. The action (22) may be easily reexpressed as where ζ = ±1. Using the Gauss law (16) and after a bit of algebra we have which leads, in the static field configuration, to the Hamiltonian of the form, We can choose λ ′ 1 = ζγ 2mκ and λ ′ 2 = ζγ mκ so that our Hamiltonian becomes The last integral vanish since B must be zero in de boundary. Then, at the minimum of the energy configurations, the self-dual equations are satisfied Notice that for the particular choice ζ = 1, we recover the supersymmetric case. This equations can be explicitly written by using the equation (17) which present an additional linear term from those found in Ref. [8]. When ψ is set to zero, the above set of equations reduces to Assuming a solution of the form φ = √ ρ b , we arrive to Differentiating with respect to x, we get the following one-dimensional Liouville type equation We propose as the solution of this equation the following series Here a n are the real coefficients of series, b is a real constant and we have renamed ρ as ρ b (x). In order to check that this is really a solution we rewrite Eq.(73 ) as follows −(∂ x ρ) 2 + (∂ 2 x ρ)ρ + υρ 2 (ρ − 1) = 0 (75) where υ = ζγ κ . When the series (74) is introduced into Eq.(75) we obtain ∞ n,m=1 − a n a m mnb 2 + n 2 a n a m b 2 + 2υa n a m sech n+m (bx) + ∞ n,m=1 a n a m nmb 2 − n 2 a n a m b 2 − na n a m b 2 sech n+m+2 (bx) − ∞ n=1 (n 2 + n)a n b 2 sech 2+n (bx) + ∞ n=1 n 2 a n b 2 + υa n sech n (bx) + υ ∞ n,i,m=1 a n a m a i sech n+m+i (bx) = 0 (76) where for arriving to this expression we have used the relation tanh 2 (bx) = 1 − sech 2 (bx) So we have an expansion of powers of sech(bx) which must be equal to zero. This implies that the coefficient of each power must vanish separately. From the coefficient of sech(bx) we obtain whereas from the coefficients of sech 2 (bx) and sech 3 (bx) we have and The method can be continued in order to determine the rest of the coefficients. Conclusion In this article we have studied a (1+1)-dimensional model introduced in the description of the supersymmetric-ultracold gases. This model and its supersymmetries was previously studied in Ref. [6,7,8]. However the problem of finding the supersymmetry algebra that generate the full theory was unresolved. In this note we started by extending supersymmetrically a model proposed by Manton, and related it to the theory of ultracold gases. Then, the correct supercharge that generate the full theory were identified and their algebra was constructed. In addition the solitonic structure was analyzed and novel solitons are found.	2014-01-18T20:17:36.000Z	2010-10-29T00:00:00.000	{ "year": 2010, "sha1": "0356da59875d63a85c8f7a16b104dad788a1841d", "oa_license": "CCBYNCSA", "oa_url": "https://ri.conicet.gov.ar/bitstream/11336/68000/2/CONICET_Digital_Nro.aa3d413a-bf1b-4f28-b7b6-df044a18ee31_A.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "0356da59875d63a85c8f7a16b104dad788a1841d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Mathematics" ] }
257912453	pes2o/s2orc	v3-fos-license	Objective measurement of retention of laparoscopic skills: a prospective cohort study Introduction: There has been an overall growth of 462% in laparoscopic procedures performed by surgical residents between 2000 and 2018. Therefore, training courses in laparoscopic surgery are advocated in many postgraduate programs. While the immediate effect is determined in some cases, the retention of acquired skills is rarely investigated. The objective of this study was to objectively measure the retention of laparoscopic technical skills to offer a more personalized training program. Methods: First year general surgery residents performed two fundamental laparoscopic skills tasks (Post and Sleeve and the ZigZag loop) on the Lapron box trainer. Assessment was performed before, directly after, and 4 months after completing the basic laparoscopy course. Force, motion, and time were the measured variables. Results: A total of 29 participants were included from 12 Dutch training hospitals and 174 trials were analyzed. The 4 months assessment of the Post and Sleeve showed a significant improvement in force (P=0.004), motion (P≤0.001), and time (P≤0.001) compared to the baseline assessment. The same was true for the ZigZag loop: force (P≤0.001), motion (P=0.005), and time (P≤0.001). Compared to the 4 months assessment, skill deterioration was present for the Post and Sleeve in the mean force (P=0.046), max impulse (P=0.12), and time (P=0.002). For the ZigZag loop, skill decay was observed for force (P=0.021), motion (P=0.015), and time (P≤0.001) parameters. Conclusion: Acquired laparoscopic technical skills decreased 4 months after the basic laparoscopy course. Compared to baseline performance, participants showed significant improvement, however deterioration was observed compared to postcourse measurements. To preserve acquired laparoscopic skills, it is recommended to incorporate maintenance training, preferably with objective parameters, in training curricula. Introduction The increase of minimally invasive surgery (MIS) is a growing trend across multiple countries and within multiple surgical specialties [1][2][3][4] . The implementation of MIS has been an essential development in surgery with many benefits such as: faster postoperative recovery, shorter hospital stay, less surgical trauma, and reduced scarring compared to traditional open surgery [5][6][7][8][9][10] . Furthermore, an increase in the demand for enhancing and training laparoscopic technical skills of surgeons and residents has also been observed [11][12][13] . Surgical residents had an increase of 462% in laparoscopic procedures between 2000 and 2018 [3] . Training in MIS techniques such as laparoscopy has proven to reduce errors during surgery and to optimize patient safety and outcomes [14][15][16][17][18][19] . Regardless of training curricula methods such as virtual reality training, video training or laparoscopic box training, assessment of technical skill is also important. Assessment and feedback on performance offer trainees with a clear goal and the degree of skill progression also increases as trainees are more motivated [20][21][22] . Optimization of MIS training, in order to reduce costs of expensive facilities, requires knowledge on effect of training and retention of trained skills. HIGHLIGHTS • Objective assessment in laparoscopic training, showed skill decay after 4 months. • This is the first study to assess skill retention with force, motion, and time. • The trainees remain better compared to their baseline assessment. • Previous studies used subjective assessment. • Maintenance laparoscopic simulation training using objective feedback is essential. Prior studies have investigated and observed surgical skill decay using subjective assessment methods and reported the importance of maintenance training [23,24] . However, no objective parameters have been priorly used to compare baseline with retention measurements. In other words, a research gap exists regarding defining laparoscopic skills retention and deterioration, using objective parameters such as force and tissue manipulation parameters. Previously, our research team validated the basic laparoscopy course (BLC) (Amsterdam Skills Centre) using objective force, motion, and time parameters [22,25] . The BLC showed improvement of basic technical laparoscopic skills of surgical residents in an early phase of the learning curve. Next to this, studies have shown that maintenance training should be part of every simulator curriculum as surgical skill decay was observed using subjective assessment methods [23,24,[26][27][28] . The aim of this study was to analyze long-term MIS skill retention, based on objective parameters in surgical novices. Study design and participants In this multicenter prospective cohort study first year Dutch surgical residents (PGY 1) that completed the BLC were included for the analysis. Ethical Review Board approval was not required and participation was on a voluntary basis. The participants received a questionnaire (Supplemental File A, Supplemental Digital Content 1, http://links.lww.com/JS9/A167) for baseline characteristics, average time since the completion of the BLC and laparoscopic experience. Protocol The BLC consists of a 3 weeks at-home laparoscopic box training with seven previously validated laparoscopic tasks [22,29] . The BLC is followed by a training day 4-6 months after the course in which the participants perform a laparoscopic cholecystectomy and appendectomy on fix for life cadaver models [30] . To assess retention of skill, data of two tasks of different complexity were analyzed (Fig. 1, Post and Sleeve and ZigZag Loop). Participants performed a baseline assessment (BLA) before the BLC, a postcourse assessment (PCA) after the BLC and a retention test at the 4-6 months training day (4MA) (Fig. 2). The objective parameters of the 4MA were compared to the BLA and the PCA. Study protocol and work was reported according to STROCSS criteria [31] , Supplemental Digital Content 2, http:// links.lww.com/JS9/A168. Subanalysis of the 4MA Analyses were performed to compare the effect of time between box training and the 4-month assessment. The participants were divided into three groups: 0-3, 4-6, and greater than 7 months. Furthermore, the effect of experience in laparoscopic procedures (0, 1-10, and > 10) and simulation training (0-150, 151-240, and > 241 min) on the 4MA was also analyzed. Systems and hardware The Lapron box trainer (Amsterdam Skills Centre, Amsterdam, The Netherlands) (File b, Supplemental File A, Supplemental Digital Content 1, http://links.lww.com/JS9/ A167) was used during the BLC. The Lapron box trainers were equipped with the ForceSense objective measuring system (MediShield B.V.) [32] . The box trainer provided in total seventeen force, motion, and time parameters of which the most relevant parameters are shown in Table A1 (Supplemental File A, Supplemental Digital Content 1, http:// links.lww.com/JS9/A167) [22,32,33] . The tasks were recorded and uploaded with the objective parameters to an online database. The Lapron box trainers were also equipped with two curved Maryland grasping forceps (Aesculap, B. Braun) for the laparoscopic tasks. Statistical analyses The objective parameters were imported from the online database into IBM SPSS statistics 28 (SPSS Inc.) for statistical analyses. The Shapiro-Wilk test was performed to test for normality and the data was not normally distributed. Nonparametric tests were chosen for statistical analyses. The Wilcoxon signed-rank test was conducted to compare the 4-month assessment with the baseline course assessment and PCA. Mann-Whitney U-test was performed to determine the effect of laparoscopic experience (number of procedures and hours of simulation training) and time since BLC completion, on the retainability of laparoscopic technical skills. Differences were considered statistically significant if P less than 0.05. Four-month assessment compared to postcourse assessment Table 1 and Figure 3 show Also, the total path length (4572 vs. 3849 mm; P = 0.015), and time (76 vs. 60 s; P ≤ 0.001) were also higher. Four-month assessment compared to baseline assessment The results of the 4MA and BLA comparison are provided in Table 2 and Effect of experience in laparoscopic procedures and simulation training The effect of experience in laparoscopic procedures and simulation training on the 4MA, is presented in Table B5 and B6 (Supplemental File B, Supplemental Digital Content 1, http://links.lww.com/JS9/A167). No significant differences, based on laparoscopic experience, were observed in the objective outcome measurements of the 4MA. Laparoscopic simulation training also showed no significant difference in the 4MA results. Effect of time between box training and retention test The effect of time between the box training and 4MA are demonstrated in Table B4 Discussion This study demonstrated that participants of the BLC show skill decay, on average after 4 months, for the Post and Sleeve task (3/ 11 parameters) and ZigZag loop (5/11 parameters) when compared to the PCA. Although, the laparoscopic technical skills of the trainees deteriorate compared to just after the course, the trainees remain better compared to their BLA. For the most difficult task (ZigZag loop), the participants even perform significantly better for all objective parameters (11/11) compared to the first measurement. No effect was observed on the 4-month assessment regarding laparoscopic experience and time between box training and training day. Participant performance after 4 months already showed an significant deterioration of laparoscopic technical skill, especially force measurements, and tissue handling parameters. However, this does not exclude that a trainee might have improved in procedural skills or knowledge. Surgical skills simulation training and assessment should not be limited to International Journal of Surgery one-off training courses but be an intrinsic part of the residents' and surgeons career and learning curve. It is important to define a clear preset proficiency level or expert bench mark to continuously analyze the learning curve of participants during the training years. Specifically, during low volume moments of the residency training program. Prior research has reported various outcomes regarding the retainability and decay of laparoscopic technical skills. Some studies showed skill retention 1-2 years after the initial measurement [26][27][28] . However, other studies have shown deterioration of laparoscopic technical skills 6 months to 1 year after the baseline test [34][35][36][37][38] . All previously conducted studies used subjective assessment. The studies performed an initial measurement on a laparoscopy box or virtual reality trainer, which was followed up by a postmeasurement at various time intervals. Next to subjective assessment, laparoscopic skills were assessed by total completion time of the task or task specific errors. This does not take into account objective parameters such as force (that mimic tissue manipulation) and the movements of laparoscopic instruments. A strong point of the current study is the objective measurement of force, motion, and time, avoiding subjective assessment/ evaluation by individual assessors. This results in no interrater variability and the assessment can be compared objectively. Also, this study had a relatively big and homogeneous inclusion of surgical residents (PGY 1) from multiple training hospitals in the Netherlands. In conclusion, the basic laparoscopic course is efficient in acquiring and retaining fundamental laparoscopic technical skills and the improvement of skill is prevalent after 4 months. To maintain the laparoscopic skills, it is desirable to consistently practice laparoscopic skills with objective force, motion, and time feedback. Ethical approval The Basic Laparoscopy Course is part of the surgical residency program, and participating in the study was voluntary (and without consequences). The study was exempt from Ethical Board review. No patient data was used; no ethics approval was required, and the study has thus not been registered with patient research registries or databases. Sources of funding No sources of funding were used. Author contribution M.R.: conception and design, drafting the article, final approval, study design, inclusions, data collection and analysis, manuscript writing and revisions. S.F.H.: conception and design, drafting the article, final approval, study design, inclusions, data collection, manuscript writing and revisions. S.R.S.: conception and design, drafting the article, final approval, inclusions, data collection and analysis, manuscript writing. H.J.B. and F.D.: conception and design, drafting the article, final approval, study design, methods, manuscript writing, manuscript revisions, supervision. Conflicts of interest disclosure All authors declare that they do not have any conflicts of interest or financial ties to disclose.	2023-04-04T06:17:22.222Z	2023-04-01T00:00:00.000	{ "year": 2023, "sha1": "d9be185bd8d71af16f8151848f1f06a197798a78", "oa_license": "CCBY", "oa_url": "https://journals.lww.com/international-journal-of-surgery/Abstract/9900/Objective_measurement_of_retention_of_laparoscopic.241.aspx", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "c9484150cde55870b13f7431f7c97bb71337e7f0", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
55121119	pes2o/s2orc	v3-fos-license	Determination of Boron in Grape ( Vitis vinifera ) by Azomethine H Spectrophotometric Method Boron content of 10 grape (Vitis vinifera) varieties was determined by using Azomethine H spectrophotometric method. The grape samples have been taken from Adana and Niğde region of Turkey. The results varied between 0.59 and 9.51 mg.kg. Consequently, the differences have been found in boron content of five regions’ (Kamışlı, Armutlubağ, Bozyer, Bağaltı, Çatılıyer) grape varieties. The highest boron content was in Armutlubağ region. Results revealed that the Turkish grape is a good natural source of boron. Introduction Boron is one of the trace elements in nature, occurring only in minute concentrations in natural systems.Yet, it is one of the most important trace elements for micronutrients of growing plants [9].Boron is necessary for vascular plants, diatoms and some species of marine algal flagellates although it is apparently not required by bacteria, fungi, green algae or animals [5]. Boron deficiency in plant may result in reduced growth yield loss, and even death, depending on the severity of deficiency.However, excess boron is toxic to plants [10]. Even though grape include boron is known, there are no data available for boron content of Turkish grapes.In a study, the boron content of red grape is determined as 0.50 mg boron in a 10 5 mg sample [4]. Because boron in plants is dependent on the availability of boron in the soil, the same food crop can vary greatly in boron content depending on where it is grown [2].Boron in food, sodium borate and boric acid are well absorbed from the digestive tract.It has been proposed that boron contributes to living systems by acting in directly as a proton donor and that it exerts an influence on cell membrane structure and function [2] and is involved in enzymatic reactions. Boron functions closely with calcium and vitamin D in the preservation of bone mass and the prevention of bone demineralization and stimulates immune system and inflammatory and hormonal responses [8].The optimal dose of boron for prevention of osteoporosis, osteoarthritis and proper physiological function appears to be between 3-6 mg.day -1 [2].Fruits, legumes, nuts, sea vegetables and vegetables are the most important dietary sources of boron [6]. Thus the determination of boron in plants is becoming increasingly important.In literatures a number of methods have been reported to determination of boron [10] such as spectrophotometry, potentiometry, chromatography, flame atomic emission and absorption spectrophotometry, ICP-OES, ICP-MS, mass spectrometry (MS) and neutron activation analysis [7].Among all these methods, colorimetric and ICP-OES methods have been extensively applied B determination in plant samples.Colorimetric methods, in general, suffer from numerous interferences and have poor sensitivity and precision.In ICP-OES, besides matrix interferences, the two most sensitive emission lines for B suffer strong spectral interference from Fe.The ICP-OES is not adequately sensitive for some nutritional work involving low B concentrations [7]. ICP-OES and spectrophotometric Azomethine H method methods have been applied for determination of boron in hazelnut.The comparison of the methods indicated that there was no significant difference at a confidence level of 95%.The values found by spectrophotometry and ICP-OES indicating that both methods provide sufficiently accurate results.[8]. There are a number of specific reagents for colorimetric determination of boron. In this work, azomethine H spectrophotometric method was used for determination of boron in grape with seed. The azomethine H method worked in this system is a good method with high sensitivity. Furthermore, it has been purposed that boron content of the same plant in different regions is compared in this study. Materials Ten grape (Vitis vinifera) varieties used in this study, were obtained from Kamışlı, Armutlubağ, Bozyer, Bağaltı, Çatılıyer regions of Turkey at harvest of October 2004.Total sample quantity for all varieties was 10 6 mg each and 10 5 mg was taken from this batch.The samples were grinded with their seeds using a blander. All chemicals used were of analytical grade unless otherwise stated.The aqueous reagents were prepared in distilled water.To avoid possible contamination due to contact with borosilicate glass, all reagent solutions are prepared using plastic ware.Boric acid standard solution (1000 mg L -1 ) was used for standardization. Methods Three grams of blend grape were weighed in crucibles.Due to the high volatility of boric acid, pH was increased up to approximately 7.0 using 0.1M NaOH.Samples were dried for 12 h at 75°C in oven and the dry samples were kept in furnace for 3 h at 525°C until ashed.Ashes were extracted with 10 mL of 2M HNO 3 and were heated on a hot plate.After dissolution, the contents were filtered by filter paper and diluted to a final volume of 50 mL with distilled water.This final solution was used for analysis.All analysis was made in triplicate. Spectrophotometric method The method used for the spectrophotometric determination of micro amounts of boron is based on the boron-catalyzed condensation of salicylaldehyde with H acid.Under the optimum conditions, the wavelength of maximum absorbtion of the yellow complex was found as 412 nm.Boron concentrations were measured in 1.00 cm sample cells, at 412 nm, according to Azomethine H method [3]. Instruments A Shimadzu double beam UV-VIS spectrophotometer, Model UV-2101 PC, was used for azomethine H method and measurements of pH were made with an Inolab pH meter. Statistical methods For the statistical analysis we choose the analysis of variance (ANOVA) in Statistical Analysis System (SPSS 11.0 for windows).The significance of differences between mean values was determined by a multiple range test (LSD; Least Significant Difference).For this reason alpha (α) was preferred to be 0.05 which corresponds to a confidence of level of 95%. Results and discussion The absorption spectra of azomethine H are measured against the reagent blank (Fig. 1.). As the maximum absorption of azomethine H is found at 412 nm, the wavelength of 412 nm was chosen for the spectrophotometric determination of boron in the samples. Previously reported azomethine H methods have measured the developed color at pH values ranging from 7.5 and 7.8 [3].Within this pH range, boron-salicylaldehyde chelate takes form.Then the condensation is completed within 15 min at 25 °C and 2.2 pH value.However in the absence of boron and at alkaline pH, the salicylaldehyde ion, itself, exhibits appreciable absorption at approximately 420 nm (Fig. 2.).Adjustment of the blank pH between 2.0 and 2.4 for color measurement avoids this interference.The tolerance for Ca, Mg and Fe which are present in great amounts in plants may interfere when boron is determined directly in some samples.We found that ascorbic acid EDTA addition to the reaction system can be very effective in masking these ions and greatly improve the selectivity.The amount of EDTA in the buffer masking reagent (2%) increased to extend the tolerance levels for these ions.In this study, azomethine H spectrophotometric method has been applied for determination of boron in white and red varieties of five different regions.Boron contents of samples in these regions have been compared by using SPSS computer program.A comparison of results is given in Table 1, Fig 3 and The table shows that some grape varieties have rather rich boron content and therefore can be used as nutritional source for boron. Both red and white grape varieties regarding boron content, the richest and poorest species are at Armutlubağ region and Bağaltı region, respectively.On the other hand, from nutritional point of view, it is known that the hard-crusted fruits are the most important boron sources (~12 mg kg -1 ); these are followed by fruits (~ 4 mg.kg - ) and then vegetables (~ 1.7 mg kg -1 ) to complete the sequence [1]. The results found in this study that the maximum level for boron content is 9 mg.kg -1 (Armutlubağ region) and this is followed by 7 mg.kg - (Kamışlı region) and then 5 mg.kg -1 (Bozyer region).Therefore these regions' grape is rich enough and important source for boron in human nutrition.Consequently, any differences have not been found between boron content of red grape and white grape varieties, however the differences have been found in boron content of five regions' grape varieties. Conclusion Spectrophotometric azomethine H method has adequate sensitivity and accuracy for boron determination in grapes.Azomethine H method is the most common and powerful technique for boron determination.UV-VIS spectrophotometers are easily available in most laboratories and can be confidently and economically used for boron determination in grapes. The results show that the boron content of grapes in different regions in the same area of Turkey, have been found. Fig 1 . Fig 1. Absorption spectrum of condensation product (Azomethin H) in the presence of 1 mg/L boron, corrected for reagent blank, 1 cm cells. Fig 4 . Fig 4. Boron content of white grapes in different regions. Table 1 : Boron content of some grapes	2018-12-05T12:26:29.493Z	2016-06-01T00:00:00.000	{ "year": 2016, "sha1": "cc6a83cc9f9fefeb490b0fb534dd3a7250a70329", "oa_license": "CCBY", "oa_url": "http://www.eurasianjournals.com/pdf-77002-13180?filename=Determination%20of%20Boron%20in.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "cc6a83cc9f9fefeb490b0fb534dd3a7250a70329", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "extfieldsofstudy": [ "Chemistry" ] }
8146250	pes2o/s2orc	v3-fos-license	RALI System Description for CL-SciSumm 2016 Shared Task . We present our approach to the CL-SciSumm 2016 shared task. We propose a technique to determine the discourse role of a sentence. We differentiate between words linked to the topic of the paper and the ones that link to the facet of the scientiﬁc discourse. Using that information, histograms are built over the training data to infer a facet for each sentence of the paper ( result , method , aim , implication and hypothesis ). This helps us identify the sentences best representing a citation of the same facet. We use this information to build a structured summary of the paper as an HTML page. Introduction One's task in research is to read scientific papers to be able to compare them, to identify new problems, to position a work within the current literature and to elaborate new research propositions [8]. This implies reading many papers before finding the ones we are looking for. With the growing amount of publications, this task is getting harder. It is becoming important to have a fast way of determining the utility of a paper for our needs. A first solution is to use web sites such as CiteSeer, arXiv, Google Scholar and Microsoft Academic Search that provide cross reference citations to papers. Another approach is automatic summarization of a group of scientific papers dealing with the subject. This year's CL-SciSumm competition for summarization of computational linguistics papers proposes a community approach to summarization; it is based on the assumption that citances, the set of citation sentences to a reference paper, can be used as a measure of its impact. This task implies identifying the text a citance refers to in the reference paper and a facet (aim, result, method, implication and hypothesis) for the referred text. We are building a system that given a topic, generates a survey of the topic from a set of papers. That system uses citations as the primary source of information for building an annotated summary. Our system must be able to identify the purpose/polarity/facet of a citation. to direct the reader towards the more relevant information. The summary is built by selecting sentences from the cited paper and the citations. This process uses a similarity function between sentences. The resulting summaries are presented in HTML format with their annotations and links to the original paper. The only task that is not performed by our system is finding the text referred to by the citation. We intend to use the information already found by our system (facet of citations and sentences) to complete that task. We had already some experience in dealing with scientific papers and their references, having participated to Task2 of the Semantic Publishing Challenge of ESWC-2014 (Extended Semantic Web Conference) on the extraction and characterization of citations. A short review of previous work follows in Sect. 2. We will summarize the task in Sect. 3 and the techniques for extracting information in Sect. 4. Finally, Sect. 5 will show our results. Previous Work There has been a growing attention towards the information carried by citations and their surrounding sentences (citances). These contain information useful for rhetorical classification [18], technical surveys [14] and emphasize the impact of papers [12]. Qazvinian [16] and Elkiss [6] showed that citations provide information not present in the abstract. Since the first works of Luhn [11] and Edmundson [5] many researchers have developed methods for finding the most relevant sentences of papers to produce abstracts and summaries. Many metrics have been introduced to measure the relevance of parts of text, either using special purpose formulas [21] or using learned weights [10]. The hypothesis for CL-SciSumm task is that important sentences can be pointed out by other papers : a citation indicates a paper considered important by the author of the citing paper. Another domain for study over scientific papers is the classification of their sentences. Teufel [19] identified the rhetorical status of sentences using Bayes classifier. To find citations inside a paper, we need to analyse the references section. Dominique Besagni et al. [1] developed a method using pattern recognition to extract fields from the references while Brett Powley and Robert Dale [15] looked citations and references simultaneously using informations from one task to help complete the second task. Task Description For this year competition we were given 30 topics, 10 for training, 10 for tuning and 10 for testing [9]. Each topic is composed of a Reference Paper (RP) and some Citing Papers (CPs). The citing papers contain citations pointing to the RP. An annotation file is given for each topic. That file contains information about each citation, the citation marker and the citance. There are two mandatory tasks (Task 1A and Task 1B) and an optional task (Task 2) 3 . Task 1A : Find the part of the RP that is indicated with each citance. This will be called the referenced text. Task 1B : Once the referenced text is identified, we need to attribute a facet to it. A facet is one of these : result, method, aim, implication and hypothesis. Task 2 : Building a summary for the RP using the referenced text identified in Task 1A. Both the training and the developing set of topics contain expected results for these tasks. The next section will describe how our system performs on the test set. Our Approach For the first task, we have to find the referenced text and its facet. We hypothesized that the referenced text should be sentences sharing the same facet as the citance. We use that fact to reduce the set of sentences to choose from for the reference. This is why we execute Task 1B on all the sentences of the RP and all the citances prior to Task 1A. We now present how we determine the facet of a citance, then the facet for sentences in the RP and finally the referenced text. Task 1B : Facet Identification Our goal is to be able to use our system for papers from different domains, without having to train them again. Toward that objective, our system only uses words that are not domain specific. Patrick Drouin [3,4] compiled such a list of words in his Transdisciplinary scientific lexicon (TSL). This lexicon comprises 1627 words such as acceptance, gather, newly, severe... We will denote the set of words from the lexicon using w ∈ L. We trained two systems, one to attribute a facet to sentences in the RP and one to attribute a facet to citances. We determine the word distribution for each facet using an histogram. We only use words appearing in the TSL. This computation yielded a sum of each words present in all referenced text for each facet. The facet with the highest score is chosen for that sentence. For training our system, we extract the reference sentences from each annotation with their assigned facet. Each sentence is tokenized using the NLTK library in Python. Only words from the TSL are kept. Our dataset consists of pairs of list of words with a facet : We build a profile (h f ) for each facet using a histogram. For each word in the lexicon, we compute the number of times it appears in sentence paired with the a specific facet. When a word appears more then once in a sentence, all its occurrences are counted. Once the histogram is built, we use it to find the facet of new sentence. First, we extract the words that are part of the lexicon from the sentence, yielding the list of words p. Then a score s f for each facet is computed by adding the profile of each word for that facet. The facet that scored the highest value is assigned to the new sentence. Looking closely at the results for the profile, we saw that some words have a negative effect on finding the facet. To find a better sublist of words to use within the TSL, we used a genetic algorithm that uses a population of lists of words. A genetic algorithm starts with an initial population (set of possible solutions) and tries to find better solutions by applying small changes to existing solutions. In our case, a solution is a subset of words L i of L. The initial population is built using random subsets. To build the next generation, we use three different techniques : 1. Adding a random word to an existing solution : Removing a random word from an existing solution : Combining two subsets of existing solutions : Once enough solutions are built for the new population, each solution is tested using cross-validation with the histogram. The list that performed best in the task is kept for the next generation. We use the same technique over the dataset consisting of the citance texts and their facets. Task 1A : Finding the Sentences Referred to by Citances Having determined the facet of sentences in both the RP and citances, we are now ready to assign referenced text to citances from the CPs. Our hypothesis is that a citance should have the same facet as the text it refers to. We extract Q f the subset of sentences from the RP that have the same facet f as a citance c i . To choose the sentence of RP referred to by the citance, we look for the sentence from Q f that is the most similar with the citance c i . sim mcs (P 1 , P 2 )= 1 2 (hs(P 1 , P 2 ) + hs(P 2 , P 1 )) (1) We use the similarity function sim mcs defined by Mihalcea, Corley and Strapparava [13]. This similarity function between sentences P 1 and P 2 (Equation 1) averages two values, the similarity from P 1 to P 2 , and the similarity from P 2 to P 1 . The similarity from one sentence P i to the other P j is computed by first pairing each word from the first sentence w ∈ P i with a word in the second one v ∈ P j . A word is paired with the one that is the most similar to it (Equation 3). For each pair (w, v), the value of the similarity is weighted by the Inverse Document Frequency of the first word idf w (Equation 2). The average of these weighted similarity values is computed to yield the similarity between P i and P j . We use only words that are Noun, Verb, Adjective and Adverb for this comparison. The POS_tagger of NLTK was used to assert the tag of each word. Since we believe that the domain of the paper is important to compute that similarity, we use all words, not only the ones that are part of the TSL. Mihalcea et al. [13] reported that within the set of possible metrics to compare words, the one proposed by Zhibiao Wu et Martha Palmer [20] yielded good result (denoted sim wup ). This metric is also available with the NLTK package. To use that metric, we transform each word into their synonym group synset using WordNet. The IDF was computed for each synset. The computation was done over the set of all the documents contained in the ACL Anthology Network 4 . Task 2 : Summarization Multiple source summarization adds three problems [17] : 1. Redundancy : a paper will often be cited for the same reason over and over, resulting in many citances having the same subject. 2. Identifying important differences between sources : our goal will be to find those citances/references that bring new information and important information to the summary. 3. Coherence : since sentences come from many sources, we want to ensure that the summary forms an unified whole. For Task 2, we choose to use the Maximal Marginal Relevance (MMR) proposed by Jaime G. Carbonell et Jade Goldstein [2]. Their technique is presented in Equation 4, in which R is the list of possible sentences and V is the summary. They propose to use the title of the research paper as the starting query Q. At each iteration, their algorithm adds a sentence s i to V . Sentences are choosen so that they bring new information to the summary (Points 1 and 2) and it must have a certain amount of similarity with the query (Point 2). λ must be adjusted to balance between adding a sentence very similar to the query and a sentence very different from the ones already in the summary V . We use the same metric (sim mcs ) as for task 1A to compare sentences. We divided the summarization process in two steps : adding sentences from the citance (R = CT) and adding sentences from the paper (R = RP). In the first step, the algorithm chooses sentences from the set of citances until it reaches 150 words. For that part, we use λ = 0.3 to give priority to similarity with the query, trying to remove meaningless citances. Since citances have been identified as bringing new information not present in the original paper, we believe it is important to keep them in the summary. Then, the summary is completed (to 250 words) using sentences choosen from the RP. Here, we use λ = 0.7. Since sentences are choosen in the RP, most of them are about the same subject, we want to give priority to sentences that are more different. The summary is built in an XML format. Each sentence is identified with its position (the id of the paper it was extracted from, the sid and ssid attributes inside the XML source files). The citances contain the id of the referred paper. This information will enable to point a reader towards the corresponding paper. To help analyse the summaries, our software builds an HTML page containing the extracted information (see Fig. 1). Task 1 We present our results for facet attribution to citance and reference text. The set of data we receive is divided in two : the training set contains 197 sentences distributed over the citances and 247 sentences over the reference text; the development set contains 273 sentences distributed over the citances and 330 sentences over the reference text. We first train our system using the training data (T) and then we retrained it using both set training and developing set together (TD). In each case, we test the result over both sets. We show the result for simple training of the histogram and for the training using the genetic algorithm (gen_T) to select the list of words to consider. We also trained our histogram without limiting to the words in the TSL for comparison purpose. For the genetic algorithm, we let it run over 25 generations. Each generation started with 1 000 lists of words. 9 000 lists are added using the proposed mutations, bringing the number of lists to 10 000. The result of these experiments are presented in Table 1 and Table 2. We see that, using the training set T gives good result on itself but lower result when we apply it on the development set. After training with both set TD (Test + Development), the result over the development set raises at the expense of the result for the training set. For citance, the genetic algorithm yields better result over the training set only. It does not help to get better histograms. Considering that fact, we ask ourselves if it is possible to Once we had identified the facets, we ran our script for finding the reference text. It was able to reach an F1 score of 0.095 over the training set and 0.052 over the development set (table 3). We reduced the search space for the referred text using the facet of the citance. Since the identification of the facet is not perfect, this reduction might remove a sentence we are looking for. In the future, we have to test our approach with all sentences, instead of the reduced set, to see if this reduction of space causes a problem more than helps the solution. Figure 1 shows the HTML interface we have generated for showing the result of our system. It allows for selecting different topics. The top of the page lets us choose between the different topics that were summarised. Each topic will present, on the left side, the text of each CPs and RP. The sentences have been divided and citance identified. The right side contains the different summaries that our software builds (using different values of λ) and the gold standard summary. Each paper links to its pdf version on the ACL Anthology 5 . Task 2 On the left side of the top part of the figure we see the RP divided in sentences. On the right side, there is a summary built by choosing five sentences from the set of citances using a λ of 0.3. These sentences where selected to be as different as possible by the MMR algorithm. The bottom screen shoot (Fig. 1) presents one of the CP on the left. The citance and citation are colored to be easy to identify. The third sentence from the top was selected by the algorithm for the summaries. Conclusion We presented the use of distinguishing between topic and non-topic (TSL) words for determining the facet of sentences in a paper. This technique is useful because it lets our system work on paper in a domain independent way. We obtained good results with a simple histogram. We still have to test our histogram over other domains, to see if they also yield good results. Our experiments with a genetic algorithm to refine the list of used words did not show any improvement. We presented our interface for browsing the results of our system. That interface presents RP, CPs and summaries with links to the original paper. This interface helps the reader browse through a topic.	2016-06-21T08:51:46.632Z	2016-06-01T00:00:00.000	{ "year": 2016, "sha1": "d365f35c82f946fb114045f596447427cb5bc2b1", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "ACL", "pdf_hash": "d365f35c82f946fb114045f596447427cb5bc2b1", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
258512620	pes2o/s2orc	v3-fos-license	Development and Validation of Microsatellite Markers for Barnyard Millet Obtained by Partial Genome Assembly : Barnyard millet ( Echinochloa esculenta L.) is grown for human consumption as well as fodder. Barnyard millet is the second most important small millet after finger millet. An Ion S5 Next Generation Sequencer (NGS) was used to sequence the 400bp DNA library of barnyard millet to obtain a draft genome and for mining of microsatellite markers. The de novo assembly yielded assembled reads of 59,67,79,933 bp with 11,39,481 contigs. A total of 46,157 SSRs were identified from 11,39,481 contigs examined. The number of sequences containing SSR were 41,591 and the number of sequences containing more than 1 SSRs were 3,867. Fifteen SSR markers were validated among the 30 accessions of barnyard millet. The average percentage polymorphism of markers was 66.54% with average Polymorphism Information Content (PIC) of 0.28 and average SSR primer index (SPI) of 0.57. These microsatellite markers can be used for linkage mapping and genes/QTLs tagging for genetic improvement of target traits in barnyard millet and related crops. Millets are said to be orphan crops as they stand second after staple cereals crops, but they provide food security to farmers and consumers residing in harsh hot climatic regions of Asia and Africa (Wilson and VanBuren, 2022).Pearl millet and Sorghum are the major millet crops followed by minor millets like foxtail millet, finger millet, proso millet, kodo millet, barnyard millet.India has second largest germplasm collection of barnyard millet in the world after Japan (Singh et al., 2021).Barnyard millet has two species "Japanese barnyard millet (Echinochloa esculenta) and Indian barnyard millet (Echinochloa frumentacea)."The two species have different chromosome numbers (2n = 54 and 2n = 36, respectively), and hybrids between them are sterile.Barnyard millet in Japan is cultivated in areas where climatic and edaphic conditions are unsuitable for rice cultivation (Yabuno, 1987).In India, barnyard millet production of 0.147 mt and productivity 1034 kg ha -1 , respectively (Renganathan, 2020).Genetic improvement following conventional approach has resulted development of six high yielding varieties of barnyard millet in India.Besides conventional breeding research on application of molecular markers in millet improvement in also gaining pace.Importance of millet as climate resilient crop and as source of genes(s) for coping with climatic adversities has drawn attention of researchers in the recent years to understand the millet genomics for genetic improvement (Wilson and VanBuren, 2022). Molecular markers use is a highly precise approach for development of cultivars with desired traits and microsatellite markers are preferred to establish marker-trait association.Microsatellites are repetitive segments of DNA scattered throughout the genome in noncoding regions between genes or within genes (introns), like short tandem repeats (STR), simple sequence repeats (SSRs), or simple sequence length polymorphism (SSLPs) (Litt and Luty, 1989).Microsatellites are defined as 2-8 bp repeats by some researchers (Armour et al., 1999), whereas others classify them as 1-6 or even 1-5 bp repeats (Schlotterer et al.,1998).The most common types of microsatellites are repeats of mono-, di-, tri-, and tetra nucleotides; however, repeats of five (penta-) or six (hexa-) nucleotides are also frequently categorized as microsatellites.The enrichment of genomic libraries through selective hybridization or primer extension has historically served as the foundation for microsatellite identification and isolation.Finding microsatellite repeats in DNA datasets like EST sequences is an alternative strategy.The discovery of hundreds of microsatellite sites in the genome of a target species is made possible by new methods like Next Generation Sequencing (NGS), which can provide a huge number of high quality genome sequences quickly and inexpensively.The microsatellite markers can be used for QTL mapping and development of linkage map (Tomar et al., 2017).Hence in the current study, partial sequence of barnyard millet genome was obtained using NGS.The contig sequences created by partial de novo assembly were further mined for microsatellite locations to design SSR markers which were validated on barnyard millet genotypes to classify them as crop specific SSR markers. DNA extraction and partial genome sequencing Barnyard millet genome sequencing was performed with DNA extracted from a genotype CO(KV)2, provided by ICAR-Indian Institute of Millets Research (IIMR), Rajendranagar (Hyderabad, Telangana, India).Fresh young leaves of the accession Co(KV)2, were used for DNA extraction as per the protocol described by Desai and co-workers (Desai et al., 2021) for little millet.The genomic DNA fragment library of around 400bp was amplified using emulsion PCR (Ion OneTouch™ 2 System, Thermofisher Scientific, USA), followed by enrichment of template positive bead recovery.The sequencing of ~400bp fragments was carried out in Ion S5 sequencer following the protocol laid down for the sequencer. Genome assembly The presence of contaminated DNA sequences in the barnyard reads were verified by BLASTing it against a database of potential contaminant DNA (fungi, bacteria and virus).Any kind of contaminating sequences were removed from the analysis.The CLC trimmer function (default limit = 0.05) (CLC Genomics Workbench 8.2 software, CLC Bio, Aarhus, Denmark) was used to eliminate the adapters sequence used in the Ion S5 sequencing process.Assembly was also performed on CLC Genomics Workbench software using the parameters Length Fraction (LF) and Sequence Similarity (SIM) between DNA reads, as described by the CLC Genomics Workbench software, with maximum stringency (0.50 LF and 0.80 SIM).The minimum contig length parameter was set to 100 bp. MISA (MIcroSAtellite; http//pgrc.ipkgatersleben.de/misa)was employed for SSR mining and identification.The minimum number of repeats used for selecting the SSRs was six for di-nucleotide repeats, five for trinucleotide repeats, and three for tetra-, penta-, and hexa-nucleotide repeats.Primers for SSRs were designed using Batch primer 3.0 with the following criteria primer lengths of 14-23 bases, GC content of 45-60%, annealing temperature of 50°C to 60°C, and PCR product size of 200bp and above. Genome Assembly After the quality filtration (mean quality score >=20) using CLC workbench as described in the materials and methods section, all the reads were used for assembly preparation.Data were pooled for runs of Ion S5 and then used for assembly.Assembly was carried out by CLC workbench V8.2 de novo assembler.The de novo assembly yielded assembled reads of 59,67,79,933 bp with a number of contigs count of 11,39,481.The maximum and minimum length of contigs was 25,557 bp and 100 bp respectively, while the average contig length of 524 bp.The N25, N50 and N75 contig sizes were weighted median values and defined as the length of the smallest contig in the sorted list of all contigs where the cumulative length from the largest contig to contig is at least 25%, 50%, and 75% of the total length respectively.In the present experiment, N25, N50 and N75 contigs size were found to be 1,672 bp, 848 bp and 413 bp, respectively (Table 1). SSR mining A total of 46,157 SSRs were identified from 11,39,481 contigs examined using MISA tool (Table 2).The number of sequences containing SSR were 41,591 and the number of sequences containing more than 1 SSR were 3,867.Moreover, the SSRs in compound form were about 2,444.Among these potential SSRs, five types of repeated motifs dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide were identified, of which dinucleotide and trinucleotide were the most abundant SSRs.The number of di, tri, tetra, penta and hexa-nucleotide SSRs were 25,191, 17,630, 2,748, 474, and 114, respectively (Table 3).Among the SSRs identified, the most dominant dinucleotide repeat motif was AG/CT (11,388) followed by AT/AT (7820) and AC/GT (5031), the most dominant trinucleotide repeat motif was CCG/CGG (4,664) followed by AGC/CTG (3,418) and AGG/CCT (1,854) while the most dominant tetra-nucleotide repeat was AGAT/ ATCT (389) followed by ATCC/ATGG (Fig. 1). SSR primer design and validation A set of 15 primers were designed from the 46,157 SSRs identified with the product size varying between 300 to 700 bp (Table 4).The primers were designed from the different contigs assembled from the reads.The validation of these primers was carried out on 30 genotypes of barnyard millet (Table 5). Out of the 15 primers, 13 primers gave an amplification at the expected base pair with the total number of 21 amplified bands.Based on the sequence data produced by the de novo assembly technique, expected product sizes for each microsatellite marker were calculated.We verified that the polymorphic loci's size ranges included the size of the expected product.This was true for 9 out of 13 primers making 69.23% of markers to produce amplicons within the range of the predicted sizes.No markers showed amplicons that were 90% bigger or smaller than anticipated.Out of the total number of bands amplified by the 13 SSR primers, 16 were polymorphic and 5 were monomorphic with an average polymorphism of 1.23 (Table 6).The average percentage polymorphism was 66.54% with average polymorphism information content (PIC) of 0.28 and average SSR primer index (SPI) of 0.57. The capacity to discover and create microsatellite markers for molecular breeding, is undergoing a true revolution.A new paradigm of microsatellite development has been put to the test by research teams with access to an NGS facility, and continual decline in prices for obtaining next-generation sequencing data has provided easy access for obtaining genomic sequences.Shotgun pyrosequencing of DNA or enriched libraries were primarily employed in the majority of the initial articles published reporting the use of next-generation sequencing technologies for the production of microsatellite markers.Transcriptome sequencing and assembly were the first applications of Ion S5 sequencing followed by mining of genic SSR markers [ (Hamid et al., 2020;Hamid et al., 2019;Hamid et al., 2018;Radadiya et al., 2021;Rathod et al., 2020a;Rathod et al., 2020b)].The genomic information generated by nextgeneration sequencer for microsatellite markers in millets can be used for the breeding millets against biotic and abiotic stresses (Desai et al., 2021;Satyavathi et al., 2022).In the present era of genomics, in year 2012 foxtail genome information was released.Later in year 2017, pearl millet, finger millet and barnyard millet genomes were announced, and lastly in year 2019 proso millet genome was released (Krishna et al., 2022).The barnyard millet draft genome size is estimated to be of 1.27 Gb covering more than 90% of genome coverage, but the genome annotation is still in the early phase (Krishna et al., 2022).Information on genomic sequences is limited for application purpose, for genetic improvement of the crop there is need to have set of molecular markers specific to barnyard millet.Cross transferability of SSR markers from other crops has been successful in barnyard millet, most of the EST-SSR of foxtail were transferred successfully (Kumari et al., 2013), foxtail genomic SSRs were also found to show positive amplifications (Pandey et al., 2013;Babu et al., 2018a) observed 100% transferability of finger millet genomic-SSRs and 71% transferability of rice genomic-SSRs.Whereas, Babu et al., (2018b) observed that Conclusion Mining of SSR in crops is essentially needed for strengthening the genetic improvement programmes. Robust markers panel development leads to create a dense linkage map for establishing marker-trait association to tag genes of interest.The thirteen markers developed in the present study using the partial genome of barnyard millet cultivar CO (KV)2 can be used for knowing genetic polymorphism in the available germplasm.Further, more SSR markers can also be mined from the available partial genome of barnyard millet obtained in the study as there are 41591 sequences containing SSRs.These markers can be used in barnyard millet for the development of linkage map, QTL mapping and the characterization of the available germplasm.Above this, the transferability of these markers can also be studied in other millets crops. Fig. 1 . Fig. 1.Distribution of microsatellite motif types and tandem repeat numbers in Barnyard millet genome. Table 1 . Statistics of barnyard millet partial genome sequence generated with Ion Torrent S5 Table 2 . The statistics of genomic data assessed for SSR mining Table 4 . Primers sequences of the SSR developed for banyard millet Table 6 . Size, number of amplified bands, percent polymorphism and PIC obtained by 13 SSR primers in the 30 genotypes of barnyard millet S = Shared; U = Unique; T = Total Polymorphic bands; PIC = Polymorphism Information Content; SPI = SSR Primer Index = Number of Bands × PIC	2023-05-06T15:15:12.486Z	2023-04-01T00:00:00.000	{ "year": 2023, "sha1": "6e9134040cffe04db4ac5baf85d8acabfc52038c", "oa_license": "CCBYNCSA", "oa_url": "https://epubs.icar.org.in/index.php/AAZ/article/download/134630/49884", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "be83f001f82e0ab30f095721b550dbdc0ca0efc5", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "extfieldsofstudy": [] }
207669618	pes2o/s2orc	v3-fos-license	Association Analysis of NALCN Polymorphisms rs1338041 and rs61973742 in a Chinese Population with Isolated Cervical Dystonia Background. A genome-wide association study (GWAS) demonstrated a possible association between cervical dystonia (CD) and a sodium leak channel, nonselective (NALCN) gene. However, the association between NALCN and CD was largely unknown in Asian population. The present study was carried out to examine the associations between the two single nucleotide polymorphisms (SNPs) rs1338041 and rs61973742 in the NALCN gene and CD in a Chinese population. Methods. In a cohort of 201 patients with isolated CD, we genotyped the two SNPs rs1338041 and rs61973742 using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). We also included 289 unrelated, age- and sex-matched healthy controls (HCs) from the same region. Result. No significant differences were observed in either the genotype distributions or the minor allele frequencies (MAFs) of the two SNPs between the CD patients and the HCs. There were no significant differences between early-onset and late-onset CD patients, between patients with and without a positive family history of dystonia, or between patients with and without tremor or sensory tricks. Conclusion. Lack of association between the SNPs of NALCN and CD suggests that the SNPs of NALCN do not play a role in CD in a Chinese population. Introduction Dystonia is a movement disorder characterized by involuntary sustained or intermittent muscle contractions affecting one or more sites of the body causing abnormal, often repetitive movements, postures, or both [1]. Cervical dystonia (CD), the most common form of late-onset focal dystonia, is characterized by sustained or intermittent neck muscle contractions causing abnormal head movements and postures [2,3]. Up to now, more than 20 loci (from DYT1 to DYT25) have been identified to be the causative loci of primary dystonia [4] after the first identified TOR1A (DYT1) gene linked to early-onset primary dystonia [5,6]. Among them, CIZ1 (Cip1interacting zinc finger protein; DYT23), ANO3 (anoctamin 3; DYT24), and GNAL (guanine nucleotide binding protein, alpha activating activity polypeptide, olfactory type; DYT25) genes have been discovered for CD [7][8][9]. However, mutations of these genes mainly are seen in familial CD patients [4,10]. Although most of patients with late-onset dystonia often seem to be sporadic patients, it appears to have a strong genetic basis [11]. Unfortunately, at present, the genetic architecture of late-onset dystonia remains largely unknown [10]. A recent genome-wide association study (GWAS) has been performed on British patients with focal CD. In this study, although no loci reached a statistically significant association with CD, the value of the single nucleotide polymorphism (SNP) rs1338041, which is located in an intron region of sodium leak channel, nonselective (NALCN) gene on chromosome 13, was rather low [12]. After subsequent imputation, the results showed a few clusters with potential significance. Rs61973742 within the 5 untranslated region (UTR) in the NALCN was one of the most meaningful SNPs [12]. However, a replicated study of the GWAS from Spain found no association between these two SNPs of NALCN-rs1338041 and rs61973742-with CD [13]. As we all know, GWAS is an efficient method to understand genetic underpinnings of genetic complicated disease that are not based on prior knowledge; however, false positive results may be produced in the meantime [14]. Therefore, it is necessary to confirm the finding of GWAS in other ethnic groups. In the current study, we investigated the association of the most potential significance SNPs (rs1338041 and rs61973742) of NALCN with CD in a Chinese population. Subjects. A total of 201 patients with focal cervical dystonia were recruited from the department of neurology at West China Hospital of Sichuan University. Patients were examined and diagnosed by movement disorder specialists according to the current criteria [15]. Patients carrying GAG deletion mutation of the DYT1 gene and having a known causes, including traumatic or structural brain lesions, or treatment with neuroleptic drugs or patients diagnosed with another syndrome with dystonia, such as Parkinson's disease or dystonia plus syndrome, were excluded from the study. Patients with positive family history of dystonia were defined as having one or more first-degree or second-degree family members with dystonia as previously described [16]. A total of 289 unrelated healthy individuals (59.9% women; mean age 43.35 ± 14.77 years) from the same areas of residence were recruited to the study as the healthy controls (HCs) group. None of the HCs had neurological disorders or psychiatric disorders examined by neurologists. Witten informed consent was obtained from all participants before being enrolled, and the study was approved by the Ethics Committee of Sichuan University. Genotyping. Peripheral blood samples were collected from all participants. Genomic DNA was extracted from peripheral blood leukocytes using standard phenol-chloroform procedures. Genotype for rs1338041 and rs61973742 was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Two SNPs were all amplified using a forward mismatched primer that creates a restriction enzyme digestion site and the primer sequences and the PCR conditions of two SNPs are shown in Supplementary Results The mean age of 201 patients with CD was 41.25 ± 15.76 years at examination time, and the mean age of onset of patients was 38.27 ± 16.06 years. Among all CD patients, the ratio of male to female was 77/124. Fifty patients (24.9%) had an age of onset less than 26 years, and forty-one patients (20.4%) had a positive family history of dystonia. The genotype frequency distributions in the controls did not deviate significantly from the Hardy-Weinberg equilibrium (0.522 and 0.464 for rs1338041 and rs61973742, resp.). No significant differences were found in the genotype distributions or MAFs of the two SNPs between CD patients and controls (Table 1). No differences were observed in the genotype distributions or MAFs between patients with early-onset and late-onset CD or between patients with and without a family history of dystonia (Table 2). There were no differences in the genotype distributions or MAFs in terms of tremor or sensory tricks (Table 3). Discussion This is the first study on the genetic susceptibility of the rs1338041 and rs61973742 SNPs of NALCN and CD in a Chinese population. In the current study, rs1338041 and rs61973742 SNPs were not found to modify the susceptibility to CD. The NALCN gene comprises at least 44 exons (43 coding exons) encoding NALCN, which is a voltage-independent and cation-nonselective channel, and is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability [17,18]. A previous GWAS showed the possible association between the two SNPs of NALCN and CD in 212 British resident CD patients of European descent. Given that ion channels are crucial components of cellular exciting ability and are involved in many neurological diseases, for example, ANO3, whose mutations have detected in sporadic CD, functions as calcium activated chloride channels which modulate neuronal excitability [19]. It supports that an ion channel may be a potential candidate gene for dystonia. Therefore, the British study came to the following conclusion: NALCN, whose encoded protein belongs to a Na + -leak channel, may be a plausible candidate gene for dystonia [12]. However, our current study and the Spanish study both failed to replicate the association of rs1338041 and rs61973742 with CD [12,13]. Several factors should be considered to explain such difference. First of all, in genetic analysis, ethnic population specificity must be taken into consideration as an essential factor. For example, according to the British GWAS, the minor A-allele of rs1338041 showed a doubtful risk to CD patients for the fact that the frequency of minor Aallele seemed higher in CD patients than controls, while the Spanish population and the present study both manifested a higher frequency of A-allele in controls than in patients, meaning a possible protective effect of A-allele. In the case of rs61973742, this phenomenon does not exist, the potential effects of these three available studies making no difference. Secondly, given that the clinical manifestation of dystonia is diversity, the patients recruited among studies could be different. However, our study is in accordance with previous studies on British or Spanish, both containing focal CD only [12,13]. Thirdly, a major difference is that the age of onset of CD in our patients (38.27 ± 16.06 years) was much less than the other two studies (British: 60.6 ± 10.9 years, and Spanish: 43.5 ± 15.7 years). Considering this factor, we divided our patients into two groups, early onset and late onset. However, no differences in the genotype or allele frequencies between early-onset and late-onset CD were found either. Therefore, these factors may contribute little to the differences. Finally, the sample size must be taken into consideration. The British GWAS reported a putative association of CD with SNPs in the NALCN. However, there was no single SNP that reached the statistical significance in that no single SNP passed the genome-wide significance level (defined as < 5 × 10 −8 ) after GWAS. The possible reason is that the sample size is not enough to discover some SNPs with small effect on the disease according to the author. The prevalence of primary dystonia in China was 27.0 per million persons based on the minimum estimates [20]. Therefore, the present study had a probability of 80% power to detect genetic effects at an OR of 1.85 under an additive model in our sample (two-sided, < 0.01) [13]. The Spanish study also had a strong power to find the association between SNPs of NALCN and CD [13]. To sum up, it seems that variations in the NALCN gene might not be associated with CD. The British GWAS might overvalue the positive effect of SNPs of the NALCN gene in dystonia [12]. In conclusion, the lack of association between NALCN SNPs rs1338041 and rs61973742 and CD suggested that SNPs of the NALCN gene do not play a role in Chinese CD population.	2018-04-03T02:52:11.872Z	2016-04-28T00:00:00.000	{ "year": 2016, "sha1": "1600502d113857f567e70d5eb465cad427d4c3c5", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/pd/2016/9281790.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ed1ea8482c88e41af2389f56b2c594e651b94126", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
266052613	pes2o/s2orc	v3-fos-license	The large-scale magnetic field of the M dwarf double-line spectroscopic binary FK Aqr This work is part of the BinaMIcS project, the aim of which is to understand the interaction between binarity and magnetism in close binary systems. All the studied spectroscopic binaries targeted by the BinaMIcS project encompass hot massive and intermediate-mass stars on the main sequence, as well as cool stars over a wide range of evolutionary stages. The present paper focuses on the binary system FK Aqr, which is composed of two early M dwarfs. Both stars are already known to be magnetically active based on their light curves and detected flare activity. In addition, the two components have large convective envelopes with masses just above the fully convective limit, making the system an ideal target for studying effect of binarity on stellar dynamos. We use spectropolarimetric observations obtained with ESPaDOnS at CFHT in September 2014. Mean Stokes I and V line profiles are extracted using the least-squares deconvolution (LSD) method. The radial velocities of the two components are measured from the LSD Stokes I profiles and are combined with interferometric measurements in order to constrain the orbital parameters of the system. The longitudinal magnetic fields Bl and chromospheric activity indicators are measured from the LSD mean line profiles. The rotational modulation of the Stokes V profiles is used to reconstruct the surface magnetic field structures of both stars via the Zeeman Doppler imaging (ZDI) inversion technique. Maps of the surface magnetic field structures of both components of FK Aqr are presented for the first time. Our study shows that both components host similar large-scale magnetic fields of moderate intensity (Bmean ~ 0.25 kG); both are predominantly poloidal and feature a strong axisymmetric dipolar component. (abridged) Introduction Magnetic fields play an important role in stellar evolution and are found throughout the entire Hertzsprung-Russell (HR) diagram ⋆ Based on observations obtained at the Canada-France-Hawaii Telescope (CFHT), which is operated by the National Research Council (NRC) of Canada, the Institut National des Sciences de l'Univers of the Centre National de la Recherche Scientifique (CNRS) of France, and the University of Hawaii.The observations at the CFHT were performed with care and respect from the summit of Maunakea, which is a significant cultural and historic site. (see Donati & Landstreet 2009 for a review).In particular, cool stars are thought to generate their magnetic field through dynamo action acting within their outer convective envelope, leading to a complex interplay between magnetic field generation, mass loss, and rotational evolution (see e.g.Brun & Browning 2017).Among cool stars, M dwarfs are main sequence stars covering the mass range between 0.08 and 0.6 M ⊙ , and are the most frequent type of stars in the solar neighbourhood (Lépine & Gaidos 2011).These stars are of particular interest regarding the generation of their magnetic fields, which involves a transition in their internal structure that takes place during the M dwarf regime.M dwarfs earlier than spectral types M3 and M4 or more massive than ∼0.35 M ⊙ have a structure similar to that of Sun-like stars -an inner radiative zone and an outer convective envelope separated by a tachocline.M dwarfs of later spectral types are fully convective (Chabrier & Baraffe 1997;Feiden et al. 2021), and therefore the generation of their magnetic fields may rely on nonsolar dynamo processes (see e.g.Morin 2012).Observational studies show not only that M dwarfs harbour the strongest magnetic fields among single cool main sequence stars (Shulyak et al. 2017;Reiners et al. 2022), but also that the topology of their surface magnetic fields change rapidly when crossing the fully convective boundary.Stars more massive than ∼0.5 M ⊙ harbour complex multipolar and time-dependant large-scale fields, while those below this threshold host strong dipole-dominated largescale fields displaying only minor evolution over several years (Donati et al. 2008;Morin 2008;Morin et al. 2010). Studying binary and multiple stellar systems gives us a better understanding of stellar magnetic fields.In general, it is assumed that the components of close systems were formed under the same conditions (at the same time and from the same interstellar parental cloud with the same chemical composition).This makes them well-suited targets for investigating the origin, properties, and impact of stellar magnetic fields.The present study is part of the BinaMIcS project (Binary and Magnetic Interactions in various classes of Stars; Alecian et al. 2015a,b).The aim of BinaMIcS is to study stellar magnetism under the influence of the physical processes occurring in close binary systems.The components of the selected systems cover a large part of the HR diagram, including a wide range of masses on the main sequence from cool low-mass fully convective stars to hot and massive stars.Two key aspects of the BinaMIcS project are that it targets both hot and cool stars in order (i) to analyse how potential starstar interactions may modify stellar activity with respect to single stars; and (ii) to assess the effect of tidal interactions in close binary systems on the dynamo-generated magnetic fields.Three cool binary systems have already been analysed in the framework of the BinaMIcS project: σ 2 CrB (F9V+G0V, Rosén et al. 2018) V1878 Ori (K2-3+K2-3 weak-line T Tauri stars, Lavail et al. 2020), and UV Psc (G5V+K3V, Hahlin et al. 2021). The present study focuses on the binary system FK Aqr (GJ 867 AC, HD 214479), discovered by Vyssotsky & Mateer (1952) and later studied in detail by Herbig & Moorhead (1965, HM65 hereafter).FK Aqr is a double-lined spectroscopic binary with an orbital period of 4.08322 d (HM65) consisting of two M1-2Ve dwarfs of similar mass; their mass ratio is q = 0.8 (HM65).FK Aqr is a bright (V = 9.09, HM65) and nearby (π = 115.01± 1.30 mas, Davison et al. 2014) spectroscopic binary, and is part of the quadruple system GJ 867.This is one of only four quadruple systems within 10 pc of the Sun and the only one among these systems with all four cool components (three M dwarfs and one probable low-mass M dwarf or brown dwarf; Davison et al. 2014).Indeed, the recent study by Winters et al. (2019) shows that while binaries are fairly common in the solar neighbourhood -indeed 26.8 ± 1.4% of M dwarf primaries have one or more companions in a volume-limited sample within 25 pc -quadruple and higher-order systems are much rarer, and make up only 0.3%.FK Aqr is the primary component of a widely separated visual binary.Its visual companion is FL Aqr (GJ 867 BD), a single-lined spectroscopic binary of spectral type M3.5Ve.With a visual magnitude of V = 11.45 and a separation of 24 ′′ .5, it does not contaminate our observations of FK Aqr.Taking into account the very similar parallaxes, proper motions, and radial velocities of these two binaries, Davison et al. (2014) consider them to be very likely physically associated. Both systems show flare activity (Byrne 1978(Byrne , 1979;;Byrne & McFarland 1980;Pollock et al. 1991).Pollock et al. (1991) reported that both components contribute to the observed X-ray emission with an intensity ratio A:B of about 3:1.Based on these and other studies, these latter authors concluded that GJ 867 BD flares more frequently but typically produces less-energetic flares than GJ 867 AC in the optical domain (one flare every 30 min with integrated energies typically between 10 29 and 5 × 10 30 erg for GJ 867 BD, and one flare every 4 hours with energies between 10 31 and 10 32 erg for GJ 867 AC).The light curve of FK Aqr does not exhibit a large-amplitude modulation.The first measurement reported in the literature is presented by Bopp & Espenak (1977), where ∆V = 0.06 mag.In subsequent studies, the photometric variability is reported to be ∆V < 0.04 mag (Byrne & McFarland 1980;Byrne et al. 1990).Cutispoto (1995) and Cutispoto & Leto (1997) even report a change in the shape of the light curves from their studies at different epochs, which they interpret as a change in the surface distribution of the spots. The present paper is organised as follows: a short description of the telescope and observational dataset is given in Sect. 2. Section 3 describes all the employed methods to process the data and characterise the system: radial velocities of both components are measured and combined with astrometric measurements in order to fully characterise the orbit of the system; longitudinal magnetic field values B l are computed from mean Stokes I and V LSD profiles; and the three classical chromospheric activity indicators (Hα, CaII H&K, and CaII IRT) are measured.In Sect. 4 we describe how the surface magnetic field topology of both stars is modelled from the Stokes V mean LSD profiles with the Zeeman Doppler imaging (ZDI) method.We discuss our results and provide concluding remarks in Sect. 5. ESPaDOnS spectro-polarimetry Observational data were obtained with the fibre-fed échelle spectropolarimeter ESPaDOnS (Donati et al. 2006a), which operates at the 3.6 m Canada-France-Hawaii Telescope (CFHT).In polarimetric mode, the instrument has a spectral resolving power of about 65 000 and an almost continuous spectrum coverage from the near-ultraviolet (at about 370 nm) to the near-infrared domain (at about 1050 nm) in a single exposure, with 40 orders aligned on the CCD frame by two cross-disperser prisms.The Stokes I (unpolarised light) and Stokes V (circular polarisation) parameters are simultaneously measured using a sequence of four subexposures, between which the retarders (Fresnel rhombs) are rotated in order to exchange the beams in the instrument and to reduce spurious polarisation signatures (Semel et al. 1993). The automatic reduction software LIBRE-ESPRIT (Donati & Brown 1997a) is applied to all observations as a component of the CFHT Upena reduction pipeline, which includes the optimal extraction of the spectrum, wavelength calibration, correction to the heliocentric frame, and continuum normalisation. In total, 26 polarimetric sequences were obtained for FK Aqr under the BinaMIcS project, covering the observational period 3-16 September 2014.The total exposure time per spectrum is 960 s (4 × 240 s).The observations reach a peak signal-to-noise ratio (S/N) in Stokes I between 233 and 731 at 871 nm.The journal of observations is presented in Table 1, where Col. 1 gives the date of observation, Col. 2 gives the heliocentric Julian date of the corresponding observation, Col. 3 gives the rotation cycles, Col. 4 gives the rotational phases, calculated assuming a rotational period of 4.08319598 days (this is a refined value of A77, page 2 of 19 Tsvetkova, S., et al.: A&A, 682, A77 (2024) Table 1.Observations and measurements of the longitudinal magnetic fields B l and radial velocities of both components of the close binary FK Aqr. Rot. the period, which we obtained with the PHOEBE code, which is described in detail in Sect.3.4).The remaining columns give the measurements of the longitudinal magnetic field and the radial velocity of both components of FK Aqr, which are described in the following section.(2011). PIONIER near-infrared interferometry Each observation has been adjusted with a simple binary model made of two uniform discs.The disc diameters have fixed values of 0.58 mas and 0.48 mas based on the expected apparent diameters of M2 dwarfs at a distance of 8.89 pc (from the Gaia parallax).These diameters are only marginally resolved by the baselines of VLTI, and therefore their exact values have negligible impact on the estimated separation and flux ratio. The list of best-fit flux ratio, separation, and position angle for each interferometric observation is summarised in Table 2. The typical uncertainty on the separation is 0.1 mas, and therefore the separation of a few milliarcseconds between the two components is very well resolved.On average, the flux ratio between the two components in the near-infrared H-band is f 2 / f 1 = 0.47 ± 0.01. Least-squares deconvolution method We used the least-squares deconvolution (LSD) multi-line technique (Donati & Brown 1997a;Kochukhov et al. 2010) to generate the mean Stokes I and V line profiles1 .In general, this technique averages several thousand photospheric atomic lines from one spectrum in order to increase the S/N, thereby allowing us to detect weak polarised Zeeman signatures.In the case of FK Aqr, we applied a line mask that is computed for M dwarfs and is already used over a sample of this type of star (Morin et al. 2010).The mask is created from an Atlas9 local thermodynamic equilibrium model (Kurucz 1993) with T eff = 3500 K and log g = 5 and a threshold in the line depth of 40% (Donati et al. 2008).As a result, around 5000 spectral lines were averaged per spectrum.The LSD profiles were calculated with the following normalisation parameters: normalised line depth equal to 0.645, wavelength equal to 722 nm, and an effective Landé factor equal to 1.20.The Stokes I profiles exhibit a pseudo-continuum level of below unity.This effect is observed in all M dwarfs and is believed to be caused by the contribution of molecular spectral lines that are not included in the line list; we therefore scaled it in order to bring it to unity.The profiles are presented in Fig. 1.Clear Stokes V signatures are visible and detected from all observations of both components.Their shapes are all the same for both stars: simple two-lobe shapes with negative blue and positive red lobes. The observations are phased according to the following ephemeris: A77, page 4 of 19 1. Radial velocity Radial velocities (RV) of both components of the system are measured from their LSD Stokes I profiles by fitting a Lorenzian function.The error bars contain both the uncertainty from the fit, and the stability of the ESPaDOnS spectropolarimeter, which is 30 m s −1 (Moutou et al. 2007).The measured values and their calculated error bars are given in Cols.9-12 of Table 1.We used these radial velocity measurements combined with the measurements given by HM65 (and the corresponding error bars of both datasets) to refine the orbital parameters of the system employing the PHOEBE code.This is described in detail in Sect.3.4. Astrometry of the binary FK Aqr We ran an orbital fit of the astrometric positions.There are many tools and methods available to perform such a fit.Here, we used the code already presented in Le Bouquin et al. (2017).The bestfit parameters are summarised in Table 3 and the orbital trace is shown in Fig. 2. We found that the eccentricity e is small and compatible with zero.Consequently, the argument of periastron of the secondary ω is poorly defined.Because we kept this parameter free, the time of periastron passage T is also poorly defined, as one can be converted into the other by a rotation of the circular orbit.Nevertheless, this is not a problem for this analysis, where we are mostly interested in the inclination i = 52.39 ± 0.45 deg and in the semi-major axis of the apparent orbit a = 5.635 ± 0.037 mas.These two parameters are very well constrained thanks to the dense sampling of the astrometric orbit. It is possible to compute the total mass of the system with Kepler's law by combining the apparent size of the astrometric orbit, the orbital period, and the Gaia distance (d = 8.897 ± 0.004 pc; Gaia Collaboration 2020).We found M t = 1.008 ± 0.020 M ⊙ . We also performed a simultaneous fit of the combined radial velocities (those of HM65 and those of 2014 ESPaDOnS observations) and astrometric observations using the code of Le Bouquin et al. (2017).This is presented in Appendix A. Fig. 2. Best-fit orbital solution to the astrometric observations.The orbital trace is the motion of the secondary around the primary (the primary being the brightest in the H-band).The periastron of the secondary is represented by a filled symbol and the line of nodes by a dashed line.The astrometric observations are the red ellipses (of typical size less than 0.2 mas), and the corresponding points along the orbit are the black dots.North is up and east is left. Refining the orbital parameters with PHOEBE As the ESPaDOnS spectropolarimeter has excellent stability, which allows very precise measurement of stellar radial velocities, we can use our measurements to refine the orbital solution of the close binary FK Aqr.There is only one orbital solution published in the literature, that given by HM65.To this end, we used the PHOEBE 2 binary modeling code (Prša & Zwitter 2005;Prša et al. 2016).PHOEBE uses the MIT licensed EMCEE backend function (Foreman-Mackey et al. 2013), which is a pure-Python implementation of Goodman & Weare's Affine Invariant Markov chain Monte Carlo (MCMC) ensemble sampler.We applied this EMCEE backend function to our 2014 ESPaDOnS dataset alone, to the HM65 dataset alone, and also to the two datasets combined into one. As a very first inspection of the radial velocity dataset, we applied a Lomb-Scargle period search analysis (Lomb 1976;Scargle 1982) over the combined dataset (HM65 dataset combined with our dataset from 2014 ESPaDOnS).Given the long temporal gap between the two datasets, the periodogram of the combined dataset for the orbital period displays numerous aliases of similar likelihood.Therefore, given the very small uncertainty that PHOEBE calculates for the period (below), we consider that the HM65 period is very reliable. Employing the EMCEE function of PHOEBE, we initially started with shorter chains to inspect the datasets; we ran the HM65 dataset alone, the 2014 ESPaDOnS dataset alone, and then both combined as one.We found that 30 walkers and 7000 iterations were sufficient to achieve very good values for the parameters of the orbital solution and a reduced χ 2 of the fit of 2 http://phoebe-project.org/A77, page 5 of 19 Tsvetkova, S., et al.: A&A, 682, A77 (2024) between 1.0 and 2.0, both within a reasonable amount of computation time.This allowed us to run more than 25 models.In different models, we gave the parameters and their intervals of exploration different central values.Moreover, in some models, we fixed the values of certain orbital parameters and allowed PHOEBE to work with fewer unknowns.We used Gaussian distributions.In general, PHOEBE's values of the orbital parameters were consistent with the values provided by HM65, except for the systemic velocity.The outcome of PHOEBE's EMCEE function is as follows: (1) applied to the HM65 dataset alone, PHOEBE's orbital solution matches the orbital solution given by HM65 within the error bars, and is consistent with the radial velocity precision estimated by these latter authors of 1.4 km s −1 .(2) Applied to the 2014 ESPaDOnS dataset alone, PHOEBE's orbital solution matches the orbital solution given by HM65 within the error bars, except for the systemic velocity of V γ ≃ −7.36 ± 0.02 km s −1 , which is different from the HM65 value of -8.7 km s −1 (no error bar is given in HM65) by approximately 1.3 km s −1 .(3) Applied to a combined dataset, there is again a difference between the two systemic velocities.A change in radial velocity over several decades could be explained by secular acceleration (Kürster et al. 2003) or orbital evolution of the wide system GJ 867 AC/BD (Davison et al. 2014), but none of these effects alone or combined could produce a variation of 1.3 km s −1 over a time span of 60 yr.We therefore assume that the difference is largely explained by the accuracy of the HM65 radial velocity measurements, which are based on chromospheric emission lines.Different lines (or wavelength intervals) are used in RV measurements, and there are offsets to be considered (due to slightly different line asymmetries).The systematic difference between the RVs from ESPaDOnS and HM65 may also come from this.We therefore shift the RV values of HM65 by 1.4 km s −1 (the radial velocity precision given by HM65). To achieve the final results presented in this paper, we ran the EMCEE function of PHOEBE with 30 walkers and 50 000 iterations over the combined dataset.We fixed the value of the angle i = 52.39• ± 0.45 • (derived in Sect.3.3).Thus, the parameters over which PHOEBE iterated, the priors, areq = 0.8 ± 0.2, e = 0.01 ± 0.01, V γ = −8.0 ± 2.0 km s −1 , a binary = 10.8 ± 2.0 R ⊙ , ω = 356 • ± 60 • , T 0 = 2437145.143805± 0.005, and P orb = 4.08322 ± 0.00007 d.The corner plot of the PHOEBE calculations is displayed in Fig. 3.The final results are given in Table 4.The last column of this table lists the output of PHOEBE and the error bars, and the middle column shows the HM65 orbital solution for comparison.The PHOEBE model and all the radial velocity measurements are shown in the upper panel of Fig. 4, while the lower panel of the same figure shows the RV residuals.The reduced χ 2 of the fit is 1.33 for the primary and 1.55 for the secondary.The root mean square (rms) of the residuals of the ESPaDOnS measurements is 0.5 for the primary and 0.3 for the secondary.The rms of the residuals of the HM65 measurements is 1.9 for the primary and 2.3 for the secondary. As described above, we present PHOEBE's orbital solution based on the combined dataset (archival RVs of HM65 and RVs of 2014 ESPaDOnS dataset).In this combined dataset, we shifted the RV values of HM65 by 1.4 km s −1 because of the discrepancy we noticed in the systemic velocity.For the completeness of this study, we ran PHOEBE again, this time using the original RV values given by HM65 (not corrected by 1.4 km s −1 ), and under the same conditions as we used for presenting the final orbital solution: combined dataset, 30 walkers, 50 000 iterations, and the same central values and intervals for the parameters.This did not lead to a change in the values of the orbital parameters, which are listed in Table 4, but we obtained slightly worse reduced χ 2 values: 1.69 for the primary and 1.78 for the secondary. v sin i values The projected rotational velocities (v sin i values) of both components of the binary are among the input parameters needed in order to reconstruct their magnetic maps.The catalogue of Eker et al. (2008) provides v sin i values of 7 km s −1 for both components.Houdebine (2008) gives a value of 4.7 km s −1 with an accuracy of 2 km s −1 using the method of crosscorrelation between the target stars and three template stars.Later, Houdebine (2010) measured v sin i again using observations from HARPS and ELODIE and the corresponding values are 7.17 km s −1 from HARPS and 4.7 and 7.3 km s −1 from ELODIE with an uncertainty of 0.3 km s −1 .These latter authors adopt a final value of 7.02 km s −1 . From the astrometric value of the inclination angle i and the orbital solution achieved with the PHOEBE code, we can easily calculate the masses of the two stars -0.55 ± 0.01 M ⊙ and 0.44 ± 0.01 M ⊙ for the primary and secondary, respectively.We then interpolated the values of the masses and radii of the M dwarfs given by Pecaut & Mamajek (2013) to find the radii of the two components of our system.We found values equal to R A = 0.56 R ⊙ and R B = 0.45 R ⊙ for the primary and secondary, respectively.From the values of stellar radius, rotational period, and inclination angle, we calculated v sin i values for both components, finding 5.4 ± 0.6 km s −1 and 4.4 ± 0.6 km s −1 for the primary and secondary, respectively.We use these two values in our modelling of the magnetic field topologies of the two stars as described in the following section. Mean longitudinal magnetic field B l We computed the line-of-sight component of the stellar magnetic field integrated over the visible stellar disc, B l , using the A77, page 6 of 19 first-order moment method (Rees & Semel 1979;Donati & Brown 1997a;Wade et al. 2000a,b) and the following equation: where v (km s −1 ) is the radial velocity in the stellar restframe, λ (in nm) is the normalisation wavelength (722 nm for FK Aqr), g is the Landé factor (here 1.20), and c (km s −1 ) is the light velocity in vacuum.We set the velocity boundaries of the integration window at 56 km s −1 around the line centre of the LSD profiles for both components of the binary.We compute B l values only for the rotational phases where the line profiles are completely separated (which means B l values are not computed for overlapping orbital phases, when the system is within 20% of conjunction). The measured values of B l and their corresponding uncertainties for both components of FK Aqr are given in Cols.5-8 of Table 1. The measured values of B l vary in the interval from −143 ± 17 G to −60 ± 7 G for the primary (denoted (p)) and from −368 ± 25 G to −27 ± 26 G for the secondary (denoted (s)) in Table 1 and Fig. 5 (first two plots from top to bottom).The observations are phased according to Eq. ( 1), which shows four rotational cycles.We coloured them on the plot as follows: the first cycle is denoted in black, the second in red, the third in green (only two observations from 12 September 2014, phases 0.22 and 0.26), and the fourth in dark blue (only one observation from 16 September 2014, phase 0.25). B l measurements for both stars match well with the reconstructed Zeeman Doppler maps presented in Sect.easily connect the B l variation with the main features of these maps. Line activity indicators In general, M dwarfs that are magnetically active and show emission in CaII H&K and Balmer lines are called 'dMe' dwarfs (Dyer 1954).Both components of the binary FK Aqr belong to this group.We investigated the time variability of the three classical line activity indicators in both components, which are the Hα Balmer line at 656.3 nm, the CaII H&K lines at 396.8 and 393.4 nm, respectively, and the CaII infrared triplet (IRT) at 849.8, 854.2, and 866.2 nm.Before calculating the equivalent widths (EWs) of the lines, we refined the continuum normalisation of the individual orders containing these spectral lines for each spectrum.As there are phases in which the lines of the primary and secondary component are partially or completely blended, we present the sum of the EWs of the two components at all phases.The EWs are computed by integrating over the spectral lines.The resulting EW values in Å are listed in Table 5 and displayed on Fig. 5. Hα The Hα lines of both components are in emission, as is generally true for the most active stars (Cram & Mullan 1979).Both components show the typical double-peak (self-reversal) profile for dMe-dwarfs (Worden et al. 1981;Stauffer & Hartmann 1986), showing that the line is formed in an optically thick chromosphere.This behaviour is well reproduced by chromospheric models with a high temperature gradient and therefore a very high non-thermal heating rate (Houdebine & Doyle 1994a).Robinson & Cram (1989) and Robinson et al. (1990) also observed the same type of profile for both components of FK Aqr.This self-reversal is asymmetric in all our observations, and in general is related to mass motions in the stellar atmosphere.The peak asymmetries are observed in other active single M dwarfs in both quiescent states and flaring activity (Fuhrmeister et al. 2008(Fuhrmeister et al. , 2011)). CaII H&K The CaII H&K lines of both components are in emission.The CaII K lines of the two components are not blended, which is not the case for the CaII H lines.The CaII H line of the secondary is evidently blended with the Hϵ emission line of the primary (and vice versa, the CaII H line of the primary is blended with the Hϵ emission line of the secondary, depending on the orbital phase).We therefore present the behaviour of only the CaII K lines. We noticed significant variation in the EW of the CaII K line from one rotation cycle to the next (Fig. 5 depicted as black and red dots) near phase 0.73.The four observations corresponding to the second rotation cycle (red dots on Fig. 5) were taken consecutively on September 10, 2014, within a three-hour window. The first three observations, which result in discrepant EW values, have the lowest S/Ns for the CaII K order within our data set: 11, 17, and 8 for the first, second, and third observation, respectively (while the fourth observation reaches a S/N of 28).They also correspond to the least precise B l measurements in our data set (see Fig. 5 top panel).We therefore attribute this strong apparent variability in the CaII K EW on September 10, 2014, to a poorly defined continuum at the blue edge of lower S/N spectra. CaII IRT The three lines of the CaII IRT (849.8, 854.2, 866.2 nm) of the primary are observed in absorption with emission cores.The emission cores of the lines at 849.8 and 854.2 nm reach the continuum and exceed it at all phases in our observations.The emission cores of the third line of the calcium triplet at 866.2 nm are below the continuum (or only just reach it) only in the phases of conjunction.The emission cores of the secondary are superimposed on the wings of the spectral line of the primary for all three lines of the CaII IRT. Zeeman Doppler imaging We used the ZDI method (Semel 1989;Donati & Brown 1997a;Donati et al. 2006b) to reconstruct the surface magnetic field topologies of both components of the binary FK Aqr.This method uses the rotation-induced modulation and Doppler shifts of Stokes V signatures to map the surface vector magnetic field decomposed onto a poloidal-toroidal spherical harmonics frame (Donati et al. 2006b).The iterative algorithm fits the observed LSD Stokes profiles with a set of simulated profiles corresponding to the same rotational phases.These synthetic Stokes profiles are computed from a model star, the surface of which is divided into a grid of 2000 pixels of roughly the same area.ZDI uses maximum entropy regularisation as described by Brown et al. (1991), who implement the algorithm for maximum entropy optimisation developed by Skilling & Bryan (1984).The local synthetic Stokes I line profile is assumed to possess a Voigt shape.The method also uses the weak-field assumption (see e.g.Donati et al. 2003). In the present paper, we use a version of the code ZDIPY3 developed by Folsom et al. (2018) that has been adapted to simultaneously map both components of an SB2 spectroscopic binary system (referred to as the SB version of the code hereafter).ZDIPY is Python-based and implements the same physical model, analysis principles, and coefficient definition as the code Date EW Hα of Donati et al. (2006b) described above.We considered spherical stars in the present study.This is well-suited given the high value of the ratio a A +a B R A +R B = 17 (the binary is non-interacting in the case where the ratio is greater than 10; e.g.Eker et al. 2008).The distance of the Lagrangian point L 1 from the primary of FK Aqr was calculated to be around 0.53 a when employing the classical formula given by Lagrange (where a is the separation between the two stars; Hellier 2001; Leahy & Leahy 2015), showing no departure from sphericity.A second source of non-sphericity in general could be the stellar rotation itself.The criterion for this is given by Cang et al. (2020, their Eq. ( 3)); the magnitude of the oblateness is given by the relation between the ratio R p /R e (polar and equatorial radii, respectively) and the stellar rotation rate Ω.We checked this criterion for FK Aqr and conclude that it too is negligible (R p /R e ≈ 1). The method requires several input parameters.The adopted values for the projected rotational velocities of FK Aqr are v sin i = 5.4 ± 0.6 km s −1 for the primary and v sin i = 4.4 ± 0.6 km s −1 for the secondary (both values calculated in Sect.3.5).We assume synchronous rotation; the adopted value for both rotation periods is therefore P rot = P orb = 4.08319598 ± 0.00000095 d, which is the output value of our PHOEBE analysis.According to the Zahn (1977) formalism, we indeed compute a synchronisation timescale of shorter than 50 Myr for FK Aqr.The inclination angle of the binary is set to 52.39 (derived from the astrometric analysis described in Sect.3.3) and we assume the stellar rotation axes are perpendicular to the orbital plane.The spherical harmonics expansion is limited to ℓ = 10, a value well suited to the moderate v sin i values.The linear limb darkening coefficients for both components of FK Aqr are set to 0.72 according to Claret (2004). The local line profile parameters (line strength, Gaussian, and Lorentzian widths) are adjusted to achieve the best fit between synthetic and observed Stokes I line profiles.Although the line parameters are partly degenerate, our tests show that the reconstructed magnetic maps are weakly sensitive to the precise choice of a set of line parameters (see Appendix B). Another input parameter required by the SB version of ZDIPY is the flux ratio of the two components of the binary.This parameter is not available in the literature.We therefore employed the maximum entropy method to fit the models with observations by keeping all the parameters except the flux ratio at same values.In this way, the model with the maximum value of entropy (i.e. the weakest magnetic field) gave us a value for the flux ratio equal to F s /F p = 0.34 , which we use in our final model.Finally, the SB version of ZDIPY also requires the radial velocities of both components at each observed rotational phase.We used the RV values derived from our PHOEBE orbital model. Our observations span four rotational cycles and provide a reasonably even and dense phase coverage.The ZDI model is almost able to fit our data set down to the noise level with a reduced χ 2 r of 1.7.As can be seen in Fig. 1, the model accurately reproduces the modulation of the high-quality Stokes V data along the stellar rotation and orbit.The reconstructed large-scale magnetic field topologies of the two components are presented in Fig. 6.The properties of the surface magnetic field of both stars are very similar: an average magnetic field modulus B mean ≃ 250 G, and the magnetic topologies are almost purely poloidal and dipole-dominated.The statistics of the magnetic fields of both components are presented in Table 6. The radial magnetic map of the primary features a positive polar region in the hemisphere orientated towards the observer, which is otherwise dominated by a negative polarity (Fig. 6).This feature translates into a rather strong quadrupolar component (≃22% of the reconstructed magnetic energy) for this star.This polar region of opposite polarity may seem surprising given the simple two-lobe Stokes V profiles observed at all phases in Fig. 1.We therefore further investigated the sensitivity of the appearance of this feature to various ZDI inputs.First, we explored the map evolution as a function of the target χ 2 r for our ZDI model.We found that this polar region of positive polarity weakens with increasing target χ 2 r and completely disappears for χ 2 r above 2.5 (see Appendix C).Second, we investigated the effect of the spectra taken at phases during A77, page 10 of 19 Tsvetkova, S., et al.: A&A, 682, A77 (2024) which the spectral lines of the two stars overlap.As shown in Appendix D, when removing these spectra from our model, the polar region of positive polarity weakens and correspondingly the fraction of magnetic energy residing in the quadrupolar component decreases.This shows that this reverse polarity pole is partly due to cross-talk between the magnetic maps of the two components.Finally, we note that our v sin i values are based on radii derived from stellar models suited to inactive stars, whereas several studies of active M dwarfs infer a significant radius inflation (e.g.López-Morales et al. 2007;Donati et al. 2008).We therefore tested the effect of varying our input v sin i values, and found that the polar region of positive polarity disappears when increasing the v sin i value above 6.4 km s −1 for the primary (see Appendix E).Although this value would correspond to an unrealistically large radius inflation of 30% with respect to single M dwarfs, our tests confirm that the presence of this high-latitude area of positive polarity on the primary component of FK Aqr cannot be considered a reliable feature.Glebocki & Stawikowski (1995) conclude that the possibility of a different inclination of the primary of FK Aqr to the orbital plane is highly unlikely.Nevertheless, we ran some new models, changing the inclination angle of the primary.Small changes in inclination have almost no effect.Even for very large changes in inclination, the major features of the map are largely unchanged.Additionally, we ran the single-star version of ZDIpy with the Unno-Rachkovsky implementation (Bellotti et al. 2023) on the non-overlapping profiles of the primary.This model shows almost no difference compared to the model without Unno-Rachkovsky implementation.Unno-Rachkovsky model implementation in the ZDIpy binary-star version is deferred to a future paper. Summary and discussion We conducted an observing campaign targeting the M dwarf binary system FK Aqr in the framework of the BinaMIcS project (Binary and Magnetic Interactions in various classes of Stars, Alecian et al. 2015a,b) with the aim being to study stellar magnetism under the influence of the physical processes occurring in close binaries.Combining our astrometric and radial velocity measurements with archival radial velocities, we refined the orbital parameters of the system and derived an orbital inclination of the system of 52.39 • ± 0.45 • and deprojected masses of the components of M 1 = 0.55 ± 0.01 M ⊙ and M 2 = 0.44 ± 0.01 M ⊙ . This puts both stars just above the so-called full-convection threshold, which means that their internal structure comprises an inner radiative core below a deep convective envelope.The two components of the FK Aqr system are therefore particularly interesting targets for studying how the transition from partly convective to fully convective stellar dynamos occurs in close binary systems. The system FK Aqr was observed with the spectropolarimeter ESPaDOnS (Donati et al. 2006a) and 26 spectra in total were collected in the period from 3 to 16 September 2014.We used the least-squares deconvolution (LSD) multi-line technique (Donati et al. 1997b) to generate the mean Stokes I and V line profiles, from which clear Stokes V signatures are visible from all observations.The signatures have simple shapes with negative blue and positive red lobes.We computed the line-of-sight component of the stellar magnetic field B l using the first-order moment method (Rees & Semel 1979;Donati et al. 1997b;Wade et al. 2000a,b), and find it to vary in the interval from −143 G to −60 G for the primary and from −368 G to −27 G for the secondary. The time variability of the three classical line activity indicators (Hα, CaII H&K, and CaII IRT) of both components was compared to the variability of B l .The temporal evolution of the EW of the three lines is of limited amplitude and varies almost within the error bars.In particular, we do not find evidence for rotational modulation or enhanced activity at specific orbital phases. We used the Zeeman Doppler imaging tomographic method (ZDI; Semel 1989;Donati & Brown 1997a;Donati et al. 2006b) to reconstruct the large-scale component of the surface magnetic fields of both M dwarfs.We used the ZDIPY code presented in Folsom et al. (2018), which is Python-based and adapted to binary stars.The two components host large-scale magnetic fields with similar properties.Both are largely dominated by the poloidal component (with 90 and 96% of the reconstructed magnetic energy for the primary and secondary, respectively) and are mainly axisymmetric (72 and 70% for the primary and secondary), featuring a major contribution of the ℓ = 1 poloidal dipole modes (57% for the primary and 78% for the secondary).The reconstructed magnetic map of the primary also features a significant quadrupolar component (22% of the reconstructed magnetic energy), but our tests show that this feature is sensitive to model parameters and to cross-talk between the primary and secondary at conjunction phases, and it could be significantly weaker (down to 12%).The mean large-scale magnetic field of both components is close to 250 G, and the local field modulus reaches values close to 700 G in both cases. Previous studies of the large-scale magnetic field of single rapidly rotating M dwarfs have revealed the existence of a change in the observed magnetic properties at a mass of approximately 0.4-0.5 M ⊙ , which is just above the full-convection threshold (Donati et al. 2008;Morin 2008).Stars with saturated activity (corresponding to rotation periods of shorter than ≃5 d) that are more massive than this limit exhibit large-scale magnetic fields of moderate intensity (B mean in the range 100-200 G), a significant or even dominant toroidal component (more than 20% of the reconstructed magnetic energy), and often a significant contribution of non-axisymmetric poloidal modes.M dwarfs just below this mass limit display stronger large-scale magnetic fields (400-800 G) that are almost purely poloidal, are dominated by the dipole mode (more than 60% of the reconstructed magnetic energy), and in most cases are strongly axisymmetric.The active M3 dwarf AD Leo (M ⋆ = 0.41 M ⊙ , P rot = 2.23 d) can be considered as an intermediate case: it hosts an almost purely poloidal large-scale magnetic field dominated by the axial A77, page 11 of 19 Tsvetkova, S., et al.: A&A, 682, A77 (2024) dipole, but the average field modulus takes intermediate values in the range 200-300 G (Morin 2008;Lavail, Kochukhov & Wade 2018;Bellotti et al. 2023).The secondary component of FK Aqr, which has stellar parameters very similar to those of AD Leo (M ⋆ = 0.44 M ⊙ , P rot = 4.08 d), generates a large-scale magnetic field of the same type (dipole-dominated of intermediate strength).The case of the primary appears more interesting: its large-scale magnetic field can also be classified as AD Leo type, although it is significantly more massive (M ⋆ = 0.55 M ⊙ ), and is indeed the most massive M dwarf known to host a dipoledominated magnetic field of intermediate strength.There are stars with similar parameters in the single M dwarf sample from Donati et al. (2008), namely OT Ser (M ⋆ = 0.55 M ⊙ , P rot = 3.40 d) and DT Vir (M ⋆ = 0.59 M ⊙ , P rot = 2.85 d), both of which host a clearly different type of magnetic field, which is significantly weaker and features in particular a strong toroidal component.Interestingly, the young M dwarf AU Mic (22 Myr), with mass and rotation period close to those of the FK Aqr primary (M ⋆ = 0.50 M ⊙ , P rot = 4.84 d), hosts a significantly stronger (B mean = 475 G) magnetic field featuring significant toroidal and non-axisymmetric components, although its young age renders a direct comparison less relevant (Klein et al. 2021;Donati et al. 2023). The primary component of the FK Aqr system represents the high-mass end of the AD Leo-type of magnetism.However, the role of its binary nature is not yet clear.A valuable point of comparison is provided by the short-period binary YY Gem, which is composed of two M dwarfs of almost equal mass (M ⋆ = 0.61 M ⊙ , P rot = 0.81 d).For both components, Kochukhov & Shulyak (2019) recover a large-scale magnetic field of intermediate strength (B mean = 205 and 260 G) featuring a significant toroidal component (∼30% of the magnetic energy), and with roughly half of the magnetic energy in non-axisymmetric modes; that is, the same type of magnetism as single M dwarfs with similar parameters.Simultaneously, in both systems FK Aqr and YY Gem, the primary and the secondary (with similar and almost equal masses, respectively) generate surface magnetic fields with very similar properties.Interestingly, in FK Aqr, the dipolar components of the magnetic fields of the two stars appear to be aligned, while they are anti-aligned in the case of YY Gem. The system FK Aqr also provides new constraints on the extent of the parameter domain in which rapidly rotating M dwarfs are able to generate two different types of large-scale magnetic field: strong dipolar or weaker multipolar fields.Morin et al. (2010) report this behaviour for six stars less massive than ∼0.2 M ⊙ with rotation periods of shorter than ∼2 d.This could be explained by an effect of age, long-term magnetic cycles (Kitchatinov et al. 2014), or dynamo bistability (Morin et al. 2011;Gastine et al. 2013).This behaviour is also reported by Kochukhov & Lavail (2017) for the two components of the coeval wide binary system GJ 65 AB, which lie in the previously identified domain with masses close to 0.12 M ⊙ (Kervella et al. 2016) and rotation periods of ∼0.25 d.The secondary UV Ceti hosts a strong dipole-dominated field, while the large-scale magnetic field of the primary BL Ceti is much weaker and less axisymmetric.The pair FK Aqr provides further evidence that the parameter space where the two types of magnetism co-exist does not extend above 0.2 M ⊙ , at least for stars in close binary systems. Future analyses of other M dwarf binary systems observed in the framework of the BinaMIcS project, combined with new results on the magnetism of single M dwarfs -in particular based on observations collected with the near-infrared spectropolarimeter SPIRou -will contribute to disentangling the effects of mass, rotation, age, and binarity on the dynamo-generated magnetism of main sequence stars close to the full-convection threshold. Fig. 1 . Fig. 1.Normalised Stokes I and V profiles of FK Aqr in the observational period 3-16 September 2014.Observed profiles are plotted in dashed black lines, synthetic profiles are in red lines, and blue horizontal dashed lines are the zero level.All profiles are shifted vertically for display purposes.The rotational phases of the observations are indicated in the right part of the plot next to each profile.The vertical violet (for the primary) and green (for the secondary) dashed lines in the Stokes V panel indicate the center of the line profiles. TsvetkovaFig. 3 . Fig. 3. Corner plot of the posterior distributions for the PHOEBE RV model of FK Aqr using the EMCEE MCMC sampler.The dashed black lines correspond to the 16th, 50th, and 84th percentiles. Fig. 4 . Fig.4.PHOEBE's orbital solution of FK Aqr based on RV measurements.Upper panel: RV measurements of both components are given with symbols according to the legend in the upper right corner: symbols 'p' and 's' stand for the primary and secondary, respectively.The Phoebe model is given in black lines.Lower panel: RV residuals according to the same legend given in the upper left corner.In both panels, all error bars are plotted, but in most cases they are within the symbols of the measurements. Fig. 5 . Fig. 5. Time variability of the magnetic field and line activity indicators of FK Aqr.First two plots from top to bottom show the variability of the longitudinal magnetic fields B l of the two components of the system in the period 3-16 September 2014 (where (p) and (s) stand for the primary and secondary, respectively).Observations are phased and the rotational cycles are denoted in black, red, green, and dark blue colours corresponding to the first, second, third, and fourth cycles.The last three plots show the variability of the equivalent widths of the spectral lines, given in Å.All error bars are plotted, but some of them are within the symbols. Fig. 6 . Fig.6.Magnetic maps of both components of the system FK Aqr (primary on the left and secondary on the right) for the observational period 3-16 September 2014.From top to bottom, we show the field components in spherical coordinates: radial, azimuthal, and meridional.On the right side of each subplot, we provide a colour bar showing the magnetic field strength expressed in gauss.The phases of the observations are marked on top of each radial map. Table 2 . Observations and measurements of the interferometric observations. Notes.The flux ratio is given in the H-band (1.4 − 1.7 µm).The position angle is the position of the secondary (faintest in H-band), measured north (0 deg) to east (90 deg).e min and e max are the semi-minor and the semi-major axes of the 1σ astrometric error ellipse.PA e max is the position angle of the semi-major axis of the error ellipse, measured from north (0 deg) to east (90 deg). Table 3 . Best-fit parameters of the astrometric orbit.the heliocentric Julian date of the observations and ϕ is the rotational cycle.The values of T 0 = 2 437 145.1548 ± 0.0046 and P Tsvetkova, S., et al.: A&A, 682, A77 (2024)orb = 4.08319598 ± 0.00000095 d were obtained as detailed in Sect.3.4.As FK Aqr is a synchronised close binary, the rotational periods of both components are the same.Calculated in this way, rotational phases are listed in Col. 4 of Table Table 4 . Orbital parameters derived from radial velocity measurements.Notes.Our values were derived with PHOEBE using the combined dataset which includes our 2014 ESPaDOnS dataset and the HM65 dataset.Note that different definitions of the argument of periastron in HM65 and our analysis result in a 180 • difference for the argument of periastron. Table 5 . Sum of the equivalent widths of both components of the binary FK Aqr for the lines Hα, CaII H&K, and CaII IRT (854.2 and 866.2 nm). Table 6 . Magnetic analysis of the components of FK Aqr.	2022-12-13T04:03:08.132Z	2023-12-07T00:00:00.000	{ "year": 2023, "sha1": "4a65207d17b3e9b3b4fa65e8df7f59d0ca18d948", "oa_license": "CCBY", "oa_url": "https://www.aanda.org/articles/aa/pdf/2024/02/aa47604-23.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "75cafa97cf4785884ddf0e239bb06133b8f1f089", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
27776983	pes2o/s2orc	v3-fos-license	Precision calculation of magnetization and specific heat of vortex liquids and solids in type II superconductors A new systematic calculation of magnetization and specific heat contributions of vortex liquids and solids (not very close to the melting line) is presented. We develop an optimized perturbation theory for the Ginzburg - Landau description of thermal fluctuations effects in the vortex liquids. The expansion is convergent in contrast to the conventional high temperature expansion which is asymptotic. In the solid phase we calculate first two orders which are already quite accurate. The results are in good agreement with existing Monte Carlo simulations and experiments. Limitations of various nonperturbative and phenomenological approaches are noted. In particular we show that there is no exact intersection point of the magnetization curves both in 2D and 3D. A new systematic calculation of magnetization and specific heat contributions of vortex liquids and solids (not very close to the melting line) is presented. We develop an optimized perturbation theory for the Ginzburg -Landau description of thermal fluctuations effects in the vortex liquids. The expansion is convergent in contrast to the conventional high temperature expansion which is asymptotic. In the solid phase we calculate first two orders which are already quite accurate. The results are in good agreement with existing Monte Carlo simulations and experiments. Limitations of various nonperturbative and phenomenological approaches are noted. In particular we show that there is no exact intersection point of the magnetization curves both in 2D and 3D. It was clearly seen in both magnetization [1] and specific heat experiments [2] that thermal fluctuations in high T c superconductors are strong enough to melt the vortex lattice into liquid over large portions of the phase diagram. The transition line between the Abrikosov vortex lattice and the liquid is located far below the mean field phase transition line. Between the mean field transition line and the melting point physical quantities like the magnetization, conductivity and specific heat depend strongly on fluctuations. Several experimental observations call for a refined precise theory. For example, a striking feature of magnetization curves intersecting at the same point (T * , H * ) was observed in a wide rage of magnetic fields in both the layered [3] materials and the more isotropic ones [4]. To develop a quantitative theory of these fluctuations, even in the case of the lowest Landau level (LLL) corresponding to regions of the phase diagram "close" to H c2 [5], is a very nontrivial task and several approaches were developed. Thouless and Ruggeri [6] proposed a perturbative expansion around a homogeneous (liquid) state in which all the "bubble" diagrams are resummed. Unfortunately they proved that the series are asymptotic and although first few terms provide accurate results at very high temperatures, the series become inapplicable for LLL dimensionless temperature a T ∼ (T − T mf (H))/(T H) 1/2 smaller than 2 in 2D quite far above the melting line (believed to be located around a T = −12). Generally attempts to extend the theory to lower temperatures by the Borel transform or Pade extrapolation were not success-ful [7]. Several nonperturbative methods have been also attempted including renormalization group [8] and the 1/N expansion [9]. Tesanovic and coworkers developed a theory based on separation of the two energy scales [10]: the condensation energy (98%) and the motion of the vortices (2%). The theory explains the intersection of the magnetization curves. In the first part of paper we apply optimized perturbation theory (OPT) first developed in field theory [11,12] to both the 2D and 3D LLL model. It allows to obtain a convergent (rather than asymptotic) series for magnetization and specific heat of vortex liquids together with precision estimate. The radius of convergence is a T = −3 in 2D and a T = −5 in 3D. On the basis of this one can make several definitive qualitative conclusions. Our starting point is the Ginzburg-Landau free energy: where A = (By, 0) describes a nonfluctuating constant magnetic field in Landau gauge and D ≡ ∇− i 2π Φ0 A,Φ 0 ≡ hc 2e , L c is the width (for simplicity we write expressions for the 2D case, essential 3D complications are discussed separately). For simplicity we assume a(T ) = αT c (1 − t), t ≡ T /T c . On LLL, the model after rescaling reduces to where the LLL reduced temperature a T ≡ − 4π bω is the only parameter in the theory [6]. We will use a version of OPT, the optimized gaussian series [12]. It is based on the "principle of minimal sensitivity" idea [11], first introduced in quantum mechanics. Generally a perturbation theory starts from dividing the Hamiltonian into a solvable "large" part K and a perturbation V . Since we can solve any quadratic Hamiltonian we have a freedom to choose "the best" such quadratic part. Quite generally such an optimization converts an asymptotic series into a convergent one (see a comprehensive discussion, references and a proof in [12]). Due to the translational symmetry of the vortex liquid there is just one variational parameter, ε, in the free energy divided as follows: where a H ≡ a T − ε. One reads Feynman rules from eq.(3): K determines the propagator (just a constant), the first term in V is a "mass insertion" vertex with a value of 1 4π a H , while the four line vertex is 1 8π . To calculate the effective free energy density f ef f = −4π ln Z, one draws all the connected vacuum diagrams. We calculated directly diagrams up to the three loop order. However to take advantage of the existing long series of the non optimized gaussian expansion, we found a relation of the OPE to these series. Originally Thouless and Ruggeri calculated these series f ef f to sixth order, but it was subsequently extended to 12 th (9 th in 3D) by Brezin et al and to 13 th by Hu et al [13]. It is usually presented using variable x introduced by Thouless and Ruggeri [6] x = 1 ε 2 , ε = 1 2 a T + a 2 T + 16 as follows: We can obtain all the OPT diagrams which do not appear in the gaussian theory by insertions of bubbles and mass insertions from the diagrams contributing to the nonoptimized theory. Bubbles or "cacti" diagrams are effectively inserted by a technique known in field theory [14]: Summing up all the insertions of the mass vertex is achieved by ε 2 = ε + αa H . Here α was introduced to keep track of order of the perturbation, so that expanding f ef f to order α n+1 , and then taking α = 1 we obtain f n (ε) (calculating f n that way, we checked that indeed the first three orders agree with the direct calculation). The n th OPT approximant f n is obtained by minimization of f n (ε) with respect to ε: The above equation is equal to 1/ε 2n+3 times a polynomial g n (z) of order n in z ≡ ε · a H . That eq.(5) is of this type can be seen by noting that the function f depends on the combination α/ (ε + αa H ) 2 only. We were unable to prove this, but have checked it to the 40 th order. This property greatly simplifies the task: one has to find roots of polynomials rather than solving transcendental equations. There are n (real or complex) solutions for g n (z) = 0. However (as in the case of anharmonic oscillator [12]) the best results gives a real root with the smallest absolute value. We then obtain ε(a T ) = 1 2 a T + a 2 T − 4z n solving z n = ε · a H = εa T − ε 2 . On Fig. 1 we present OPT for different orders including n = 0 (gaussian) together with several orders of the nonoptimized high temperature expansion. One observes that the OPT series converge above a T = −2.5 and diverge below a T = −3.5. The proof of convergence is analogous to that for the anharmonic oscillator, see ref. [12]. On the other hand, the nonoptimized series never converge despite the fact that above a T = 2 first few approximants provide a precise estimate consistent with OPT. Above a T = 3 the liquid becomes essentially a normal metal and fluctuations effects are negligible (see Fig. 2, 3). Therefore the information the OPT provides is essential to compare with experiments on magnetization and specific heat. If precision is defined as (f 12 − f 10 ) /f 10 , we obtain 4.87%, 1.27%, 0.387%, 0.222%, 0.032% at a T = −2, −1.5, −1, −0.5, 0 respectively. For comparison with other theories and experiments on Fig. 2 and 3 we use the 10 th approximant. The calculation is basically the same in 3D, the only complication being extra integrations over momenta parallel to the magnetic field. However since the propagator factorizes, these integrations can be reduced to corresponding integrations in quantum mechanics of the anharmonic oscillator [6,11]. The series converge above a T = −4.5 and diverge below a T = −5.5. The nonoptimized series are useful only above a T = −1. The agreement is within the expcted precision when we compare our results in 3D with ref. [15]. Now we turn to the vortex solids. Here the minimization is significantly more difficult due to reduced symmetry. Unlike in the liquid the field ψ acquires a nonhomogeneous expectation value and can be expressed as ψ(x) = v(x) + χ(x),where χ describes fluctuations. Assuming hexagonal symmetry, it should be proportional to the mean field solution v(x) = vϕ k=0 (x) with a variational parameter v taken real thanks global U (1) gauge symmetry where ϕ k (x) is the quasi -momentum basis on LLL [5]. Expanding χ where real fields A k = A * −k (O k = O * −k ) describing acoustic (optical) phonons of the flux lattice. The phase exp[−iθ k /2] defined, as in the low temperature perturbation theory developed recently [16] x , is crucial for simplification of the problem. The most general quadratic form is with matrix of functions G(k) to be determined together with the constant v by the variational principle. The corresponding gaussian free energy f ef f is The gap equations obtained by the minimization of the free energy look quite intractable, however they can be simplified. The crucial observation is that G OA (k) = 0 is a solution and general solution can be shown to differ from this simple one just by a global gauge transformation.One can set ∆ is a constants (details will appear elsewhere). The function E(k) and the constant ∆ satisfy: Observing that β k has a very effective expansion in and using the hexagonal symmetry of the spectrum, E(k) can also be expanded in "modes" E(k) = E n β n (k). The integer n determines the distance of a points on the hexagonal lattice X from the origin. One estimates that E n ≃ χ n a T , therefore the coefficients decrease exponentially with n. For some integers, for example, n = 2, 5, 6, β n = 0. We minimized numerically the gaussian energy by varying v, ∆ and first few modes of E(k). In practice two modes are quite enough. The results show that around a T < −5, the gaussian liquid energy is larger than the gaussian solid energy. So naturally when a T < −5, one should use the gaussian solid to set up a perturbation theory instead of the liquid one. The gaussian energy in either liquid (see line T0 on Fig.1) or solid is a rigorous upper bound on the free energy. We calculated the leading correction (without its minimization) in order to determine the precision of the gaussian result (see Fig. 3 for the specific heat results). We obtain 0.2%, 0.4% and 2% at a T = −30, −20, −12 respectively. In the rest of the paper we compare our results with other theories, simulations and experiments. An analytic theory used successfully to fit the magnetization and the specific heat data [17] was developed in [10]. Their free energy density is: The corresponding magnetization and specific heat are shown as a dashed lines on Fig.2 and 3 . This is seen on Fig.3 quite clearly. Instead of rising monotonously from C/∆C = 1 till melting as is predicted by OPT, their curve (dashed) first drops below 1 and only later develops a maximum above 1. In the liquid region it underestimates the specific heat. We conclude therefore that although the theory of Tesanovic et al is very good at high temperatures they become of the order 5 − 10% at a T = −3. An advantage of this theory is that it interpolates smoothly to the solid and never deviates more than 10%. Experiments on great variety of layered high T c cuprates (Bi or T l [3] based) show that in 2D, magnetization curves for different applied fields intersect at a single point (M * , T * ). The range of magnetic fields is surprisingly large (from several hundred Oe to several T esla). This property fixes the scaled LLL magnetization defined bω M . Demanding that the first two terms in 1/a 2 T expansion of m(a T ) are consistent with the exact result, one obtains When it is plotted on Fig.2 (the dotted line), we find that at lower temperatures the magnetization is overestimated. The OPE results are consistent with the experimental data [3] (points) within the precision range till the radius of convergence a T = −3. It is important to note that deviations of both the phenomenological formula eq.(10) and the Tesanovic's are clearly beyond our error bars. Therefore we conclude that the coincidence of the intersection of all the lines at the same point (T * , M * ) cannot be exact. Like in 3D the intersection is approximate, although the approximation is quite good especially at high magnetic fields. Specific heat OPE result in 2D is compared on Fig. 3 with Monte Carlo simulation of the same model by Kato and Nagaosa [18] (black circles) (and the phenomenological formula following from eq.(10), dotted line). The agreement is very good for both the low temperature and the high temperature OPT. To summarize, we obtained the optimized perturbation theory results for the 2D and 3D LLL Ginzburg -Landau model in both vortex liquid and solid phases. The leading approximant (gaussian) gives a rigorous upper bound on energy, while the convergent series allow one to make several definitive qualitative conclusions. The intersection of the magnetization lines in only approximate not only in 3D, but also in 2D. The theory by Tesanovic [10] describes the physics remarkably well at very high temperatures, but deviates on the 5-10% precision level at a T = −2 in 2D and has certain imprecise qualitative features in the solid phase. Comparison with Monte Carlo simulations and some experiments shows excellent agreement. We are grateful to our colleagues A. Knigavko and T.K. Lee for numerous discussions and encouragement and Z. Tesanovic for explaining his work to one of us and sharing his insight.The work was supported by NSC of Taiwan grant #89-2112-M-009-039.	2018-04-03T01:02:00.278Z	2001-04-16T00:00:00.000	{ "year": 2001, "sha1": "528d4e5c4e66b8f7ffb643f03793c6222829fd72", "oa_license": null, "oa_url": "http://arxiv.org/pdf/cond-mat/0104283", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "884632350f67a28c9123ea18f72cec9fa2ee0a79", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Medicine" ] }
225473684	pes2o/s2orc	v3-fos-license	Upscaling of 2D mineralogical information to 3D volumes for geoscience applications using a multi-scale, multi-modal and multi-dimensional approach Bridging the scales of geological observation is an on-going challenge to earth scientists, but recent advances in the development of 2D and 3D imaging and analysis technology has allowed a workflow to be developed that starts to make this possible. Using concepts borrowed from allied industries (petroleum geoscience and materials science), this study shows how a multi-scale, multi-modal and multi-dimensional approach can lead to a better understanding of rock’s mineralogy and texture, especially ore-bearing drill cores, which in turn can be useful for elucidating the meso- and macro-features of ore bodies, leading ultimately to improved mineral exploration success. Introduction There have been significant recent advances in the last 20 years or so in the way geologists examine rocks both mineralogically and petrographically. Traditional 2D methods, such as light microscopy and electron beam-based techniques, were for many years largely analogue, and have now thankfully been fully digitised, allowing for huge improvements in the efficiency of imaging, analysis, interpretation, and reporting of polished surfaces and/or thin slices of rock samples (Fig. 1). With the emergence of new cutting edge scanning XRF and hyperspectral imaging techniques, geoscientists are now able to scan geomaterials (such as drill cores) at increasingly larger scales than that covered by a single petrographic thin section. This has allowed us to start to bridge the different scales of geological observation, from the mega-to the nanoscale, that previously were not easily accessible to most earth scientists (Fig. 2). The emergence of cross-disciplinary activity We are living in an age of digital communication, whereby scientists and engineers can freely communicate and share their ideas. Cross-disciplinary collaboration often leads to extraordinary developments and breakthroughs. As geologists, we are taught to think in 3D, and it has always been a limitation that we have been mostly restricted to examining rocks in the laboratory using 2D surface-based technologies (SEM, EPMA, LA-ICP-MS, etc.). But this has all changed in the last decade. By using so-called workflows, borrowed and adapted from allied disciplines (including materials and biological sciences, and Figure 1. Recently developed technology allows for the automated collection of high quality, full colour, digital images of rock thin sections, both in plane and polarised light, allowing textural features such as zoning, twinning, and crystal and groundmass behaviour to be observed and recorded. petroleum science and engineering), geologists can also now image rocks using powerful X-ray computed tomography-based techniques (X-CT), and focussed ion beam-SEM technologies (FIB-SEM). Lessons from the oil industry By way of example, cuttings and cores from a hydrocarbon reservoir drilling programme are the only way to know what is actually beneath the ground. There is no chance of digging a trench, or opening up a trial pit, as in mineral exploration and mining. Making the most of every centimetre of core, or gram of ditch cutting sample, is a key driver for innovation and development in the oil industry, especially when it comes to rock and mineral analysis. Nothing is left to chance, the risks are too big, and aspects such as correct stratigraphic correlation and rock sequence prediction must be as accurate as possible in order to reduce the chances of placing a gas or oil well in the wrong place. As a result, just about every possible geo-analytical technique is used to characterise the physical properties of the overburden, cap rock, reservoir, source, and even the basement. It all starts with geophysics, moves to petrophysics, then reservoir quality, and finally petroleum engineering. Every physical attribute of a rock or mineral is exploited to further understand what is present and how it will behave at reservoir conditions -these include: mineralogy, texture, 3-D grain network, pore structure, rock density, magnetic susceptibility, radioactivity, conductivity, density, brittleness, and response to NIR, X-ray and electron beams -to name just a few parameters that are typically measured. From experience, the key to success in the petroleum industry seems to be a multi-scale, multi-dimensional, and multi-modal approach to rock imaging, analysis and interpretation. There is no one technique that can be considered all encompassing (Fig. 3). Only by the integration of technologies can a complex problem of, say, multi-phase flow in porous geomaterials, be fully understood. Even then this apparently rigorous approach is sometimes not enough. Mistakes are made. Unforeseen problems occur because of the essentially 1D nature of drilling into what are actually complex 3D sedimentary basins, often undertaken precariously on offshore rigs, typically to great depths, and where sedimentary facies can change rapidly over short lateral distances, without notice, and unconformities or faults can unexpectedly change even the most robust and cherished depositional models of the reservoir geologist. Applications to mineral exploration In the mining world, we clearly face similar but also different challenges. Having undertaken airborne or ground geophysical surveys, observations and interpretations to select a target, everything in mineral exploration still has to be ultimately ground-truthed. Potential targets need to be drilled to prove the presence (or not) of an ore-rich interval, as perhaps indicated by the physics. But once we have hit the zone of interest, do we make the most of our core? Is every length of core that is pulled from the ground fully digitally scanned before it is cut, split or sawn in half and quartered? Definitely not. Why not? Well, one reason is that the budgets and risk factors in mining are not on the same scale as in the oil industry. But accepting this, we can still learn much from each other. The concept of digital rock analysis, whereby a digital rendition of a real sample is used to visualise and model rock properties, is an excellent potential cross-over concept for mining, now widely used in the petroleum world. By creating a 3D version of a core, based on reality and not a model, it is now possible to perform virtual experiments and observations without damaging the original. With this mind, consider the case of a high-grade ore-rich exploration drill core sequence (Fig. 4). Imagine being able to visualise the 3D structure of the core, along with the minerals of commercial interest within it, and calculate their grain size, see their associations, and provide a degree of prediction of processing performance, initially without cutting open the rock. Once regions-of-interest have been found, they can then be further investigated by destructive techniques such as slabbing, crushing, or thin section preparation. Automated 2D imaging and analysis technologies can then be used to calibrate the original 3D volumes, as well as digitally capture what the grains of gold, platinum or sulphides (or whatever) actually look like in extreme detail, down to and beyond the micron-scale, and provide quantitative micro-compositional information on them as well. Even the most highly mineralised ore, containing massive sulphides and precious metals, can now be imaged and rendered into a useful 3D digital volume, which can be later interrogated, quantified, archived and retrieved, using advanced image processing and data storage know-how (Fig. 5). Figure 5. Three exploration drill cores -all containing multi-deformed pyrrhotite layers (bright phases) -within a silicate matrix (greys). Such images allow areas of special interest to be highlighted and can guide thin section area selection. Samples courtesy of Mawson Resources. Summary Multi-scale, multi-modal and multi-dimensional workflows have opened up a whole new world of analytical possibilities to us as a community. For the exploration geologist, it means that their valuable drill core intersections can now be examined and archived before it goes off for destruction (slicing with a diamond saw and crushing of half core for routine chemical assaying), thus preserving all the contained characteristics (bedding, layering, folding, mineralisation), see Fig. 6. For the mineral processing engineers, 3D liberation analysis is now a real possibility, something that has been requested by them for many years, and there is encouraging work starting to be published [2,3]. And to the ore deposit geologist, it allows for a superior textural and structural understanding of mineralised rocks, all the way from the field (mega-and macro-scale) to the laboratory (micro-and nanoscale). This workflow approach is currently being implemented at the Geological Survey of Finland in order to reduce the risks in mineral exploration; gain insights into the commercial mineralogy of ores; contribute to smarter and greener mineral processing behaviours; and to overall improve mineral deposit knowledge and understanding via an integrated 2D -4D approach. Once these workflows have been properly and securely set up, it is then possible to generate databases that can be used to create realistic ore body models that are based not only on grade, or mineralogy alone, but also on, for example, more enlightened aspects such as ore paragenesis. This in turn can lead to better predictive metallurgical models, because the geologist and metallurgist will together now have a full understanding of the nature of minerals of commercial importance, from grind size estimate, to liberation behaviour, and finally elemental deportment. This is key information that exploration companies ideally need upfront, at the time they are investing heavily in costly drilling Figure 6. Multi-scale, multi-modal, multi-dimensional workflow used to characterise exploration drill core in order to optimise the discovery of gold and cobalt mineralisation. Example courtesy of Mawson Resources, and developed in collaboration with Camborne School of Mines (University of Exeter), Bruker Nano Analytics (Berlin), and Hippo Geoscience. campaigns, as it reduces their risk and uncertainty, and improves understanding of the yet-to-be-fullydiscovered ore body (Fig. 6). Menace minerals, either from an environmental view point, or purely a practical processing aspect (poor separation potential), can be flagged up early on in the exploration life cycle, and therefore save much pain later on down the track. This information is also invaluable if it can be made available prior to blasting activities in operational mines. The future Finally, the world of material science is another much underutilised area of expertise and knowledge used by geologists. It typically involves bringing together the doers (technologists) with the knowers (scientists), which is not commonly practiced for some reason. There is much literature and experience that can be readily borrowed from technologists and brought to bear on the study of earth materialssuch as fracture analysis, crystallisation and annealing processes, and micro-chemical reactions -all of which can lead to a better understanding of minerals and rocks, their discovery, extraction, and recovery potential.	2020-08-06T09:07:23.741Z	2020-08-06T00:00:00.000	{ "year": 2020, "sha1": "88fd829f0aff7716b88c0c498ca9ac22c450cd65", "oa_license": null, "oa_url": "https://doi.org/10.1088/1757-899x/891/1/012006", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "05d259e261317ba57ceec1951e3681248d25b14c", "s2fieldsofstudy": [ "Geology", "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Computer Science", "Physics" ] }
9841253	pes2o/s2orc	v3-fos-license	Lack of Altered BECN1 Gene Expression in Iranian Patients with Acute Myeloid Leukemia Acute myeloid leukemia (AML), one of the most prevalent leukemia types in adults, demonstrates great heterogeneity in molecular and clinical terms. Hence, there is a necessity to the mechanisms involved in AML generation in order to determine optimal treatment. This cross sectional study aimed to assess changes in BECN1 gene expression in with blood samples from 30 AML patients, compared with samples from 15 healthy persons. RNA was extracted and cDNA was synthesized and Real Time PCR applied to determine BECN1 gene expression. The results showed no significant differences in BECN1 gene expression between patients with AML and normal controls (P > 0.05). It appears that expression of BECN1 does not play a significant role in genesis of AML leukemia. Introduction Autophagyisa processes associated with lysosomes, which can lead to induction of destruction and digestion of Organelles and cytoplasmic proteins. The process can cause induction of digestion of Organelles and cytoplasmic proteins physiologically in reaction to the environment and cellular stimulation. Moreover, for autophagy, destructive cellular function is presented as induction of apoptosis, along with Bcl2 loss and mTOR stimulation and there are many disputes on the two functions of autophagy process (Shi et al, 2013). Clearly, in case of declined performance of the cellular cycle, waste organelles such as mitochondria or by-products of cellular proteins remain in ectopic form and produce reactive oxygen species (ROS) or materials with oxidation property and cause dysfunction in vital performances and more importantly, cause mutations in DNA sequence and this can cause more mutations and as a result, more tumorigenesis (tumor creation). However, as it was mentioned, increased activity of the process can also help higher stability of cell, which is in line with cancerous cells property (Grander et al, 2010). Hence, there are disputes on this dual function for this process. In early researches and in rats, it is demonstrated that haploinsufficiency for these genes can lead to increased susceptibility to a variety of tumor. In later studies and on cancer samples, changes in expression of these genes compared to normal samples are confirmed. For example, changes in expression of genes in this process like Beclin1, ATG7, p63 and Bif were investigated in some prevalent cancers like breast cancer, intestine cancer and prostate cancer and reduction or increase in genes is demonstrated. Moreover, the prognosis value of the reduction or increase of expression is emphasized in prediction for risky patients (Maiuri et al, 2009). However, it should be mentioned that reported changes in gene expression are presented in form of reduction or increase in expression, which is a matter to think about and it is still ambiguous that is this paradox depended on type of cell or depended on cancer stage and this needs more investigations and further studies (Wang et al, 2008). Impairment in autophagy pathway can affect creation and formation of hematologic malignancies such as AML. For example, it has been found that prescription of some drugs with therapeutic potential can cause reduction of the division of cancer cells and increase the maturity of these cells and such effect is attributed to autophagy process induction (Wang et al, 2008). In Acute myeloid leukemia (AML), it has been shown that AML cells are mainly sensitive to autophagy stimulations and this could create this idea to mind that autophagy can be associated with cytotoxic medication of AML. Despite to this important and probable point, today, there is no comprehensive research to demonstrate status of autophagy genes in AML. As an important mediation, Beclin1can affect initiation and progression of autophagy and a variety of researches have referred to removal of BECN1 recurrence and its diversity in wide range of tumors. The main purpose of this research is to find frequency of variances in BECN1 gene expression in patients with de novo AML. On the other hand, this research investigated information of individuals with different expression variances. Materials and Methods In this research, peripheral blood samples were taken from 30 patients with de novo AML referred to 501 Army Hospital (Imam Reza) and 15 normal individuals participated in the study as control group after signing the consent letter and their blood samples were also taken. Peripheral blood sample to 4cc was mixed with 200μl of EDTA and was transferred in 15-ml falcon to laboratory of Department of Genetics of Faculty of Medical Sciences of Shahid Beheshti University. After confirmation of diagnosis of Oncology specialist, blood samples were transferred to the lab. Obtained results from real-time PCR reactions in this study have been analyzed using LinReg and REST-2009 software. Statistical results of <0.05 are considered as considerable and significant values. Data for all patients were collected for analysis. Study and patients were assigned on the basis of national/ international breast cancer protocols and approved according to local law and regulations, by the Institutional Review Boards of each participating referral hospital. The study was performed Department of Genetics of Faculty of Medical Sciences of Shahid Beheshti University, in adherence to the guidelines of the Declaration of Helsinki, and was approved by the ethics committee of this University. DNA extraction Extraction of DNA was done on patient and normal samples and as a result, density and purity of samples was obtained using biophotometer. Ten 10μl of each sample was electrophoresed on 2% agarose gel in buffer 0.5 X (TBE). Density of RNA was about 40-50μg/ml and OD260/280 was in range 1.6-1.9, which showed favorable quality of RNA. To test quality of RNA, extracted RNA was electrophoresed on 2% agarose gel. Determining quality of cDNA To determine accuracy and quality of the cDNA made, PCR reaction of ABL1 housekeeping gene was performed on all samples and the length of product was obtained to 170pb. Performing the reaction can be a reason for accuracy of cDNA synthesis. For this evaluation RNA quality was evaluated using Eppendorf spectrophotometry and beta-actin was used as housekeeping gene in PCR analysis. RNA of lymph node and breast tissue samples were showed to be of less value if either of the housekeeping gene gave signals that was at least 3.5 cycles over the optimum of lymph node and other specimen checked. Low quality RNA samples were not found. There were 30 patients with same number lymph nodes included in the final data set from this study. The RNA was undergone to reverse transcription reaction to make a cDNA before performing RT-PCR. The obtained RNA was stored in form of separate 10μl in -20 ͦ C until next processing. RNA was used in 4μg concentration, 26 ng/μl oligo dT (Biolab, New England) was added and incubated at 63 ͦ C for 5 minutes and the product transferred on ice. Then in final 25 μl reaction volume, included 1×protoscript buffer, 10mmol/L dithotheriol, 0.5 mmol/L dNTP, 10 U/μl M-MLV reverse transcriptase enzyme (Biolab, first strand cDNA synthesis kit) 0.25 U/μl RNase inhibitor (Biolab). This mix incubated in 37 ͦ C for 1 hour and after that, incubated in 92 ͦ C for 6 minutes. The obtained product (cDNA) was diluted 1:5 with distilled water and stored at -20 ͦ C. Real-Time assay was conducted for mammoglobin marker and beta-2-macroglobinas housekeeping gene. ABL 1 melting curve (internal control) The reaction was performed on normal and patient samples in duplicate form. Obtained results are resulted in form of melting curve. Beclin1 gene expression The reaction was performed on normal and patient samples in duplicate form. Obtained results from the reaction are discussed. To determine specificity of product of Real Time RT-PCR reaction Products of real time RT-PCR reaction were electrophoresed on 2% agarose gel to confirm specificity of the product through performing the reaction. Results of analysis of real-time RT-PCR raw data Raw data of RT-PCR reaction were analyzed using LinReg and REST software after determining efficiency of PCR and determining base level and the results are Investigation of changes in expression of genes involved in progression of cancer cells can play key role in advancement of diagnosis methods, medication and prognostic of different human cancers. BECN1 family genes such as BECN1 play key role in Apoptosis process. Cancer cells need escaping from inhibitory processes such as apoptosis to have uncontrolled growth (Khodapasand, 2013). As a result, it is expected that relevant gene expressions of this process such as BECN1can be changed abundantly in cancer cells. BECN1 gene is an autophagy gene that increases formation of autophagy vesicles and plays key role in protecting cells against chromosome instability. Increased level of this gene can predict optimal survival of different types of cancer (Yang et al, 2015). BECN1plays vital role in autophagy regulation, apoptosis and separation and also growth of cancer (Baspinar et al, 2013). Hence, investigating these changes and the relationship between them and different factors of disease can play key role in researches in field of cancer. Blood cancer can be the most prevalent group of cancer and it seems that it is created as a result of impairment in cell differentiation. Hence, there is a strong correlation between this type of cancer and changes in gene expressions as a result of epigenetic changes (Park et al, 2011). Acute myeloid leukemia has encompassed 15% of leukemia cases of childhood, which can be diagnosed by clinical and biological properties and has lower cure rate than acute lymphocytic leukemia. In addition to conventional chromosomal translocation, mutation of oncogenes and loss of tumor suppressor genes play key role in AML pathogenesis. For example, abnormality in special genes like FLT3 (Birg et al, 1992), Nucleophismin (NPM) (Falini et al, 2005), RAS (Ahuja et al, 1990), BECN1 (Wang et al, 2008) and mutation destruction of tumor suppressive genes like P53 (Mori et al, 2005) are common in AML cells. BECN1 is a tumor suppressor gene, which is involved in gene transcription and translation (Lankat Buttgereit et al, 2009). However, obtained results from this study show lack of selected presented as follows Figure 1. The mean value and SD of BECN1 in both groups is presented as follows: Obtained results from this research are presented here as headlines: • RNA concentration was about 40-50μg/ml and OD260/280 was in range 1.6-1.9, which shows favorable quality of RNA. • Extracted RNA on 1% agarose gel (existence of 28 s, 18s, 5s bands shows quality of extracted RNA) • PCR reaction of ABL1 housekeeping gene was performed on all samples and the length of product was obtained to 170pb. Performing the reaction could be a reason for accuracy of cDNA synthesis. • Electrophoresis of products for BECN1 gene showed that the reaction and its specificity are confirmed. • Obtained results from this study showed that BECN1 gene expression in patients showed no change and variance compared to normal samples. Discussion Currently, cancer is being considered as a genetic disease, in which changes in genome can result in impairment in expression of oncogenes or tumor suppressor genes and as a result, can cause cancer. Available data show that change in sequence of nucleotides can't be the one and only factor for change in gene expression or impairment in mechanism of gene regulation (Liang et al, 1998). One mechanism that plays vital role in creation and progression of cancer is epigenetic changes. Epigenetic (Wei et al, 2012). In this research, the main purpose has been to analyze expression changes of this gene in AML and obtained results showed lack of changes in BECN1 gene expression assumed in De novo AML. Also, obtained results from the research showed that BECN1 expression was not significantly different between two patients and normal groups (Wei et al, 2012). BECN1 gene expression has decreasing process in many other human tumors. Ding et al have investigated Beclin1 gene expression in mRNA and protein levels in 63 specimens of gastrointestinal stromal tumor. They found that BECN1 expression was reduced in 68% of specimens compared to adjacent normal gastrointestinal tissues (Ding et al, 2010;Ozpolat et al, 2007). It seems that reductive regulation o BECN1 in different types of human cancers is along with accelerated growth of tumor, metastasis and weak prognostic (Wang, 2008). At the present study, no correlation was observed between level of BECN1 expression and other clinical parameters like patients' sex, platelet count and hemoglobin concentration. For further investigations, the authors tend to implement this study in larger sample size and in frame of a big project. In larger sample size, although Beclin1gene expression shows no significant difference between patients and control group, reduction of Beclin1 gene expression was along with BECN1 mutation, more number of white blood cells, monosomies karyotype and older age (Mourgues et al, 2015). In 60-80% of patients with AML in age range below 60 years old, complete remission after induction therapy was observed. Complete remission is defined as the presence of less than 5% blasts in the bone marrow and modification of peripheral blood cell count: Neutrophil count more than 1,000 cells per microliter, the platelet count more than 100,000 cells per microliter, hemoglobin more than 10gr/dl and no presence of blasts in peripheral blood. Moreover, more than 20% bone marrow cellularity should be existed with evidence of hematopoiesis all three cell lines (Smith et al, 2004). No significant correlation was also observed between expression level of Beclin1 of patients with complete remission and those didn't enter to remission phase. Also, Beclin1 gene expression level in patients with complete remission was relatively similar to those who didn't enter to remission phase. However, BECN1 regulatory mechanisms in AML and its role in natural hematopoiesis and also probable correlation of it with outcome of patients need further studies.	2018-04-03T00:11:22.828Z	2016-12-01T00:00:00.000	{ "year": 2016, "sha1": "857f310979948b251e2770d7a7694e34a7cb4eef", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "3a02abf106d43549069097620e56e666c83c7398", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
86559202	pes2o/s2orc	v3-fos-license	Can Sodium Citrate Effectively Improve Olfactory Function in Non-Conductive Olfactory Dysfunction? Olfactory dysfunction is relatively common, affecting approximately 20 to 30 percent of adult. Including a temporary loss of the sense of smell, some articles report up to 60% of the population experienced olfactory dysfunction. Numerous clinical reports have suggested that olfactory dysfunction could affect nutritional disturbances, social anxiety and depression which consequently influence the quality of life negatively. In particular, if occupation depends on the olfactory function, the olfactory dysfunction becomes crucial obstacle. Recently, as people tends to focus on the quality of life, the number of population who are aware of olfactory dysfunction and visit out-patient clinic for diagnosis and treatment is increasing. However, effective treatment options Can Sodium Citrate Effectively Improve Olfactory Function in Non-Conductive Olfactory Dysfunction? Introduction The objective of this study was to perform a systematic review of the literature for application of intranasal sodium citrate in the patients with olfactory dysfunction to help determine the sodium citrate treatments for this condition. Two authors independently searched the data base (Medline, Scopus, and the Cochrane database) for relevant studies from inception to January 2018. Included studies were randomized controlled studies published in English comparing topical sodium citrate application (treatment group) with saline (control group) in patients who had olfactory dysfunction. Outcomes of interest included the change of olfactory identification and threshold during 2 hours post-treatment. Three studies were enrolled in the meta-analysis. Compared with control group, treatment group did not increase posttreatment score of olfactory identification [standardized mean difference (SMD)=-0.03; 95% confidence interval (CI)= -0.29-0.24; I 2 =0%] and olfactory threshold (SMD=0.18; 95% CI=-0.09-0.45; I 2 =0%) significantly. In the degree of pre-post improvement of two outcomes, although treatment group statistically showed the significant improvement in olfactory threshold (SMD=0.30; 95% CI=0.05-0.55; I 2 =17%), the clinical significance of this outcome was meaningless. Similarly, there was no significant difference in olfactory identification between two groups (SMD=0.17; 95% CI=-0.11-0.45; I 2 =22%). Unlike the recent favorable results, our summated results presented the uselessness for the local application of sodium citrate in improving patient's olfactory function. However, we also had some limitation such as small sample size and inconsistent application methods. Therefore, larger trials and standardized methodology are needed to reach more stronger and exact results. for physicians are limited, 6) and mainly focused on oral steroid or intra nasal steroid administration. 3) Calcium in vertebrate olfactory receptor neurons (ORNs) is involved in both odor-induced excitation and intracellular feedback pathways. The effect of the rise in intracellular calcium is a negative feedback action on various stages of the odor transduction mechanism, which is responsible for the down-regulation of the smell sensitivity such as olfactory adaptation. Based on the facts, previously Panagiotopoulos, et al. 7) assumed that a rise in mucosal calcium could contribute to the consequent influx of calcium inside the cell and reducing mucus free calcium levels might modulate these inhibitory effects and thereby enhance olfaction. They showed that intranasal sodium citrate, calcium sequestrant, in hyposmic patients improved olfactory function. Since his research, there have been several studies which intended to demonstrate the effect of sodium citrate on olfactory dysfunction. [8][9][10] The objective of this article is to perform a systematic review of the literature for intranasal sodium citrate in the patients with olfactory dysfunction to help determine the efficacy of sodium citrate treatments for this condition. Search strategy and selection of studies Studies published in English before December 2017 were identified from MEDLINE, SCOPUS, and the Cochrane Register of Controlled Trials, using the following search terms: "olfaction," "odor identification," "odor threshold," "calcium," "nasal mucus," "hyposmia," "anosmia," and "sodium citrate." Two reviewers, working independently, screened all abstracts and titles for relevant studies, and discarded any that were not relevant. Randomized controlled trials were eligible for review if they investigated the topical application of sodium citrate in patients who suffered from olfactory dysfunction. Studies were not eligible for inclusion if 1) patients experienced the olfactory dysfunction due to the conductive cause (e.g., nasal polyp or chronic sinusitis); 2) patients had previous history of congenital anosmia; 3) patients suffered from a systemic or malignant disease; or 4) if multiple reports were based on the same trial data. Additionally, in the event of missing or incomplete data, attempts were made to request further details of the published results from the authors directly. Studies were excluded from the analysis if outcomes of interest were not clearly reported with quantifiable data, or if it was not possible to extract and calculate the appropriate data from the published results ( Fig. 1). Data extraction and risk of bias assessment Data from the eligible studies were extracted using standardized forms and were independently checked by the two reviewers. Sodium citrate, a solution licensed and used safely in other body cavities (e.g., stomach and bladder), is known to buffer calcium ions. Topical application of sodium citrate into olfactory cleft is employed to bind the free calcium in the nasal mucus. It is assumed that sodium citrate reduce the calcium level from nasal mucosa, and subsequent decrease the negative feedback in the olfactory receptor cell response. The primary outcome for this meta-analysis was the posttreatment score of olfactory identification 9,10) or threshold 9,10) within 2 hours after nasal application of sodium citrate. Outcomes associated with sodium citrate after nasal application was compared against a control (placebo). Additionally, the outcomes regarding to change of pre-and post-treatment ol- factory identification 9,10) or threshold [8][9][10] were derived from comparison between the pre-and post-treatment values. The incidence of the adverse effect, a sodium citrate-related morbidity, was assessed as a secondary outcome. For studies which investigated the influence of sodium citrate on the improvement of olfactory threshold and identification, the following data were extracted: the number of patients, scores of parameters, and p-values recorded in the comparison between the sodium citrate and control groups. The risk of bias for each study was evaluated using the Cochrane "Risk of Bias" tool. Statistical analysis A meta-analysis was performed using "R" statistical software (R Foundation for Statistical Computing, Vienna, Austria). An outcome analysis was performed using the standard mean difference. An effect size of approximately 0.2 is considered a small effect, while 0.5 is considered a medium effect and 0.8 a large and clinically significant effect. A funnel plot and Egger's test were used simultaneously to detect publication bias. Additionally, the Duval and Tweedie's trim and fill method was used to adjust for missing studies and to correct the overall effect size according to publication bias. Additionally, sensitivity analyses were performed to estimate the influence of each individual study in the overall meta-analysis results. Results Three studies, which included a total of 267 participants, were included and reviewed in this study. Risk of bias assess-ments and study characteristics are described in Table 1. Comparison of the use of sodium citrate versus controls: post-treatment olfactory identification and threshold In the patients who received sodium citrate (sodium citrate group) and those who received placebo (control group), there were no statistically significantly different in the posttreatment score of olfactory identification [SMD=-0.03; 95% confidence interval (CI)=-0.29-0.24; I 2 =0%] and olfactory threshold within 2 hours (SMD=0.18; 95% CI=-0.09-0.45; I 2 =0%) (Fig. 2). Based on the degree of improvement in olfactory identification, there was no significant difference between two groups (SMD=0.17; 95% CI=-0.11-0.45; I 2 =22%). By contrast, the improvement in olfactory threshold (SMD= 0.30; 95% CI=0.05-0.55; I 2 =17%) was significantly higher in the patients who received sodium citrate (sodium citrate group) than in those who received placebo (control group) (Fig. 3). There was no statistically significant inter-study heterogeneity (I 2 ＜50%) in these outcomes. Egger's test regarding these outcomes was not available due to small number in the enrolled studies. Duval and Tweedie's trim and fill analysis showed there was no difference between observed and adjusted values. These results showed that the selected studies were not biased. Change in olfactory measurements after sodium citrate Sodium citrate did not increase the olfactory identification (SMD=0.02; 95% CI=-0.24-0.29; I 2 =0%) and olfactory threshold (SMD=0.12; 95% CI=-0.18-0.55; I 2 =0%) after application significantly. There was no statistically signifi- cant inter-study heterogeneity (I 2 ＜50%) in these outcomes (Fig. 4). Egger's test regarding these outcomes was not available due to small number in the enrolled studies. Duval and Tweedie's trim and fill analysis showed there was no difference between observed and adjusted values. These results showed that the selected studies were not biased. Sensitivity analyses Sensitivity analyses were performed to evaluate whether the pooled estimates of the change of olfactory threshold and identification were different by omitting a different study each time and repeating the meta-analysis. The results of these sensitivity analyses were all consistent with the above outcomes (data not provided). Discussion Recent studies revealed the role of calcium in olfactory pathway. Odor molecules bind to olfactory receptor cell in the olfactory cleft. G protein-coupled cascade occurs, resulting in increase of cystic adenosine monophosphate (cAMP). cAMP opens cyclic nucleotide-gated channels and then calcium influx occurs. Increase in intracellular calcium acts not only as a second messenger of axon, but also plays inhibitory role. Mucosal calcium gives negative feedback on multiple stages of the olfactory passage. 7,[11][12][13] Through negative feedback, adaptation to long-term odorant exposure is possible. This mechanism could give an interesting idea for a treatment option for olfactory dysfunction by reducing mucosal calcium level and restraining the negative feedback. 14,15) Therefore, there have been som sodium citrate e previous studies which support positive results regarding the administration of functioning as a calcium buffer on olfactory dysfunction. Assessment of the olfactory function can be generally divided into 3 different components such as odor threshold (the perception of odors at low concentrations), odor discrimination (the nonverbal distinction of different smells), and odor identification (the ability to name or associate an odor). Although there has been no objective assessment of olfactory function in clinical situation, several psychophysical tests assessing olfactory performance have been used for the assessment of each of these components. 16) Sniffin' Sticks are a validated psychophysical test that is based on pen like odordispensing devices, and previous studies have verified that it shows high test-retest reliability and validity in measuring the olfactory sensitivity. 17) A series of threshold smell tests including phenyl ethyl alcohol was also previously validated and showed the high test-test reliability. 18) This study performed the meta-analysis with previous studies related to sodium citrate and olfactory dysfunction and used the validated psychophysical test to assess the degree of olfactory function. Additionally, the comprehensive evaluation of several components in olfaction, especially including olfactory thresholds, provides the most significant approach to the measurement of olfactory disfunction. 16) Therefore, we measured the olfactory identification and threshold as the olfactory outcomes. Our results showed that the posttreatment score of olfactory identification and threshold within 2 hours were not significantly changed in sodium citrate group compared with the control group. Although the degree of treatment-related change in olfactory threshold was statistically significantly higher in the sodium citrate group than in control group, the effect size regarding the olfactory threshold was small (SMD=0.3) and there was no significant difference in olfactory identification between two groups. Additionally, in sodium citrate group, the change in olfactory identification and threshold after treatment was also not statistically significantly identified. The common representation of the SMD is Cohen's d, which suggests that a smaller effect size indicates that a treatment is clinically less effective. An effect size (SMD) between 2 means of around 0.3 is considered a small effect (possibly clinically non-significant), an effect size of around 0.5 is considered a medium effect, and an effect size of 0.8 or greater is considered a large and clinically significant effect. 19) The only significant effect size for the measurements regarding the treatment-related change in olfactory threshold were no more than 0.3, which meant that these effect sizes were clinically insignificant during treatment periods. These results consistently showed that the application of sodium citrate in patients with olfactory dysfunction would not be helpful to improve their olfaction. In addition, as researchers' comment, although there were no serious adverse effect related to the application and patient seemed to be tolerable, local trivial adverse effects such as rhinorrhea, nasal obstruction or throat irritation were frequent and the treatment effect short-lived, with only lasting 2 hours. 9) Given the therapeutic effect size and halflife period and frequent minimal side effects, this treatment would not be recommended to the patients clinically. Panagiotopoulos, et al. 7) firstly performed the pilot case B series study and reported that sodium citrate showed the very high effectiveness on the improvement of olfactory function in patients, of which p-value was less than 0.0001. However, since then, the next three randomized controlled studies could not demonstrate as high therapeutic effectiveness as the previous one did, of which p-value were mostly larger than 0.01. Consequently, our studies which included the next three studies could not identify the usefulness of local application of sodium citrate in patients with olfactory dysfunction. For this discrepancy, there were some reasons. Firstly, Panagiotopoulos, et al. 7) conducted the repeated identification testing on during 3 days. The repeated same test could make participants find the right answer skillfully and the results tend to be biased. 9) Secondly, Panagiotopoulos, et al. 7) used a 12-item identification test, which is less sensitive and specific than the full 16-item test that other researchers utilized. 9) Thirdly, combined testing of two components of olfaction could be more reliable approach to detect the olfactory function and threshold test presented more distinct properties than the identification tests. 16) In previous other studies, olfactory function test included two components such as threshold and identification. However, Panagiotopoulos, et al. 7) concluded on the basis of only identification test. In particular, compared to olfactory threshold, olfactory identification has higher possibility of learning effect. 20) It is possible that learning effect increased identification correction rate as the tests repeats, which may lead to the incorrect results. 9) Fourthly, the effect of calcium in the olfactory function would be more complex than expected previously. In addition to the adaptation of the ORN to odor stimuli (negative) feedback inhibition, all basic transduction processes in the ORN are mediated by calcium. The generation of the receptor action potential, its amplification, and the termination of the receptor potential are the share of calcium itself. 21) Therefore, only depletion of calcium in mucus could explant the olfactory improvements or related outcomes. Although the results of this study offer the uselessness for the local application of sodium citrate in improving patient's olfactory function, we also had some limitation. Firstly, included studies use different sodium citrate administration method. Different concentration of solution and different type of the nozzle may have caused bias. [22][23][24] Secondly, dividing the participant with the degree of olfactory dysfunction such as anosmia and hyposmia or more detailed may be helpful since anosmia group is less likely to gain olfactory function improvement. 25) Thirdly, since sodium citrate is expected to have temporary effect of the sodium citrate, there has been no long-term study to analyze until now. Lastly, this meta-analysis included only three studies with a total of 267 patients. Further study with large, randomized, controlled clinical trial should be performed to provide more evidence on the efficacy of sodium citrate. Conclusion This study demonstrated that local application of sodium citrate in the patients with olfactory dysfunction did not improve olfactory function effectively but could cause frequent but trivial local adverse effect such as transient nasal or throat discomfort. Considering that calcium itself has multifunction on olfaction, not limited to negative feedback, we recommend that further well designed studies be needed to verify the clinical effectiveness of sodium citrate.	2019-03-28T13:33:43.605Z	2019-02-19T00:00:00.000	{ "year": 2019, "sha1": "c3fc82655f30760433d48288d76276e99d697fea", "oa_license": null, "oa_url": "http://kjorl.org/upload/pdf/kjorl-hns-2018-00766.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "0411920e3fe3cbd21c50602450dd8ab3530155df", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
52099279	pes2o/s2orc	v3-fos-license	‘It’s not good to eat a candy in a wrapper’: male students’ perspectives on condom use and concurrent sexual partnerships in the eastern Democratic Republic of Congo ABSTRACT This paper reports on fieldwork carried out in 2011 with aim to investigate young men’s perspectives about condoms use, concurrent sexual partnerships and sex in the context of HIV/AIDS. This study employed a qualitative approach to collect data from 28 boys aged 16–20 from two urban and two rural high schools in South Kivu province. Four focus group discussions and 20 individual interviews were conducted among them. The findings showed that most students identified condoms as unsafe and untrustworthy. Reasons given for the mistrust of condoms were related to the belief that condoms do not give enough protection from Sexually Transmitted Infections, HIV and pregnancies. Most participants believe that condoms have a ‘small hole’ or are unreliable and are therefore not effective in prevention. They also mentioned that condoms encourage inappropriate sexual activity. They prefer flesh-to-flesh sex rather than protected sex using a condom. However, a few participants acknowledged the importance of condom use. Despite the risk of HIV transmission, boys believe that it is appropriate for them to have concurrent sexual partnerships. They justified the concurrent sexual partnerships as a way of ensuring that they cannot miss a girl to satisfy their sexual desire. Given the boys’ failure to use condoms and their strong inclination to concurrent sexual partnerships, there is a need for heath groups and stakeholders within the area to increase awareness about condoms’ effectiveness and improve knowledge dissemination on Sexually Transmitted Diseases and how they are prevented. Introduction Condoms are strongly mistrusted and are based on the perceived lack of protective efficacy (Maticka-Tyndale, 2012). These include beliefs that condoms do not fully protect against STIs or HIV (because they slip, break or allow infectious agents to pass through) or even that condoms increase the risk of HIV transmission (because they are fabricated with the virus in them or with holes in them as a plot to exterminate the people of sub-Saharan Africa, or that the virus is small enough to pass through condoms) (Maticka-Tyndale, 2012, p. 63). Similarly, several studies in Nigeria (Okonkwo, 2010), South Africa (MacPhail & Campbell, 2001), Malawi (Chimbiri, 2007), Kenya (Maticka-Tyndale & Kyeremeh, 2010;Maticka-Tyndale et al., 2005), Democratic Republic of the Congo (DRC) (Bosmans, Cikuru, Claeys, & Temmerman, 2006;Kandala, Lukumu, Mantempa, Kandala, & Chirwa, 2014;Kayembe et al., 2008a;Kidman, Palermo, & Bertrand, 2015) found that participants believe condoms cannot prevent infection. Such a belief can present a major impediment to condom use. Many of the adolescents, from the study conducted in Kinshasa and Bukavu, doubted the reliability of condoms, and girls who would use them were regarded as sexual easy givers (Bosmans et al., 2006). A number of studies in the DRC find that regular condom use depends on a range of factors, including the knowledge that condoms are ineffective in preventing infection with HIV (Carlos et al., 2015;Kayembe et al., 2008aKayembe et al., , 2008bKidman et al., 2015;Van Rossem, Meekers, & Akinyemi, 2001). In another study in Kinshasa and Bukavu participants indicated that condoms did not offer full protection (Bosmans et al., 2006). A study conducted in South Kivu finds that social constructions of masculinity not only actively encourage the idea that 'men cannot control their sexual urges', but also hinder women's negotiation ability to negotiate safe sex and refuse it (Mulumeoderhwa & Harris, 2013). Condoms supply limitations A Survey conducted in the DRC indicates that only 20% of Congolese women from 15 to 49 have used contraceptive methods (31% in cities and 15% in villages) (Congo Democratic Republic DHS, 2013-2014. This may actually result from the lack of investment and maintenance of public health services, combined with a massive withdrawal of multilateral and bilateral cooperation in 1992 and on-going war since 1996, resulted in the collapse of the health system of the DRC, which was left solely in the hands of non-governmental organisations (NGOs), churches and private assistance (Van Herp, Parqué, Rackley, & Ford, 2003). In Bukavu, for example, the decision not delivering condoms is in accordance with the policy of Catholic Church. Those who have responsibility for the peer education programme and Health Department of the diocese fiercely refuse to supply condoms, particularly to girls living on the street and surviving as sex workers. They also do not consider condoms as a means of protecting them from unwanted pregnancies, HIV or other sexually transmitted infections, but rather as a persuasion to continue their way of life (Bosmans et al., 2006). Sociocultural barriers and strict conformity to religious doctrine can prevent adolescents from receiving appropriate and comprehensive sexuality education and cause peer education officers to block condom supplies (Bosmans et al., 2006, p. 8;Parker et al., 2013). In fact, close collaboration between religious leaders and HIV prevention programmes is necessary to ensure success (Carlos et al., 2015;Muanda, Ndongo, Messina, & Bertrand, 2017;Parker et al., 2013). Multiple partnerships Although condom has been a main factor explored in the literature, several other factors are hypothesised to influence effective prevention of HIV and Sexually Transmitted Infections. A study using cluster sampling conducted in the eleven provinces of the Democratic Republic of the Congo among 8617 adolescents, indicates that adolescents reported having at least two sexual partners; males had more partners (62.2%) than females (35.3%) (Kayembe et al., 2008a). Another study conducted in Kinshasa also finds that most participants were already sexually experienced, and have had concurrent sexual partners while not using condoms consistently. The low rate of condom use and the high percentage of young people reporting concurrent sexual partners suggest that Congolese teenagers and young adults remain a group highly at risk of STIs and HIV transmission (Kayembe et al., 2008c). Concurrent sexual partners and inconsistent condom use can seriously hinder the effective fight against HIV/AIDS (Carlos et al., 2017;Lusey, Sebastian, Christianson, Dahlgren, & Edin, 2014). Thus, concurrent sexual partnerships can increase an individual's risk for acquiring HIV after the inconstancy of condom use (Carlos et al., 2017;Steffenson, Pettifor, Seage, Rees, & Cleary, 2011;Tarkang, 2015). The assumptions mentioned above may also hinder the prevention of HIV. To our knowledge, this is the first study in South Kivu that has investigated the attitudes on condoms, concurrent sexual partnerships and the context in which adolescent sexual activity is negotiated. Drawing on qualitative data, this study takes into consideration the sociocultural context in which personal and collective experiences, knowledge, attitudes and behaviours are constructed and negotiated. The aim of this study is to investigate young men's perspectives about condoms, concurrent sexual partners and sex in the context of HIV/AIDS. Studying adolescents Given that it is important to investigate the attitudes of men towards condoms and concurrent sexual partnerships, actually, considering men in projects on violence against women is important and enables to reduce 'problems brought by immoderate manhood and harmful concepts of masculinity' (Adomako & Boateng, 2007). The UNFPA (2006, p. 5) similarly maintains that reducing gender-based violence would benefit from the support and involvement of males since 'men are increasingly adopting notions of "masculinity" that restrain their humanity, and put their partners and themselves at risk'. This study focuses on adolescent young men, and it is justified by the realisation that the process of becoming an adolescent, as experienced by them is, in most cases, characterised by the differentiation of behaviours and controls that impinge differently on men. Adolescents provide a perspective on how gender norms are socialised and a sense of how early such socialisation occurs. Adolescents are in the liminal position of not quite children or adults. They should be approached because they are old enough and able to explain how chronicle events, including violence that is pertinent in their lives. Theoretical framework The present study was guided by gender and power theory. Gender and power theory, which focuses on the social norms associated with relationships between men and women, may inform our understanding of the use of condoms and concurrent sexual partners (Connell, 1987). Some scholars extended Connell's theory into public health to include behavioural and biological risk factors as explanations for women's increased risk for HIV. Their model includes alcohol and drug use and high-risk concurrent sexual partners who have been linked to sexual intercourse (Bal, Mitra, Mallick, Chakraborti, & Sarkar, 2010;Versteeg & Murray, 2008). Others DePadilla, Windle, Wingood, Cooper, and DiClemente (2011) validated Wingood and DiClemente's model with empirical data demonstrating the relationship between theoretical constructs of gender and power and condom use (Wingood & DiClemente, 2000). Pulerwitz, Amaro, De Jong, Gort maker, and Rudd (2002) found that in the construct of sexual relationship, power may account for failure in the use of condoms. Additionally, gender and power theory outlines norms governing social and sexual relationships between men and women (Connell, 1987). It provides a practical tool for explaining how gender norms and beliefs are associated with HIV risk and sexual behaviour (DePadilla et al., 2011;Hahm, Lee, Rough, & Strathdee, 2012;Teitelman, Tennille, Bohinski, Jemmott, & Jemmott, 2011;Weine et al., 2013;Wingood & DiClemente, 2000). Gender and power theory establishes that women's limited economic position and dependence on men for financial support (Connell, 1987;Wingood & DiClemente, 2000), diminishing their ability to negotiate safer sex or condom use (Hardee, Gay, Croce-Galis, & Peltz, 2014;Wingood & DiClemente, 1998). Women's lack of negotiating power within sexual relationships due to male control, power or coercion (Connell, 1987;Wingood & DiClemente, 2000), may put women in a more vulnerable position when it comes to HIV risk (Ackermann & De Klerk, 2002;Jewkes & Morrell, 2012;Raiford, Seth, & DiClemente, 2013;Riley & Baah-Odoom, 2012;VanderDrift, Agnew, Harvey, & Warren, 2013). The broader social principles governing gender norms and expectations often define sexual behaviour for women and outline normal female behaviour within relationships (Connell, 1987;Wingood & DiClemente, 2000).Thus, gender norms may lead to the expectation that women are 'passive' receivers in sexual relationships, that women might not or should not carry condoms, or expectations that women should defer to men in making decisions about sexual behaviour (Connell, 1987;Wingood & DiClemente, 2000), which leads to greater HIV risk for women ( Richardson et al., 2014). This study posits that gender and power may play a significant role in men's dominance over and oppression of women, thus exposing women to Sexually Transmitted infections and HIV. Method A qualitative research method was employed to investigate young men's perspectives about condoms, concurrent sexual partnerships and sex in the context of HIV/AIDS. The study has also employed grounded theory, one of the qualitative approaches. Qualitative research is an examination process of understanding based on distinct methodological traditions of investigation that explore a social or human problem. The researcher builds a complex, holistic picture, analyses words, reports detailed views of participants, and conducts the study in a natural setting (Creswell, 1998, p. 15). One of the features of qualitative research is that it involves systematic discovery. Its objective is to produce knowledge of social events by understanding their meaning to people, investigating and documenting how people exchange ideas with each other and how they understand and interact with the world around them (Ulin, Robinson, Tolley, & McNeill, 2002, p. 26). By employing this method, we were able to understand participants' attitudes and behaviours. In fact, the qualitative researcher needs to attentively watch incidents and actions as they occur without any action affecting them or interference (Babbie & Mouton, 2001, p. 270). This study employed two of the qualitative methods for data collection which are namely: focus groups and individual interviews. Focus groups and individual interviews were conducted with boys. The research assistant and I conducted focus groups and individual interviews. In a focus group, we played the part of being a guide, but within the group, a multitude of interpersonal dynamics occurred and it was through this group interaction that data were generated (Morgan, 1993). We played a low-key role and gradually introduced a limited number of questions at appropriate times. For example, one focus-group question asked whether they thought it was it important to use condoms. If yes/not explain why. The related interview question was 'If your girlfriend or wife asked you to use a condom, what would you think?' Other questions included in our research were: (1) Is it OK to have more than one girlfriend at a time? If yes/not explain why. (2) How many girlfriends do you have at the present time? (3) Can you have a relationship (with your girlfriend) without sex? If yes/not explain why. Focus groups can help participants develop a point of view that goes beyond their individual context and thus may change 'personal troubles' into 'public issues'. The group process can also nurture collective identity and provide a point of contact to initiate grassroots transformation. Although focus groups can, in theory, simply reflect or monitor change, there is always the potential for the focus group process itself to initiate changes in respondents' thinking or understanding, through exposure to the interactive process (Barbour, 1999). Focus groups can bring significant support to sensitive research. They can be useful in enabling access to particularly sensitive research populations and giving voice to sections of the community that frequently remain unheard (Farquhar, 1999). We chose focus group discussions as a key tool of data collection because of our interest of understanding of attitudes and behaviour as a social phenomenon which are strongly influenced by peer norms. The individual interview was chosen as a supplementary technique in this study to get opinions that could be difficult to divulge in focus group discussions. Four focus group sessions were conducted in Swahili, the language spoken by the participants, and lasted between 90 and 120 minutes. The interviews took place during the two weeks that followed each focus group, and lasted approximately 60 minutes. This approach was vital to gaining access to the perspectives of participants who could be ostracised by their peers because of their views or become extremely passive in the focus groups. The interviews also enabled us to personalise the issues raised in the focus groups, to probe responses where relevant, and helped to explore further and confirm themes emerging from the focus group interviews. That being said, the specific benefit of focus groups derives from the interaction, discussion and exchange of ideas among the participants. The use of direct quotations from participants is a way of guarding against a dominating individual, as was following up in the interviews. I transcribed the verbatim audio from the tape recorder from Swahili to English for later analysis. Data were translated, reread and back-translated from Swahili to English. Participants Fieldwork and data collection were carried out in 2011 in four secondary schools in Bukavu and Kavumu. Bukavu, the capital of South Kivu province, is overcrowded with people who have moved from elsewhere seeking greater security. The population was about 800,000 in 2012. Kavumu is a rural area located some 50 km to the north of Bukavu. Given the widespread destruction and deterioration of basic infrastructure throughout the province, people in Kavumu have difficulty in accessing basic social services such as education, sanitary drinking water, and primary health care. Such services are somewhat better in towns, but are limited in both quantity and quality. Most of Kavumu's houses are built with flat timber and others with mud and timber. In terms of sampling, the four schools were chosen for reasons of convenience but are broadly typical of high schools in the province. A total of 28 young men participated in the study. The 28 participants (the number was chosen to allow one focus group of seven boys at each school) comprised about a quarter of the students at the schools who were in their final two years of study and were between 16 and 20 years old. The school's principal and one teacher, from each school of our data collection, chose from grades 11 and 12 the male students who participated in the study. In choosing a sampling, the goal is to select a group of participants who are purposefully located to express themselves on the topic under exploration (Gerson & Horwitz, 2002). Five participants from each focus group were invited to voluntarily participate in individual interviews. The five out of seven focus group participants were chosen for the reason of convenience and that in individual interviews, we may have 20 boys. These individual interviews were conducted with 20 volunteers from the 28 focus group participants. The majority of participants in this study were from the Bashi ethnic group. Data analysis Data from focus groups and individual interviews was analysed using thematic and grounded theory analysis to identify the themes that emerged. This method faces the risk that a researcher may deliberately or unintentionally inject her/his own biases into the results and reach inaccurate conclusions; that is, it may fail in terms of internal validity. Careful action was taken in order to prevent this from happening by having two people to carry out the focus groups and interviews, and two other people to help with the recording and scribing. We drew attention to certain information in description (Huberman & Miles, 1994). We read the entire data in depth in search of meanings and patterns, and wrote down ideas, reflective notes (Creswell, 1998;Huberman & Miles, 1994), and formed initial codes during the analysis process. We engaged in axial coding-causing condition, context, and consequences. We also engaged in open coding-categories, properties, and dimensionalise properties (Bodgan & Biklen, 1992), made contrasts and comparisons (Huberman & Miles, 1994), and developed coding categories. We developed a guide for coding the focus groups and interviews, and reread to identify themes as they emerged. During the coding process, we retained the individual extracts of data and the dominant stories (Braun & Clarke, 2006). We reduced information by sorting material into categories (Bodgan & Biklen, 1992), and noted patterns and themes. We related to categories by building a logical chain of information (Huberman & Miles, 1994). By interpreting data, we engaged in selective coding, development of stories, and developed a conditional matrix. Finally, we presented a visual model and propositions. We aimed at reporting experiences and relying on voices and interpretations of participants through extensive quotes, presented themes that represent words used by participants, and advance evidence of different views on each theme (Creswell, 1998, p. 76). Vicsek (2007) suggests often citing in thematic analyses not only isolated manifestations, but also fragments of discussions containing several contributions. In the process of preparing this article, we had a number of discussions concerning the codes that emerged under each theme. The themes we identified arose largely from the data that we collected through questions asked during the focus groups and individual interviews, which, in turn, arose from our research objectives. The views expressed in this study were very similar between both rural and urban respondents. There were strong similarities in views expressed in the focus groups and interviews and only slight contradictions were observed in individual interviews where some participants disagreed with the dominant views from the focus groups. Participants, from urban area, indicated that the women's initiative to suggest condoms should not be perceived as a sign of having sexual relations with a number of partners. Ethical considerations All procedures necessary in studies involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical clearance for this study was granted by University of KwaZulu-Natal's Ethics Committee and permission was also obtained from the South Kivu Department of Education and from the schools where the study was conducted. Research procedures were explained to participants, stressing that their participation is voluntary. No one refused to participate. Contrary to that, participants were very engaged during the focus groups and interviews. Informed written consent was obtained from all individual participants, and parental permission was obtained for participants under the age of 18. Confidentiality and anonymity were strictly respected, and pseudonyms were used in order to identify the responses. This study reports the respondents' comments in their own words and the quotations presented are, unless otherwise noted, representative of the beliefs articulated by the majority of participants. Results This study investigated young men's perspectives about condoms, concurrent sexual partners and sex in the context of HIV/AIDS. The following themes and sub-themes emerged from the study: 'you cannot eat a candy in a wrapper', condoms convey 'have sex and satisfy your sexual urge', condoms are ineffective, a girl who suggests a condom is untrustworthy, and concurrent sexual partnerships help make a choice of spouse. 'You cannot eat a candy in a wrapper' Young men socially construct their ideal of true love in unprotected or 'flesh-to-flesh' sex. Most participants, during focus group discussions, indicated that young men prefer flesh-to-flesh sex rather than protected sex using a condom: … Nowadays' youth often say: 'you cannot eat a candy in a wrapper, which means skin-to-skin'. It is not any youth who agrees to use condoms. (Bahati,18,urban boy) There are boys who say that they cannot eat a candy in a wrapper, and that they must have flesh-to-flesh sex. Such boys may think that a girl does not love him if she suggests him to use condom. (Iste,18,rural boy) Most participants had a strong preference for 'fleshto-flesh' sex because condoms were perceived to reduce sexual pleasure. Therefore, they promote beliefs such as 'you cannot eat a candy in a wrapper'. This adage is seen as a motive to promote unsafe sex. Boys' preference for sex without condoms may be one of the contributing factors to the spread of Sexually Transmitted Infections and HIV. Condoms convey 'have sex and satisfy your sexual urge' Most participants perceive condoms to encourage inappropriate sexual activity. In effect, they believe that condom promotion campaign and sell entail young people to have sex: The fact of selling condoms encourages the widespread of sexual violence. It is as if people are conveying a massage saying: 'have sex and satisfy your sexual urge'. This can discourage men to hold their sexual urge until they get married, because they know that they cannot get diseases. (Gustave, 18, rural boy) I think that condoms encourage people to become sexually active. Condoms convey the message to people that they cannot get HIV or pregnancy. This has become a vehicle to encourage sex. (Leopold, 20, rural boy) I would like to say that condom is bad because it encourages sexually activities in the country. People are using condoms because they know that the condom protects, that is why we advise those who make condoms to reduce their production or stop making them. (Clever,17,rural boy) Most participants believe that the commerce of condoms encourages sexual activities in the community. Thus, they strongly suggest the cessation of their production. For boys, the selling and supplying of condoms portray a message that invites men to have sex. Condoms are not only difficult to access privately but there are also problems to negotiate their use as women lack the power to do so. For some participants, the suggestion of condom use should not be mentioned in the household setting. Boys' beliefs are actually linked to religious practices as they emphasise that the Bible forbids men to pour sperm on the ground. For married men the condom is not an option, they are not allowed to use it, because the Bible forbids men to pour sperm down [everybody laughs]. (Clever,17,rural boy) Most Christian churches in the country discourage the use of condoms. They instead promote abstinence and monogamy as ways of preventing HIV/AIDS. Condoms are ineffective There is a widespread belief that condoms have a 'small hole' or are unreliable and are therefore not effective in preventing pregnancy, HIV and Sexually Transmitted Infections: It is not good to use condoms, because a condom has a small hole that is invisible to the eyes. Once you use it, you should know that you put yourself in risk of getting HIV. Because disease can enter through that hole, particularly when you ejaculate the sperm passes through it and enters a woman. That's why, I say that condoms can cause unwanted pregnancy; because once you use it [condom], you expose yourself to danger. (Aristote, 17, urban boy) It is not ok to use condoms. It seems that research has revealed that there is a small hole; I forgot the name that scientists call it. First of all, the person who uses condoms, if we look among youth, he is not confident of himself. (Bisimwa, 17, urban boy) … Although the condom has small holes there are other sicknesses that it can prevent. I insist that it is bad to use condoms because they are made by the machine. Therefore, it is possible for condoms to have holes that are invisible. (Baraka,17,urban boy) Most participants believe that when people use condoms they deliberately put themselves at risk of contracting Sexually Transmitted Infections as condoms have small invisible holes. Therefore, condoms can only prevent some diseases. Condoms are further described as ineffective because humans make them. Participants also indicate that people who use condoms lack confidence. This clearly demonstrates how immoderate masculinity can put young people at risk of contracting HIV and Sexually Transmitted Infection. The condom's ineffectiveness may also be attributed to its fabric. Therefore, young men think condoms can easily break during sex: I think that it is useless to use condoms because you can wear condom yet you still get disease or get pregnant. A condom is made with a soft tissue that is not strong, when the skin scratches it, the condom can break. Concerning sexually transmitted diseases, it is difficult to protect from them, because I realized that even if you use thousand condoms at once, the way that they are built can cause cancer. You can get sicknesses or pregnancy though you wear them, they can prevent absolutely nothing. (Bahati,18,urban boy) The widespread distrust of condoms causes young men to believe that condoms can cause cancer due to the way they are built. Participants might have referred to substances containing condoms. A few participants believe that the condom can slip off into the vagina during sexual intercourse: It is not good to use condom because you may wrongly wear it, and therefore stuck in the vagina, and causes trouble. (James, 18, rural boy) A girl who suggests a condom is untrustworthy In response to a question about their use of condoms, most of the participants were clear that the decision to use condoms was a male prerogative. That is, females cannot initiate the condom use. During individual interviews, the majority of young men commented that if their girlfriend or wife asked them to use a condom, they would interpret it as a lack of trust in them or would suspect her of being unfaithful: If my fiancé or my girlfriend asks me to use condom whereas we always have sex without the condom, I can really doubt of her. I am going to think that she cheated on me. (Espoir, 19, urban boy) Let's suppose that your girlfriend suggests you to use condom, for me it is not ok. I can think that she is sick or she avoids falling pregnant. (Aristote, 17, urban boy) If I were married then my wife insists on using condom whereas I do not like, I may suspect her to hide something that she does not like me to know. I can really investigate to know what is going on. (Toussin, 18, rural boy) The majority of participants interpret their female partners' request to use a condom as an implication of their partners having sexual relations with other men outside of the relationship. There is actually a certain stigma that is associated with women's suggestion to use condoms, as this signifies either infidelity or experience in initiating sex, both often considered to be unacceptable characteristics for women. Other boys, albeit a minority, spoke in favour of condom use. According to them, women's request to use condom should not be perceived as a sign of having had sexual relations with a number of partners: The women's initiative to suggest condoms should not be considered as if she had sexual relations with a number of partners. On the contrary, young people should be encouraged to carry condoms because there are cases that happen when you are not expected. For example, you are a girl walking in the bush or during the night, you may meet men who rape you but if you have got condoms you may plead with them and suggest them to use them so that you may not get diseases or get pregnant of a child who will not know his father. (Baraka, 17, urban boy) … Some young people like to drink beer, we know that there are some beers you drink and arouse your sexual urge. Maybe you go with your girlfriend to the nightclub. Although you do not like to have sex with her you get it [urge] once you are under the influence of alcohol. That is why we must carry condoms wherever we go to protect ourselves against STIs and HIV. We have to carry them [condoms], and consider them as weapons. (Issa,19,urban boy) Some participants resisted the perception that viewed women requesting condom use as the practice of having sexual relations with a number of partners. During individual interviews, they acknowledge the importance of condoms, and advise their peers to use them in order to prevent Sexually Transmitted Infections, HIV and pregnancies. However, girls' agency in sexual negotiation may seem difficult. They find it extremely difficult to bargain the use of condoms, especially when, at the same time, they are expected to show a lack of experience in sexual matters. Concurrent sexual partnerships as an acceptable norm before marriage Most participants are very clear that it is appropriate for them to have more than one girlfriend. They will thenby being able to compare girls-be more able to make a good choice of a marriage partner. There is at least some implication that, when he marries, he will be faithful to his wife. It is ok to have two girlfriends. It is a must to have two girlfriends because you are going to compare them. Meanwhile, you can ask yourself: 'Is this or that other one who deserves to be married?' I think having many girlfriends depends on every individual, and according to what he looks for. (Toussin, 18, rural boy) Boys must have many girlfriends because it is during youth period that you plan your future life so that you may say at end 'my wife is like other women.' If you have dated many girls, you must have discovered that women are all the same because you marry once and live with one wife. (Patrick, 17, rural boy) An eighteen years old boy cannot only have one girlfriend. He must examine girls' characters because girls are hypocrites. That is why he has to make an effort to have at least two girlfriends before he gets married, he must examine their characters to choose one. (John,17,rural boy) The concurrent sexual partnership practice is seen as a weighing mechanism that facilitates a boy's choosing a spouse among his girlfriends. Most participants base the choice of a spouse on a comparative paradigm. Young men especially avoid marrying girls who easily accept sex or have sex with other men. The spouse's choice is actually based on her reputation. Through the expression 'my wife is like other women', boys convey that a young man who has had more than one sexual partner will remain faithful to his wife as a result of his previous sexual activities and understanding. He may have acquired the sense of contentment, and therefore finds no reason to have sex outside his marriage. Resorting to the second girlfriend for sexual gratification Not all boys' girlfriends, it should be noted, may be sexual partners. During the individual interviews, when the interviewer asked participants about the number of girlfriends they had, their answers included the following: It is ok to have many girlfriends because in these days boys are more focused on sex. Now do you imagine if somebody is looking for sex, and does not find it? He has a girlfriend who does not like it. Then, he is obliged to look for another girl who can gratify his sexual urge. (Josi,18,urban boy) Some participants view concurrent sexual partnerships as a middle ground strategy implying that the boy eventually keeps a girlfriend just in case his primary girlfriend 'breaks his heart'. They view the necessity of such a practice for desire for sexual activity. They believe that it is irrational for the man to have only one girlfriend because if she does not agree to have sex, he may have no one to provide it. Sex is viewed as the substance around which a relationship generates its subsistence. Some participants also see benefit in having one girl for a trusting relationship and another one essentially for sex. There is an implication that a boy may have several girlfriends but in the event marry someone else: We must have two girlfriends at the same time … You must have one that you trust and know that she cannot deceive you, and the other one to have sex with her or to speak unnecessary things with her. (Aimer,19,rural boy) A few participants, during the individual interviews, contest the idea of having more than two girlfriends: It is not ok to have more than two girlfriends … You surely encounter financial problem because nowadays' girls like boys to take them out. When your girlfriend tells you so, you are obliged to take her out … and you find spending money to all these girls. (Derrick,19,urban boy) In fact, it is not good to have more than two girlfriends, but it happens that when you are in relationship with a girl, her girlfriend falls in love with you. Although you love your girlfriend so much but because her girlfriend gives you money, then, you find yourself turning your attention to her. She buys you shoe polish and other things you need. Finally, if you like to have sex with her, she does not have time to say no. (Iste,18,rural boy) For some participants, the effort of a young man to keep one girlfriend may come to an end when his girlfriend refuses to provide sex, then he eventually finds another girl who not only provides it, but also is even ready to financially assist him. Some boys may keep the second girlfriend for financial gain. Money is also mentioned as a limiting factor for boys to engage in concurrent sexual partnerships. Girls want boys to take them out. It is worth mentioning that the majority of young people are unemployed which may limit some young men to entertain current sexual partners. Most participants view peer pressure as an important factor that encourages sex. Further individual interviews showed that waiting is possible, but peer pressure makes it very hard for young men to have a relationship without sex: I sometimes think that my classmates and boys we grew up together will look down on me because I do not have sex with my girlfriend. The girlfriend also may laugh at me. Today or tomorrow she is going to say that I was not her boyfriend. Consequently, many friends of mine had sex with girls who have got HIV/ AIDS and impregnated them because of such a belief. (Iste, 18, rural boy) It happens when you are with your friends; they start talking about their girlfriends and how they had sex with them. You are influenced when you find that you are the only one who has never slept with a girl. You may sometimes tell them that you have never slept with a girl, they start giving you strategies to apply for sex. You soon apply them and succeed to have sex with her. After sleeping with her, she may get pregnant but when you tell them what happened, they laugh at you though they are the ones who advised you to sleep with her. Maybe her parents may send you to jail after denying that pregnancy; your friends continue to laugh at you. (Bashengezi,18,urban boy) The interviews on this issue brought out the tension of believing one thing as an individual but practising something else because of peer pressure and socially constructed expectations. Boys who experience low self-esteem may rely on others for affirmation. This may lead them to search for self-esteem in sexual encounters. Participants also view peer groups as the training ground where young men discuss sexual persuasive strategies. Despite the encouragement, peers laugh at the boy who impregnates a girl. And if he has a relationship without sexual involvement the girlfriend may also look down on him. From the outset, there is seemingly a good deal of frustration for the boy. In sum, the findings have shown strong link between both resistance to condom use and concurrent sexual partnerships in association with HIV risk. Participants justified the concurrent sexual partnerships as a way of ensuring that they cannot miss a girl to satisfy their sexual desire. The sexual activity particularly in these conditions may increase an individual's risk for acquiring HIV. Discussion Qualitative methods were applied to investigate the perspectives of young men about condoms, concurrent sexual partnerships and sex in the context of HIV/AIDS. Results from a series of four focus groups and 20 individual interviews with rural and urban young men suggest that while young men mistrust condoms, they have concurrent sexual partners. If we consider participants' perspectives, several important points emerged that help understand the reasons why many young men feel reluctant to use condoms. In response to questions regarding condoms, participants reported that they do not like to use condoms because they are unreliable, ineffective, and have a small hole that is invisible. Consequently, when people use condoms during sexual encounters they may run the risk of contracting sexual diseases. Young men's stigma about condoms might have increased due to the lack of effective awareness and prevention campaign. Barker and Ricardo (2005) corroborate that young men demonstrate uncertainty and lack of confidence regarding the use of condoms. Condoms are thought to be ineffective or defective. Most participants, in the current study, also believed that condoms can easily break or slip off into vagina during sex. This concurs with a study conducted in Nigeria, in which respondents reported that the inconsistency of condom use is additionally due to widespread distrust of condoms based on respondents' usage experience, which indicates that 'condoms burst, tear, and leak'. Consequently, condoms do 'not offer you 100% protection' during premarital sex. Respondents associate condom unreliability with both user error and poor product quality (Okonkwo, 2010). In the CADRE's (2007) survey in South Africa, participants believed that condoms could practically do little to prevent infection. Using condoms entails the possibility of condom failure with the result that a person remains vulnerable as the condom can burst and cause an infection. Crosby, Charnigo, Weathers, Caliendo, and Shrier (2012) and Sanders et al. (2012) find that among the most common errors are condom breakage, slippage, and leakage, which are the primary errors that reduce condom effectiveness. Sanders et al. (2012) argue that the slippage during withdrawal may reflect the user error of not holding the edge of the condom during withdrawal. Some participants in the current study also believed that condoms can cause cancer. Versteeg and Murray (2008), in their study among men and women on condom use in South Africa, found that some participants believed condoms could cause sickness. The practice of having sex without condoms was also motivated by males' belief that having sex with condoms can diminish pleasure during sexual intercourse. Therefore, most participants subscribed to beliefs such as 'you cannot eat candy in a wrapper', which encourages them to deliberately engage in unprotected sex ('flesh-to-flesh') as a sign of celebrating their maleness. They reiterated the desire to have 'flesh-to-flesh' sex, i.e. sex without a condom. These attitudes and their impact on risk-taking behaviour may be linked to the spread of HIV. Participants therefore, indicated they wanted 'flesh-to-flesh' or 'skin-toskin' sex. They also mentioned that the commercialisation and distribution of condoms encourage sexual promiscuity in the community. A number of studies in South Africa have discussed the above assumption. Versteeg and Murray (2008) indicate that participants believed condoms decrease sexual pleasure and hinder men to perform positively during the sexual act. Ackermann and De Klerk (2002) find that men mentioned that condoms decrease sexual gratification. Hunter (2002) demonstrates, in his study conducted in KwaZulu-Natal, that dominant masculinity sometimes shapes men's violent control over women, and encourages men to insist on 'flesh to flesh' sex. He also notes that men convince women that condom use leads to 'bad sex', and is associated to 'unfaithful' behaviour. They believe that true love is represented by inyama enyameni ('flesh-to-flesh' sex). MacPhail and Campbell (2001) claim that the concept of masculinity is connected to the belief of unprotected sex, 'flesh to flesh', as being more pleasurable. Hunter (2002), and Wood, Lambert, and Jewkes (2008), indicate that in South Africa dominant masculinities construct men's excessive control over women and their insistence on having 'flesh to flesh' sex. This also corroborates the findings of Njue, Voeten, and Remes (2011) in Kenya, in which young men mentioned trust, discomfort and the reduction of pleasure as reasons for not using condoms. For most girls, not choosing to use a condom was connected to their limitation to demand its use, intimacy and pleasure, and mistrust about its safety. Sanders et al. (2012), in their study in the United States, maintain that the ineffectiveness of condoms may not only compromise condom effectiveness, but may also discourage condom use if people become frustrated or have less pleasurable experiences as a result of their use. However, Crosby, Graham, Yarber, and Sanders (2010) argue that problems with erection reduced pleasure (for both partners) and failure of the female partner to orgasm were noticed more frequently among men reporting a lack of time for condom application. The girl's request to use a condom was viewed with distrust. A number of participants commented that if their girlfriend or (future) wife asked them to use a condom, they would interpret it as a lack of trust in them. They would also suspect her of being unfaithful. Women who suggest condoms are viewed as carriers of disease. The girl's request to use a condom is also seen as a lack of love towards the male partner. Participants perceived certain threats to reputations of women suggesting condoms, especially when she demonstrates having the skill and confidence to negotiate condom use that implies sexually activity and experience in initiating sex, with potentially negative associations. Women are thus unable to insist on condom use due to the lack of communication regarding sexual matters and power in interpersonal relationships. These inequalities may reduce female voice in sexual negotiation. In such situation, the possibilities for women to insist on condoms are very limited. Therefore, having sex and the control around it only depends on the men's will. Most of the participants' perceptions clearly described how the imbalance of gender power impedes women's power to negotiate safe sex. Gender power disparity significantly hinders their ability to negotiate and suggest condoms, and further exposes both partners to Sexually Transmitted Infections and HIV. Ackermann and De Klerk (2002), in their study in South Africa, indicated that if a woman suggests the use of condom, men accuse of being unfaithful or hiding sexually transmitted diseases. This also corroborates with Hallman's (2004) study conducted among young women and men aged 14-24 years in KwaZulu-Natal, which finds that young men consider women who suggest condom use as loose. The simple talk about condoms use could lead to psychological, corporal and economic abuse. Various studies conducted in sub-Saharan Africa found that sex among adolescents often results in very little negotiation or communication. Condom use is the mostly difficult point among youth since it refers to uncleanliness and infidelity, and many girls describe a fear of punishment for initiating a conversation on this issue (Varga, 1997). MacPhail and Campbell's (2001) study conducted in South Africa, also finds that these sexual behaviours are reinforcing among peers. Young men are criticised for using a condom and, as a result, may decide to not use it again. A study conducted in Kinshasa and Bukavu finds that condom use was related to promiscuity rather than being seen as an act of accountable behaviour based on respect for the adolescent's own well-being and that of his or her girlfriend or boyfriend (Bosmans et al., 2006). Langen (2005) observed similar patterns in her research in South Africa and Botswana, where gender power disparity significantly reduces women's power to suggest condoms to their boyfriends. Some participants mentioned that they are reluctant to use condoms because condoms are man-made, and the Bible forbids men to waste sperm. This indicates that religious thinking is one of important factors behind young men's beliefs on condom use. This corroborates Muanda et al.'s (2017) study conducted in other three provinces of the DRC in which some participants reported such beliefs. Bosmans et al. (2006) confirm that sociocultural barriers to discuss sexuality and the promotion of condom use were reinforced by strict obedience to Catholic doctrine. Religion was used as the justification for reducing the 'ABC' approach (abstinence, be faithful, condom use), whereby abstinence was promoted among adolescents as the only responsible way of behaving. The Refugee Council (2004) argues that in the DRC, contraception is not often used, particularly in rural areas, and that some religious leaders are opposed to it because they believe that it will encourage immorality. Brummer (2002) maintains that condoms were seen as 'not natural' or people doubted of their ability to guaranteeing safe sex. However, a few participants, in the current study, advised young men to leave religious beliefs aside, and only focus on how to prevent themselves from the risk of contracting sexually transmitted diseases. It is clear that the majority of participants deliberately ignored the risk of contracting Sexual Transmitted Infections, and have strong inclination to concurrent sexual partnerships and inconsistent condom use. Most participants actually justified the concurrent sexual partnerships as a way of ensuring that they cannot miss a girl to satisfy their sexual desire. Some participants saw the benefit in having one girl for a trusting relationship and another essentially for sex. Concurrent sexual partnerships may encourage the spread of Sexually Transmitted Infections especially in the context of South Kivu as men reject condom use. CADRE (2007), in a South African cross-sectional survey, found that concurrent sexual partnerships give hope and provide a supportive structure that ensures that sensitive support is always available. In particular, having another partner provides a cushion of support, should a current relationship end. Wood et al. (2008), in their study conducted in South Africa among young people, found that it was common for young people to have more than one partnership at a time whether casual or more serious (and an entire slang terminology was used to describe sexual partners positioned differentially in the hierarchy of an individual's relationships). Rakgoasi (2010), in his study in Botswana, found that concurrent sexual partnerships were necessary if partners refuse to have sex with them, especially if the reasons for their partner's reluctance towards sex are not clear to them. To many men, they then engage in concurrent sexual partnerships, which are needed to meet their need for sex; two girlfriends are enough, largely because of financial commitments. Participants in the current study also reported that peer pressure makes it very hard for them to have a relationship without sex. They implied that a relationship between a man and a woman must involve sex so that when this ends she cannot consider him useless or not enough of a man, and thus bad mouth him when she is among her peers. An ethnographic study conducted in North Western Tanzania describes how one of the participants had a long relationship with his girlfriend, which only involved talking, joking and caressing, until his male friends encouraged him to have sex with her (Wamoyi, Wight, Plummer, Mshana, & Ross, 2010). The study conducted in Kenya by Njue et al. (2011) finds that peer influence was a great driving force for risky sexual behaviours. Wood et al. (2008) demonstrate how in South Africa the control of women became an important aspect of successful masculinity among young men, which is proved through their ability to have the most desirable girlfriends, and to control their girlfriends. Masculinity was constructed as an on-going challenge among male peers. Sexual conquest was considered a sign of status mostly attained by wooing, trickery and the use of force. Barker and Ricardo (2005) argue that men often aim to demonstrate their masculinity before their male friends and social group within restricted ideas of what it means to be a man. They describe a sense of being monitored and watched to see if they meet the standard versions of masculinity. Other men and women assess the achievement of masculinity. Most participants viewed concurrent sexual partnerships as an opportunity to make a choice of spouse from among their girlfriends. The practice may assist them in making a better choice in terms of marriage. They stated that they need to maintain concurrent sexual partners because of the idea that having concurrent sexual partners would allow them to compare and select the partner who is most suitable for marriage. Character is seen as a more reliable criterion for selecting wives. Participants believed that a girlfriend's appearance and character determine trustworthiness. Women who reject the sexual advances of other men, and who are viewed as 'controlled', gain value as the best future spouses. Although participants indicated the importance of concurrent sexual partners, when they want to marry, they do not marry girls with whom they have had sex. They mentioned that they choose their future spouses on the basis of their [girls'] reputation. This may result in the girl's hypocrisy. As a reason for responding negatively to requests for sex, girls often say no although they may desire it, they are afraid their boyfriends will afterwards despise them, consider them prostitutes, and recount their sexual endeavours to their peers (Mulumeoderhwa & Harris, 2014). A study in South Africa reports that participants support concurrent sexual partners as a way of ensuring that men cannot miss a girlfriend if one 'breaks your heart', and this practice also helps men make the choice of a future spouse (Mulumeoderhwa & Harris, 2013). Limitations of the study Considering that this study took place among 28 students in four schools in one province of the DRC, it would be inappropriate to generalise its findings. Young women were not included in the study which weakens the paper; we admit that their voice would have made the paper much stronger. In the future, other researchers need to investigate the girls' attitudes on condoms and concurrent sexual partners, and also the role that churches play in influencing the condom use. It can be hoped that those concerned with reducing gender inequality in the DRC and elsewhere will find the research insightful. Recommendations Given the boys' failure to use condoms and their strong inclination to concurrent sexual partnerships, there is a need for heath groups and stakeholders within the area to increase awareness about condoms' effectiveness and improve knowledge dissemination on Sexually Transmitted Diseases and how they are prevented. However, this is not the place to discuss how such attitudes could be cost effectively spread; that would require a separate study. Peacock and Levack (2004), Chege (2005) and Petersen, Bhana, and Mckay (2005), among others, have reviewed various efforts of 'constructive male involvement' to reduce levels of gender violence. These findings also underscore the need for prevention efforts targeting fidelity which may foster sexual behaviour change, and ultimately curb the spread of HIV. Conclusion This study has shown up that the strong mistrust of condoms and strong support for concurrent sexual partnerships may hinder the effectiveness of the fight against HIV/AIDS. The study has also found that negotiation regarding condom use is missing in many sexual relationships involving young people in South Kivu, and condom use remains unpopular in male-female relationships. Most participants had strong reservations on condoms and were positively inclined to the notion of having concurrent sexual partnerships. The study has also demonstrated that the imbalance in power relations between male and female partners in heterosexual relationships may hold a grip over the ability of young women to either refuse sex or negotiate the use of condoms. Thus, the boys' failure to use condoms and their strong inclination to concurrent sexual partnerships can significantly increase the spread of Sexually Transmitted Infections and HIV. Disclosure statement No potential conflict of interest was reported by the author.	2018-08-29T23:34:40.840Z	2018-01-01T00:00:00.000	{ "year": 2018, "sha1": "e7d5754644f093c3124368ac3538f00ccdaf98f7", "oa_license": "CCBY", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/17290376.2018.1516160?needAccess=true", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "e7d5754644f093c3124368ac3538f00ccdaf98f7", "s2fieldsofstudy": [ "Sociology" ], "extfieldsofstudy": [ "Psychology", "Medicine" ] }
57192893	pes2o/s2orc	v3-fos-license	A curious case of ankylosing spondylosis and motor neuron disease: A mere coincidence or correlation? Ankylosing spondylitis (AS) is a chronic (progressive) painful inflammatory rheumatic disease with genetic predisposition. Genetic susceptibility and common expression cause susceptibility to other inflammatory diseases such as psoriasis, ulcerative colitis, and Crohn's disease. However, cases of motor neuron disease (MND) in patients of biologically treated patients of AS have been rarely reported. AS does not follow the same course in everyone; even among affected members of one family, the outcome varies. Here, we present a case of an unusual AS without expression of human leukocyte antigen-B27 genetic marker who subsequently develops amyotrophic lateral sclerosis the most common form of MND. This mere correlation of one noncurable disease with one potentially treatable chronic rheumatological condition adds our knowledge to existing literature. Introduction Ankylosing spondylitis (AS) is an inflammatory disorder of unknown etiology that primarily affects the axial skeleton though peripheral joints and extraarticular structures are frequently involved. It is more common in males and usually begins in the second and third decades. AS belongs to the spondyloarthritis (SpA) family of diseases, which share several clinical, genetic, and immunologic features. [1] AS is distinguished in this family by involvement with sacroiliac (SI) joint inflammation or fusion and with more prevalent spinal ankylosis. [2] Amyotrophic lateral sclerosis (ALS) is the most common form of progressive motor neuron disease (MND). It is the prime example of neurodegenerative disease and is arguably the most devastating of all neurodegenerative disorders. AS is associated with extraarticular manifestations; the most common extraarticular manifestations are represented by uveitis, bowel disease, lung, heart, skin, bone, and kidney involvement. Many epidemiological studies have found higher incidences of extraarticular manifestations to be a consequence of uncontrolled systemic inflammation. [3] This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com The goals of treatment of AS and axial SpA are to reduce symptoms, maintain spinal flexibility, and reduce functional limitations and complications. The mainstays of treatment have been nonsteroidal anti-inflammatory drugs and exercise, with the additional use of slow-acting antirheumatic drugs in patients with peripheral arthritis. Treatments with biological agents such as tumor necrosis factor α (TNF-α) inhibitors have revolutionized treatment of AS but not without serious side effects. Cases have been reported where ALS has been developed in TNF-α inhibitor treated patient of AS. [4] Here, we have experienced ALS in a known case of AS who had no history of any biological treatment. Case Report Eight years ago, a 31-year-old male patient presented with symptoms of dull back pain, insidious in onset, felt in the lower lumbar and gluteal region, accompanied by morning stiffness which initially improved with activity. Within a few months, the problems became persistent and bilateral. With the passage of time, he experienced neck pain and stiffness with restricted mobility. He was diagnosed with having AS based on available criteria. Then, after 8 years, at the age of 39 years, he experienced weakness of all four limbs which started gradually and was progressive for the past 1 year. It started with the right upper limb followed by involvement of the right lower limb. Both left upper and lower limb were also involved sequentially within a few months with progressive wasting and atrophy of muscles incapacitating him to perform his daily works. There was also a history of slurring of speech and occasional choking along with flickering movement of the muscle. Bladder and bowel functions were normal. The patient is nondiabetic but hypertensive. There was no history of chronic obstructive airway disease, tuberculosis, or trauma. There was no history of fever, abdominal pain, convulsion, skin changes, diminished vision, ptosis, redness of the eye, or any episode of bloody diarrhea. He denied any substance or illicit drugs abuse. Family history was insignificant. On clinical examination, general survey was unremarkable. Higher function and cranial nerve examinations were normal. The neck was stiff with restricted movements in all directions. Other signs of meningeal irritations were absent. The cranium was normal, but the spine was curved with concavity anteriorly. In motor system, there was gross atrophy of muscles of the shoulder and pelvic girdles and distal limb muscles without any sensory loss. There was gross atrophy of the tongue, and fasciculation was also seen. The tone was increased. Power was 3/5 in proximal group of muscles in all four limbs with severe weakness in distal muscles with bilateral claw hands [ Figure 1]. Deep tendon reflexes in both the upper and lower limb were exaggerated bilaterally. Plantar reflex was extensor bilaterally. Glabellar tap and pout reflexes were present. Cerebellar signs were absent. Movements were restricted in all directions in cervical, dorsal, and lumbar spines. Modified Schober test was positive. Small joints of hands were swollen without any signs of inflammation. There was clawing of fingers and toes. No extraarticular manifestation was present. Investigations of routine blood were not remarkable apart from high erythrocyte sedimentation rate of 72 mm. Liver function and lipid profiles were normal. C-reactive protein was positive. Rheumatoid factor and anti-cyclic citrullinated peptide both were negative. Urine and stool analysis were normal. Both antithyroglobulin and anti-microsomal antibodies along with thyroid profiles were in normal range. Analysis of human leukocyte antigen-B27 (HLA-B27) was negative. Electrocardiography showed normal sinus rhythm. Echocardiogram was normal. Electromyography and nerve conduction velocity study of all four limbs revealed chronic diffuse alternating hemiplegia of childhood disease with secondary motor axonopathy. Dual-energy X-ray absorptiometry scan of hip and left forearm showed osteopenia (T score is 1.3 and 1.2, respectively) and lumbosacral spine was normal (T score 0.3). Chest X-ray showed increased reticular markings. X-ray of the cervical and dorsolumbar spine revealed gross ligamentous calcification of both anterior and intraspinal ligaments along with the loss of cervical and lumbar lordosis [ Figure 2]. Shoulder and bilateral knee joint X-rays both were normal. Hip X-ray showed left hip arthritis. Juxtaarticular osteopenia was seen in X-ray hand and wrist. X-ray of SI joint revealed mild sclerosis bilaterally. Magnetic resonance imaging of the spine revealed ankylosis of lower thoracolumbar vertebra with calcified intervertebral discs. Discs were fused in all direction with height reduction. No evidence of cord compression and significant compromise of neural foramina were present. Based on the clinical presentations, examinations, and radiological features, this patient was diagnosed with a case of seronegative spondyloarthropathy, i.e., AS and amyotrophic lateral sclerosis. He was put on methotrexate 10 mg once a week with folic acid and calcium supplementation. He was already Discussion The patient was presented features of inflammatory back pain with additional features of AS including typical posture and sacroiliitis. However, HLA-B27 was negative in this patient. HLAs are cell surface proteins, helps the body to fight illness by presenting peptides derived from foreign proteins or from the body's own proteins, to T lymphocytes and other cells of the immune system. [5] Usefulness of HLA-B27 typing as an aid to diagnosis of AS has been discussed in depth by Khan. [6] A group has reported 93%-94% HLA-B27 positivity rate among AS patients seen in Delhi (Northern India). [7] As a result, it has been a standard exercise in clinical practice to carefully exclude the mimics of AS if the patient proves negative for HLA-B27 gene. The list of causes of back pain is long. Briefly, however, if clinically a patient has "spondylogenic" and "inflammatory" features, the possibilities are narrowed down to AS, infections (tuberculosis, etc.,), and some of the rare metabolic conditions (e.g., ochronosis). These conditions were excluded by clinical methods and investigations discussed above. The diagnosis of AS before the occurrence of irreversible damage is difficult. This delay is most likely due to the low awareness among non-rheumatologists of AS or SpA and the fact that radiological proof of sacroiliitis is a late feature of the disease. [8] AS can almost always be readily diagnosed on the basis of history, physical examination, and X-ray findings. A knowledge of the presence of HLA-B27 can sometimes be valuable as an aid to the diagnosis, although the prevalence of HLA-B27 and the strength of its association with AS vary markedly in different ethnic and racial groups. [8] In the absence of any other diagnostic possibility, this patient was treated as AS. However, during the ensuing follow-up, he was fortuitously found to have MND that was confirmed by clinical examination and various investigations. Once MND was detected, the main diagnostic focus shifted to whether the patient had two separate diseases occurring together by coincidence or whether there was a link between them. An association of MND and AS has been very sparsely reported in the literature. Prognosis is related to disease severity. Some cases may have times of active inflammation followed by remission while others never have times of remission and have acute inflammation and pain, leading to significant disability. As the disease progresses, it can cause the vertebrae and the lumbosacral joint to ossify, resulting in the fusion of the spine as noticed in our patient. Our patient has no extraarticular manifestation of AS. MND, or ALS, is a neurodegenerative disorder of unknown etiology. Several cases have been reported where mimickers of MND have been diagnosed or even cured with available treatment. [9] Every attempt should be made to exclude potential treatable mimickers of MND. TNF-α inhibitor treatment has been implicated as a potential etiology of ALS in preexisting rheumatological conditions in few reports. [4] However, here, we find the development of ALS in a known case of AS who had no history of any biological treatment. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.	2019-01-22T22:24:07.831Z	2018-10-01T00:00:00.000	{ "year": 2018, "sha1": "ddf5ce04460a49a5535b4df4482eaec550794c73", "oa_license": "CCBYNCSA", "oa_url": null, "oa_status": null, "pdf_src": "WoltersKluwer", "pdf_hash": "1dc8c30a0d8db2fb83802630a824136f5e729915", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
250157294	pes2o/s2orc	v3-fos-license	Lanthanide-Complexed Esters for Single-Well SOR Measurements The main objective has been to improve the prevailing single-well chemical tracer push-and-pull technique, SWCTT, for measurement of residual oil saturation (SOR) in defined pay zones in a single well test [1,2,3] after water injection on the following subjects: • Improvement in tracer detection limits by a factor > 1000 • On-site or even on-line detection of tracer signal in true time • Reduction in the needed amount of tracer by a factor of > 1000 • Reduced footprint on production platforms during tracer operation The intended audience and technology users are oil and service companies. Objective and target audience The main objective has been to improve the prevailing single-well chemical tracer push-andpull technique, SWCTT, for measurement of residual oil saturation (SOR) in defined pay zones in a single well test [1,2,3] after water injection on the following subjects: • Improvement in tracer detection limits by a factor > 1000 • On-site or even on-line detection of tracer signal in true time • Reduction in the needed amount of tracer by a factor of > 1000 • Reduced footprint on production platforms during tracer operation The intended audience and technology users are oil and service companies. Introduction The prevailing technique for measurement of SOR after water injection in a single well tracer test consists of the following operational points: 1. Injection: Inject a water-based ester solution as a pulse out to some 5-10 m from the well (as a doughnut). The ester is water/oil partitioning, and governed by a partition constant K which is a function of pH, temperature, water salinity, molecular content of residual oil etc.: Here, Tr is the symbol of the ester tracer. If the experimental conditions are constant, the K-value is constant. Oil partitioning of the ester means that the movement of the ester is lower than the water itself because the partition equilibrium demands that some ester, at any time, to be present in the stagnant residual oil. The most used esters today are ethyl-and propyl-esters. 2. Well shut-in: The injection is stopped, and the well is shut in for 2-4 days (mainly depending on temperature and type of ester) in order for the ester to hydrolyze, i.e. react with the surrounding water. This reaction is continued until approximately 50 % of the ester is estimated to be hydrolyzed. The hydrolysis reaction is proceeding according to the chemical reaction below: Ester + Water  Acid + Alcohol 3. Back-production: After the shut-in period the back-production of the remaining ester and the produced alcohol (and acid) starts. The alcohol is a pure and passive water tracer and does not partition into oil. The volumetric distribution of the alcohol at the end of the shut-in period is, ideally, identical to the distribution of the remaining (ca. 50%) ester, i.e. on back-production they "start on the same starting line". Thus, on back-production, the alcohol follows passively the water phase while the remaining ester will partition into the water-contactable residual oil and be delayed with respect to the water flow. 4. Sampling/analysis: The back-produced water is sampled frequently as a function of time, and the concentration of ester and alcohol in each sample is analysed largely by gas chromatography (GC). Most of the time, samples are shipped from the production site to a laboratory situated elsewhere for analysis. Establishment of tracer production curves is performed based on analytical results. 5. Result interpretation: Interpretation of the tracer production curves can take different routes. The most correct procedure is probably to establish the first moments of the distributions of the two recorded production curves. The distance between the times represented by the first moments (or alternatively produced water volumes for the first moments, see Figure 1 below) of the two distributions is proportional to the water-contactable SOR. Problem areas which may obstruct the results from ideality (not further treated in this project): • Fluid drift in the reservoir during shut-in: This point is normally taken care of by injection of a so-called cover tracer throughout the injection period. • Hydrolysis of the ester also during injection and back-production: Must be corrected for by modelling. Improvement areas addressed in this project: • New esters with different analytical expression: Development of new esters based on lanthanide complexes is in focus for extensively improved lower detection limit (LOD) where the detection principle is based on laser-(or light-) induced fluorescence. The fluorescence is caused by the lanthanide ion present, and it is specific and strong (selective and sensitive). • On-line analysis: This fluorescence method offers possibility for on-site or even online continuous analysis of back-produced tracer compounds. • Effective removal of acid catalysis rate problem on the hydrolysis: Ester hydrolysis rate is catalyzed by both acid and base. The lowest rate is in the range pH 6-7. The acid produced during hydrolysis may continuously modify the hydrolysis rate if injected amount of tracer is high (as in the case of ethyl-and propyl esters) and if the buffering capacity of the surrounding water and rock is low. Reduction in tracer amount by a factor of > 1000 may effectively remove this problem. Fig.1. Principle illustration of back-produced passive tracer (produced alcohol) and water-oil partitioning tracer (remaining ester) and the distance between their first moments which is proportional to watercontactable remaining oil saturation. • Modification of near-well formation properties: Any potential modification of chemical properties in the near-well probed volume due to injection of massive amount of tracer (read: ester) is removed due to low tracer amount. • Accuracy: Thus, the sensitivity and accuracy of the method is expected to improve. • Environmental impact and footprint: The low amount of tracer (a few hundred grams) leads to much lower potential environmental impact, to easier logistics and to a reduced space requirement (footprint) on the production platform. The content of this project is inspired by our experience from conducting single-well SORmeasurement operations on the Norwegian Continental shelf. Methodological Approach Wanted complexation agent properties Due to their unique electronic configuration ([Xe]4fn), the trivalent ions of the lanthanides (Ln 3+ ) display long-lived and narrow-line fluorescence emission bands in the UV-visible region under UV excitation [4,5], see Figure 2. The fluorescence is element-specific and strong. It is therefore of interest to examine the possibility to utilize this strong and unique fluorescence for analytical purposes. This requires the development of an ester with a lanthanide atom incorporated as a complex. Several molecular complexing agents for lanthanides were evaluated, all of which should exhibit the following properties: • Form strong and thermally stable complex with Ln3+ • Contain at least one carboxylic acid group to enable esterification with a selected alcohol • Show molecular charge neutrality and bipolarity of the Ln 3+ -complex • Enable separation and detection by combining HPLC (or UPLC) with time-resolved fluorescence detection • Show relatively long-lived fluorescence (phosphorescence) on the scale of milliseconds to enable discrimination of short-lived fluorescence from various watersoluble oil components. Synthesis Two of the molecular complexing agents considered, DTPA and DOTAGA, are pictured in the Figure 3 above. Esterification of Ln-DTPA resulted, not unexpectedly, in a multi-ester complex that proved difficult to use at a later stage due to a complex pattern of hydrolysis. The desired molecular complex should, ideally, contain only one ester group. Hence, DOTAGA was selected as one of the most promising complexing agent candidates. This molecule has one of the five carboxylic acid groups in the end of a longer hydrocarbon chain. The esterification is expected to take place mainly at that position, and a monoester should be possible to obtain as a major rection product. Esterification was finally carried out in either DMSO or alcohol solution. Water could not be used because of hydrolysis. The Ln 3+ complexation could be performed both before and after the esterification process to create DOTAGAmonoethyl-(or monobutyl-) ester-Ln complex, -the preferred method being complexation before esterification. The argument is that in this case, three of the carboxylic groups would bind to the central Ln 3+ -ion, thus neutralizing the positive charge. Hence, these groups will be less available for esterification. Various reaction products were created, but they could be separated by HPLC/UPLC techniques. The configuration of one of the most interesting molecules is given in Figure 4 to the left which is the DOTAGAmonobutyl-ester-Tb complex. All further details about the synthesis and molecular separation techniques are given in two technical reports [6,7]. Separation and analysis Analytical methods based on HPLC/UPLC combined with mass spectrometry (MS) and timeresolved fluorescence (TRF) were developed for characterization of the products of the synthesis. Figure 5 shows an ion chromatogram of the molecule pictured in Figure 4 while Figure 6 shows examples of HPLC-separation analyzed/detected with TRF. Fluorescence lifetime: Measurements of fluorescence lifetime showed that the DOTAGA-monoethyl-ester-Tb complex displayed a lifetime of 2.2 ms in DMSO. The lifetime increased to 2.7 ms with temperature treatment from 25-70 °C, indicative of formation of an even stronger complex. This lifetime is very suitable for TRF to discriminate against water-soluble oil components with fast lifetimes. Hydrolysis rate Hydrolysis rate was measured at the three different temperatures 50, 70 and 100 °C. Results are shown in Figure 7. These rates are suitable for SWCTT. Water/oil partitioning A desired value of the partition coefficient would be approximately K ≈ 3-6. Table 1. The results have shown that this tracer is exclusively hydrophilic despite the attempt of increasing its affinity to the oil phase by esterification. The salinity and composition of the oil phase seems to have no influence on the partitioning for this compound. The explanation is probably that the 4 th carboxylic group was not protonated at the conditions of the experiment. Thus, the ester molecule would have an electric charge of 1-. Such a charged molecule will not partition into an oleic phase. This charge must be neutralized. Accordingly, results suggest that this project has not yet reached its final goal because an ester with sufficiently phase-partitioning properties was not produced. Validation Due to the negative results in the final phase partitioning tests, there is, so far, no validation to be done. Otherwise, the ester would have been subject to dynamic laboratory experiments simulating the near-well formation with known oil saturation. Further, one would seek possibility of performing a push-and-pull test in a suitable field pilot project. These types of experiments will have to await a positive outcome of possible future experiments with respect to the phase-partitioning behavior. Conclusions and recommendations The outcome/results of all the defined tasks and work packages were positive except the last crucial one which deals with sufficient water/oil partitioning. Hence, at the present stage, the technology as such is not ready for field implementation. Future work What is needed is to mask or neutralize the negative charge (1-) of the ester on order to make the molecule more lipophilic. There are, perhaps, two ways to meet this challenge: 1. Apply a liquid ion exchanger or ion-pair reagent (tertiary or quaternary amine) to further complex the ester molecule. In this process the charge is neutralized. However, only experiments can disclose if the resulting partition coefficient is suitable and within the desired range of K ≈ 3-6. 2. The "backbone" in the DOTAGA complexing agent molecule is the DOTA molecule with 4 carboxylic groups. Another modified version of this molecule is PCTA (3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1(15),11,13-triene-3,6,9-triacetic acid) illustrated in Figure 8. One of the 4 -COOH-groups have been replaced with a noncharged ring system. This molecule is now considered as a possible water/oil partitioning tracer for interwell SOR (PITT) examinations. A modified version of this molecule, in the butyl ester form and complexed with terbium, may look like the illustration in Figure 9. This ester molecule is non-charged and should show a fair degree of oil partitioning. However, it has not yet been synthesized, and must only be regarded as a qualified speculation at this point in time. Spin-off A spin-off of this project is based on the findings that the synthesized and tested DOTAGAmonoethyl-ester-Tb and DOTAGA-monobutyl-ester-Tb molecules offer the possibility to be used as temperature probes in the near-well reservoir formation. This is because of a suitable hydrolysis rate and because they do not partition into the oil phase. This possibility has not been further studied and qualified in the present project.	2022-07-01T15:10:17.100Z	2021-11-30T00:00:00.000	{ "year": 2021, "sha1": "5b7b7615b230d3afa5d10296e35296fd85a63ac7", "oa_license": "CCBY", "oa_url": "https://ebooks.uis.no/index.php/USPS/catalog/view/208/173/826-2", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "180e039d56a89e3b4afd9389f5de94ecb0b2b3e3", "s2fieldsofstudy": [ "Chemistry", "Engineering", "Environmental Science" ], "extfieldsofstudy": [] }
4317118	pes2o/s2orc	v3-fos-license	Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution Summary Prolonged shedding of human coronavirus in hematopoietic cell transplant recipients was associated with initial high viral load, high-dose steroids, and myeloablative conditioning regimens. No substantial intrahost evolution of viral genomes occurred over time. Respiratory viruses are associated with prolonged shedding, higher rates of lower respiratory tract disease, and mortality in hematopoietic cell transplant (HCT) recipients. Development of novel therapeutics and effective infection prevention has been critically important, especially for well-established respiratory viruses such as respiratory syncytial virus, influenza virus, and parainfluenza viruses [1][2][3][4]. With new molecular diagnostics widely available, similar concerns have been raised for other respiratory viruses including human coronavirus (HCoV) [5]. In addition to the demonstration of frequent prolonged shedding of HCoV after HCT [6], recent data suggest that common HCoVs (229E, OC43, NL63, and HKU1) are important respiratory pathogens related to significant mortality in HCT recipients [7]. Data on host and virologic factors associated with prolonged shedding, including genome evolution within a host, may provide a rationale for the development of antiviral therapy at various stages, but are currently lacking. Therefore, we examined HCT recipients to define viral and host factors associated with prolonged HCoV shedding in the upper respiratory tract and examine evolution of viral genomic sequences over time by metagenomic next-generation sequencing (mNGS). Study Design We reviewed HCT recipients with HCoV detected in nasal samples by multiplex respiratory viral polymerase chain reaction (PCR) at the Fred Hutchinson Cancer Research Center. Subjects were required to have a negative viral PCR test within 2 weeks of the last positive virology testing performed. If the interval between consecutive positive tests was beyond 2 weeks, strain identification was performed using both samples to confirm the strains were the same. The subjects were identified from 2 cohorts (Supplementary Figure). The first cohort included patients whose nasal samples were collected and tested for clinical purposes when respiratory symptoms were present from March 2009 through June 2016. The second cohort came from a prospective surveillance study of HCT recipients undergoing transplant from December 2005 and February 2010 [8]. Standardized respiratory symptom surveys and multiplex respiratory PCR tests were performed weekly during the first 100 days posttransplant, then every 3 months through year 1 posttransplant and whenever respiratory symptoms occurred between days 100 and 365 posttransplant. Only subjects with respiratory symptoms were selected for the current study, and no duplicated subjects were analyzed. Separately, mNGS was conducted when ≥4 positive samples with cycle threshold (Ct) values of <28 were available irrespective of presence of respiratory symptoms from the above-mentioned prospective surveillance study of HCT recipients. Demographic and clinical data were collected from the database and medical chart review. The study was approved by the Institutional Board Review at Fred Hutchinson Cancer Research Center. Laboratory Testing and Definitions HCoV detection and viral load were determined from nasal specimens by quantitative reverse-transcription PCR as part of a multiplex PCR used to detect 12 respiratory viruses. Strain-specific PCR was performed using saved nasal samples according to a previously published protocol [9]. mNGS was performed on DNAse I-treated RNA extracts from 0.45-uM filtered nasal specimens using "tagmented" (transposon-mediated fragmentation) cDNA libraries with 15-20 cycles of PCR amplification when ≥4 samples with Ct values of <28 were available [10]. Sequence reads were trimmed using cutadapt and aligned to a concatenation of the 4 HCoV reference genomes (NC_002645, NC_005831, NC_006577, and KF530069) using Geneious version 9.1 [11]. The duration of shedding was defined as time between the first positive and first negative sample. Prolonged shedding was defined as the duration of shedding ≥21 days, which was described to be a median shedding duration of HCoV during the first 100 days after HCT [6]. Highest daily steroid dose and lowest cell count in the 2 weeks prior to first HCoV detection were recorded. Conditioning regimen was categorized into myeloablative and nonmyeloablative/reduced intensity based on the definition previously described [12]. Statistical Analysis Univariable and bivariable logistic regression analyses were performed to evaluate associations between virologic and host factors and prolonged shedding. Only the first episode of HCoV infection per subject was used for the outcome analyses. Variables with P ≤ .2 in the univariable models were candidates for bivariable models. Kruskal-Wallis test was performed to compare continuous values among more than 2 groups. Twosided P values <.05 were considered statistically significant. All statistical analyses were performed using SAS 9.4 for Windows (SAS Institute, Cary, North Carolina). Outcome Analyses Initial high viral load (Ct value below the median) was associated with prolonged shedding with the lowest P value (<.01) by univariable analysis. Univariable and bivariable logistic regression analyses indicated that initial high viral load was associated with prolonged shedding consistently in all models ( Table 2). High-dose steroid use (≥1 mg/kg/day) prior to HCoV diagnosis and myeloablative conditioning regimen were associated with prolonged shedding in the bivariable analyses. Four patients started viral shedding prior to transplant; therefore, we separately analyzed 40 patients who started shedding after transplant, and the results remained similar (data not shown.) Whole-Genome Sequencing Whole genomes of OC43, NL63, and HKU1 were consecutively sequenced in samples from 4 HCT adult subjects and 1 pediatric subject where samples were available for 19 to 132 days following the first positive sample (Table 3). Engraftment occurred in 4 patients prior to the start of shedding. No majority consensus variants were recovered for any patient <30 days after onset of shedding. Single-nucleotide variants accumulated at a rate of approximately 1 variant per 3-4 weeks, consistent with previous estimates of the HCoV molecular clock ( Figure 2) [13,14]. No single-nucleotide polymorphisms (SNPs) of OC43 and HKU1 were recovered in patients 4 and 5, respectively. One adult patient (patient 3) developed lower respiratory tract disease in the setting of high-dose steroid use for acute graft-vs-host disease during prolonged shedding. Bronchoalveolar lavage was performed at day 73 after starting the shedding, from which Aspergillus fumigatus was detected in addition to HCoV OC43. One 18-year-old pediatric patient (patient 4) had 3 different HCoV strains detected in succession over a period of 5 months. DISCUSSION In this study, we demonstrated a significant association between prolonged shedding of HCoV and initial high viral load in transplant recipients. In addition, prior high-dose steroid use and myeloablative conditioning regimen appear to be associated with prolonged shedding. The duration of shedding appeared to be similar across all 4 HCoV strains. No drastic intrahost evolution of viral genomes occurred in this immunocompromised population with prolonged shedding. Severe acute respiratory syndrome and Middle East respiratory syndrome (MERS) coronaviruses are recognized as highly human-pathogenic coronaviruses causing fatal lower respiratory tract disease [15][16][17]; however, there are no established antiviral therapies [18,19]. Recent data suggest that lower respiratory tract disease caused by 4 other HCoV strains (229E, OC43, NL63, and HKU1) was also associated with significant respiratory support and mortality in immunocompromised hosts [7]. The unmet need for the development of antiviral therapy against HCoV is expected to expand as immunocompromised populations grow. We found that initial high viral load, prior high-dose steroid use, and myeloablative conditioning were important factors associated with prolonged HCoV shedding in HCT recipients. Duration of viral shedding is often used as an endpoint at early stages of clinical trials for new antiviral drugs [20,21]. Stratification based on risk factors is critical to avoid imbalances due to host and viral factors in randomized trials, which might otherwise mask true differences of experimental agents. Genetic variability of HCoV OC43 at the community level and intrahost heterogeneity of MERS coronavirus have been reported [22][23][24][25]. Such variability might have important implications in viral disease pathogenesis, and the study of viral genome evolution within a host can provide vital information important in developing and assessing antiviral agents [26]. For example, development of antiviral drug resistance during individualized therapy that is associated with poor outcome has been described extensively with influenza virus [27][28][29]. Grad et al reported intrahost genome evolution of respiratory syncytial virus over time in an infant with severe combined immune deficiency who underwent a bone marrow transplant [30]. The viral population diversity dramatically increased after engraftment, which appeared to reflect dynamic response to immune pressure from host immunity. To our knowledge, no previous data exist describing how the HCoV genome evolves within a host over time. In the current study, engraftment occurred in 4 of 5 patients sequenced prior to the onset of shedding. Interestingly, no SNPs were recovered <30 days after the onset of shedding even after immune reconstitution. Variants accumulated starting at 1 month after the onset of shedding (1-6 changes over time), consistent with the previously estimated evolution rates of HCoV [13,14]. Given the relatively slow evolution rate of coronaviruses, these observations could be promising from the standpoint of antiviral resistance and therapeutic development [13,14]. Due to their exceptionally large RNA genomes, coronaviruses are known to encode highly processive polymerases as well as proofreading exoribonucleases that temper viral genome evolution relative to other RNA viruses [31][32][33]. Further epidemiological and biochemical work is required to characterize the functional impact of the variants recovered here. (Table 3). Both nonsynonymous and synonymous variants are depicted relative to the day 0 genome recovered. Majority consensus nonsynonymous changes at allele frequency >50% are indicted in red, while majority consensus synonymous changes at allele frequency >50% are shown in green. Allele frequencies for variant sites are depicted when the majority allele had <95% frequency. Nonsynonymous changes are shown for the amino acid of a given gene, while synonymous changes are shown for the nucleotide of that gene. Allele frequencies alone are shown when the majority consensus base is not a variant relative to the day 0 genome but the majority allele frequency at that base is <95% (in flux). Abbreviations: HE, hemagglutinin esterase; M, membrane protein; N, nucleocapsid protein. The main limitation of this study was the relatively small sample size; thus, bivariable logistic regression analyses were performed instead of multivariable analyses to evaluate risk factors for prolonged shedding. Similarly, although no particular strain appeared to be associated with prolonged shedding, strain identification using saved samples was successful in only 70% of the patients, which limited our ability to detect small difference of shedding duration among each HCoV strain. Further studies with larger sample sizes will help to clarify the distinct association between particular HCoV strains and prolonged shedding. Finally, our cohort included 4 patients who had documented HCoV shedding prior to transplant. Considering the unmeasured influence of their different backgrounds on our analyses, we separately analyzed 40 patients who started shedding after transplant. Only univariable logistic regression analysis could be performed due to the small sample size, with similar results. This is the first study to evaluate risk factors associated with prolonged shedding of HCoV by quantitative and strain-specific reverse transcription PCR as well as intrahost genomic evolutions by metagenomic RNA sequencing in transplant recipients. Our study provides critical information to develop antiviral therapies and design randomized trials with viral load endpoints. In addition, as the duration of shedding is an important determinant of viral infectivity and transmissibility, predictive factors for prolonged shedding may provide useful information for effective infection control, such as the expected duration of isolation. Further studies are needed to validate the risk factors including particular HCoV strain for prolonged shedding. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.	2018-04-03T01:41:05.350Z	2017-05-30T00:00:00.000	{ "year": 2017, "sha1": "da9053f430ca250740d6edc3316d6a2aca6f997b", "oa_license": "implied-oa", "oa_url": "https://europepmc.org/articles/pmc5853311?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "da9053f430ca250740d6edc3316d6a2aca6f997b", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
9300791	pes2o/s2orc	v3-fos-license	Beneficial Role of Antioxidants on Clinical Outcomes and Erythrocyte Antioxidant Parameters in Rheumatoid Arthritis Patients Background: This study aims to investigate the effect of antioxidants supplement on clinical outcomes and antioxidant parameters in rheumatoid arthritis (RA). Methods: The pre-post study was conducted on 40 female patients with RA in 12 weeks that taken daily one Selenplus capsule contained 50 μg selenium, 8 mg zinc, 400 μg vitamin A, 125 mg vitamin C, and 40 mg vitamin E. About 5 mL venous blood sample was taken from all participants and disease activity score (DAS) was determined by DAS-28 formula and high-sensitive C-reactive protein (hs-CRP). Glutathione peroxidase (GPX) and superoxide dismutase (SOD) were measured by spectrophotometric kit and catalase (CAT) was measured by Abei method. Total antioxidant capacity (TAC) was determined by spectrophotometric kit. Distribution of the variables was assessed using histogram with normal curve as well as Kolmogorov-Smirnov test and data were analyzed with paired t-test for differences between pre-post data using SPSS software version 13.5. Conclusions: Our findings showed that antioxidants may improve disease activity significantly, but it did not affect the number of painful and swollen joints and increased erythrocyte antioxidant levels. Antioxidants may be useful for controlling of clinical outcomes and oxidative stress in RA. INTRODUCTION Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that about 0.5%-1% of world population are affected by its complaints. [1]The prevalence of RA in females is 3 times higher than males.The RA affects blood vessels, heart, lungs, muscles, and joints, resulting in bone deformity and osteoporosis. [2]everal studies have reported that oxidative stress and production of oxygen-free radicals have important role in RA development [3,4] and epidemiologic studies have revealed a reverse relationship between dietary intake of antioxidants and RA incidence [5,6] and due to reduction of intake and absorption of dietary antioxidants in RA patients, the levels of blood antioxidants are decreased too.The antioxidants supplements such as vitamin E, [7,8] vitamin C, [4,9] and selenium [10,11] may control the disturbance of lipid peroxidation and loss of antioxidants markers in patients with RA.Vitamin E can interact with nitric oxide and may trigger the gene expression of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD) enzymes, [12] vitamin C may demolish the peroxides of macrophage activities, zinc may strengthen the immune system, [13] and selenium has an important role as a cofactor of GPX enzyme in reduction of oxidative stress. [14]n past 30 years, controlled trials have conducted to compare the effect of dietary antioxidants and antioxidant-rich diets in controlling of RA clinical outcomes; [15,16] however, they could not find a clear statement about antioxidants in RA prevention and treatment due to difference in study period, dose, and different types of antioxidants. [12]Regarding to integrity of antioxidant defense system and few clinical studies on combined antioxidant supplements in RA, the aim of this study is to evaluate the effect of combined antioxidant supplements as daily oral capsule on clinical outcomes and antioxidant parameters in female patients with RA for 3 months. Study design and patients A pre-post clinical trial was conducted on female patients with RA for 12 weeks.The study group was selected from 400 registered RA patients in Sheikh-al-Rais and Sina clinics of Tabriz University of medical sciences, Iran according to inclusion criteria.The inclusion criteria were RA diagnosis by rheumatologist according to American College of Rheumatology guidelines-1987, 40-60 years old, no change in treatment approach in past 2 months. The exclusion criteria were diabetes mellitus, hypertension, thyroid disorders, liver and kidney failure, Cushing syndrome, severe infection, gastric illnesses, smoking, and exposure to daily smoking at home.We followed-up the intake of daily supplement use and type and dose of medications by regular phone calls, so change in type and dose of drugs and antioxidant supplement resulted in omission from study. We asked from selected patients to take daily a "selenplus" capsule (Eurovital pharmaceutical company, Germany) that contained 50 µg selenium, 8 mg zinc, 400 µg vitamin A, 125 mg vitamin C, and 40 mg vitamin E. The supplement has been given to patients without trading label.After explanation of the study risks and benefits, written consent form was taken from all subjects.Registration code of the local ethics committee of Tabriz University of Medical Sciences is 8912 and registration number in the registration center for clinical trials in Iran is IRCT138901183655N1.In the case of any side effects, patients could leave the study and dose of each antioxidant nutrient was at the Recommended Dietary Allowances amount. Procedure At the beginning of the study, accurate clinical examination such as counting the swollen and painful joints was done by a rheumatologist and the validated DAS-28 form was filled to calculate disease activity index [17] according to following formula. [18]: Also, dietary intake questionnaire including food frequency questionnaire (FFQ) and 24 h recall questionnaire for 3 days (2 working days and 1 weekend) were completed by an expert nutritionist.The FFQ was composed of 168 food items that assessed the frequency of the intake in day, month, season, and year semiquantitavely.The recall questionnaire was a dietary detailed form in 6 parts: Breakfast, lunch, and dinner with three snacks that filled by face to face interview about the type and amount of the food items.Dietary intake of energy, macronutrients, and antioxidant micronutrients analyzed by nutritionist III software (MAM soft research Co, USA 1993).The weight of patients was measured by digital scale (after calibration and without shoes), the height was measured by stadiometer (after attachment of 4 points of body to the wall) and the body mass index calculated by Quetelet formula.The patients were followed-up every 2 weeks by phone calls and the measurements were repeated after 3 months. Five milliliter fasting venous blood samples (8-12 h after fasting) were taken from all participants and were kept in -70°C freezer (Snider's, Germany) until conducting biochemical measurements.Biochemical measurements including GPX and SOD were measured by spectrophotometric kit (Ransel, Randox laboratories ltd, UK) and autoanalyzer apparatus (Abbott, model Alcyon 300, USA) and CAT was measured by Abei method. [19]TAC was determined by spectrophotometric kit (Randox TAC kit, Randox laboratories Ltd, UK).Serum high-sensitive C-reactive protein (hs-CRP) was quantified by photometric kit (Pars Azmoon Company Ltd, Iran). Statistical analysis SPSS software version 13 (SPSS for windows, Chicago, IL, USA) was used for statistical analysis.Distribution of data was tested by Q-Q plot and Kolmogorov-Smirnov test.For parametric data, paired t-test and in case of nonparametric data, Wilcoxon signed rank test was used.The linear regression model was used for adjusting confounding factors such as dietary intake of some selected nutrients.P < 0.05 was defined significant. RESULTS A total of 39 patients sustained in the study after 12 weeks.The baseline characteristics and dietary intake have been reported in reference no.20. [20]One was left in reason of unrelated medical problem.Table 1 indicates basic characteristics of the subjects at the start point of trial and the median of the duration of the disease was 72 months [Table 1].The pharmacotherapy regimen did not change during the period of the study in the selected patients and any changes in the dose and type of the drugs resulted in the omission of the study.Dietary intake of energy and selected nutrients during 12 weeks of intervention did not differ significantly [Table 2] and in the linear regression findings, no significant linear relationship between dietary antioxidants values with biochemical indices was observed. In our study, DAS-28 score and serum hs-CRP have changed during 12 weeks of intervention (P < 0.01), while the number of swollen and painful joints did not change significantly [Table 3].The antioxidant markers of patients including TAC, GPX, SOD, and CAT increased significantly after 12 weeks supplementation (P < 0.01) [Table 4]. DISCUSSION In our study, antioxidants supplement for 12 weeks reduced significantly serum hs-CRP and DAS-28 score.The literature review indicates that zinc and selenium supplementation have been used in RA remission and prevention for several years [21] and the similar results of these studies were resulted from multicomponent antioxidants and nutrients as Koracevic et al., [22] showed concurrent supplementation with 37.5 mg vitamin E, 150 mg vitamin C, 1.4 g eicosapentaenoic acid, 0.2 g docosaenoic acid, and 0.5 g gamma linolenic acid could not significantly reduce the number of swollen and painful joints. [22]As the results of alike studies revealed intake of simultaneous antioxidant micronutrients can have a helpful effect against RA progress. [22]In another similar study, 300 mg vitamin C, 5 mg zinc, 25000 International Unit vitamin A for 12 weeks reduced the disease activity (P < 0.0001). [23]Also, Pretez et al., [10] study showed that 12 weeks selenium supplementation decreased the number of swollen and painful joints; however, the results were not statistically significant.Some studies have used higher doses of one antioxidant although they did not observe significant improvement in clinical outcomes. [24]It seems that the reason of these findings is due to no increase in antioxidants levels in polymorphonuclear leukocytes and antioxidant defense system in blood cells. [25]As Onal et al., [26] study indicated the pharmacotherapy in patients with RA results in lower levels of zinc and selenium and higher levels of copper in red blood cells, so intake of oral drugs such as corticosteroids and chloroquine elevates the required amount of the antioxidants to suppress inflammatory-like substances. In our study, erythrocyte antioxidant markers including TAC, GPX, SOD, and CAT increased significantly during 12 weeks supplementation due to probable direct effect of oral antioxidants on antioxidants levels.Similarly, Shinde et al., [27] have shown that 400 mg vitamin E and 500 mg vitamin C could increase the reduced form of erythrocyte glutathione (P < 0.001), probably because vitamin E is the most important fat-soluble antioxidant and protects the cell membranes against oxidative stress just as vitamin C preserves cytosol and membranes of free radicals activity. [28]Furthermore, the results of VanVugt et al., [29] study indicated that 400 mg alpha-tocopherol, 10 mg lycopene, 5 mg alpha carotene, 10 mg lutein, and 200 mg vitamin C for 12 weeks increased plasma levels of vitamin E, lycopene, lutein, alpha-carotene, and vitamin C and reduced F2-isoprstanes as the oxidative stress marker.Shah et al., [30] illustrated that there is a strong association among the disease activity with antioxidant enzymes markers and they have showed that production of reactive oxygen substances can disturb the immune defense system and modulate inflammation processes to reduce the antioxidant molecules in blood cells.It seems that mixture of antioxidants help to reduce required dose of pain killer drugs and diminish the complaints of disease. Since autoimmune diseases such as RA are accompanying with reduction of cellular immune level that results in high coincidence of other chronic diseases, healthy antioxidant-rich diet can improve immune system and compensate the inadequate intake of micronutrients, especially antioxidant-rich supplies of RA patients in northwest of Iran. [27,28]Also, consumption of antioxidant micronutrients in the form of dietary items or supplements may be helpful in enhancement of enzymatic and nonenzymatic antioxidants due to strengthen the antioxidant defense system of the body. [29,30]Lack of the control group is the major limitation in this pre-post clinical trial due to limited financial support.One of the strengths is the high response rate of participants (97.5%) and low loss to follow-up during the intervention.Also, mild to moderate severity of RA was considered as a criterion of the study and the dietary intake of antioxidants was supposed as confounding factors. CONCLUSIONS The combined antioxidant supplement may improve DAS-28 score significantly, but it did not change the number of painful and swollen joints statistically significant during 12 weeks, while it could increase TAC, GPX, SOD, and CAT levels.It seems that supplementation with antioxidants may be useful as a complementary treatment in control of clinical outcomes and oxidative stress in patients with RA. Table 1 : The basic characteristics of subjects in the study Table 2 : Dietary intake of selected nutrients before and after 12 weeks intervention Table 3 : The changes of clinical outcomes in subjects of study before and after 12 weeks intervention Table 4 : The changes of erythrocyte antioxidant parameters in subjects of study before and after 12 weeks intervention *Mean±standard deviation, paired t-test, † Significant differences in P<0.01	2017-04-19T01:29:50.891Z	2014-07-01T00:00:00.000	{ "year": 2014, "sha1": "3cf57bff4f11483f0684af4d6e191e90896a7ce1", "oa_license": "CCBYNCSA", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "9e8ea46865a566ab1a1f23bb5599823b6e647b8d", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
259766485	pes2o/s2orc	v3-fos-license	When does the chaos in the Curie-Weiss model stop to propagate? We investigate increasing propagation of chaos for the mean-field Ising model of ferromagnetism (also known as the Curie-Weiss model) with $N$ spins at inverse temperature $\beta>0$ and subject to an external magnetic field of strength $h\in\mathbb{R}$. Using a different proof technique than in [Ben Arous, Zeitouni; 1999] we confirm the well-known propagation of chaos phenomenon: If $k=k(N)=o(N)$ as $N\to\infty$, then the $k$'th marginal distribution of the Gibbs measure converges to a product measure at $\beta<1$ or $h \neq 0$ and to a mixture of two product measures, if $\beta>1$ and $h =0$. More importantly, we also show that if $k(N)/N\to \alpha\in (0,1]$, this property is lost and we identify a non-zero limit of the total variation distance between the number of positive spins among any $k$-tuple and the corresponding binomial distribution. INTRODUCTION The Curie-Weiss model is a mean-field model for ferromagnetism in statistical mechanics.It is described by a sequence of probability measures, the Gibbs measures µ N , on the sets {−1, +1} N .These measures are parametrized by a positive parameter β > 0 known as the inverse temperature and a real parameter h ∈ R interpreted as strength of an external magnetic field.Given such β > 0 and h ∈ R, the Gibbs measure takes the following form: The normalizing constant is called the partition function of the model. There is a vast literature on the Curie-Weiss model with the main asymptotic results summarized in the textbooks [5], [11] and [14]; see also the papers [6,10,12].The order parameter of the model is called the magnetization and is defined by where P N := P N (σ ) := \|{i = 1, . . ., N : σ i = +1}\| is the number of positive spins.Let µ N • m −1 N denote the distribution of the random variable m N under the probability measure µ N .The first-order limiting behavior of the magnetization is given by which indicates a phase transition at β = 1 in the absence of the external magnetic field (h = 0).Here, =⇒ denotes weak convergence, δ x is the Dirac measure at x and m(β , h) is the largest in absolute value solution to This solution is unique and positive if h > 0, unique and negative if h < 0, and it is equal to zero if h = 0 and 0 < β ≤ 1.If h = 0 and β > 1, equation ( 4) has two non-zero solutions m(β , 0) and −m(β , 0). For β > 1, still assuming h = 0, there is a conditional central limit theorem for )), conditioned on m N > 0 (respectively, m N < 0).In this case the limiting expectation is 0 and the limiting variance is v 2 β ,0 .Finally, if h = 0, there are no phase transitions as β varies in (0, ∞) and √ N(m N − m(β , h)) converges to the centred normal distribution with variance v 2 β ,h . 1.1.Propagation of chaos and the main results.For the time being, fix k ∈ N and pick any k-tuple among N spins.The propagation of chaos paradigm for Gibbs measure states that for a mean-field model as the Curie-Weiss model the marginal distributions of the k spins become asymptotically independent.This shall be investigated in the sequel.Since the family of random variables (σ i ) N i=1 is exchangeable under the Gibbs measure µ N , without loss of generality we may pick the first k spins and consider their marginal distribution µ (k) N,β ,h .Let P k := \|{ j ∈ {1, . . . k} : σ j = +1}\| be the number of positive spins among the picked ones.Note that P k completely determines µ (k) N,β ,h , hence we might as well study the distribution of P k under µ N , denoted by µ N • P −1 k .Intuitively, if h = 0 and 0 < β < 1, as N → ∞, the first k spins should indeed be asymptotically independent and take values ±1 with the same probability 1/2.This implies that the distribution of P k should be close to the binomial distribution with parameters k and 1/2.This can formally be written as follows.Let Bin (n, p) denote a binomial distribution with parameters n ∈ N 0 and p ∈ [0, 1]: Throughout the paper we shall slightly abuse the notation and write mixtures of distributions by simply writing a mixing distribution instead of a parameter which is being mixed.For example, for any distribution Recall that the total variation distance d TV between two probability measures M 1 and M 2 on R is defined by where B(R) is the Borel sigma-algebra.Recall also that k ∈ N is fixed for the time being.The 'propagation of chaos' phenomenon for the Curie-Weiss model tells us that, if h = 0 and 0 < β < 1, then lim On the other hand, for h = 0 and β > 1, it is known that Note that the approximating distribution is a mixture of two binomial distributions, one with success probability (1 + m(β , 0))/2 and the other one with (1 − m(β , 0))/2.Finally, it is known that for h = 0 (and arbitrary β > 0), relation (8) holds with 1/2 replaced by Assume now that k = k(N) depends on N and lim N→∞ k(N) = ∞.The aim of the this note is to answer the question whether the analogues of ( 8) and ( 9) hold true in this case and if not, what is a threshold on the growth of k(N) such that the limit in (8) or (9) becomes non-zero and what is the value of the limit.Note that for k(N)/N → 0, that is k = o(N), propagation of chaos has been explicitly shown to hold for β = 1 and h = 0 in [4] (note that the authors consider relative entropy rather than total variation distance and that their model also covers h = 0 implicitly).Our answer is provided by Theorem 1.1 below, which is our main result.To simplify its formulation, let us introduce additional notation. For m ∈ R and v 2 > 0 put That is to say, t → ϕ(t; m, v 2 ) is the density of a Gaussian distribution with mean m and variance v 2 .Define 2 ) dt (11) to be the total variation distance between two centred Gaussian distributions, which can easily be calculated in terms of the error function.Then our main result reads as follows. Let us now informally discuss the case when α > 0. For simplicity, we consider (13).The limit on the right-hand side is non-zero, which suggests that there is a residual dependence between the k(N) spins under the Gibbs measure.The reason for the non-zero limit is the fact that the distribution of P k(N) and the corresponding binomial distribution satisfy central limit theorems with different variances, the variance of P k(N) being strictly larger, which comes from the fact that the spins are positively correlated under the Gibbs measure.The distance between these normal distributions appears on the right-hand side of (13). In Theorem 3.5, we shall determine a mixed binomial distribution which approximates the distribution of In some sense, this describes the residual dependence between the spins under the Gibbs measure. Remark 1.2.The exchangeability of the measure µ N has been used to investigate the Curie-Weiss model for example, in [17, Section 5.2] and [2].In particular, an explicit representation of µ N as a mixture of Bernoulli measures (valid for each fixed N) can be found in [17,Theorem 5.6].A general propagation of chaos principle stating that the distribution of k entries in a finite exchangeable vector of length n can be approximated by a mixture of i.i.d.distributions can found in [7]. The paper is organized as follows.Our proof relies on local limit theorems for the magnetization m N and also for the total number of positive spins P N under µ N .In some regimes those are known.We collect the corresponding results in Section 2 below.The proofs of these local limit theorems, which we have not been able to locate in the literature, are given in Section 4. The proof of Theorem 1.1 is given in Section 3, including the statement of residual dependence.Two auxiliary technical results related to calculations of the total variation distance are presented in Section 5. LOCAL LIMIT THEOREM FOR THE MAGNETIZATION Denote by N (m, v 2 ) a Gaussian distribution with mean m and variance v 2 , so This correction term appears below in the local limit theorems for m N , since Nm N always has the same parity as N. and the following local limit theorem holds true: and the following local limit theorem holds true: For some values of (β , h) the above local limit theorems can be extracted from the vast literature on the Curie-Weiss model.For example, in the high-temperature regime β ∈ (0, 1) and for every h ∈ R, (16) has been proved in [19,Theorem 4.5 and Eq. (4.4)].If h > 0 and β > 0, then ( 16) can be found in [1, Theorem 2.14 and Lemma 1.1].The missing case h < 0 and β > 0 in Proposition 2.1 can be derived by the same methods.Finally, if h = 0, a local limit theorem for a multi-group Curie-Weiss model in the hightemperature regime β ∈ (0, 1) has been derived in [13].Quite (un-)expectedly, we have not been able to locate Proposition 2.2 in the literature, because of the non-standard approximation by a mixture of normal distributions.We shall give an elementary proof based on the Stirling approximation in Section 4. We shall actually need local limit theorems for P N rather than m N .They follow immediately from Propositions 2.1 and 2.2 using the obvious relation R, for some absolute constant C > 0, which is a consequence of the mean value theorem for differentiable functions.The above bound allows us to neglect the correction term δ N appearing in local limit theorems for m N . and the following local limit theorem holds true Corollary 2.4.Assume that h = 0 and β > 1.Then and the following local limit theorem holds true PROOF OF THEOREM 1.1 We embark on a simple observation which is a consequence of exchangeability of the spins under the Gibbs measure µ N .Given that P N = i ∈ N 0 , the conditional distribution of P k is hypergeometric with parameters N, i and k denoted hereafter HyperG(N, i, k).Recall that In other words, the distribution of P k(N) can be represented as the following mixture of hypergeometric distributions: The family of hypergeometric distributions possesses the following property which is of major importance for us.Proof.For 0 ≤ j ≤ k, Alternatively, we can argue probabilistically: If each of n balls is colored black or white with probability p and 1 − p, respectivelly, and then a sample of k balls is drawn at random from n balls, then the number of black balls in the sample has binomial distribution with parameters (k, p). The subsequent proof of Theorem 1.1 proceeds according to the following scheme: STEP 1.We approximate L (P N ) by an appropriate mixed binomial distribution Bin (N, L (W )), where W is a random variable taking values in [0, 1] and L (X) denotes the distribution of a random variable X. The approximation is understood in the sense of the d TV -distance which, as we shall show, converges to 0. To accomplish this step we employ the local limit theorems for P N provided by Corollaries 2.3 and 2.4. It turns out that in a role of the mixing distribution W we can take a beta distribution (or a mixture of two beta-distributions) with properly adjusted parameters. where the infimum is taken over all pairs (X,Y ) of random variables such that X is distributed according to M 1 and Y is distributed according to M 2 .STEP 3. We derive a local limit theorem for Bin (k, L (W )). STEP 4. We calculate the total variation distance between Bin (k, L (W )) and the three binomial distributions appearing in Theorem 1.1 by using local limit theorems for binomial distributions in conjunction with another well-known formula for d TV : If measures M 1 and M 2 are supported on Z, then see Propositions 5.1 and Proposition 5.2 below. Our implementation of Steps 1-3 relies on the next proposition.For α, β > 0, Beta (α, β ) denotes a beta distribution with the density where B is the Euler beta-function.In what follows we assume that all auxiliary random variables are defined on some probability space (Ω, F , P). Proposition 3.2.Assume that (Θ N ) N∈N is a sequence of N 0 -valued random variables that satisfy a local limit theorem of the form: for a fixed integer K ∈ N, a collection of positive weights p 1 , . . ., p K satisfying Suppose that k = k(N) is a sequence of positive integers such that (12) holds for some α ∈ [0, 1] and put For N ∈ N, let X N be a random variable with a mixed hypergeometric distribution HyperG(N, L (Θ N ), k(N)) and Y N,k(N) be a random variable with the mixed binomial distribution Bin (k(N), ∑ K j=1 p j Beta (γ j,1 N, γ j,2 N)), that is, Remark 3.3.We shall use this proposition with Θ N = P N and K = 1, p 1 = 1 (in conjunction with Corollary 2.3) or K = 2, p 1 = p 2 = 1/2 (in conjunction with Corollary 2.4). Remark 3.4.Let us provide an informal explanation of Proposition 3.2.Consider for simplicity the case K = 1, p = 1.Then, (21) states that Θ N satisfies a local limit theorem with asymptotic centering Na 1 .One is therefore tempted to approximate Θ N by the binomial distribution Bin (N, a 1 ), however the variance of this distribution, which equals Na 1 (1 − a 1 ), is strictly smaller than the asymptotic variance Nσ 2 1 appearing in (21) due to the assumption σ 2 1 > a 1 (1 − a 1 ).Instead, we approximate Θ N by a mixed binomial distribution that artificially blows up the variance until the variances match.The informal explanation of this is that both distributions satisfy a local limit theorem with the same centering and normalization.In the case k(N) = N, we have Θ N d = X N and Proposition 3.2 states that the total variation distance between the distribution of X N and Bin (N, Beta (γ 1,1 N, γ 1,2 N)) converges to 0. Moreover, in the formal limit σ 2 1 ↓ a 1 (1 − a 1 ), we retrieve Lemma 3.1. Proof of Proposition 3.2.We start by noting that, for fixed j = 1, . . ., K, the pair (γ j,1 , γ j,2 ) is a unique solu- tion to the following system of equations On the left-hand side of the first equation we recognize the mean of Y N,N , whereas the left-hand side of the second equation is equal to the variance of Y N,N up to a term O( 1).This suggests that the distribution of Θ N is close to the distribution of Y N,N .In fact, assume that we have proved lim According to (19) there exists a sequence of pairs To this end, we shall check that Y N,N satisfies exactly the same local limit theorem as Θ N , that is, We shall actually prove a stronger result for later considerations, namely where By the very definition of Y N,k(N) it suffices to prove (26) only in the case K = 1, p 1 = 1 and j = 1.We find it instructive to first prove a central limit theorem for Y N,k(N) of the form which explains the formula for the variance σ 2 α,1 in (26).Let B N be a random variable with the betadistribution Beta (γ 1,1 N, γ 1,2 N) and C (1) N be independent random variables with gamma-distributions with parameters (γ 1,1 N, 1) and (γ 1,2 N, 1), respectively.Using a representation for the beta-distribution, see [15, Theorem 3], and the central limit theorem for C (1) N and C (2) where (γ 1,1 +γ 1,2 ) 3 .Moreover, let (U k ) k∈N be a sequence of independent copies of a random variable with the uniform distribution on [0, 1].It is well-known that in the Skorokhod J 1 -topology on D[0, 1], where (W (t)) t∈[0,1] is a standard Brownian bridge.By independence and our assumption k(N)/N → α, we have a joint convergence which is a.s.continuous at the limiting point in (31) we arrive at which is equivalent to (28), since Var (W (a 1 )) = a 1 (1 − a 1 ).Fix ε ∈ (0, min(a 1 , 1 − a 1 )) and note that where the penultimate equality follows from the law of total variance, and the last equality is a consequence of lim N→∞ Var( √ NB N ) = s 2 ; see (29).The above estimate in conjunction with a standard tail estimate for the normal law implies that ( 26) is equivalent to The latter can be deduced from the following explicit formula together with the asymptotic relation for the beta-function, which is uniform in x, y ∈ [δ , δ −1 ], for every fixed δ ∈ (0, 1).Out choice of ε ensures that all the arguments of the beta-functions in (33) lie in [δ k(N), δ −1 k(N)] for a sufficiently small δ > 0. The uniform asymptotic relation ( 35) is a consequence of Stirling's formula with a uniform estimate of the remainder; see, for example, Eq. (5.11.10) and (5.11.11) in [18].The proof of Proposition 3.2 is complete. Remark 3.6.The theorem above describes the residual dependence in the propagation of chaos phenomenon via a beta-binomial distribution, which in turn has a simple interpretation as follows.Consider a Pólya urn which initially contains γ 1 (β , h)N positive spins (white balls) and γ 2 (β , h)N negative spins (black balls).Balls are drawn one at a time and immediately returned to the urn together with a new ball of the same color.The number of white balls drawn after k(N) trials has the beta-binomial distribution Bin (k(N), Beta (γ 1 (β , h)N, γ 2 (β , h)N)).Thus, Theorem 3.5 tells us that the number of positive spins under the Gibbs measure is close in distribution to the number of white balls drawn from the above Pólya urn after k(N) trials. CASE h = 0 AND β > 0. In this case we again apply Theorem 3.5.From (26) it follows that Combining this with the de Moivre-Laplace local limit theorem and using Proposition 5.1 we arrive at (15).] that the total variation distance between the distribution of (ξ 1;n , . . ., ξ k;N ) and the standard normal distribution on R k converges to 0. On the other hand, for k(N)/N → α with α = 0, the total variation distance between these distributions converges to a non-zero limit which has been identified in [9, Theorem 1.6 (b)]; see also Eq. (2.12) on p. 403 in [8].All these results are similar to what we know about the Curie-Weiss model.There is, however, one important difference: An approximation by a variance mixture of normal distributions is not possible if α > 0 in the setting of ξ N .A much more general result has been shown in [9, Theorem 2.3 (b)].Let us give a short informal argument.Let (ζ 1 , . . ., ζ k ) be a random vector with the standard normal distribution on R k and let R > 0 be a mixing variable independent of the ζ i 's.We ask whether it is possible to approximate (ξ 1;N , . . . ,ξ k;N ) by R • (ζ 1 , . . . ,ζ k ) in the total variation distance.By rotation invariance, it suffices to consider the distance between the squared radial parts; see Eq. (2.4) on p. 402 in [8].Since 1 On the other hand, the squared radial part of R k has a chi-square distribution with k degrees of freedom, and we have where we used that E(χ 4 k ) = 2k + k 2 .If we want to match expectations, we need and we cannot match the variances since 2α > 2α(1 − α).This is in sharp contrast to the case of the Curie-Weiss model, for which Theorem 3.5 shows that the larger variance of P k(N) can be artificially matched by a mixed binomial distribution. PROOF OF PROPOSITION 2.2 Recall that we work under the assumptions h = 0 and β > 1, which imply m := m(β , 0) ∈ (0, 1).For simplicity we also assume throughout this proof that N = 2n is even.The case of odd N can be treated similarly.For every −n ≤ ℓ ≤ n, we have For further use we record the asymptotic formula for the partition function: if β > 1, then where ], which can be derived from Théorème B in [3] by specializing to the Curie-Weiss model or from (3.19) in [16] by choosing p = 1; see also [ is even and attains two local maxima at ℓ ≈ nm and ℓ ≈ −nm.This can be checked by calculating the ratio of its two consecutive values.Using the aforementioned symmetry and the standard tail estimate for the normal density, we see that (17) To check that the indices outside C n are negligible we use the same reasoning as in [16]; see pp. STEP 2 . Lemma 3.1 implies that the µ N -distribution of P k is close (in a sense of the d TV -distance) to the mixed binomial distribution Bin (k, L (W )).Formal verification of this employs the well-known characterization of the d TV -distance has the same distribution as Θ N (respectively, Y N,N ), for every N ∈ N. Therefore, d TV (L (X N ), HyperG(N, L (Y N,N ), k(N))) =d TV (HyperG(N, L (Θ N ), k(N)), HyperG (N, L (Y N,N ), k(N))) =d TV (HyperG(N, L (Θ ′ N ), k(N)), HyperG (N, L (Y ′ N,N ), k(N))) → 0. But this immediately yields the claim since HyperG(N, L (Y N,N ), k(N)) has the same distribution as Y N,k(N) by Lemma 3.1.Thus, it remains to prove (24). Remark 3 . 8 . Here we give a comparison of our findings with some known results on the uniform distribution on a high-dimensional sphere, which bears some similarities with the Curie-Weiss model in the context of the propagation of chaos phenomenon.For every N ∈ N, consider a random vector ξ N = (ξ 1;N , . . . ,ξ N;N ) which is uniformly distributed on the sphere√ NS N−1 of radius √ N in R N .Itis a classical result of Maxwell-Poincaré-Borel that, for every k ∈ N, the distribution of any k components of ξ N converges weakly to a k-dimensional standard normal distribution, as N → ∞.Moreover, if k = k(N) depends on N such that k(N)/N → 0, then it has been shown in [8, Section 2	2023-07-12T06:42:41.952Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "d45d19bd0e89bf3ac8388f53293bfa7abad94d48", "oa_license": "CCBY", "oa_url": "https://projecteuclid.org/journals/electronic-journal-of-probability/volume-28/issue-none/When-does-the-chaos-in-the-Curie-Weiss-model-stop/10.1214/23-EJP1039.pdf", "oa_status": "GOLD", "pdf_src": "ArXiv", "pdf_hash": "d45d19bd0e89bf3ac8388f53293bfa7abad94d48", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics", "Physics" ] }
15160656	pes2o/s2orc	v3-fos-license	Neurobiological Correlates in Forensic Assessment: A Systematic Review Background With the increased knowledge of biological risk factors, interest in including this information in forensic assessments is growing. Currently, forensic assessments are predominantly focused on psychosocial factors. A better understanding of the neurobiology of violent criminal behaviour and biological risk factors could improve forensic assessments. Objective To provide an overview of the current evidence about biological risk factors that predispose people to antisocial and violent behaviour, and determine its usefulness in forensic assessment. Methods A systematic literature search was conducted using articles from PsycINFO, Embase and Pubmed published between 2000 and 2013. Results This review shows that much research on the relationship between genetic predisposition and neurobiological alterations with aggression is performed on psychiatric patients or normal populations. However, the number of studies comparing offenders is limited. There is still a great need to understand how genetic and neurobiological alterations and/or deficits are related to violent behaviour, specifically criminality. Most studies focus on only one of the genetic or neurobiological fields related to antisocial and/or violent behaviour. To reliably correlate the findings of these fields, a standardization of methodology is urgently needed. Conclusion Findings from the current review suggest that violent aggression, like all forms of human behaviour, both develops under specific genetic and environmental conditions, and requires interplay between these conditions. Violence should be considered as the end product of a chain of life events, during which risks accumulate and potentially reinforce each other, displaying or triggering a specific situation. This systematic review did not find evidence of predispositions or neurobiological alterations that solely explain antisocial or violent behaviour. With better designed studies, more correlation between diverse fields, and more standardisation, it might be possible to elucidate underlying mechanisms. Thus, we advocate maintaining the current case-by-case differentiated approach to evidence-based forensic assessment. Introduction Violent crime is a complex problem without simple solutions. Given the prevalence of violent criminality in our society, [1][2][3] an understanding of the predictive and causal factors of violence is needed to improve assessment of criminal responsibility, risk assessment and management practices. What factors put individuals at risk for developing violent behaviour and committing a crime? What factors promote resiliency and protect individuals from re-offense? In the last 50 years, much has been learned about psychosocial risk factors that predispose people to violence. [4,5] However, psychosocial and biological causes of crime are inseparably entwined and are constantly interacting. Over the past two decades, on the tails of the genome project and a revolution in brain imaging, scientists across the world have tried to solve the enormous jigsaw puzzle of the biology of violent and criminal behaviour. These efforts have advanced our knowledge about and understanding of the biological factors and mechanisms involved in violent and criminal behaviour. [6,7] Nevertheless, forensic (risk) assessment is still mainly based on psychosocial risk factors. [8][9][10] The challenge now is to integrate these innovative neurobiological and genetic findings with current criminal assessment practices. When an individual suffers from a severe mental disorder that leads to a crime, it is generally agreed in most jurisdictions that he or she cannot be held criminally responsible and should be exempt from penal consequences. [7,[11][12][13] Psychiatrists and psychologists are often called upon to render the expert opinions needed for legal determinations of criminal responsibility and risk for recidivism [14,15]. Currently, forensic assessment is predominantly focused on psychosocial factors, however, till date, risk assessment instruments do not include biological risk factors. [8][9][10] We do know that psychosocial factors interact with biological factors in shaping (violent) behaviour. [6] Research shows that a small proportion of offenders, approximately 6%, account for the majority of all crimes [16] and that 5% of families account for more than 50% of all arrests. [17] With the increased knowledge of biological risk factors, interest is growing to include (more) information about biological risk factors in forensic assessments. In recent years, neuroscientific evidence, e.g. neurogenetics [18] and neuroimaging, has begun to be used to document a person's tendency towards aggression as was done in the case of the serial killers Brian Dugan [19] and Bradley Waldroup [20] and two recent murder cases in Italy. [11,20,21] This may yield several benefits. First, biological risk factors would lead to more objective measures of criminal responsibility or risk assessment of violent behaviour since they are thought to be less prone to manipulation. Second, assessing biological risk factors may reveal new information that could not be previously determined, such as assessing the possible role of a specific brain damage in criminal behaviour. Third, assessing information on biological risk factors would provide more information on the interaction between social and biological risk factors and their relationship with violent behaviour. In summary, a better understanding of the neurobiology of violent criminal behaviour would help to provide insight into whether and how assessment of biological risk factors could improve forensic assessment. This systematic review aims to provide an overview of the current evidence about biological risk factors that predispose people to antisocial and violent behaviour and determine its usefulness in forensic assessment. As a framework to review the available literature, we adopt a biosocial model of violence as used by Raine. [6] Thus, we focus on evidence from genetics and interaction with pre-and post-natal environments, as well as related areas such as neuroanatomy, neuropsychology and neurology, neurophysiology, neurochemistry and endocrinology. This multi-disciplinary approach offers additional insight into the criminal mind and the underlying causes of violent behaviour to assist in forensic assessment. Our hypothesis is that a general model that holds on a population level is, as of now, not evidence based. Forensic assessment remains to be done on a case-by-case base. Though several pieces of knowledge can be connected, an overall picture that leads to a general understanding of criminal violence is not yet possible. First, we present brain (dys-)functioning and behavioural effects with a special focus on brain anatomy and neurotransmitters. Subsequently, we review genetic and environmental influences on antisocial behaviour, as well as possible correlations with risk factors. The relevant models and theories for each section will be discussed, including supporting evidence for each one. Finally, we consider the possible implications for forensic assessment and address research challenges. Data sources Two literature searches were conducted in the electronic databases of PsycINFO, Embase and Pubmed. The first search concerned reviews and/or meta-analyses published between 2000 and 2013; the second search retrieved empirical research published between 2010 and 2013. We selected publications by using a query based on keywords concerning criminality, aggression, antisocial behaviour or psychopathy in combination with either neurosciences or genetics. The exact query is provided in the appendix. Inclusion. In both searches we included publications using the following criteria: a) published in peer-reviewed journals, b) written in English, and c) included offender populations. We excluded papers that were: a) case reports, books, conference abstracts, letters, b) written in languages other than English, c) published before the dates mentioned, d) animal studies, or e) concerned only paedophilia/paraphilia due to the likeliness of these being caused by mechanisms other than violent criminality. Selection. All selected publications were assessed for relevance based on both title and abstract. The articles were judged for inclusion by two independent researchers based on content. The reviews and/or meta-analyses were divided into two groups. The first group of articles related to a criminal or forensic context. The second group of articles addressed types of behaviour, i.e. physical aggression and violence that are most relevant to the criminal justice system in terms of personal damage for the victim and serious legal consequences for the perpetrator. Twenty articles that were deemed essential but were not found in the systematic search were added. This is shown in figure 1. Brain and criminology Various disciplines study brain functioning and the effect dysfunction has on behaviour, but each has a distinct perspective and aim. The following section aims to correlate the findings across these research fields to facilitate a more thorough understanding of the possible relationships between criminal behaviour, neuroanatomy, brain biochemistry and neuropsychology. We first present neuroanatomy, including the morphological structures of the brain that are found to be relevant in imaging studies. Next, we discuss forensic neuropsychology -this encompasses the integration of psychological findings with neurology by performing tests that specifically target an area of the brain. [9,22] Finally, an overview of the related neurotransmitters and hormones is presented. A schematic summary of the evidence from the different research fields can be found in a series of six tables. Neuroanatomy One of the challenges of neurocriminology is to trace biological markers of sociopathy with brain imaging research. Brain-imaging techniques identify physical deformities and functional abnormalities that may predispose some individuals to violence. This has led to theories of neuroanatomical deviations and criminal behaviour. In the following paragraphs, these theories and evidence for them are discussed. However, first we provide a brief overview of the most important structures mentioned in research on violent criminal behaviour and the relationships between these structures. (See Table 1). Another important aspect in the instigation of behaviour is processing social information in one's environment. Signals that can indicate a threat, like posture, facial expression or screaming, are directed to the lateral nucleus of the amygdala. [2,26,31,[38][39][40] The signals project to the basal nuclei, where they are integrated with perceptual information originating from the orbitofrontal cortex. [2,24,41] This can lead to a behavioural response via the central nucleus, the hypothalamus and brainstem. [2] Thus, the orbitofrontal cortex is thought to integrate the cognitive activity of the total prefrontal cortex into the emotional limbic system. [25][26][27]37] In this manner, the prefrontal cortex restricts impulsive, disinhibited behaviour and volatile emotions [2,25,26,31]. Dysfunction or neuroanatomical deviations of one or several of the above-described structures have been shown to be related to violent criminal behaviour. However, before we describe these studies, it is important to distinguish between two types of violent aggressive behaviour. Reactive, [42][43][44][45][46][47][48] emotional [45] or impulsive [2] aggression is a reaction to events, often driven by emotion. Instrumental [42][43][44][45]47,48] or premeditated [2] aggression, however, is cold and calculated. Since reactive behaviour is influenced primarily by emotions, and the instrumental is not, Figure 1. Selection of publications. Out of the 3508 found articles, 126 were used for this article. doi:10.1371/journal.pone.0110672.g001 distinct brain areas are expected to be involved in the various forms of aggression [2,42,44,46,48]. Prefrontal cortex. The frontal lobe dysfunction theory states that violent and reactive aggression is a consequence of deficits in the frontal brain, mainly the prefrontal cortex. [44,44,49,50] Supporting evidence for this theory comes from various research areas. First, research conducted with PET shows reduced functioning of the prefrontal cortex in offenders, [6,33,37,51,52] and reduced activity in violent patients. [36] The association is stronger in murderers with a benign social background, than in those with a bad home background, [6] as expected based on the social push theory (which will be discussed later on). Frontal abnormalities have also been found using EEGs on aggressive subjects in various populations (like violent criminals [53]), with PET in forensic psychiatric patients [31,53] and in an MRI study of antisocial patients. [3,53] One study distinguished between the various regions of the prefrontal cortex, and specifically found reduced activity in the ventrolateral part of the prefrontal cortex -relevant for social behavior [54] -in aggressive subjects [44]. Second, studies on brain metabolism show that in general, a reduced glucose metabolism in the prefrontal regions can be found in violent offenders. [32,55] Specifically, it was found that murderers [56] and violent psychiatric patients [31,33] have a lower prefrontal cortex metabolism than controls. This finding has been replicated in impulsive murderers for whom a rise in the metabolism of subcortical regions was also found, as expected, since inhibition by the prefrontal cortex is reduced. [2] However, murderers in that same group who planned their crimes did not have a lowered metabolism in their prefrontal cortex. [2] When the findings for predatory and affective murderers were separated, it was clear that affective murderers had lower prefrontal metabolic activity than predatory murderers, who resembled controls [32,53]. Third, a study examining N-acetyl aspartate, considered a marker of neuronal integrity, [3,57] showed that violent patients had less N-acetyl aspartate in their prefrontal cortex compared to controls. [3,32] More importantly, the frequency of violence was inversely correlated with the levels of N-acetyl aspartate. [3] A lower phosphate metabolism was also found in the prefrontal cortex of the violent patients [3,32]. In general, deficits in the prefrontal area, mainly the ventromedial part, have been found to be related to poor control of reactive violence. [2,31,45,46,48] Instrumental violence, on the other hand, is thought to be associated with the dysfunction of both the ventromedial prefrontal cortex and the amygdala. [46] In addition, individual differences appear to be important. Personal differences in the ability to modulate emotions have been shown to be linked to prefrontal activation. [2] This might be relevant in understanding vulnerability to violence and aggression [2]. Amygdala. The amygdala is comprised of thirteen nuclei, together forming one structure. [54] The integrated emotion systems model hypothesises that deviant social behaviour, such as violence, is the result of inhibited emotional development caused by amygdala dysfunction. [28,35,[58][59][60] This is supported by a study in murderers, that showed a lowered amygdala activity, compared to age-and sex-matched controls [42]. Moreover, the integrated emotion systems model states that amygdala damage leads to impaired interpretation of emotions. [23,61] This results in diminished empathy, failure to recognise fearful expressions and impaired passive avoidance learning, all of which have been documented in psychopaths [25,58]. These studies lead to the conclusion that prefrontal cortex function [3,53] or size [1] and amygdala function [3] are related to violent aggression. It is possible that both the frontal lobe dysfunction theory and the integrated emotion systems model are true, and reinforce each other. It is also possible that the former theory explains reactive aggression and the latter explains instrumental aggression. Hippocampus, temporal lobe, anterior cingulate cortex. In addition to the amygdala and prefrontal cortex, other brain structures have also been shown to differ in criminal subjects compared to the general population. The hippocampus is part of the limbic system and is involved in memory. The hippocampus has been shown to function abnormally in violent offenders [3] and subjects who commit murder [6,55] and is structurally distinct in psychopaths [6]. The temporal lobe contains the hippocampus and plays a key role in the formation of explicit long-term memory that is modulated by the amygdala. A 20% reduction of the temporal lobe was found in aggressive psychopaths, [62] and functional abnormalities of the temporal lobe were found in violent psychiatric patients. [33] Sexual offending might also originate in the temporal lobe. [37,63,64] However, this goes beyond the scope of this review. The anterior cingulate cortex is a limbic region involved in response selection, behavioural regulation, inhibition, [65,66] and empathy. [26] Interestingly, anterior cingulate cortex hemodynamic activity predicts re-arrest; higher activity leads to better inhibitory control and recurrence rates that are half of those with low activity. [65] If this correlation is replicated, it could possibly lead to a better tool for risk analysis in combination with known psychosocial risk factors, as scepticism remains regarding the sensitivity and specificity of emerging neurobiological markers as independent tools [61]. Hemispheres. Apart from findings concerning specific brain areas, much research is directed at structural dysfunction in the left or right hemispheres. The left Hemisphere Activation Hypothesis states that psychopaths have problems shifting from left hemisphere activity to right hemisphere activity, and specifically processing information in the left hemisphere. [49] Support for this hypothesis is offered by deficits in the left dorsolateral prefrontal cortex, [49] which is associated with attentional control. [27] This makes sustaining attention in the left hemisphere more difficult. [49] Deficits in the right orbitofrontal cortex and anterior cingulate cortex support this since they are involved in the ability to change to right hemisphere activity [49]. In one study, both affective and predatory murderers had higher subcortical right hemisphere functioning than controls, but affective murderers also had lower left and higher right prefrontal functioning. [31] In another study, violent psychiatric patients were found to have increased or abnormal left limbic activity. [33] Offenders who were victims of child abuse have been shown to have reduced right temporal cortex functioning, [6] which is associated with conduct disorder. [26] Reduced volume of the right temporal cortex has been found in psychopathic patients [52]. General deficits in the right hemisphere have been proposed as well. During abstract processing, which is thought to be based in the right temporal lobe, a dysfunction in the right hemisphere was found in incarcerated psychopaths. [56] In antisocial and violent populations, poor right hemisphere functioning has also been observed [32]. Overall, several brain areas appear to be deficient in violent individuals. An overview of findings is given in table 1. However, it is unknown whether these deficits always result in violent behaviour per se, since this was not investigated in the studies mentioned. The evidence that brain deficits are related to violent behaviour is mainly based on case reports such as the one on Phineas Gage (this case will be discussed in more detail later on), which have been shown to be untrue or only partly true. [21] Moreover, case reports that show no violent behaviour in persons with brain deficits are also available. [21] Therefore, more research should be done on how brain deficits or alterations are related to the actual instigation of violent behaviour. In this respect, it may be more fruitful to examine how altered brain function is related to violent behaviour. Neuropsychology and neurology The brain areas described above have distinct functions. In the following paragraphs, we describe additional evidence for a relationship between altered brain function and the propensity for violent behaviour. A schematic overview of the evidence is given in table 2. Lesions in or dysfunction of the prefrontal cortex [37] or the frontal lobe in general [51] lead to impaired executive functioning. Impaired executive functioning is associated with antisocial [6,68] and aggressive [51,53] behaviour. More importantly, low executive functioning can predict aggressive behaviour in boys with a paternal history of substrance abuse. [53] This could possibly help determine the risk for recidivism in general [53]. Inhibition. Another structure that has been shown to be significant in violent behaviour is the anterior cingulate cortex. The anterior cingulate cortex [65] and the serotonergic neurons in the prefrontal cortex are thought to be important for behavioural inhibition. [37] Indeed, prefrontal damage, especially orbital damage, [8,37,68] does lead to lower inhibition and pseudopsychopathic behaviour [8,69]. Empathy. Several brain regions are involved in the instigation of empathy. Lesions in the orbitofrontal cortex [53], prefrontal cortex, [41] amygdala [41,54] or anterior cingulated cortex [41] are related to a lack of empathy. A lack of empathy is indeed often found in offenders. [37,70] These abnormalities in the emotional regulation circuitry are thought to lead to reactive aggression and the violence seen in these individuals [2]. Psychophysiology. Differences in physiology between the offending and general population have been found, long before brain-imaging techniques existed. In this respect, one of the most replicated observations in aggressive anti-socials and psychopaths is low autonomic arousal in rest, measured by resting heart rate and skin conductance [41,45,52,55]. It is likely that low autonomic arousal is related to or a result of anatomical and functional deviances in violent offenders. For example, it is proposed that reduced noradrenergic functioning and reduced right hemisphere functioning would explain the low autonomic arousal found in violent criminals. [32] Moreover, autonomic arousal is also controlled by the amygdala, [28] which has been found to be less functional in murderers. Therefore, low arousal may be a marker for amygdala dysfunction [39]. Although the origin of low autonomic arousal is of interest, forensic risk assessment would particularly benefit from knowledge about how low autonomic arousal may be related to or predict (violent) criminal behaviour. Several theories are of interest. In accordance with the notion that low autonomic arousal may not only be related to but may also predict criminal behaviour, several studies have shown a link with future criminal offenses, [32,52,55,72,73] aggression [6,32,55,62,71,72] -especially instrumental aggression [42] -and antisocial behaviour. [6,32,41,55,58,62] It has even been shown that low autonomic arousal is predictive of children growing up to become offenders. [32,55,66] In one study, aggressive children had lower heart rates than nonaggressive children (p,0.001), and children with lower heart rates were rated as aggressive more often than those with high heart rates (p,0.003). [71] Therefore, autonomic arousal may be an interesting marker to improve risk assessment in future. A downside to using autonomic arousal as a marker is that as of yet it is unknown what the cut-off point for increased risk would be. Neurotransmitters, hormones, and toxins Neurotransmitters and some hormones are important for communication between neurons in the brain and thus, they are of importance in the instigation of behaviour. Therefore, researchers have sought the origin of criminal behaviour in a disturbed balance between some of these neurotransmitters or hormones. Since toxins influence the levels of these neurotrans- mitters, they could also be of significance. A schematic overview of the evidence is provided in table 3. Serotonin. One of the most replicated findings is the relationship between serotonin and aggression. Numerous studies have shown that low levels of serotonin are associated with both reactive and instrumental aggression [2,7,8,10,25,31,34,41,43,45,51,66,[74][75][76][77][78] and impulsivity. [7,10,22,25,31,34,37,43,51,72,77,79] In addition, low serotonin levels [31,55] and reduced levels of 5-hydroxyindoleacetic acid, a serotonin metabolite, have been found in aggressive or violent populations. [2,31,41,43,52,74] Furthermore, a negative correlation between the serotonin 5-HT1A receptor and aggressive behaviour has been established. [79] In impulsive aggressive subjects, reduced serotonin transporter availability was found in the anterior cingulated cortex. [78] Moreover, one study showed that low levels of serotonin predicts recidivism. [7] 5-hydroxyindoleacetic acid levels have been found to predict aggression two to three years in the future in boys with conduct-disorder and recidivists. [2] Antidepressant drugs that act on serotonin, like SSRI's that cause serotonin levels to go up, can reduce violent behaviour in some individuals, [10,33,45,80,81] especially those with high impulsive aggressiveness. [48] Although the abovementioned studies show that aggression and violence are related to low levels of serotonin, other results seem to indicate the opposite. Metabolic enzymes such as monoamine oxidase A (MAO-A) also contribute to aggression because they function to alter neurotransmitter levels. Since MAO-A catalyses the deamination of serotonin, reduced MAO-A activity will lead to higher levels of this neurotransmitter. [57,66,74,82,83] However, MAO-A deficiency, resulting in higher levels of serotonin, has been shown to increase reactive aggression [20,74,84] and low activity to increase criminal behaviour. [7,84] This is called the serotonin paradox. One author argues that the change in behaviour due to MAO-A is actually a consequence of secondary effects, and cannot be explained by its effect on neurotransmitters alone. [74] Taken together, the abovedescribed results do show that the relationship between serotonin and aggressive or violent behaviour is more complicated than is sometimes presented in the courtroom. [81] An individual riskassessment on the basis of serotonin levels is not supported by evidence. Noradrenalin. Although the relationship between serotonin and aggression and violent behaviour seems strong, there is also evidence that other neurotransmitters are involved. For example, noradrenalin levels, a neurotransmitter involved in the inhibition of memory storage and experiences, [48] in plasma and cerebrospinal fluid are positively correlated with impulsivity [31] and affective aggression. [45] This does not provide much information about the exact site of noradrenalin release, but makes drugs counteracting noradrenergic function interesting for preventing aggressive behaviour [31,45]. In summary although evidence for the effects of the serotonin system on violent aggression is strongest, several other neurotransmitters seem to affect aggression and violence. To make the situation even more complicated, the neurotransmitter system also has interactions with other systems in the body, such as the endocrine system. Hormones. One of the most studied relationships is that between the stress system and aggression. Since the prefrontal cortex contains some of the highest levels of cortisol receptors of the primate brain, low levels of stress hormone will alter the turnover of various neurotransmitters. [1] Adrenocorticotropic hormone (ACTH) is produced when cortisol is suppressed, and it increases serotonin metabolism. [31] This results in lower serotonin levels. [31] Cortisol itself seems to be inversely correlated to levels of serotonin. [92] As such, low cortisol levels were associated with sensation seeking [41] and decreased sensitivity to punishment [39], but also with aggressive behaviour in boys, [10,41,43,55,93] adolescents [39,55,93] and adults [33,72,73,93]. However, similar to the serotonin system, these relationships are not unequivocal since high levels of stress hormone have also been found to be related to aggressive behaviour. [94] The key seems to be that the production of cortisol is deregulated. A second important hormone in relation to violent aggression is testosterone. Plasma testosterone levels have been associated with childhood and adult delinquency, [6,93] antisocial behaviour, [64] aggression [2,6,10,33,55,66,72,93,95] and dominance, [25] but a correlation with social success has also been suggested. [93] These correlations have not always been well replicated. [93] The effect of testosterone on aggression is not visible in young children, possibly because aggression in childhood does not increase dominance as it does in adulthood. [6] In 9-11 year old boys, the association between testosterone and aggression has been documented [55]. During development, testosterone induces or inhibits cell death, guiding the brain to typical male pathways. [43,74,93] Later, it stimulates neural pathways associated with aggression. [74] Testosterone receptors have in fact been found throughout the limbic system. [43] The association between testosterone and aggression has been confirmed by users of anabolic steroids. [74] In tests, testosterone injections led to a shift in sensitivity from punishment to reward. [39] Hypogonadal adolescents receiving testosterone became more aggressive physically, but not verbally. [93] This could be explained by changes in musculature as well [93]. Testosterone and cortisol inhibit each other's production. [25,39,58] This means it is possible that the findings of the effects of cortisol are actually due to testosterone, or the other way around. The triple balance of emotion model states that the hyposensitivity for punishment and the hypersensitivity for reward found in psychopaths [40] could be explained by a high testosterone-to-cortisol ratio. [25,39,52,58] Indeed, testosterone increases sensitivity to reward, [25,39] and low cortisol stimulates the hypothalamic-pituitary-gonadal axis, reducing sensitivity to fear [39,58]. A third group of hormones related to antisocial behaviour are the thyroid hormones. T 3 and T 4 have been related to antisocial behaviour. [41] T 3 has also been specifically linked to recidivism [41]. Other substances. Both the endocrine and neurotransmitter system are influenced by substances in the body other than hormones. For example, the connection between alcohol and violence is well documented. [34,91,96] Over half of all violent crimes occur under the influence of alcohol. [91] Alcohol's mechanism of action is thought to be dependent on the function of GABA A , [48] 5-HT and N-methyl-D-aspartate receptor (NMDA)receptors. [91] A lowering of tryptophan, thought to be parallel to the level of brain serotonin, has been documented two hours after alcohol consumption in a normal population [34]. In addition, alcohol inhibits the capacity of the prefrontal cortex, leading to impaired executive functioning. [1,97] This makes it a disinhibiting factor, leading to acting out what was previously inhibited. [10,70,92,97] Alcohol is an aggravating factor in domestic violence, [33,51] and increases the chances of committing homicide [98]. Another substance that may affect neurotransmitter levels is cholesterol. Low cholesterol has been linked to aggression. [2,55] In community samples of psychiatric patients or criminal offenders with low cholesterol levels, an increase in violence was found. [55] A possible explanation for this observation is that low cholesterol leads to lower serotonin levels [55]. To summarize, the various neurotransmitter systems in the central nervous system have complex interactions with each other and with other systems in the body such as hormones and toxins. This makes it hard to understand how aggression and violence are regulated in individuals. Gene-gene interactions can also be expected to occur. [102,107] Given the complex interplay of neurotransmitters, the effects of genetic polymorphisms can be corrected or aggravated by other genetic polymorphisms [102,107]. However, aggressive behaviour is not caused by genetics alone. The 'social push'-theory states that genes need a particular social environment to result in specific behaviour. [6] Antisocial personalities, for example, develop due to biological factors, but lead to antisocial behaviour more often if the social situation predisposes, or pushes the individual to that behaviour. [42,56] On the other hand, if the social environment does not require antisocial behaviour to achieve what is wanted, antisocial behaviour might not develop despite an unfortunate biological background [42,52]. This also means that the correlation between antisocial behaviour and biological risk factors becomes weaker in cases of poor social backgrounds, like a broken home. [42] This is because when the environment does not push an individual towards a negative behaviour (such as someone who has been reared in a benign social environment), but the antisocial behaviour comes to expression anyway, genetic factors have played a larger role in the instigation of the antisocial behaviour. [73] When the environment pushes too hard, like in very poor social backgrounds, every individual is influenced, resulting in a weak correlation between genetic factors and the actual behaviour [42]. To study whether it is genetic makeup or the environment that causes specific behaviour, twin and adoption studies are often used. [40,45,69,82,103,105,109,[111][112][113] This is because monozygotic twins share identical genetic material, and dizygotic twins share on average 50% of their dissenting genetics, [40,82,101,103,112,114,115], but both share an environment. [103] Subtracting the differences between these groups allows estimations of the contribution of environmental versus genetic factors when behavioural differences are measured. [101,103,114,115] Twin studies have, for example, shown the relevance of both genetics and environment for the development of antisocial behaviour, violence and aggression [55,111]. The adoption method compares the correlation between adopted children and their adopting parents with the correlation between adopted children and their biological parents. [101] This also results in an estimate of the contribution of genetic and environmental factors [101]. One of the most studied genes in research on gene-environment interactions is MAO-A (see table 4). A MAO-A deficiency has been shown to increase reactive aggression, [20,74,84] and its low activity increases criminal [7,84] and antisocial behaviour. However, this last result was found especially in males when the subject had also suffered from childhood maltreatment. [10,20,76,86,106,114] In criminal settings, those who had a promoter sequence resulting in low MAO-A activity and who had been maltreated as a child were overrepresented. [20,116] Both examples illustrate that the effect of MAO-A is dependent on environmental factors, so the environment and genetics interact [57,108,116]. Gene effects rarely influence behaviour directly. MAO-A, for example, may have a role in the difference between male and female levels of violence since the MAO-A gene is encoded on the X chromosome. [7,96,106,112,113] The documented correlation between high testosterone levels and low MAO-A activity, and resulting aggression, supports the hypothesis of further testosterone-induced suppression of the MAO-A gene. [113] The promoter region of the MAO-A gene does in fact contain glucocorticoid/testosterone response elements. [20] Testosterone competes with cortisol for binding, but leads to less transcription than cortisol binding does [20]. Males have been found to have less connectivity between the orbitofrontal cortex and the amygdala, [113] lower functional connectivity between the ventromedial prefrontal cortex and the amygdala, [20] lower orbitofrontal activity [113] lower cingulate cortex activation, [113] and a larger amygdala. [117] This does not explain, however, the difference between male and female proclivity for violence. This example makes clear that the change in behaviour due to the MAO-A gene is actually a consequence of secondary effects, and cannot be explained by its direct effect on neurotransmitters alone [74]. Prenatal environmental factors. The prenatal period is an important time for development of the brain and influences function and the way actual behaviour is instigated later on. Exposure to several addictive substances used by the mother during this period influences brain development. [6,32,69] A schematic overview of the evidence is given in table 5. Substance exposure. Prenatal alcohol exposure can cause structural deficits in the corpus callosum, [55,67] and cerebellum in the infant. [67] It also impairs the infant's memory [67] and executive functioning [67] and lowers IQ. [55,67] Though the physical signs diminish in adolescence, the neuroanatomical differences remain. [67] These changes may explain why it is also found that prenatal alcohol exposure increases the risk for conduct disorder [6]. For nicotine a dose-response relationship between the number of cigarettes smoked during pregnancy and violence has been found. [6,55] Prenatal nicotine [6,32,55] and carbon monoxide [32,55] exposure is thought to disrupt the development of the noradrenergic system, [6] possibly via enhancing the muscarinic 2 (M 2 )-receptor, [32] leading to diminished sympathetic nervous system activity. This could explain the observation of low autonomic arousal in violent and antisocial individuals and criminals [6,32]. Prenatal cocaine exposure is also associated with increased delinquency, but these results are debated [55]. Nutrition. Apart from addictive substances, basic nutrition during pregnancy influences the development of the baby and later behaviour. Like the well-known effects of folic acid on preventing spina bifida, other nutrients influence the development of aggressive behaviour. Women who suffered nutritional deficits during the first and second trimester of their pregnancy gave birth to children who had antisocial personality disorder more often than the general population in two studies. [32,66] In addition, heavy metals like copper have also been shown to influence later behaviour. High copper in the neonatal brain is associated with abnormalities in the hippocampus, [55] which is associated with violence. A low zinc to copper ratio was found in males with a history of assaultive behaviour [32]. In addition to the use of addictive substances or nutrition by a mother, birth complications are also a risk factor for prenatal development. Both of these factors could be seen as markers, although the exact mechanisms of these factors are not clear. [43] For example, a significant interaction between maternal smoking and delivery complications has been documented [43]. Birth complications. Birth complications such as anoxia, preeclampsia and forceps delivery lead to increased risk for antisocial and criminal behaviour through brain dysfunction. [6,32] The hippocampus is particularly susceptible to hypoxia and anoxia. [6,55] It is clear that birth complications interact with psychosocial risk factors, like maternal stress, poor parenting and an unstable family environment [32]. In summary, prenatal development seems to affect brain development and as such affects behaviour, which in some cases results in violent behaviour later in life. Minor physical anomalies may be considered as markers of deviant brain development during pregnancy. [6] Features like low-set ears, adherent ear lobes and a furrowed tongue are anomalies that have been described [6] and shown to predict violent offending in unstable home situations. [6,32] A schematic overview of these factors is given in table 5. Postnatal environmental factors. Although the prenatal environment has an effect on brain development, the post-natal environment also shapes brain functioning and gene expression. In the following paragraphs, several examples show how the environment may interact with genes or brain development to influence the development of aggressive or violent criminal behavior. Age. An explanation for the robust observation of age as a risk factor for criminal behaviour is found in the development of the prefrontal cortex. [6,32,90,109] A first explanation states that since the myelinisation of the prefrontal cortex continues into a person's 20s or even 30 s, it simply cannot cope with the executive demands of adulthood placed upon an individual after adolescence [6,7,32]. A second explanation is found in the accessibility of the means, opportunity and motive for aggressive behaviour. [47,57] During adolescence, people first experience significant physical strength and cognitive challenges, are less inhibited by supervision and experience pressure to perform both in school and relationships [6,57,90]. The combination of these hypotheses offers more insight. The changing environment of adolescence requires increased executive functioning, which relies on the prefrontal cortex. [6] Overload of the prefrontal cortex results in impaired development, leading to antisocial behaviour. A stable, supportive environment may offer protection from this harmful overload [6]. A third explanation is offered by the influence of testosterone. The high-risk periods of adolescence and young adulthood overlap with a testosterone curve in many cultures. [75] So the peak occurrence of violence at these ages could be caused by testosterone. [75] The peaks in sensation seeking, possibly related to testosterone and cortisol, are also seen during these time periods [90]. The highest risk of violent behaviour is indeed found in persons in their late teens and early twenties. [4,8,32,57,75,90,99,118,119] This holds for both the general population and people who are mentally ill [4]. As another example, sexual abuse as a child has been associated with later alcohol dependence. [124] This is a risk factor leading to violence. [124] A reduction in child abuse by 50% can be achieved by simple home visits during the first two years of child rearing. [57] Community-based programs also improve self-reported wellbeing of the parents. [57] Given the influence child abuse has on developing antisocial and offensive behaviour, these programs could lower crime rates. Socioeconomic status. The lower socioeconomic classes, as measured by SES, are overrepresented among criminals, and a direct correlation has been found. [8,17,70,78,100,119,123] Sub factors of the SES classification, like poverty, [3,17,55] unemployment [52] and school failure [55,99,120,125] have also been found to correlate with criminal behaviour. As an explanation for this finding, the increased stress caused by low economic status has been mentioned. [78,88] In addition, serotonin response correlates with a SES-score, therefore, a lack of serotonin could confound the correlation between low SES-scores and criminality as well. [78] However, the increased need for and acceptance of violence are also mentioned. In adolescents, it was found that subjects with either high or low social status were more inclined to use physical aggression at school. Middle economic status was a protective factor [78]. Low IQ. Low IQ-scores, [10,17,57,99,119,120,125] especially for verbal intelligence, [43,50,99] form a risk factor for delinquency and antisocial behaviour. [10] One explanation is the expected lower achievement in school, possibly leading to exclusion, poverty and antisocial behaviour. [57] The increased risk of getting caught if one has a low IQ, or an inherent neurobiological correlate between IQ and delinquent behaviour could also account for this finding. Gang membership. The association between gang membership and delinquency has been established in multiple studies, for both male and female gangs. [10,99] Neurobehavioural deficits, such as a history of head injury or intermittent explosive disorder are found in gang members more often than in controls. [6] When corrected for other risk factors before and after membership, this association still exists. [10] It may be that individuals with neurobehavioral deficits are more likely to become a gang member (because of traits like sensation seeking) or gang membership affects brain functioning. Nutritional influences. Postnatal nutritional factors and antisocial or violent behaviour are correlated. [55] For example, protein under-nutrition leads to antisocial personality disorders. [32,55] Serotonin depletion, due to tryptophan under-nutrition (the limiting amino-acid which is used for serotonin) caused aggression under laboratory conditions, compared to well-fed controls. [2] This was also found in rats and monkeys [55]. Iron deficiency has been found in aggressive children and those with a conduct disorder. [55] In children with attention deficithyperactivity disorder (ADHD), both a behavioural and cognitive improvement were found when iron was supplemented. [55] High serum copper levels and high hair levels of manganese, lead and cadmium have been found in aggressive persons. [55] For some of these metals, this effect was only found in combination with low calcium levels [55]. Though not well understood, the relationship between these metals and behaviour is thought to lie in neurotransmitters. [55] The influence of metals on behaviour is debated though, since few studies have been conducted, not all results have been replicated and no prospective study or study taking other risk factors into account has been published [55]. Brain damage. The example of Phineas Gage is often used to illustrate the effects of brain damage. His prefrontal cortex was selectively damaged by an iron spike. [1,3,6,21,24,29,35,37,39,70] Though he survived, his behaviour changed after the accident; he became more aggressive and socially inappropriate. [1,3,21,29] Head injury is found in offenders more often than in the general population, [15,35,51,100] and those with prefrontal damage exhibit aggression more often than those without [13,44,53]. The exact location of the injury influences the changes in behaviour. [50] Dorsal lesions lead to pseudo-depression, marked by apathy and impaired long-term planning; [37,68] orbital lesions lead to more superficial emotional responses and pseudopsychopathy. [8,37,68] Whether prefrontal cortex damage leads to criminality or socially less accepted behaviour, is not yet predictable [1]. The age of injury also has an influence. [50] When the prefrontal cortex injury occurs before adolescence, it leads to diminished executive functioning and what is called 'acquired sociopathy'. [1] When the injury happens in adulthood, however, more impulsive and uncontrolled emotional behaviour results, but executive functioning is not reduced. [1] The age at the time of brain damage also predicts the age for the start of the criminal career of offenders [66]. However, as mentioned before, there are also case reports of people suffering from the same brain injuries as offenders, who do not show violent aggressive behaviour [21]. Overall, several environmental factors are involved in aggressive behaviour and criminality, some more understood and replicated than others. A schematic overview is given in table 6. Discussion The aim of this paper was to review evidence of biological risk factors that predispose individuals to antisocial and violent behaviour, and to discuss their use for forensic assessment. Several aspects that complicate comparing research in this area must be mentioned to understand the usefulness of the reviewed evidence. First, much research in this field is performed on psychiatric patients or normal populations, not on offenders. Although the number of studies using groups of offenders grew between 2000 and 2013, there is still a great need to understand specific offender subgroups. Even if studies use offenders, most groups studied fail to represent the entire imprisoned population. Different studies each select different offender groups thus making the results less valid. [50,53,69,105,120] Defining the studied population is difficult, and different choices are the cause of many differences between studies. In addition, many of the groups studied are simply too small to draw any meaningful conclusions that extend to all offenders, [3,32,50] or to find reliable results that can be replicated. Second, most studies, specifically neuroimaging studies, compare groups of offenders with other groups of individuals. Forensic (risk) assessments mainly focus on a relationship between deviances and violent behaviour shown by a single individual when committing a crime. Therefore, the forensic field is in need of research showing how alterations in genes, brain, or psychophysiology influence violent behaviour in a specific individual at a specific moment in time. Third, apart from research on gene-environment interactions, studies on the relationship between neurobiological deficits and violent behaviour that also take psychological or sociological evidence into account are scarce. Most reviewed primary research focuses on only one of the fields related to violent aggressive behaviour, not on the interaction between these fields. Violent aggression, like all forms of human behaviour, [112] does not only develop under specific genetic and environmental conditions, but rather it requires an interplay between the two. [7,69,76,95,101,103,108,110,126] Violence should be considered as the end product of a chain of events over the course of a person's development, during which risks accumulate and potentially reinforce each other. [57] This research gap should be bridged. Fourth, the interaction does not lead directly to violent aggressive behaviour, but to sensation seeking, impulsivity or low harm avoidance. Evidence of alterations that solely explain violent behaviour was not found. Therefore, it is unlikely that genetic or neuroscientific tools will be used as independent tests in forensic (risk) assessments. Fifth, studies that do relate neurobiological deficits to behaviour use a variety of aggressive or antisocial behaviours that are not necessarily of use for forensic assessment, which is mainly interested in physical or violent aggression. How violence, aggression and delinquency are defined and quantified differs in every test; and self-report scales are unreliable. [96,105,120,126] In addition, the distinction between violent reactive and instrumental aggression is not always clear, although these forms of violence are likely to have very disparate neurological backgrounds [2,10,44,76,101]. Sixth, different studies use a variety of techniques and methods. Neuroanatomical studies focus on imaging single subjects so it is hard to place the subject in a context where violence is likely to be triggered. Neuropsychological studies, on the other hand, often use large populations and are able to test subjects in more ecologically valid situations. Specifically, in imaging studies, the various regions of the frontal cortex are usually not considered separately. [27,35,44] Also the nuclei of the amygdala are not measured separately. [54] This leads to generalisation, simplification and reduced power, since only some of these regions might actually be linked to deviant behaviour. Also, testing levels of substances in subjects differs per study. The circadian rhythm of cortisol is not always taken into account. [72,93] Various time periods between samples and circumstances make studies hard to compare. To conclude, with better designed studies and more standardisation, comparing studies would be easier and it might become possible to link behaviour to underlying mechanisms [53]. Conclusion The influence genes and deviances in brain development have on the development of violent aggressive behaviour, and in which situation, needs further research before genetic and brain imaging information can be used in forensic assessments or in court. [11,20,76] Though most mechanisms are not elucidated, some of the findings may in time be used to estimate risk of recidivism in combination with psychosocial assessment tools. This means better tools for neurologically based assessment might become available as the knowledge develops. As the developmental profile of brain areas and their vulnerabilities are being discovered, key moments to modulate specific environmental factors for persons with a high-risk genetic profile will become possible. [114] For example, some findings can be used to more accurately assess risk of criminal behaviour on an individual basis. However, there is an important ethical difference between using neurobiological assessment tools in the case of suspects and convicted offenders versus in the general population or subgroups, such as children or adolescents. Even in case of the former group, offender rights might be at stake [65]. On a more general level, knowledge of nutrition could be used to improve our society or correctional facilities, and help prevent future encounters with forensic facilities. Better guidance during the most difficult years of adolescence and home visits can diminish chances of a harmful overload of the prefrontal cortex and decrease chances of child abuse. And obviously, brain damage should be avoided. Reducing those criminogenic risk factors reduces the likelihood of engaging in criminal activity, both directly and via reduced triggering of gene-environment interactions. [103] In the future, new information from neuroscience, when integrated into the information already available from sociological and psychological assessments, could contribute to the development of better risk assessment tools, treatments and cures for offenders, reducing recidivism as well [16,21,63,66]. This review underlines the importance of maintaining a caseby-case differentiated approach to evidence-based forensic assessment that takes into account the individual psychosocial development, and neurobiological and genetic risk factors contributing to violent crime.	2017-03-31T05:20:28.421Z	2014-10-20T00:00:00.000	{ "year": 2014, "sha1": "2ea257d68f9857d55de8eccc3fa2f623e2385e60", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0110672&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2ea257d68f9857d55de8eccc3fa2f623e2385e60", "s2fieldsofstudy": [ "Psychology", "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
118595488	pes2o/s2orc	v3-fos-license	Generalizing thawing dark energy models: the standard vis-\`a-vis model independent diagnostics We propose a two parameter generalization for the dark energy equation of state (EOS) $w_X$ for thawing dark energy models which includes PNGB, CPL and Algebraic thawing models as limiting cases and confront our model with the latest observational data namely SNe Ia, OHD, CMB, BOSS data. Our analysis reveals that the phantom type of thawing dark energy is favoured upto $2 \sigma$ confidence level. These results also show that thawing dark energy EOS is not unique from observational point of view. Though different thawing dark energy models are not distinguishable from each other from best-fit values (upto $2\sigma$ C.L.s) of matter density parameter ($\Omega_m^0$) and hubble parameter ($H_0$) at present epoch, best-fit plots of linear growth of matter perturbation ($f$) and average deceleration parameter ($q_{\rm av}$); the difference indeed reflects in best-fit variations of thawing dark energy EOS, model-independent geometrical diagnostics like the statefinder pair $\{r,s\}$ and $Om3$ parameter. We are thus led to the conclusion that unlike the standard observables ($\Omega_m^0$, $H_0$, $f$, $q_{\rm av}$), the model-independent parameters ($r,s,Om3$) and the variations of EOS (in terms of $w_X-w_X'$ plots) serve as model discriminators for thawing dark energy models. INTRODUCTION Late time cosmic acceleration at the present epoch has almost been a de facto phenomenon since the late nineteens. Advances in cosmological observations during the past two decades reveal strong evidences in favour of this accelerated expansion of the universe. These evidences have been brought forthà la independent astrophysical observations like Supernovae Type Ia (SNe Ia) luminosity distance modulus as a function of redshift Riess et al (1998); Perlmutter et al (1999); Davis et al (2007); Riess et al (2007); Wood-Vasey et al (2007); Kowalski et al (2008); Kessler et al (2009); Riess et al (2009) ;Amanullah et al (2010); Suzuki et al (2012), Observational Hubble Data (OHD) Jimenez et al (2002); Abraham et al (2004); Simon et al (2005); Gaztanaga et al (2009) ;Stern et al (2010); Moresco et al (2012); Zhang et al (2012) , Cosmic Microwave Background (CMB) Shift Parameter Ratra et al (1999); Podariu et al (2001); Komatsu et al (2009Komatsu et al ( , 2011; Bennett et al (2012) and Baryon Oscillation Spectroscopic Survey (BOSS) Data Sanchez et al (2012). A good deal of attempts have been taken to explain this accelerated expansion assuming the presence of some exotic fluid, namely dark energy, in huge abundances in the universe. Though there exists a lot of dark energy models (see for example Bento et al (2003); Bentoet al (2006) Dutta et al (2011); Hiranoet al (2011) and references therein) with standard as well as exotic ideas; the canonical and non-canonical scalar fields are the most promising candidates till date. Of late, Robert R. Caldwell and Eric V. Linder Caldwell et al (2005) categorized these scalar field models in two broad classes namely "freezing" and "thawing" dark energy, based on the asymptotic behavior of the scalar field potential. In thawing models, dark energy equation of state wX initially remains at −1 and deviates from −1 near present epoch whereas just the opposite behavior of wX is witnessed in freezing models. Thawing models, in which we are interested in the present article, are broadly classified into two categories: (i) quintessence (for which wX moves to w 0 X > −1), and (ii) phantom (where wX is less than −1). A third possibility has also been explored in Clemson et al (2009) ;Scherrer et al (2008a,b); ; Gupta et al (2009) ;Sen et al (2010) which lead to both quintessence and phantom behavior of wX . In these slow-rolling scalar field models with nearly flat potential, initially the kinetic energy of the field is much smaller than the potential energy. This is because of the initial large Hubble damping which keeps the field nearly frozen at wX = −1 at earlier era i.e., in radiation and matter dominated eras. Due to the expansion of the universe, energy density of the universe decreases. After the radiation and matter dominated eras, the field energy density becomes comparable to the background energy density of the universe resulting in the deviation of the field from its frozen state, thereby leading to deviation of wX from −1. Slow-roll scalar field thawing models can be characterized by different relations between wX and the scale factor a of the universe. Some typical examples of CPL parametrization (Eq. (1)) Chevallier et al (2001); Linder et al (2003), PNGB models (Eq. (2)) and Algebraic thawing models (Eq. (3)) are included in the work by E. V. Linder Linder et al (2008). The corresponding equation of state parameterizations are respectively given by, where F is a parameter which is inversely proportional to the symmetry breaking energy scale and p and b are two free parameters. In the present work, we propose a two parameter generalization for this thawing dark energy models as where f (a) is an arbitrary function of scale factor a. In this article, we have chosen f (a) as f (a) = c/a n , where c and n are two arbitrary parameters. In this context we would like to mention that choice of f (a) can be made otherwise and it would be interesting to see if there exists any observational constrain on the form of f (a) which is beyond the scope of this article. With the chosen form of f (a) = c/a n for n = 1 and c = 1 our proposal exactly overlaps with CPL thawing dark energy model Linder et al (2008). For n = 1 and 1 < c < 3, our proposal leads to PNGB thawing dark energy model Linder et al (2008) which have been studied exclusively for scalar fields dark energy with PNGB potential Frieman et al (1995); Kaloper et al (2006); Dutta et al (2007); Rosenfeld et al (2007). For suitable choice of the parameters n and c, our model can approximately reflect Algebraic thawing Linder et al (2008) as well. As it turns out, all the existing (and probably, upcoming) thawing dark energy models fall in this broad minimal parametrization with different values of the parameters n and c. So, rather than proposing individual models, it is quite reasonable to construct a minimal generic form of parametrization, analyze it and search for possible constraints on the parameters from present-day observations. This is the primary objective of the present article. Along with this view, we also draw some comparisons among the results obtained for different values of n (i.e., for n = 1, n = 1.5 and n = 2) with different fixed values of c and vice-versa. We further provide justification for this proposed generalized form of thawing dark energy model against the other existing thawing models by comparing them with ours. Moreover, we constrain our model by latest Supernova Type Ia Data from Union2.1 compilation Riess et al (1998);Perlmutter et al (1999); Davis et al (2007); Riess et al (2007); Wood-Vasey et al (2007) Sanchez et al (2012). For such analyses we have five parameters in total namely c, n, w 0 X , Ω 0 m and H0 (where Ω 0 m and H0 are matter density at present epoch normalized to critical density and Hubble parameter at present epoch respectively). Since the value of Ω 0 r (the normalized radiation density at present epoch) is very low we do not treat it as a parameter and consider Ω 0 r = 5.05 × 10 −5 Beringer et al (2012) for numericals. Our analysis also helps in comparing the standard diagnostics with model independent ones, and reveals the pros and cons of each one. The major conclusions of the paper are as follows: • Existing thawing dark energy models Linder et al (2008) can be generalized in the form of Eq. (4) as we have presented in this article. Our minimal generalization of thawing dark energy models (Eq. (4)) with two parameters n and c leads to different existing thawing models namely CPL (n = 1, c = 1), PNGB (n = 1, 1 < c < 3) and the Algebraic thawing (suitable choices of n, & c). • Results obtained for different n values (with different values of c) barely differ from the observational point of view. Other way around, we can say that values of n (with different values of c) can hardly affect the best fit values as well as the 1σ & 2σ C.L.s of matter density parameter Ω 0 m and Hubble parameter at the present epoch H0. Also the best fit plots for redshift evolution of average deceleration parameter qav and the growth of matter perturbations in terms of evolution of growth factor f with redshift z (best fit plots) remain unaffected when the values of n and c are altered accordingly. Therefore it is difficult to provide a unique dark energy EOS wX for the thawing dark energy models as different values of n with different values of c lead to the same cosmological dynamics. • The best fit values and the 1σ & 2σ C.L.s of EOS at the present epoch w 0 X does leave little trace on model discrimination for thawing dark energy. Here we discuss the fact that the values of n and c can be constrained by wX − w ′ X (w ′ X = dw X d ln(a) ) plots Caldwell et al (2005) for thawing dark energy models. More importantly, best fit wX −w ′ X plots can also serve as a model discriminator for the thawing dark energy models. The non-linear wX −w ′ X plots can be realized for values of n other than 1 with different values of c. This is an important issue as PNGB and CPL parameterizations can result only in linear wX − w ′ X plots and recent works on scalar field dark energy models point towards the nonlinear wX − w ′ X plots Ali et al (2009). • Most importantly, the model-independent parameters like statefinder pair {r, s} Sahni et al (2003) and the so called Om3 Sahni et al (2008) parameter do play a crucial role in discriminating among different dark energy models. Study of these parameters in the context of our generalized thawing model, therefore, reveals the fact that unlike the standard parameters mentioned in 2nd major conclusion above, these parameters indeed serve as model discriminators for different thawing dark energy models i.e., these parameters can identify the different values of n as well as c in our generalized model. The paper is organized as follows. In the next Sec. we propose the generalization for the thawing dark energy models and mention the standard as well as the model independent parameters. The Sec. 3 briefly describes the various observational data we used. In the Sec. 4 we present the results obtained by the analyses of the various observational data. In the Sec. 5 we discuss our results and put forward the conclusions of the present work. Generalized thawing dark energy EOS We propose a minimal two parameter generalization for thawing dark energy EOS wX as, where f (a) is an arbitrary function of scale factor a of the universe. We study the dynamical universe with radiation, matter and thawing dark energy obeying the proposed EOS wX with f (a) = c a n . The proposed choice of f (a) here, for the generalized thawing model is motivated by the following findings: i) for n = 1 and c = 1, our model is exactly same as CPL parametrization (Eq 1). ii) for n = 1 and c = F (F being the parameter described in Sec. 1), our model is exactly same as PNGB model (Eq 2). iii) Algebraic thawing case (Eq 3) can also approximated for certain choices of c and n in terms of b and p. iv) for values of n other than 1, generalized thawing dark energy EOS takes the form where w 0 X is the value of wX at the present epoch. Expansion of wX (a) about a = 1 gives, In order to test the validity of our generalized model we show in Fig. 1, the theoretically predicted wX − w ′ plots for different thawing models that arise for different values of n and c (we will put constrains on this wX − w ′ X plane with direct observational data later in this paper). We find from Fig. 1, theoretically obtained wX − w ′ X plane for different combinations of c and n in our model satisfy the allowed regions for the same Caldwell et al (2005). In Fig. 1, the left plot is for quintessential thawing with w 0 X = −0.9 and the right one is for the case of thawing originated in phantom scenarios with w 0 X = −1.1. For n = 1 with c = 1 (dotted) we get CPL thawing (Eq. (1)) and for n = 1 with c = 2, 3 (solid and dot-dashed respectively) we get PNGB thawing (Eq. (2)). The plots in black in Fig. 1 indicate these two models in the wX − w ′ X plane. The orange plots are for the Algebraic thawing model with p = b = 1 (dotted lines), p = b = 2 (solid lines) and p = b = 6 (dot-dashed lines). The results with higher values of n are shown by the green (n = 1.2) and blue plots (n = 1.5). The dotted, solid and dot-dashed lines in these cases corresponds to c = 1, 1.2, 1.5 respectively. Theoretical constraints on the models parameters n and c In this section we discuss the constraints on the model parameters of our genralized thawing dark energy EOS as proposed in the work by Caldwell et al Caldwell et al (2005). In Fig. 2 red region shows the allowed region of the parameter space (n, c) which is allowed for thawing dark energy with our generalized EOS. It is also necessary to point out that our generalized EOS can represent dark energy models other than thawing. The region of (n, c) parameter space except the red zone represents these models. This allowance of n and c values in Fig. 2 is also reflected in Fig. 1. The standard and model independent parameters As is well-known, any dark energy model must at least probe three parameters directly from observations: i) the present value of equation of state (EOS) for dark energy (w 0 X ) ii) the present value of matter density (Ω 0 m ) iii) the Hubble parameter today (H0). Nevertheless, dark energy model building today is tightly constrained by several observations, which, taken together, leave out a very narrow window through which the model should pass. So, from today's perspectives, apart from the above three good old parameters, the supplementary parameters which one needs to address are the following: The statefinder pair {r, s} Sahni et al (2003) serves as a geometrical diagnostic to probe the properties of dark energy in a model independent manner. This pair {r, s} has been studied extensively in the earlier works Panotopoulos et al (2008) For the late universe (z < 10 4 ), which is well approximated by the presence of matter and dark energy, the statefinder pair {r, s} can be expressed as, where a is the scale factor of the universe and ΩX is the dark energy density parameter. In the late universe we have Ωm +ΩX = 1, Ωm being the matter density parameter. For ΛCDM model, it can be checked that the statefinder pair {r, s} takes the value r = 1 and s = 0. Any deviation in r from 1 and s from 0, indicates the existence of varying dark energy in the universe. The Om parameter proposed by Sahni et al Sahni et al (2008), is another tool to distinguish the dynamical dark energy from the cosmological constant. The uncertainty in matter density parameter allows significant errors in cosmological reconstruc- Figure 1. Plots depicting generalized thawing EOS in terms of w X − w ′ X plane for different parameter values as obtained from theoretical predictions. The left figure is for w 0 X = −0.9 and the right figure corresponds to w 0 X = −1.1. Black (orange) plots are for CPL, PNGB (Algebraic) thawing models for w 0 X = −0.9 and w 0 X = −1.1. The dotted, solid and dot-dashed black lines are for c = 1 (CPL) and c = F = 2, 3 for the PNGB thawing case and for Algebraic thawing case they are (in orange) for p = b = 1, p = b = 2 and p = b = 6. The blue and green curves are for our generalized thawing EOS. Green (dotted, solid, dot-dashed) lines are for n = 1.2 (c = 1, 1.2, 1.5). Similarly blue (dotted, solid, dot-dashed) lines are for n = 1.5 (c = 1, 1.2, 1.5). The area between solid red lines (overlapped with dotted and dot-dashed black lines) is the allowed thawing region Caldwell et al (2005). tions of dark energy. Om parameter can in practice differentiate between the models, independent of the matter density parameter. The Om diagnostic has been studied well in the earlier works Lu et al where h(z) = H(z)/H0. It can be easily seen that for cosmological constant Om(z1, z2) = 0 and when z1 < z2, Om(z1, z2) > 0 (Om(z1, z2) < 0) represents the case of quintessence (phantom) Sahni et al (2008). This is how Om evaluated at two different redshifts (z1 and z2) can help in distinguishing the dark energy model. Needless to mention that this procedure is independent of Ω 0 m and H0. The three-point diagnostic Om3 Sahni et al (2008) is defined by, For ΛCDM model Om3 = 1. Another dimensionless parameter, which is useful for determining the beginning of cosmic acceleration in dark energy model, is the average deceleration parameter qav, defined as Sahni et al (2008), where q(t) is the deceleration parameter. We use Eqs. (8,9,11,12) for evaluating the statefinder pair {r, s}, Om3 and qav for the case of our generalization of thawing dark energy model. Further more, we investigate the growth factor f in the context of this proposed generalized thawing EOS. For this purpose we assume the generalized thawing dark energy models proposed here, are decoupled from the cold matter sector. This would lead to the effect that the galaxy cluster formation is not directly influenced by the existence of dark energy. But the presence of dark energy alters the Hubble expansion rate which affects the growth of inhomogeneities in the cold matter sector. In the linear regime of matter perturbations, the evolution of the inhomogeneities are governed by the relation Wang et al (1998) where δ = δρm/ρm is the matter density contrast with ρm being the matter density. The growth factor f is defined as Wang et al (1998), . Eq. (13) can be written in terms of growth factor f (defined in Eq. (14)) as, The growth equation can be expressed in terms of the redshift z by the relation ln a = − ln(1 + z). The growth factor is well approximated by the ansatz Wang et al (1998) where γ is termed as "growth index". The growth factor f is affected by dark energy models via Ωm(z). COMPILATION OF COMBINED DATASETS For the purpose of putting constraints on the generalized thawing dark energy EOS, we use the latest Supernova Type Ia (SNe Ia) Data from the Union 2.1 compilation Suzuki et al (2012) SNe Ia, 25 from OHD, and 1 each from CMB and BOSS). We make a combined χ 2 analyses of the data sets comprising of all 607 data points to constrain our model parameters w 0 X , Ω 0 m and H0, as well as to confront with the model-independent parameters mentioned in Section 2. This makes our analysis robust. Union 2.1 compilation of Supernova Type Ia Data Luminosity distance (dL) measurement of distant supernovae with redshifts z is the first observational data to probe the current acceleration of the universe and the dark energy properties as well. The most recent compilation of the Supernova Type Ia Data is given by Union 2.1 dataset Suzuki et al (2012). The data is tabulated in terms of distance modulus µ(z) with redshift z. The distance modulus can be written as where DL(z) = H0dL(z) (speed of light in vacuum is normalized to unity) and µ0 = 42.38 − 5 log 10 h with h given by H0 = 100h Km.Sec −1 .Mpc −1 . χ 2 of SNe Ia data is given by, Marginalizing over the nuisance parameter µ0, one gets the χ 2 as, where A, B and C are given by, Observational Hubble Data (OHD) Measurements of Hubble parameters from differential ages of galaxies provide another way to probe the late time acceleration of the expanding universe. Jimenez et al Jimenez et al (2002) first utilized this idea of measuring Hubble parameter through the differential age method. Simon et al Simon et al (2005) Table 1. The χ 2 function for the analysis of this observational Hubble data can be defined as (2002) CMB Shift Parameter Data CMB shift parameter R, to a great extent, is a model independent quantity extracted from CMB power spectrum. It is given by where z * is the redshift value at the time when photons decoupled from matter in the universe. z * can be calculated as (with Ω b being the baryon density parameter) where the functions g1 and g2 read as χ 2 CMB is defined as, From WMAP 9 year results Bennett et al (2012), we use R = 1.728 ± 0.016 at the radiation-matter decoupling redshist z * = 1090.97 . The χ 2 for the BOSS data is defined as Combined χ 2 analyses Combining all the datasets from Sections (3.1) -(3.4), comprising of altogether 607 data points, the combined χ 2 can be evaluated as: In what follows, we minimize this combined χ 2 with the observational data sets and search for possible consequences by confronting our generalized model directly with observations. In the case we consider all the dark energy models i.e., thawing as well as non-thawing that can arise from our generalized EOS the total χ 2 will be function of n, c, Ω 0 m , w 0 X , H0 when we consider combined data sets consisting of SNe Ia, BAO, OHD and CMB Shift parameter data. Marginalized χ 2 in general is defined as Nesseris et al (2005); Perivolaropoulos et al (2005), where the χ 2 (ps, θ) is marginalized over the parameter θ in the range θ1 < θ < θ2. DATA ANALYSIS AND RESULTS In this section our primary objective is to make a combined χ 2 analysis for our generalized model as proposed in Eq (4) with SNe Ia, OHD, CMB and BOSS data for the evaluation of the parameters space and their 1σ and 2σ confidence level (C.L.) limits. We further study these cases to compare between the results for n = 1 and other values of n with the different values of c. Our results are tabulated in Table 2, 3 and 4. There are five parameters in this generalized thawing model and they are n, c, w 0 X , Ω 0 m and H0. We fix the values of n at 1, 1.5, 2 with different values of c so that we can compare different thawing models and find the best fit values of other three parameters by χ 2 analyses. The results of χ 2 analyses for PNGB and CPL models are furnished as Case I below and the χ 2 analyses results for other thawing models with n = 1.5 and n = 2 are presented as Case II and Case III respectively. Case I: n = 1 (CPL & PNGB) In what follows, we describe the results obtained for CPL and PNGB cases which can be obtained from the proposed generalization of wX (Eq. (4)) with n = 1. The χ 2 analyses results for n = 1 with different values of c are tabulated in the Table 2. These are the cases of PNGB (1 < c < 3) and CPL (c = 1) thawing dark energy models. Here we choose the values of c to be 1, 1.5, 2. It is seen from Table 2 that best-fit results (w 0 X ) point towards the existence of phantom type thawing dark energy in the universe. As the parameter c goes on taking higher values the phantom nature gets enriched i.e., the deviation of w 0 X from −1 goes on increasing. During this change of EOS (wX), the value of matter density parameter at present epoch and present epoch value of the Hubble parameter remain unaltered. Also needless to mention here that the values of total χ 2 remain unchanged as is evident from Table 2. In Fig. 3, the 1σ and 2σ contours of the different observables e.g., w 0 X , Ω 0 m and H0 for n = 1 with different values of c are shown by light blue and dark blue shaded regions respectively. The " * " in the plots represents the best fit values obtained by χ 2 minimization (Table 2). Here one can see that the phantom kind of thawing dark energy is more favoured than the quintessence type upto 2σ C.L. Here we investigate the other thawing model that can be originated for n = 1.5. The χ 2 minimization results obtained for n = 1.5 with c = 0.5, 1, 1.5 are tabulated in Table 3. Here also the best-fit results suggest that the nature of thawing dark energy is of phantom kind and as c increases the deviation of w 0 X from −1 gets increased. One also sees from Table 3 that the best fit values of present epoch matter density parameter Ω 0 m remain unchanged as the values of c changes. It is also observed that the best fit values of the Hubble parameters H0 at the present epoch also have hardly undergone any changes in these cases. Like the previous case χ 2 remains unchanged. In Fig. 4, the best fit values (obtained from χ 2 minimization) are shown with " * " symbol and the 1σ and 2σ contours for different observables e.g., w 0 X , Ω 0 m and H0 are given by light blue and dark blue color shadings respectively. Here one can observe that the phantom type of thawing dark energy is more favoured over the quintessence upto 2σ confidence level. Case III: n = 2 Moving onto the n = 2 thawing scenario, here the results for n = 2 with c = 0.5, 1, 1.5 are presented in Table 4. Like the previous two cases discussed above, it is also evident here that the best-fit w 0 X points towards the phantom nature of thawing dark energy present in the universe. Also it is seen that w 0 X decreases with the increasing value of c leaving no significant signatures in the best-fit values of Ω 0 m and H0. Also the χ 2 in this case remains unchanged like the previous two cases. As in the previous two occasions, best fit values (obtained from χ 2 minimization) and 1σ, 2σ contours are denoted by " * " and light blue, dark blue color shades respectively in Fig. 5. Here also it is easy to figure out that the thawing dark energy can be of both quintessence as well as phantom kind (more favoured). Now we compare the results for different values of n with a particular value of c. For c = 1, one can figure out from the Tables 2, 3, 4 that as n value increases from 1 to 2, w 0 X shifts from −1.009 to −1.011 indicating the enhancement of phantom nature of thawing. The present values of matter density parameter Ω 0 m and Hubble parameter H0 remain unchanged in these cases. The same analogy goes for c = 1.5 case. From the above discussions this is apparent that all the three thawing models (that can be represented by a single form proposed in this work (Eq. (4))) produce identical Ω 0 m and H0 values at least upto 2σ C.L. In Fig. 6, the growth factor f is plotted against the number of e-foldings N = log(a) for different best fit values of w 0 X , Ω 0 m and H0 obtained in the Tables 2, 3, 4. The left (right) panel is with the initial condition f (N = −7) = 0.8 (f (N = −7) = 0.9)) for n = 1, 1.5, 2 with different values of c as described in Case I, Case II and Case III in this section. The evolution of the growth factor f is identical in all the cases suggesting the formation of the same large scale structure in all cases of thawing considered here (i.e., for CPL and PNGB (n = 1), Algebraic thawing for n = 1.5 and n = 2). Therefore the growth factor f does not serve as a model discriminator but acts as a supplementary probe to confirm correct estimation of cosmic structures formed. Fig. 7 depicts the best-fit variation of w ′ X with wX as obtained using the best fit values of w 0 X from the Tables 2, 3 and 4 for different combinations of n and c. The plots show that we can indeed have non-linear behavior of wX along with the linear behavior for the generalized thawing dark energy model from observations. Comparison of these plots with our theoretical predictions, as done in Fig. 1 will be interesting. Therefore Fig. 7 goes over Fig. 1 which was only a theoretical prediction. As it turns out from this figure, the wX − w ′ X plane indeed serves as a model-discriminator for different thawing dark energy models. Model-independent diagnostics In Fig. 8 we show the best-fit variations of the statefinder parameters {r, s} with redshift z for n = 1 case (with the best-fit values of w 0 X , Ω 0 m and H0 presented in the Tables 2) which is known as CPL for c = 1 or PNGB for other values of c. The dashed, solid and dotted plots are for c = 1, 1.5, 2 respectively. These plots bear the clear signatures of thawing as one can see that for higher values of z, the statefinder r tends to 1 and the statefinder s to 0. This is because wX = −1 as z increases and since in present epoch wX deviates from −1, r and s also deviates from 1 and 0 respectively. The same features are also observed in the cases of n = 1.5 and n = 2 in Fig. 9 and Fig. 10 respectively. In Fig. 11, we show the best-fit variation of the Om3 parameters with the redshift z3 while z1 and z2 are kept at z1 = 0.2 and z2 = 0.57 for the best-fit values of w 0 X , Ω 0 m and H0 presented in the Tables 2, 3, 4. The plot at the extreme left of Fig. 11 shows the variation of Om3 parameter for n = 1 with c = 1 (dashed), c = 1.5 (dotdashed) and c = 2 (dotted). The middle and the right plots of Fig. 11 show similar variations for n = 1.5 and n = 2 respectively with c = 0.5 (dashed), c = 1 (dotdashed) and c = 1.5 (dotted). As Om3 is a three point diagnostic, we need three redshift points to measure its value. We fix two redshift points z1 and z2 with z1 = 0. Figure 7. Plot of best-fit w X − w ′ X plane. The region between the two red lines is allowed w X − w ′ X plane for thawing model Caldwell et al (2005). The black, the blue and the orange lines corresponds to the thawing models arising out of the generalized EOS (Eq. (4)) for n = 1, 1.5, 2 respectively. The dotted, solid and dotdashed lines corresponds to c = 1, c = 1.5, c = 2 for the case of n = 1 and c = 0.5, c = 1, c = 1.5 for the case of n = 1.5 and n = 2. (2012) and allow z3 to be a variable. All the variation starts from a point where z3 = z2 that leaves Om3 = 1 and the immediate deviation of Om3 from 1 to less than 1 suggests the phantom nature of dark energy which is here the varying phantom thawing dark energy. In Fig. 12 the best-fit variation of average deceleration parameter qav has been plotted with the best fit values of the parameters w 0 X , Ω 0 m and H0 obtained in the Tables 2, 3, 4. It is seen from the plots that all of them overlap with each other. It is thus evident that average deceleration parameter is not capable of being a model discriminator, but it does indicate the transition period from the deceleration to acceleration phase. In this case this transition occurs nearly at the redshift z ∼ 7as is evident from the best fit plots. Observational constraints on the model parameters In this section we present the result for marginalized contour of n and c (Fig. 13) with the other parameters w 0 X and Ω 0 m marginalized over the ranges −1.7 < w 0 X < −0.2 and 0.1 < Ω 0 m < 0.9. In Fig. 13 the light blue and the dark blue shades represent the 1σ and 2σ contours. We also present the marginalized contour of w 0 X and Ω 0 m (Fig. 14) with the model parameters n and c marginalized over the ranges 0.1 < n < 3 and 0.1 < c < 20. In Fig. 14, the areas enclosed by the smaller inner contour and the bigger outer contour represents respectively, 1σ and 2σ allowed regions. In performing so the fact that we have included thawing dark energy models as well as dark energy models which are not thawing, is evident from the Fig. 2. We also would like to mention that in this process we have used the type Ia supernova data, baryon oscillations spectro-scopic survey data and the cosmic microwave radiation shift parameter data. The values of n and c leading to thawing dark energy models with our generalized dark energy EOS (Eq. (4)) is described in the subsection II A and II B. From Fig. 13, one can notice that present day data does not put any strong constraints on the dark energy models, i.e., claiming that dark energy is thawing is not parhaps completely justified from the observational point of view. In other words, data does not restricts us to thawing dark energy models only or present day data is insufficient to favour any particular class of dark energy models at present. DISCUSSIONS AND CONCLUSIONS In the present work, we proposed a two parameter generalized EOS, wX , for thawing dark energy models and studied the dynamics of spatially flat FRW universe containing radiation, matter and dynamical dark energy. This proposal of ours is a minimal generalization of thawing dark energy EOS and is given by, This leads to wX (a) = −1 + (1 + w 0 X )a c for n = 1 and for other values of n, wX (a) = −1 + (1 + w 0 X ) exp[ c (n−1) (1 − a (1−n) )], where the scheme is that each value of the parameters n and c defines a specific thawing model, tuning them will lead to a second model, and so on. We have also demonstrated that this minimal generalization scheme is quite apt as it naturally goes over the wellknown thawing dark energy models such as CPL, PNGB and Algebraic thawing, for suitable choice of the two parameters n and c. We have elaborately discussed the cases with n=1, n=1.5 and n=2 for different values of c (c = 1, 1.5, 2 for n = 1 and c = 0.5, 1, 1.5 for both the cases of n = 1.5, 2). We have shown that though the parameter c is very important for the slope of the wX (a) vs. a plot, it barely changes the dynamics of the universe. This is quite evident from the average deceleration parameter (qav(z)) vs redshift z plot (Fig. 12), growth parameter plots (Fig. 6) etc and also from the present values of matter density parameter Ω 0 m and the Hubble parameter H0 as well. In this context it is therefore very important to mention that fine tuning of c does not, at all, effectively change the observables like the values of density parameters and the Hubble parameter at the present epoch (vide Tables 2, 3 and 4 for best-fit values and Figs. 3, 4, 5 for 1σ and 2σ C.L.s). Here it is necessary to mention that in spite of treating the present epoch value of radiation density parameter Ω 0 r as a parameter in the numerical analysis, we have chosen its value to be Ω 0 r = 5.05 × 10 −5 Beringer et al (2012). This is because of the small value of Ω 0 r which will not change the total density parameter upto four decimal places and therefore not considering it as a parameter will not affect density parameters Ω 0 m or Ω 0 X significantly. Also we would like to conclude that different values of n would lead to same cosmological dynamics for a particular value of c which is evident from average deceleration parameter plot (Fig. 12) and growth factor plots (Fig. 6). These plots clearly demonstrate that it is hardly possible to distinguish between the results for different thawing models (related to different values of n and c). It is necessary here to mention that in calculating growth factor f , we have considered those thawing models that do not modify the Newton's constant G. There exists a class of non-minimally coupled scalar field models that give rise to thawing and modify the Newton's constant G as well (see Ali et al (2012), Hossain et al (2012)). In those cases, no generic form for effective Newton's constant G eff exists as the modification depends on the nature of nonminimal coupling. Therefore we exclude those thawing models in our proposal of generalized thawing EOS (Eq. (4)). The analysis thus reveals a very crucial information about the general class of thawing dark energy models, namely, different thawing dark energy models can not be distinguished with the present-day values of matter density, Hubble parameter, the growth factor plots and the average deceleration parameter plots. The importance of our analysis further lies in the fact that this is a quite generic conclusion, since our proposition does take into account within itself almost all the thawing dark energy models. So, we claim that one indeed needs to go beyond these parameters in order to distinguish among thawing dark energy models. These distinguishers come in the form of geometrical diagnostics like statefinder pair r, s and the Om3 parameters. Even though observational data for these parameters are lacking till today, the analysis succeeds in giving some important predictions which, we believe, may show a direction of which way to proceed in near future. These statefinder pair r, s and the Om3 parameter are shown in Figs. 8,9,10,11 respectively. We have also shown in Fig. 7 that the best-fit wX − w ′ X plots can, as well, serve as another discriminator for these thawing models. Nevertheless, it is also shown in Fig. 7 that wX − w ′ X plots are non-linear for n = 1.5 and n = 2. For the existing thawing models (e.g., PNGB and CPL cases), wX − w ′ X plots are strictly linear. In a recent work, Ali et al Ali et al (2009) have found this kind of nonlinear wX − w ′ X plots arising from scalar field models. So our generalization can also produce them for values of n other than 1 and they are also favoured well by the recent cosmological observations. Moreover from Fig. 7, it can be noted that as n takes higher values, only lower values of c are allowed for thawing dark energy models. From the Fig. 2 lead to the thawing models with our generalized form of dark energy EOS (Eq. (4)). Therefore it easy to note that the values of n studied in Tables 2, 3 i.e., n = 1 and some of n = 1.5 lead to thawing but others (Table 4) are not thawing which is also reflected in the Fig. 7. It leads us to also conclude that the values of Ω 0 m and H0 are same for the thawing as well as the non thawing dark energy models (Tables 2, 3 and 4 and the Figs. 3, 4, 5). Therefore the beauty of the parametrization lies in its form which generalizes the thawing models as well as includes other dark energy models which gives us the opportunity to study all the models together in the context of present day observational data. Also From the Tables 2, 3 and 4, one can see that the χ 2 values are a bit low. This is because the error bars in the data sets namely type Ia supernova data, baryon oscillation spectroscopy data, hubble parameter data and the cosmic microwave background shift parameter data are large with respect to this generalized model and in the definition of χ 2 as the error bars appear in the denominator, we get the a bit low value of χ 2 . If the error bars are reduced in the data sets better results can be obtained and we hope to have well constraints on the model parameters in this generalized model in near future. As mentioned, this is a minimal generalization with the two parameters c and n and one boundary condition given by wX (z = 0) = w 0 X , z being the redshift. There may exist other generalizations with more than two parameters. So selection can be made on the basis of Akaike Information Criterion (AIC) Akaike et al (1974) and the Bayesian Information Criterion (BIC) Schwarz et al (1978) that are defined as, AIC = −2 ln(L) + 2p ′ , (31) BIC = −2 ln(L) + p ′ ln N , where L is the maximum likelihood value which is given by exp(−χ 2 min /2), p ′ is the number of model parameters and N is the number of data points used to find the minimum value of the χ 2 denoted by χ 2 min . We show the ∆AIC and ∆BIC values in Table 5. Usually from statistical analysis, it is inferred that the models having ∆BIC in the range 0 − 2 are strongly supported, models with ∆BIC > 2 are moderately supported, and those with ∆BIC > 6 are unsupported from perspective of a given data. However, in cosmology, with the rapid increase of the number of data points N (we remind the reader that we have used combined dataset), Eq (32) shows that the ∆BIC value is always going to increase with introduction of new model parameter(s) p ′ . This does not essentially mean that the models with least number of parameters are always favored by observations, though it may appear to be so. For example, we know ΛCDM model (with the least number of parameters) Li et al (2010) fits the SNe Ia data only in the low redshift region i.e, for z << 1, and in this vein, most of the models pay the price just because they have additional parameters, though they, in fact fair well with observations. On this note it should be mentioned that, as demonstrated in Liddle et al (2006) the above information criteria should better be replaced by Bayesian Evidence calculation, which gives a value after integrating over all probable states, and hence, does not suffer from any such limitations of AIC or BIC. Hence, nowadays, most of the cosmological models are relying more on Bayesian Evidence calculation, rather than ∆AIC or ∆BIC calculation. We hope to address this issue in near future. We are in the era of precision cosmology. Observational data are improving day by day. But these are the error bars that the data come with makes the constraints on the models poor. Therefore it is very necessary to reduce the error bars which can improve the constraints on the model parameters further. We used Type Ia supernova data, Baryon Oscillation spectroscopic survey data, observational hubble data and the cosmic microwave shift parameter data to constrain the models parameters. Among all these data supernova data influences the analysis the most i.e., the constrained parameters space depend on supernova data to a great extent. Supernovae data are not that precise at the present moment as it comes with large error bars. Improving supernova data can probably give us the improved and satisfactory results in future.	2013-10-21T08:41:24.000Z	2012-10-09T00:00:00.000	{ "year": 2012, "sha1": "cece10c705447bcd012f64541ad71a9e4f7a3f2f", "oa_license": null, "oa_url": "https://academic.oup.com/mnras/article-pdf/437/1/831/18462466/stt1941.pdf", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "cece10c705447bcd012f64541ad71a9e4f7a3f2f", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
22386134	pes2o/s2orc	v3-fos-license	cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity. Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V(max) and an increase of K(m) for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 mum cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action. Cytochrome c oxidase (COX), 1 the terminal enzyme of the mitochondrial respiratory chain, reduces oxygen to water and pumps protons across the inner mitochondrial membrane. COX contains 13 subunits per monomer, three of which are encoded by the mitochondrial genome. COX has been shown to be the rate-limiting enzyme of oxidative metabolism under physiological conditions in a variety of human cell types (1) and in a mouse cell line with a mutation in COX subunit I (2). The functional mammalian enzyme has been crystallized as a dimer (3) and shows three features, described below, that are found in key metabolic enzymes: allosteric regulation, isoforms, and phosphorylation. COX activity is regulated by small molecules such as ATP and ADP (4 -6), and the thyroid hormone T2 (7). COX contains skeletal muscle/heart ("heart-type") and nonskeletal muscle ("liver-type") isoforms of subunits VIa, VIIa, and VIII that have been known for the past 2 decades (reviewed in Ref. 8). We have recently discovered three additional isoforms: a lung-specific isoform of subunit IV, a third isoform of subunit VIII, and a testes-specific isoform of subunit VIb (9 -11). Although it is clear that protein kinases and phosphatases are crucial in cellular signaling, little is known about their role in the regulation of the respiratory chain complexes. There have been three studies of COX phosphorylation: Steenaart and Shore (12) examined mitochondrial proteins phosphorylated by endogenous kinases in the presence of [␥-32 P]ATP and identified COX subunit IV; Miyazaki et al. (13) showed that COX subunit II can be phosphorylated by nonreceptor tyrosine kinase c-Src in osteoblasts and found a positive correlation between COX activity and c-Src kinase activity (see also Ref. 14); and Bender and Kadenbach (15) incubated isolated COX from cow heart with commercially available protein kinase A (PKA), cAMP, [␥-32 P]ATP, and an ATP-regenerating system and observed the major signal for subunit Vb, along with signals for subunits II and/or III. The latter group later proposed that subunit I is also phosphorylated (16). These experiments demonstrate the possible modification of COX by serine/threonine-specific PKA in vitro, but no attempts were undertaken to show that PKA acts on COX in vivo. We show here that cAMP-dependent COX phosphorylation occurs at subunit I under physiological conditions, mediated by an endogenous tyrosine kinase. Furthermore, we were able to assign cAMP-dependent phosphorylation to tyrosine 304 of COX subunit I and show that such phosphorylation leads to strong COX inhibition. An example of a function served by this mechanism is COX inhibition through the elevation of cAMP levels in liver cells triggered by glucagon during starvation periods, a condition where energy usage should be restricted. EXPERIMENTAL PROCEDURES Isolation of Cytochrome c Oxidase-Chemicals were purchased from Sigma unless otherwise stated. Mitochondria were isolated from cow liver and heart tissue as described (17) with several modifications. All steps were performed at 4°C or on ice. Tissue (250 g) was ground and further homogenized with a commercial blender, applying a 5-fold volume of buffer A (250 mM sucrose, 20 mM Tris (pH 7.4), 2 mM EDTA). Importantly, this buffer was supplemented with 10 mM KF, 2 mM EGTA, and 10 mM theophylline to obtain phosphorylated COX, and the ground tissue was incubated with buffer A for 20 min at 4°C before proceeding with the next step. The suspension was centrifuged (650 ϫ g, 10 min) and the supernatant was collected through cheesecloth. The pellet was homogenized and centrifuged one more time to increase the yield of mitochondria. Combined supernatants were centrifuged (14,000 ϫ g, 20 min). The mitochondria were washed by resuspending the pellet in 2.5 liters of buffer A using a Teflon homogenizer (150 rpm, 4 strokes) and centrifuged at low speed (300 ϫ g for 5 min) to remove contaminants. The supernatant was centrifuged (14,000 ϫ g, 20 min) to collect mitochondria, which were washed two more times by resuspending and centrifuging (14,000 ϫ g, 20 min). The mitochondrial pellet was resuspended in buffer A and adjusted to a final protein concentration of 20 mg/ml. Per 1 ml of isolated mitochondria, 250 l of buffer B (1 M KH 2 PO 4 , pH 7.4) were added. In order to solubilize mitochondrial proteins, 2 ml of 20% Triton X-114 per 1 g of heart mitochondrial protein were added dropwise with stirring. For liver mitochondria, only half the volume of detergent was added to prevent the extraction of COX. The suspension was centrifuged for 30 min at 195,000 ϫ g. The membrane fraction-containing pellet was resuspended with a Teflon homogenizer in 100 ml of buffer C (200 mM KH 2 PO 4 , pH 7.4) and centrifuged for 25 min at 195,000 ϫ g. The pellet was resuspended in 50 ml of buffer D (200 mM KH 2 PO 4 (pH 7.4), 5% Triton X-100) as above, applying three strokes and centrifuging for 25 min at 195,000 ϫ g. The pellet was resuspended in 100 ml of buffer C, applying 10 strokes and centrifuging for 20 min at 195,000 ϫ g. The COX-containing supernatant was collected, and COX was extracted from the pellet by repeating the previous step two times. Combined supernatants were diluted with 4 volumes of distilled H 2 O and applied to 300 ml of DEAE-Sephacel column (fast flow; Amersham) equilibrated with buffer E (50 mM KH 2 PO 4 (pH 7.4), 0.1% Triton X-100). The column was washed with 800 ml of buffer E, and proteins were eluted using a linear gradient from 50 mM to 1 M KH 2 PO 4 (pH 7.4, 0.1% Triton X-100). COX usually elutes at an ionic strength of about 250 mM KH 2 PO 4 (heart) or 300 mM KH 2 PO 4 (liver). Eluted fractions were analyzed by spectrophotometer, and COXcontaining fractions were combined. Sodium cholate was added (1% w/v) with stirring, and pH was monitored and adjusted to pH 7.4 with NaOH. Fractionations were performed with 28% ammonium sulfate for at least 6 h, 37% for at least 1 h, and 45% for 5 min; proteins were collected after each step by centrifugation for 15 min at 27,000 ϫ g. Precipitated proteins were dissolved in buffer F (50 mM KH 2 PO 4 , pH 7.4) and stored at Ϫ80°C after determination of COX concentration (18) and purity (17) by spectrophotometer. Preparation of Regulatory Competent COX and Enzymatic Activity Measurements-In order to obtain COX that shows regulatory properties, cholate, which is tightly bound (3) at nucleotide binding sites (5), has to be removed, and cardiolipin removed during COX isolation has to be replaced (19). Three M COX was resuspended in 0.5 mM ATP, 120 M cow heart cardiolipin, 50 mM KH 2 PO 4 (pH 7.4), 1% Tween 20, 10 mM KF, and 2 mM EGTA. The suspension was dialyzed against the same buffer without cardiolipin for at least 14 h. COX activity was determined in a closed 200-l chamber containing a micro-Clark-type oxygen electrode (Oxygraph system; Hansatech). Measurements were performed at 25°C in 50 mM KH 2 PO 4 (pH 7.4), 1% Tween 20, 10 mM KF, 2 mM EGTA, 20 mM ascorbic acid, and 5 mM nucleotides as indicated, using 150 -300 nM COX and increasing amounts of cow cytochrome c from 0 to 40 M. All measurements performed in the presence of 5 mM ATP included an ATPregenerating system as described (20). Oxygen consumption was monitored on a computer and calculated using the Oxygraph software. Turnover is defined as oxygen consumed (M)/(s⅐COX (M)). COX Activity Measurements Using Cultured Cells or Frozen Tissue-HepG2 cells were grown to 95% confluence in modified minimum essential medium at 37°C and a 5% CO 2 atmosphere, supplemented with 10% fetal bovine serum, 1000 units of penicillin/streptomycin. Cells were washed with PBS, harvested by scraping in the presence of 10 ml of PBS, collected by centrifugation (50 ϫ g, 5 min), and washed again with PBS. The cell pellet was resuspended in incubation buffer (250 mM sucrose, 10 mM MgSO 2 , 20 mM K-HEPES (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, and 2 M oligomycin) and incubated in the presence or absence of glucagon (2 nM) or forskolin (5 M) at room temperature in the dark for 2 min or 20 min. Cells were collected by centrifugation, and 500 l of chilled solubilization buffer (10 mM K-HEPES (pH 7.4), 40 mM KCl, 1% Tween 20, 2 M oligomycin, 1 mM phenylmethylsulfonyl fluoride, 10 mM KF, 2 mM EGTA) were added, followed by cell disruption on ice with an ultrasonicator (micro tip, 4 ϫ 8-s pulses). Cell debris was removed by centrifugation (2 min, 16,000 ϫ g), and the supernatant was used for respiration measurements. Frozen tissue from cow liver or lung (50 mg) was partially disrupted in 500 l of incubation buffer using a Teflon microtube pestle applying 5 strokes (liver) or 10 strokes (lung), resulting in smaller cell clusters but leaving most cells intact. Cells were incubated and sonicated as described above. Protein concentration was determined with the DC protein assay kit (Bio-Rad). Western Analysis-SDS-PAGE of COX was carried out as described (21). Protein transfer time on a nitrocellulose membrane was 45 min to allow efficient transfer of the larger COX subunits. Anti-phosphoserine and anti-phosphothreonine antibodies are sets of four (1C8, 4A3, 4A9, and 16B4) and three (1E11, 4D11, and 14B3) individual monoclonal antibodies (Calbiochem), respectively, whereas a single anti-phosphotyrosine antibody was used (4G10; Upstate Biotechnology, Inc., Lake Placid, NY). Western analysis was performed with a 1:2000 dilution of anti-phospho antibodies, and signals were detected using horseradish peroxidase-conjugated secondary antibodies (ECL Western blotting detection kit; Amersham Biosciences). ATP Assay-Frozen cow lung tissue (40 mg) was partially disrupted in 800 l of incubation buffer with a Teflon microtube pestle by applying 10 strokes. Fifty l of this cell suspension were transferred to each of eight tubes containing 50 l of incubation buffer, four tubes also containing theophylline to a final concentration of 0.1 mM. Cells were incubated at 4°C for 20 min followed by the addition of 275 l of boiling buffer (100 mM Tris (pH 7.75), 4 mM EDTA) and immediately transferred to a boiling water bath and lysed for 2 min. The tubes were centrifuged, and 5 l of the supernatant were utilized to determine the ATP concentration using the ATP bioluminescence assay kit HS II (Roche Applied Science) according to the manufacturer's protocol. Data were standardized to the protein concentration: 100 l of a 20% SDS solution were added to each boiled sample containing precipitated proteins and cell debris and ultrasonicated for a total time of 8 min. Protein concentration was determined as described above. MS/MS Analysis of Bovine COX Preparation-A 5-g fraction of cow COX isolated in the presence of theophylline, KF, and EGTA (see above) was digested in 70% formic acid with 10% cyanogen bromide for 2 h in the dark. After digestion, peptides were diluted with three volumes of water and dried completely in a speedvac (Thermo Savant, San Jose, CA). Samples were then reconstituted in 0.1% acetic acid and introduced into an LCQ Deca quadrupole ion trap mass spectrometer (Thermo Electron, San Jose, CA) using a variation of the "vented column" approach as described (22). Peptides were loaded onto a precolumn (360-m outer diameter ϫ 75-m inner diameter; 2 cm of 5-m Monitor C18; Column Engineering, Ontario, CA) at a flow rate of 4 l/min with a Surveyor autosampler and HPLC pump (Thermo Electron). Peptides were eluted to the mass spectrometer using an analytical column (360-m outer diameter ϫ 75-m inner diameter; 12 cm of 5-m Monitor C18) and a linear gradient (0 -50% B in 30 min, A ϭ 0.1 M acetic acid, B ϭ 0.1 M acetic acid in acetonitrile; flow rate ϳ200 nl/min). The mass spectrometer performed cycles of one MS scan followed by MS/MS scans (collision energy ϭ 35%; 3-Da window; precursor m/z Ϯ 1.5 Da) of the three most abundant species in the initial MS scan. Dynamic exclusion was utilized with a repeat count of 1 and an exclusion time of 1.5 min. Data Base Analysis-Tandem mass spectra were assigned to peptide sequences from the bovine NCBI nonredundant protein data base using Bioworks 3.1SR1 (Thermo Electron) and the SEQUEST algorithm (23). Search parameters specified a differential modification of ϩ80 Da to serine, threonine, and tyrosine residues (phosphorylation) and Ϫ18 Da to methionine (homoserine lactone) and a static modification of Ϫ30 Da to methionine (homoserine). Phosphopeptide spectra were manually verified. COX Shows cAMP-dependent Phosphorylation at Subunit I and Also Can Be Phosphorylated at Subunits II and IV-We have modified an established protocol for the isolation of COX from liver and heart tissue to obtain increased amounts of regulatory competent COX (see "Experimental Procedures"). For COX phosphorylation studies on the isolated enzyme, we included in all solutions during COX purification potassium fluoride, a nonspecific inhibitor of protein phosphatases, and EGTA, a calcium chelator, preventing activation of calcium-dependent protein phosphatases. Theophylline, a weak phosphodiesterase 4 inhibitor (24), was also included to increase cellular cAMP concentrations. We isolated liver COX side by side in the absence (referred to as "standard conditions") and presence of KF, EGTA, and theophylline. For comparison, we also isolated COX from heart tissue under standard conditions. COX subunits separated by SDS-PAGE and probed by Western analysis with monoclonal anti-phosphoserine, -threonine, and -tyrosine antibodies revealed three findings ( Fig. 1): (a) COX subunit IV showed a strong signal in all three enzyme isola-tions with the anti-phosphotyrosine antibody and weaker signals with both the anti-phosphoserine and -threonine antibodies; (b) liver subunit II reacted with the anti-phosphotyrosine antibody, in agreement with a recent report showing that subunit II can be phosphorylated (13); and (c) subunit I of the liver enzyme produced a strong signal with the anti-phosphotyrosine antibody when purified in the presence of theophylline, whereas no signal was observed in its absence (Fig. 1, arrows). Heart COX isolated in the absence of theophylline did reveal a weak signal with the anti-tyrosine antibody. Under the conditions applied for liver COX, only subunit I shows detectable differences in tyrosine phosphorylation upon increasing intracellular cAMP levels. No changes in serine or threonine phosphorylation were observed. COX Subunit I Is Phosphorylated at Tyrosine 304 -COX isolated in the presence of theophylline was digested with cyanogen bromide, which resulted in only the partial digestion of subunit I, with a sequence coverage of 6%. Based on analysis by nano-liquid chromatography/electrospray ionization/MS/ MS, the fragment DVDTRAYFTSATM, which occurs in subunit I, was identified as containing a phosphate group on tyrosine 304 (Fig. 2). The phosphorylation site was unequivocally assigned by fragment ions b6, b7, b8, y6, y7, and y8. The sequence of the peptide was definitively assigned by b2, b5, b6, b7, b8, b9, b10, b11 2ϩ , b12 2ϩ y3, y4, y5, y6, y7, and y8. Tyr 304 is located on subunit I helix VIII, close to the intermembrane space (3). Helix VIII is in contact with the heme a 3 group, the reaction center where electrons are transferred to oxygen as their final acceptor. This helix also contacts helix II of catalytic subunit II (see "Discussion"). COX Phosphorylated at Subunit I Is Inhibited and Shows Pronounced Allosteric Control-To investigate the functional implications of COX subunit I phosphorylation, we measured enzymatic activity by titrating COX with increasing amounts of cytochrome c. Measurements were performed in the presence of allosteric inhibitor ATP, activator ADP, or AMP. Liver COX isolated in the presence (phosphorylated at subunit I) or absence (not phosphorylated at subunit I) of theophylline showed clear differences in kinetics (Fig. 3, Table I). First, V max of subunit I phosphorylated COX is lower compared with unphosphorylated COX. Second, enhancement of the allosteric response was seen in the phosphorylated COX, such that little or no activity is present below 10 M cytochrome c ( Fig. 3A; see also Hill coefficients in Table I). Both AMP and ADP are seen to activate COX, but activation mediated by ADP is stronger. Glucagon and Forskolin Treatment Leads to COX Inhibition-The peptide hormone glucagon acts as a strong physiological signal for increasing intracellular cAMP levels in liver by binding to G-protein-linked receptors, followed by activation of adenylyl cyclase (25). In order to relate our findings on the isolated enzyme to cellular physiology, we tested the effect of glucagon on COX activity with both intact human liver carcinoma HepG2 cells and cow liver tissue. We avoided artificially inducing the insulin pathway by scraping instead of trypsinizing HepG2 cells (26). Measurements using fed human HepG2 cells revealed strong inhibition of COX activity after treatment with glucagon (Fig. 4A). Similar results were obtained with cow liver tissue (not shown). To determine whether actual starvation would cause the same effect on COX, we analyzed starved cells. These cells showed sigmoidal kinetics in the presence of the allosteric inhibitor ATP and the absence of glucagon (Fig. 4B). This tendency was further increased after preincubation with glucagon. Thus, we show for the first time that the glucagon receptor/G-protein/cAMP pathway strongly inactivates COX. Elevating intracellular cAMP levels by direct activation of adenylyl cyclase with forskolin revealed a similar effect on human HepG2 cells (Fig. 4C) and cow liver tissue (not shown) as observed with glucagon. FIG. 1. Western blot analysis with anti-phospho antibodies of cow heart (H) and liver (L) COX. Cow liver COX was isolated side by side in the presence (ϩ) or absence (Ϫ) of theophylline. COX subunits were separated by SDS-PAGE and transferred to a nitrocellulose membrane; blotting time was 45 min. which allows efficient transfer of the larger COX subunits I, II, III, and IV as indicated. Anti-phosphotyrosine (left blot), anti-phosphothreonine (middle blot), and anti-phosphoserine (right blot) antibodies were used (see "Experimental Procedures" for details). Subunit IV shows a signal with all three antibodies. Subunit II shows a signal with the anti-phosphotyrosine antibody; heart COX subunit II shows tyrosine phosphorylation if exposure time is increased (not shown). Subunit I of the liver enzyme, which was isolated in the presence of theophylline, shows a signal with the anti-phosphotyrosine antibody similar to heart COX, whereas liver COX isolated without theophylline shows no subunit I phosphorylation (arrows). FIG. 2. Nano-liquid chromatography/electrospray ionization/ MS/MS spectrum of DVDTRApYFTSATM. Peptides were eluted into the mass spectrometer with an HPLC gradient (0 -50% acetonitrile, 0.1 M acetic acid in 30 min). The mass spectrometer acquired the top three data-dependent electrospray ionization MS/MS spectra. An asterisk indicates the neutral loss of water. The methionine residue was modified to homoserine lactone. In order to test whether the cytosolic component of the cAMP signaling pathway is required for COX phosphorylation or whether cAMP can exert an effect on COX in isolated mitochondria, for example by directly activating a tyrosine kinase present in the intermembrane space, we incubated isolated mitochondria with cAMP as described (15). In contrast to a previous study (15), no differences in COX kinetics were observed using mitochondria isolated from fresh or frozen liver tissue in the presence or absence of cAMP (data not shown). Theophylline Leads to COX Inactivation in the Lung-We tested the effect of the phosphodiesterase 4 inhibitor theophylline on cow lung tissue, applying a final concentration of 100 M, based on its use as an anti-asthma drug (24). Interestingly, even at these low concentrations, COX was inhibited, showing an enhanced sigmoidal tendency (Fig. 5A). In the presence of ADP and ATP, we observed a 25 and 60% inhibition at maximal turnover in the presence of theophylline. At low cytochrome c substrate concentrations, theophylline leads to almost full COX inhibition in the presence of ATP. We then assessed whether COX inhibition by theophylline leads to decreased energy levels. We found that ATP levels are significantly decreased (by 26%) under these conditions (Fig. 5B). DISCUSSION We show here cAMP-dependent COX phosphorylation of subunit I and its effect on COX activity. To do so, we combined enzyme activity measurements with mass spectrometry and Western blot analysis, all on enzyme preparations isolated by modifying previously used protocols. We were able to conclude that COX is switched off upon cAMP-dependent tyrosine phosphorylation of catalytic subunit I such that full or strong inhibition is present up to substrate concentrations of 10 M cyto-chrome c, even in the presence of allosteric activator ADP. This novel mechanism, involving an endogenous mitochondrial tyrosine kinase, overrules COX regulation by allosteric effectors AMP, ADP, and ATP. Our modifications of previously used protocols enabled us to isolate and analyze COX with regulatory properties. The major modifications were as follows. (a) Cholate, which is added as a detergent during COX isolation and binds strongly to COX, was removed by dialysis in the presence of nucleotides, which probably compete for the same binding sites (3,5). (b) N,N,NЈ,NЈ-Tetramethyl-p-phenylenediamine, which facilitates electron transfer between molecules, was not used in COX assays, since it prevents cooperativity. N,N,NЈ,NЈ-tetramethyl-p-phenylenediamine-mediated electron transfer bypasses the association/ dissociation reactions of cytochrome c and COX by reducing the COX-cytochrome c complex (27). (c) Dodecylmaltoside was avoided, since it has been suggested to monomerize COX (28) and thus prevent cooperativity (29). (d) Cardiolipin, which is important for COX function (30) and has been found in the crystal structure (31), was added in our assay. (e) Calcium, a frequently used buffer component during mitochondria isolation, was not used, and endogenous calcium was chelated with EGTA. Calcium is believed to be a key signal for mitochondrial activation (32), and it has been proposed to act on COX via activation of calcium-dependent phosphatases (15,33). Our experiments support the presence of a calcium-dependent phosphatase, since calcium addition to isolated liver COX phosphorylated at subunit I is not sufficient to activate COX (data not shown). (f) Endogenous phosphorylation status was protected by the use of phosphatase inhibitors. The latter two points are especially important regarding respiration measurements, because signaling networks that act on COX are disrupted during isolation of mitochondria or COX, leaving mitochondrial kinases and phosphatases uncontrolled. From COX isolated in the presence of theophylline, we have unambiguously identified COX subunit I residue Tyr 304 in the intermembrane space as being phosphorylated (Fig. 2), which differs from the previously postulated cAMP-dependent phosphorylation of COX subunit I residue Ser 441 (16). Our finding suggests a novel signaling pathway involving a tyrosine kinase. Most signals conveyed by cAMP are mediated through phosphorylation reactions catalyzed by serine/threonine-specific PKA (34), and several studies have suggested that PKA activity Fig. 3. c The Hill coefficient was determined as described (29). is present in mitochondria of yeast, cell lines, and mammalian tissues (35-41, 42, 43). However, central for PKA-mediated phosphorylation under physiological conditions are protein kinase A-anchoring proteins (AKAPs), which localize PKA to cellular compartments in close proximity to its targets (44); S-AKAP84 and AKAP121 have been shown to mediate localization of PKA to the outer side of the outer mitochondrial membrane and to be crucial for cAMP-dependent signaling to the mitochondria (45)(46)(47). Also, in this study we found that incubation of isolated mitochondria with cAMP did not change COX activity. These findings are consistent with our proposed signal transduction pathway, in which PKA does not act directly on COX but rather conveys a signal through the outer mitochondrial membrane that leads to activation of a tyrosine kinase present in the intermembrane space. A considerable number of tyrosine kinase candidates are available; more than 100 have been predicted (48), for most of which the cellular localization or function remains to be elucidated. The effect of subunit I phosphorylation on liver COX activity is displayed in Figs. 3-5. The operational definition of subunit I phosphorylation is isolation in the presence of theophylline, since probing the separated subunits with anti-phospho antibodies shows this to be the only variable detectable. Stimulation by ADP of the unphosphorylated enzyme follows a saturation curve that remains linear to about 12 M cytochrome c, where activity is Ͼ70% of that at 40 M cytochrome c (Fig. 3). By contrast, the phosphorylated enzyme shows sigmoidal behavior such that activity at 12 M cytochrome c is less than 20% compared with saturating substrate concentrations. Thus, substantial allosteric regulation of activity by reversible phosphorylation could take place at the substrate concentrations of 6 M cytochrome c reported for normal mitochondrial heart muscle particles (49). Similar effects were observed in preparations inhibited or stimulated with ATP and AMP, respectively (Fig. 3, Table I). Based on the activity of subunit I phosphorylated COX, we conclude that our isolation conditions yield an enzyme that is phosphorylated on one or both subunits I per dimer, because no respiration was apparent at low cytochrome c concentrations, whereas subunit I unphosphorylated COX was active already at very low cytochrome c concentrations (Fig. 3). Other treatments that would increase phosphorylation were tested in a cell culture system and found to have the effect on activity and cytochrome c concentration dependence predicted by the theophylline results. This is seen when cAMP levels are raised by glucagon addition (Fig. 4, A and B) or activation of adenylyl cyclase with forskolin (Fig. 4C). Glucagon-promoted COX inhibition is physiologically consistent, since under food deprivation conditions energy usage should be restricted and, in periods of starvation, such as during sleep, glucagon, the most potent hormone increasing intracellular cAMP levels in liver cells, is released from the pancreatic ␣-cells. Previous attempts to detect effects of glucagon on COX activity in hepatocytes revealed no significant effects (e.g. Ref. 50), most likely because the phosphorylation status was not preserved due to the presence of calcium and absence of phosphatase inhibitors in the isolation or incubation buffers. In addition, differences in enzyme kinetics might have been missed, since most studies have only determined V max . The differences in V max (Ͼ30 M cytochrome c) for subunit I unphosphorylated COX are between 32 and 52% higher compared with phosphorylated COX (Table I), whereas the effect at a physiologically more relevant cytochrome c concentration of 6 M (49) are dramatic, since COX is switched off when phosphorylated. Tyrosine 304 of subunit I is conserved in all eukaryotes that we have examined and is present in some bacteria (Fig. 6). Bacteria lack hormonal signaling cascades, although cAMP is known to be a potent signal in bacteria. Thus, it will be interesting to determine whether the analogous prokaryotic tyrosine residue can be phosphorylated. Tyrosine 304 is located close to the heme a 3 -Cu B reaction center, on a helix that is in contact with the other catalytic subunit, subunit II, and facing the interface region of the two COX monomers (Fig. 7). Consequently, the effect of phosphorylation is plausible. The Hill coefficient, which can be interpreted as a value for cooperativity of substrate binding sites, is clearly increased in the case of subunit I phosphorylated COX (Table I), which shows sigmoidal reaction kinetics (Fig. 3). Phosphorylation of Tyr 304 at the interface of the two monomers might therefore enhance the monomer-monomer interaction. For dimeric COX with only one cytochrome c binding site per monomer, one would expect Hill values not higher than 2. Interestingly, we obtained higher values for the subunit I phosphorylated COX (3.1-4.0) compared with values around 2 for COX not phosphorylated at subunit I. Our higher Hill values would be consistent with the presence of a high and a low affinity cytochrome c binding site per monomer (51,52), for which there could be sufficient space FIG. 6. Sequence alignment of cytochrome c oxidase subunit I. COX I sequences were aligned with the program MegAlign. Selected eukaryotic (upper part) and prokaryotic representatives (lower part) are shown. The phosphorylation site (boxed) is conserved in more than 100 eukaryotes analyzed but not conserved in prokaryotes. FIG. 7. Cytochrome c oxidase crystal structure from cow heart. Crystallographic data (31) were processed with the program Swiss PDB viewer. Shown is a side view of dimeric cytochrome c oxidase. The membrane region is indicated by dotted lines. Catalytic subunits I and II are highlighted in yellow and blue ribbons, respectively. The introduction of the phosphate group to Tyr 304 to the right monomer and the rotation of the phosphotyrosine side chain, which in the crystal structure points to the membrane center, was performed using the program Hyperchem. A geometrically optimized structure for the phosphotyrosine side chain was obtained applying 500 cycles of the "steepest descent" algorithm, producing a conformation (highlighted in space fill) that is accessible from the intermembrane space. Tyr 304 is located on helix VIII, which contacts catalytic subunit II and the heme a 3 -Cu B reaction center (shown in sticks). The modeled phosphorylated Tyr 304 points to the left monomer. according to computer modeling (53). 2 An interesting sidelight was examination of theophylline in the context of its use as an anti-asthma drug, for which the molecular mechanism remains to be clarified (24). Theophylline leads to COX inhibition in the lung at a similar concentration to that used in therapy. Concomitantly, ATP levels are significantly decreased (Fig. 5B). Since airway constriction during asthma requires energy, theophylline-promoted COX subunit I phosphorylation, which leads to inhibition of COX and therefore aerobic metabolism, could represent at least a component of theophylline action. If this explanation is correct, the observed effect would be expected to be more profound when substrates for the glycolytic pathway become limited.	2018-04-03T01:30:01.848Z	2005-02-18T00:00:00.000	{ "year": 2005, "sha1": "1a2d341d22914785706f0bcf25892e61a75c0249", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/280/7/6094.full.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "fd257846314827efce7da195f2cd367f96569975", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
231802362	pes2o/s2orc	v3-fos-license	Improved Communication Efficiency for Distributed Mean Estimation with Side Information In this paper, we consider the distributed mean estimation problem where the server has access to some side information, e.g., its local computed mean estimation or the received information sent by the distributed clients at the previous iterations. We propose a practical and efficient estimator based on an r-bit Wynzer-Ziv estimator proposed by Mayekar et al., which requires no probabilistic assumption on the data. Unlike Mayekar's work which only utilizes side information at the server, our scheme jointly exploits the correlation between clients' data and server' s side information, and also between data of different clients. We derive an upper bound of the estimation error of the proposed estimator. Based on this upper bound, we provide two algorithms on how to choose input parameters for the estimator. Finally, parameter regions in which our estimator is better than the previous one are characterized. I. INTRODUCTION With the development of modern machine learning technology, more powerful and complex machine learning models can be trained through large-scale distributed training. However, due to the large scale of the model parameters, in each iteration of the distributed optimization, the exchange of information between distributed nodes incurs a huge communication load, causing the problem of communication bottleneck. We focus on distributed mean estimation, which is a crucial primitive for distributed optimization frameworks. Federated learning [1] is one of such frameworks, in which clients participating in joint training only need to exchange their own gradient information without sharing private data. To alleviate the communication bottleneck, gradient compression [2]- [8] and efficient mean estimator [9]- [15] have been investigated to reduce the communication load. Recently, [16] studied distributed mean estimation with side information at the server, and proposed Wyner-Ziv estimators that require no probabilistic assumption on the clients data. In parallel, distributed source compression has been widely studied in classical information theory. For example, [17] first studied the setting of lossy source compression with side information in the decoder. Channel coding can obtain practical coding for distributed source coding [18], [19], but the main bottleneck lies in the expensive computational complexity of coding and decoding. This work is supported by NSFC grant NSF61901267. In this paper, we study practical schemes for distributed mean estimation with side information at the server. The motivation is based on the fact that the server could store publicly accessible data, and also at each iteration, the server has already received data sent by clients at previous iterations, which can be viewed as side information. Rather than using random coding with joint typicality tools such as in [17], [20], which is impractical to implement, we follow the work in [16] which proposed a Wyner-Ziv estimator based on coset coding. Unfortunately, they only utilized the side information at the server, but failed to exploit correlation between clients' vectors. In fact, in many scenarios such as stochastic gradient descent, data between different clients may have a high correlation since they wish to learn a global model. Inspired by Wyner-Ziv and Slepian-Wolf coding, we propose a practical scheme based on the coset coding and jointly exploit the side information at the server and correlation between clients' data. Note that in our scheme we must address an ambiguity problem not existing in [16] or in the classic Wyner-Ziv coding. In more detail, since each client compresses its data and sends it to the server, the server only observes a lossy version of clients' vectors. This ambiguity cause mismatch information at the clients and server. Using the lossy version of clients' data at the server may even deteriorate the estimation. We summarize our contributions as follows: 1) We propose a new estimator that improves the estimator in [16] by jointly exploiting the side information at the server and the correlation between clients' data; 2) We derive an upper bound of estimation error of the proposed estimator; 3) We provide two greedy algorithms on how to choose input parameters for the estimator, and characterize the parameter regions in which our estimator has a tighter upper bound than of the previous estimator. II. PROBLEM SETTING Consider the problem of distributed mean estimation with side information, as depicted in Fig. 1. The model consisting of n clients and one server, where each Client i ∈ [n] {1, . . . , n} observes data x i ∈ X ⊂ R d and the server has access to side information y = (y 1 , . . . , y n ), y i ∈ Y ⊂ R d , for some alphabets X , Y and positive integer d ∈ N. The · · · x 1 x n Side Information (y 1 , . . . , y n ) Fig. 1. Distributed mean estimation with side information server wishes to compute the empirical mean, i.e, Note that the side information y could stem from some publicly accessible data or the server's guess of x (x 1 , . . . , x n ) in the previous iterations. We focus on non-interactive protocols and study the r-bit simultaneous message passing (SMP) protocol similar to that in [16]. The r-bit SMP protocol π = (π 1 , . . . , π n ) consists of n encoders {Ψ i } n i=1 and one decoder Φ, of mapping forms: Each Client i ∈ [n] uses the encoder Ψ i to encode x i into an r-bit message, i.e., m i = Ψ i (x i , U ), where U denotes a shared randomness known by all the server and clients. The Client i then sends the message m i to the server. Assume the message m i can be perfectly received by the server. After receiving all messages m (n) = (m 1 , . . . , m n ), the server uses decoder Φ to producex asx The performance of the r-bit SMP protocol using protocol π with inputs x and y, is evaluated by the mean squared error (MSE), i.e., Instead of using any probabilistic assumption on input data and side information, we use the Euclidean distance between vectors to measure correlation among the data and side information. More specifically, let x i and y i be at most ∆ i and the distance between x i and x j be at most ∆ ij , i.e., Since the distance is symmetric with ∆ ij = ∆ ji , it's sufficient to only consider ∆ ij with i < j. Let ∆ s (∆ 1 , · · · , ∆ n ), ∆ c (∆ 12 , · · · , ∆ (n−1)n ). (7) We are interested in the performance of protocols when ∆ s and ∆ c are both known to clients and server. Define the optimal r-bits protocol with the minimum MSE as π * , and the corresponding MSE as E π * (x, y). Our goal is to find practical and efficient r-bits SMP protocols, and derive tighter upper bounds on E π * than the previous results. III. PREVIOUS WORK In [16], the authors proposed a SMP protocol based on an r-bit Wyner-Ziv quantizer Q WZ . The quantizer Q WZ contains an encoder mapping Q e WZ the same as (2) and a simplified decoder mapping Q d WZ : {0, 1} r ×Y → R d . Each Client i ∈ [n] first uses the encoder Q e WZ to encode x i and then sends the encoded message m i to the server. The server uses the decoder Q d WZ to produce estimatex i aŝ and then computes the sampling means aŝ The quantizer Q WZ achieves the following upper bound on MSE. Theorem 1 (Upper bound given in [16]). For a fixed ∆ s and r ≤ d, the optimal r-bits protocol π * satisfy for all x and y satisfying (6). Now we introduce the quantizer Q WZ , as it is closely related to work. Since all clients use the same quantizer, only the common quantizer is described. We first describe a modulo quantizer Q M for one-dimension input x ∈ R with side information h ∈ R, and then present a rotated modulo quantizer Q M,R (x, h) for d-dimension data. Finally, the r-bit Wyner-Ziv quantizer based on Q M and Q M,R is given. 1) Modulo Quantizer (Q M ): Given the input x ∈ R with side information h ∈ R, the modulo quantizer Q M contains parameters including a distance parameter ∆ where \|x − h\| ≤ ∆ , a resolution parameter k ∈ N + and a lattice parameter . Denote the encoder and decoder of Q M as Q e M (x) and respectively. The encoder Q e M (x) first computes x/ and x/ , and then outputs the message Q e The message m has length of log k bits, and is sent to the decoder. The decoder Q d M produces the estimatex by finding a point closest to h in the set Z m, = {(zk + m) · : z ∈ Z}. 2) Rotated Modulo Quantizer (Q M,R ): Given the input x ∈ R d with side information h ∈ R d where x − h 2 ≤ ∆, the input parameters for Q M,R include a distance parameter ∆ , a resolution parameter k ∈ N + , a lattice parameter , and a rotation matrix R given by where W is the d × d Walsh-Hadamard Matrix [21] and D is a diagonal matrix with each diagonal entry generated uniformly from {+1, −1} by using a shared randomness. After the rotation, every coordinate , has zero mean sub-Gaussian with a variance factor of ∆ 2 /d, i.e., The quantizer Q M,R first preprocesses x and h by multiplying both x and h with a matrix R, and then applies Q M for each coordinate. Denote the encoder and decoder of Q M,R as 3) The r-bit Wyner-Ziv Quantizer (Q WZ ): Note that in the quantizer Q M,R the input x is encoded into d binary strings of log k bits each, leading to a total number of d log k bits. In the r-bit Wyner-Ziv quantizer, the encoder first encodes x using the same encoder as Q e M,R (x), and then uses a shared randomness to select a subset S ⊂ {1, . . . , d} of these strings with \|S\| = r/ log k , and finally sends them to decoder. The decoder uses the same decoder as Q d M,R to decode the entries in S. Denote the encoder and decoder of Q WZ as Q e WZ (x) and A. New Protocols Note that in (8) only y i is used as side information to assist the estimation for x i at the server. In fact, apart from y i , the side information {y j } j =i and other clients' data {x j } j =i could also be correlated to x i , and thus can be jointly utilized to reduce the transmission load. The main challenge is that {x j } j =i cannot be perfectly known by the server, and thus using the estimate {x j } j =i as side information for x i may even deteriorate the estimation. Our protocol is based on a set of r-bit new quantizers, denoted by {Q , and L Πi is a chain parameter need to be designed and has a form of L Πi : y Πi 1 → x Πi 1 → x Πi 2 · · · → x Πi l , with x Πi l = x Πi and l being the length of chain. Given a set of chains are estimated in an order x Π1 , . . . , x Πn . For the input x Πi , the corresponding quantizer Q Given any quantizer Q, denotes its estimate for input : Without loss of generality, we assume that the estimation order Π is an identity permutation, i.e., Π i = i, i ∈ [n]. With this assumption, the chain L Πi can be written as where for all t = 1, . . . , l − 1, and the decoder i already has i − 1 estimates: The encoder is same as Q e WZ , i.e., Client i first applies the encoder Q e M,R (x i ) to encode x i , then uses a shared randomness to select a subset S ⊂ {1, . . . , d} of these strings with \|S\| = r/ log k , and finally send them to decoder. The decoder Q d Pro,i chooses an element in M i as the "side" information h for Q d M,R , where We emphasize that here the "side" information h could be the estimate of other client's data, rather than the literal side information y i used in the Wyner-Ziv quantizer Q WZ . Given the chain L i in (14), denote ∆ i1 and ∆ isis+1 as weight parameters of subchains y i1 → x i1 and x is → x is+1 , respectively. The choices of ∆ i1 and ∆ isis+1 is based on (13), and follows a way similar to that in the quantizer Q M,R . For By recursively using (16), we can obtain the estimatê x QPro,i =x QPro,i l for the input x i . In our protocol, the parameters h and ∆ depend the design of chains {L i } n i=1 , S is generated by the shared randomness, and k, can be freely assigned. When the length chain L i is l = 1, the quantizer Q Li Pro reduces to the Wyner-Ziv quantizer Q WZ if the chosen chain is L i := y i → x i . 2) Selection of Chains: Next we give two algorithms on how to choose chains {L i : i ∈ [n]}. Construct i chains {L i (j) : j ≤ i} as follows. 12: return Chains Algorithm 2: Note that Algorithm 1 is simple and fast, but may not find good chains to improve the MSE in (10). Therefore, we are interested in finding good chains and the corresponding region of (∆ c , ∆ s ) such that the upper bound of MSE is smaller than (10). We illustrate our idea with a special case where the length of each chain is less than 2. For Client i, consider y t → x t → x i and y i → x i with t < i. By Remark 3 (described later in Section IV-B), we select the chain as follows: If (∆ t , ∆ ti , ∆ i ) ∈ R 2 is in the region R 2 defined in (23), then we use the chain L i as y t → x t → x i , otherwise we use the chain y i → x i . Now we look for good chains according to R 2 . Without loss of generality, let ∆ 1 ≤ · · · ≤ ∆ n . Starting from Client 1, firstly, generate a chain of length of 1 for Client 1, and then traverse the remaining clients to verify whether the corresponding distances are in R 2 . If the distances are in R 2 , then construct a chain of length of 2. For the remaining clients whose chains are empty, renumber them and repeat the above process until every client has a nonempty chain. We formally describe the method in Algorithm 2. B. New Upper Bound of MSE Define the following quantities: Given a chain L i and a specific parameters assignment, the following lemma gives a recursive inequality about the upper bound of the error when using the quantizer Q Li Pro . Deleta clients in N ode from C 14: return Chains Lemma 1. For a given chain L i : y i1 → x i1 → x i2 · · · → x i l , when using the quantizer Q Li Pro with the following parameters: for all t ∈ [l] and c t (n) max{ 576t 2 e , 3n+ 36 e 2/3 }. Proof. See the proof in Appendix A. By properly scaling and choosing an appropriate k, we obtain a more concise form in the following corollary, whose proof is given in Appendix B. Corollary 1. If setting log k = log(2 + √ 12 ln n) , we have and D i i l satisfies that for l = 1, D i i1 = ∆ 2 1 and for l > 1, Theorem 2. For some fixed (∆ s , ∆ c ), and d ≥ r ≥ 2 log(2+ √ 12 ln n) , and µd = r log k , the MSE is upper bounded by where log k = log(2 + √ 12 ln n) and D i i l is given in (21). Remark 1. We improve the upper bound of MSE in (10) when ) times of that in (10). Remark 2. For each permutation Π on [n], since each Client Π i can choose L Πi from i candidate chains, there are n! different assignments {L Πi : i ∈ [n]}. Thus, the number of all strategies will not exceed (n!) 2 . Denote the chain corresponding to each strategy as L i , i ∈ [(n!) 2 ]. From Theorem 2 and Remark 1, we obtain that when (∆ c , ∆ s ) ∈ i∈[(n!) 2 ] R L i , our upper bound is tighter than that in (10). Remark 4. From (23), we observe that to choose a chain whose length is larger than 2, there must exist at least one pair . Otherwise, our estimator turns to be the Wyner-Ziv estimator in [16]. The condition 5∆ 2 t < ∆ 2 i seems a stringent assumption at the first glance. In fact, since our quantizer is for specific vectors x and y, and the Euclidean distance x i − y i ≤ ∆ i and x j − y j ≤ ∆ j , for i, j ∈ [n] can vary greatly. Also, one can spend additional bits on better estimating ∆ t and ∆ ti such that they are smaller enough to satisfy (23), and this additional bits cost on estimating ∆ t and ∆ ti can be omitted when n is relatively large. V. PROOF OF THEOREM 2 Now we first introduce a lemma and then derive an upper bound of MSE for any r-bit quantizer. VI. CONCLUSION In this paper, we studied the distributed mean estimation with limited communication. Inspired by Wyner-Ziv and Slepian-Wolf coding, we proposed new estimator by exploiting the correlation between clients' data. In the future work, we aim to find a more efficient estimator and apply it to more generalized distributed optimization framework. A. Proof of Lemma 1 Our quantizer is based on Q M,R , similar to Q WZ . We first introduce the following lemma, whose proof is similar to that in [16]. With a slight abuse of notation, we write Q Li l Pro as Q Pro . Lemma 3. Fix ∆ i > 0. Then, for µd ∈ [d], we have Proof. where we use the independence of S and R in the third identity and use the fact that R is unitary in the final step. Since where we use the independence of S and Q M,R in the last identity, we have The following lemma is given in [16], which shows that Q M is unbiased under certain conditions, and the error will not exceed . Lemma 4. (see [16]) Consider Q M described in III-1 with parameter set to satisfy k ≥ 2( + ∆ ). (26) Then, for every x, h ∈ R such that \|x − h\| ≤ ∆ , the output Recall from Section IV-A that for a chain y i1 → x i1 → x i2 · · · → x i l , the server estimates x i l by usingx Q L i Pro,i ,i l−1 as h and ∆ i1 + l−1 s=1 ∆ isi (s+1) as the parameter ∆ in Q M,R . By Lemma 3, we consider the quantizer Q M,R . Lemma 5. If R given in (12) satisfies that for j ∈ [d] and Proof. We use induction for l. where we set = By Lemma 4, we have So, for t and k ≥ 4, we have Thus, where we set i1 = For the random matrix R given in (12), for every z ∈ R d , the random variables Rz(i), i ∈ [d], are sub-Gaussian with variance parameter \|\|z\|\| 2 2 /d. Furthermore, we need the following bound. Lemma 6 (see [16]). For a sub-Gaussian random Z with variance factor σ 2 and every t ≥ 0, we have We now handle the α it (Q M,R ) and β it (Q M,R ) separately below. Firstly, we consider t > 1. Since R is a unitary transform, we have We consider the first term. By (27), we have where in the final steps we use the fact that (a 1 +· · ·+a n ) 2 ≤ n(a 2 1 + · · · + a 2 n ). For the second term on (28), we get A c s ) A c s )	2021-02-05T02:15:29.086Z	2021-02-04T00:00:00.000	{ "year": 2021, "sha1": "490c88151960df22f26be9e2ad206bc7acf01ab2", "oa_license": null, "oa_url": "http://arxiv.org/pdf/2102.02525", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "490c88151960df22f26be9e2ad206bc7acf01ab2", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
266083079	pes2o/s2orc	v3-fos-license	How exposure to patient narratives affects stereotyped choices of primary care clinicians In this paper, we examine whether patient narratives alter the impact of stereotyping on choice of primary care clinicians: in this case, the common presumption that female doctors will be more attentive to empathic relationships with patients. 1052 individuals were selected from a nationally representative Internet panel to participate in a survey experiment. Participants were given performance data about 12 fictitious primary care physicians, including a randomized set of narrative feedback from patients. We compared the choice of clinician made by participants who value bedside manner and were exposed to narratives in the experiment, compared to those valuing bedside manner who had not had this exposure. We estimated multivariate logistic regressions to assess whether exposure to patient comments that “disrupt” stereotypes influenced choice of physicians. Participants who saw patient comments and had previously reported caring about bedside manner had a 67% higher odds of choosing a female physician than those participants that did not see a patient comments, controlling for the content of the narratives themselves. When participants were exposed to patient comments that disrupt gendered stereotypes, they had a 40% lower odds of choosing a female physician. Simple exposure to patient narratives that do not clearly disrupt gendered stereotypes increased the likelihood of choosing a female clinician by priming attention to relational aspects of care. However, when the content of a sufficient proportion of patient comments runs counter stereotypes, even a minority of narratives is sufficient to disrupt gendered-expectations and alter choices. Introduction Over the past two decades, policy makers around the world have increasingly sought to leverage patient choice among healthcare providers to improve health system performance [1][2][3][4].Although these initiatives promoted choice of hospitals as well as clinicians, they largely focused on encouraging selection among primary care providers [2,5].This emerging policy emphasis raises a variety of questions about heretofore understudied aspects of medical consumerism [6,7]. The U.S. was a front-runner in promoting consumer choice in health care, dating back to the 1970s [8].This reflected, in part, shifting ideological preferences among policymakers.But it was also a byproduct of a fragmented insurance system that frequently disrupted relationships between clinicians and patients whenever Americans switched jobs or had their employer change health insurers.It has been estimated that roughly 30% of all Americans need to find a new physician each year, including both primary care and specialists, significantly more common than in most other market democracies [9].Beyond these circumstances, having the option of choosing a physician was also thought to enhance patients' trust in their physicians by promoting a better match between patient preferences and clinician proclivities [10].Enhanced trust in turn was shown to be a foundational element of more resilient patient-physician relationships, enhancing the fidelity of communication, compliance with prescribed treatments and the probability that patients would see their clinicians as acting in their best interest of the patient [11]. However, the anticipated virtues of choosing one's clinician can be compromised when, as is often the case, patients have limited information to guide their choice.Performance metrics are not consistently available for most individual clinicians, since for many clinicians limited sample sizes make it infeasible to calculate reliable outcome-based metrics for their panel of patients that are specific to particular conditions (e.g., asthma, diabetes, hypertension) [12,13].The measurement challenges become particularly daunting for primary care clinicians, since their heterogeneous mix of patients (many of whom have neither acute nor chronic conditions) make common condition-specific metrics irrelevant for most of their patients [14].Furthermore, patients may view the reliability and trustworthiness of external sources of information with varied degrees of skepticism, especially those that offer sketchy documentation about their methods, provenance, or reporting practices [6,15].These multiple shortcomings have made it difficult to induce consumers to use or rely upon websites that present comparative performance information about clinicians [7,16]. When consumers have limited access to trusted measures of quality, they are likely to substitute inferences about quality based on observable physician "attributes".In other words, they will rely on some form of stereotyping to guide their choices.Past research on consumer selection of clinicians suggests that a number of factors observable through on-line directories may come into play, including the provider's age, race, gender (typically a binary classification), and place of training [17][18][19][20].How closely these attribute-driven choices mirror more fully informed choice will depend on how much these stereotypes match actual clinician practices and the quality of care they provide [21].But however close this is, one might still be concerned that stereotype-infused choices risk reifying forms of bias that could be pernicious in other ways. The challenges of promoting informed selection of clinicians have been evident for the better part of a quarter century [19].But within the past decade, around the world, a new source of web-based information has become more widespread: patients' narrative accounts of their interactions with particular clinicians [22][23][24].By 2015, narrative comments had become the source of information about clinician quality most frequently consulted by American consumers [22,25]. Past research documents that consumers find narrative accounts useful [26]; exposure to narratives has been shown to influence which clinicians are selected [19,27].But none of this previous research offers clues about how the growing availability of patient narratives might alter the impact of stereotypes in selecting a doctor. In this paper, we report on an experimental study that offers some unique insights into the circumstances that foster stereotyping in medical consumerism and the potential for patient narratives to buffer these impacts.More specifically, we derive from the extant literature on the psychology of stereotyping some hypotheses that help us examine whether and how the common presumption that female doctors will be more attentive to empathic relationships with their patients shapes consumer choices of primary care clinicians in the absence of metrics that directly measure empathy [19,28,29].In this experimental context, we can then test (through randomized differential exposure to patient narratives) the extent to which patient comments that run consistent or counter to the stereotypes about clinicians' "bedside manner" have the capacity to alter the impact of stereotypes on consumer choices made when shopping for a new physician. These gender stereotypes will not apply to all clinicians.Nor will they be equally salient to all patients selecting a new provider.It is only for those patients who value aspects of clinician practice that are not readily observable, and whose understanding of gender stereotypes suggests that knowing this attribute about a clinician will help to predict their practices, that one would expect gender to influence choice of a new doctor [30].Studying these influences in an experimental context allows us to take into account these complex interactions, while controlling for a variety of other factors that are known to influence how people select a clinician [31]. These comments are randomly assigned through the experimental design to particular clinicians (and thus, to particular clinician genders).Therefore, their introduction could expose any given consumer to narrative content that either reinforces stereotypes or cause consumers to question their reliability [32].Participants were randomly assigned to different groups (also known as experimental arms); each group/arm was exposed to patient narratives presented in a different format.We make inferences about the impact of stereotyping and the mediating effects of patient narratives based on (a) consumers' initial expressed preferences (before seeing the website) prioritizing certain aspects of clinician quality, (b) changes in consumer preferences after exposure to the website, (c) the differential exposure across experimental arms to consumer feedback relevant to these preferences, and (d) consumers' choices among the clinicians available to them. In the final sections of the paper, we explore some of the policy implications of this interplay between specific forms of evidence about practitioner performance and consumer stereotyping of clinicians.Through stereotyping, individuals make inferences about the expected quality of particular clinicians.Whether this leads to more or less effective sorting and matching of patients with clinicians depends on both the predictive accuracy of the stereotype and consumers' ability to downplay stereotyping (or otherwise adjust their choices) in the presence of conflicting evidence.Whether reducing the impact of stereotypes improves or worsens patient-clinician matching is therefore a complicated question.We will explore these complications and consider extrapolations to other forms of potential matching that have relevance to contemporary medical consumers and health policy, including racial concordance between patients and clinicians, concerns related to age bias in clinical decision-making, and other related phenomena. Background and conceptual framework "Doctor-shopping" has been studied since the 1960's.Before comparative information about clinicians was widely available, patients routinely based their choices on word-of-mouth referrals from friends, family, and other physicians [33,34].Although sources of information have changed over time, the patterns of what patients' value when selecting a physician has remained relatively stable: a combination of technical abilities and socio-emotional aspects of the patient-physician relationship [25,35,36].Patients vary in the relative importance they assign to technical skills compared to interpersonal qualities such as "bedside manner" [37][38][39]. Previous research also suggests that most consumers strike a different balance between technical skills and relational capabilities when selecting a specialist, compared to a primary care clinician.Preferences for specialists more heavily favor technical aspects of quality, whereas choices among primary care providers give greater emphasis to interpersonal ability [21,40,41].The greater importance of interpersonal aspects for choices of primary care clinicians makes sense, since relationships with primary care providers are more likely to be sustained over time and more likely to involve discussions of potentially stigmatized or embarrassing behaviors, enhancing the importance of interpersonal abilities.By contrast, interactions with specialists are more frequently one-off encounters or otherwise short-term interactions [42]. Although metrics of technical quality are only unevenly available for clinicians, metrics assessing empathic interactions (a.k.a."bedside manner") are almost entirely absent [36].Consequently, information about the aspects of clinician performance that many people value the most in primary care settings is largely unavailable.Some consumers can fall back on the anecdotal accounts conveyed word-of-mouth, but this will offer little guidance for those with limited social networks or who have recently moved to places where they know few people [9].Nor can patients always rely on referrals from other clinicians when selecting a new primary care provider, as they often do when selecting a new specialist [43,44].Taken together, these considerations suggest that stereotypes play their most substantial role when consumers are selecting a new primary care provider. Potential impact of patient narratives on stereotype-infused choices of clinicians Social psychologists have long recognized that stereotyping is an inescapable result of the cognitive burdens created by a complex social world [45].Stereotypes are typically shaped by mass media messaging and other social interactions, as well by individual exposures to members of the stereotyped group [46].Although stereotypes can sometimes embody negative or demeaning characterizations, our analyses here focus on the decision-making processes related to stereotyping in general, not to the content of any prejudiced thoughts and negative attitudes toward any subset of clinicians [46,47]. Stereotypes related to gender have been documented for a wide range of social relationships, extending beyond clinical settings and the helping professions.Broadly, these stereotypes emphasize a more "compassionate" representation for women, a more "action-oriented" representation for men [48].The extent to which these stereotypes shape choice behavior will often depend on how frequently or powerfully they are "activated" by input that makes them feel relevant to the decision at hand [49].Exposure to comments that describe interactions within clinical settings may, in these ways, either reinforce or erode the perceived relevance to choice of clinicians or alter the perceived validity of the stereotype itself. Upon first consideration, growing public exposure to patient narratives about interactions with clinicians might be expected to reduce the impact of gendered stereotypes on choices among clinicians.In the absence of information about clinicians' empathy, stereotypes should lead patients who care about empathy to prefer female providers, all else equal.But patient narratives are rich in content about empathy and other relational aspects of healthcare, conveyed in 30-40% of all reported experiences [25,36,42].By providing concrete information about relationships between patients and clinicians, this evidence ought to disrupt simplistic dualities of gendered stereotyping.We will refer to this as the "naïve learning" hypothesis regarding narratives and stereotyping-not to convey skepticism about the prediction, but to note that this reflects a simple model of learning and updating of prior assumptions related to gender. Hypothesis #1: Naïve Learning: Consumers who have patient narratives available for judging clinicians will be less influenced by stereotypes in their choice of clinicians. However, a second, countervailing alternative, could also come into play.Narratives convey information about relational aspects of care that would not otherwise be available.But narrative content is not always the easiest information to "process" cognitively-in particular, to identify which clinicians in a given choice set might be the most empathic or otherwise most capable of nurturing relationships with a patient [28,50].Exposure to patient narratives (many of which talk about empathic relationships with clinicians) might thus increase the salience of relational aspects of care, without providing consumers with enhanced ability to choose based on these aspects.Under these circumstances, the narratives would likely activate the stereotype of compassionate female doctors, without conveying evidence that might disrupt the scope or impact of the stereotyping. Hypothesis #2: Activated Stereotypes: Consumers who have patient narratives available for judging clinicians will display greater influence of stereotypes on choices among clinicians.Stereotype activation will have the opposite influence from naïve learning; which of these two processes will be more influential over choice cannot be predicted a priori. Our review of past research on stereotyped reasoning presented above also suggests a different way in which the introduction of patient narratives could alter choices.To the extent that consumers display motivated reasoning-that is, are predisposed to "defend" their prior beliefs in the face of conflicting evidence-a modest number of exceptions embodied in patient comments may prove insufficient to disrupt established stereotypes, leaving the initial impact of stereotyped reasoning largely unchanged.This suggests that there will be threshold effectsinfrequent disruptions of stereotypes leave prior beliefs intact, but frequent disruptions may overturn those prior conceptions. Hypothesis #3: Motivated Reasoning: Consumers who have patient narratives available for judging clinicians will display no differences in the influence of stereotypes on choices among clinicians, unless the available evidence strongly contradicts the stereotypes. In short, the literature on stereotyped reasoning suggests that the growing availability of patient narratives could, depending on the underlying causal pathways shaping decision-making, yield a range of potential changes consumer behavior.Some would reduce the impact of stereotypes, others increase them.Later in the paper, we will return to these different pathways to consider how policy might more generally leverage the growing availability of patient narratives to influence the impact of stereotyping on medical consumerism.We next consider a particular test of these impacts on gender stereotyping in choices of primary care physicians. Study participants The participants involved in this study were selected from the Growth for Knowledge (GfK) probability-based-Internet Knowledge panel, which is representative of the US population in terms of sociodemographic characteristics, Internet usage, and health status [51].The Knowledge Panel from which respondents are drawn is composed entirely of panelists from the United States.Following the conventions of this on-line panel, a random sample of 2025 panelists were invited to participate in the study, which was described to them as, "The purpose of the study is to learn how people choose a doctor as their regular source of medical care and advice.The study is being conducted by researchers at several major universities and research organizations including Yale and UCLA."Participants were recruited to this study between the Summer of 2014 and the Summer of 2015. All members of the panel are offered a modest non-monetary incentive to participate in surveys and complete 1-4 studies each month.52% of the individuals invited to participate in this study accepted their invitation [56].All participants underwent an informed consent procedure approved by IRB's at Yale University and the RAND Corporation.Electronic consent was obtained from all participants for inclusion in this study.We did not have access to information that could identify individual participants at any time point during or after the study. Experimental design and procedure After agreeing to join the study, participants completed a pre-experiment survey.They answered questions about their real-life health experiences, including their experiences making choices among clinicians.This information was collected to ensure that randomization had successfully allocated participants with comparable levels of prior experience to each arm of the experiment.Respondents were also asked to describe their preferences regarding clinician characteristics.All told, participants were asked about the importance of a dozen clinician attributes.Among these was one attribute involving clinicians' relational capabilities: if a physician being "warm, caring and a good listener" was important to them, an attribute we anticipate being most closely linked to gender stereotypes.(A matching question was included in the post-choice survey to assess whether exposure to the website had altered preferences). Roughly a week after completing this survey, respondents were invited to return to the study and participated in the choice experiment.(The week-long delay was designed to ensure that responses on the pre-choice survey did not induce a consistency bias in respondent's choice of clinician or expressed rationale for the choice.)The various stages of the experiment are outlined in Fig 1 .A total of 85% of those who completed the pre-choice survey returned to participate in the choice experiment.Choosing among clinicians called for logging onto a "SelectMD" website of our own design, on which participants were given information about 12 fabricated primary care physicians and asked to select their preferred clinician among these options.Clinicians were listed in random order on the website.Participants expressed few concerns with the functionality of the website; 90% reported that the site was "somewhat easy or very easy to use" [35].Participants generally spent between 5 and 7 minutes on the website before selecting a preferred clinician. All participants were exposed to an identical distribution of quantitative performance measures across the 12 fabricated clinicians.These measures included star ratings (1-5 stars, with five stars representing the highest-rated clinicians) associated with three aspects of quality: (a) a rating for effective communication drawn from patient experience surveys, (b) a rating for the clinician's ability to deliver care consistent with norms of good practice, drawn from clinical records and (c) a rating for how comprehensively the clinician's practice used safetyenhancing protocols (Fig 2).Consistent with data observed in real-world clinician choice websites, the quality and safety ratings were loosely correlated (r = 0.15), the communication ratings uncorrelated with the other two metrics for quality. The star ratings were balanced in several ways with respect to physician gender (Table 1).Aggregating across the three categories, the top-rated clinicians based on star-metrics (all with 12 stars in total) included two female and two male clinicians.Male and female clinicians were equally likely to be top-rated (five star) and bottom-rated (one star) across the three quantified ratings.Female doctors had an average performance rating of 9.1 while male doctors had an average performance rating of 8.8 (Table 1).Because our prior research on consumer choice revealed that a number of consumers used a satisficing strategy across multiple dimensions of performance (e.g., avoid selecting a clinician who was below average on any rated dimension), we also balanced by gender clinicians who were at or above average (3+ stars) across all three performance categories.Doing so also allowed us to avoid respondents who may have made hasty choices in choosing a physician. By contrast to the fixed configuration of star ratings, exposure to specific patient comments was randomized.Comments were drawn from a pool of 142 real-world comments harvested from internet sites, grouped into four different categories of clinician performance, one of which was empathy/emotional rapport [35].When the narratives were randomized and assigned to each clinician, pronouns were adjusted in the text of each relevant comment to match the gender of the clinician to which the narrative had been assigned.Any given website user was exposed to an average of 12 comments, drawn from four categories of clinician performance (Fig 3).The research team assigned a valence (ranging from negative to positive on a five-point scale) to each comment; these assigned valences were validated against scores assigned by members of the public to the same comment set.Five members of the research team, drawn from different disciplines (psychology, management, and economics) independently rated the valence for each comment and the modal score was then assigned to that comment.(This valence was never reported to website users, but had to be inferred by them from the content of the narrative comment.)Here again, consistent with observed patterns from real-world websites, the valence of the aggregated patient comments for each clinician was loosely correlated (r = 0.15) with the star rating derived from the patient experience survey scores. Because the specific comments (and associated valences) were randomized for each participant, any one person visiting the SelectMD website could encounter patient comments related to empathy/emotional rapport that were fully consistent with gender stereotypes, fully inconsistent with those stereotypes, or a mix of consistent and inconsistent narratives.And since the number of narratives in the empathy/emotional rapport domain was also randomly varying, the strength of the "signal" conveyed by the narratives (however consistent or inconsistent) also varied across participants, subjecting stereotypes to different levels of challenge from the content of the narratives [50]. In keeping with a broader experimental design, participants were randomized into one of seven arms [52].In all but one of these arms, participants were given the ability to read patients' comments about each clinician (Table 2).The arms varied in terms of the formatting used to present the patient comments and the capacity that website users had to sort among the comments.The names and genders of physicians were visible to participants in all arms of the experiment (Figs 2 and 3).After participants selected a clinician on the website, they completed a post-choice survey asking about their experiences on their website, their decision-making process, and satisfaction with the choices given to them.This included questions about whether (a) respondents had noticed that there were patient comments on the website (not all had) and (b) how effectively they were able to extract information from these comments to compare across clinicians (not all were).As part of this sequence of questions, they were asked about which attributes of physicians mattered to them most while making their selection.This question was worded identically to the question about preferences included on the pre-choice survey. Key analytic variables and constructs The primary dependent variable in this study was the choice of clinician.A binary outcome variable was constructed using the gender of the chosen clinician, to identify when a female clinician physician was chosen.A value of one denotes a female physician; a value of zero denotes a male physician (no value-laden inferences are intended by this coding). Past research on patients' assessing clinicians based on their gender suggest that stereotypes will alter choices only for those patients who value the aspects of clinician practice that are related to gendered expectations-in this case, those who place a particularly strong value on "bedside manner".Because this was assessed in the pre-choice survey (a week prior to participating in the experiment), these preferences were not influenced by the content of the website.23 percent of respondents placed bedside manner among the top three (out of the dozen possibilities) attributes that they valued in a clinician. Our initial analyses of the impact of narratives compare the choices made by those who value bedside manner and were exposed to narratives in the experiment, compared to those valuing bedside manner without this exposure.But "exposure" is a relatively complex categorization.For purposes of our analyses presented below, the control group (that is, those not exposed) was constructed as an amalgamation of three subsets of participants.The first group includes participants randomized to the Arm 1 of the experiment, that is, those individuals who did not have any patient narratives incorporated into their version of the SelectMD website (N = 174).The second group includes participants from Arms 2 through 7 (all of whom narratives on their version of the website) who reported in the post-choice survey that they did not actually access the comments on the website (N = 246).The third group includes participants who reported having seen the comments but felt unable to identify from the narratives which doctor was best in the choice set using the narratives were also added to the augmented control group (N = 124).Consequently, our baseline tests of the impact of patient narratives on stereotyped decision-making among those who both saw and felt able to interpret the content of the narrative accounts.From a total sample of 1,052 participants, 564 participants were in the treatment group while 488 were in the control group. Constructing the control and treatment groups in this manner provides a meaningful test for a particular form of narrative impact-influencing choices among those who felt they could derive meaning from the patient comments.Because there are other plausible ways to define narrative exposure (each having different interpretations regarding narrative impact), we will explore some alternative definitions of treatment and control groups later in the paper. Our earlier review of the literature on motivated reasoning suggests that the potential impact of patient narratives will also depend on the informational content of the narratives-in particular, the extent to which they convey experiences that run consistent with or counter to the gendered stereotypes.Because comments are randomized for each participant, some will have received a mix of narratives related to bedside manner that reinforce stereotypes, others a mix of comments that include a few narratives that are inconsistent with gender stereotypes (and therefore weakly disruptive of the stereotypes), yet others a mix of comments that disproportionately represent male clinicians as caring/empathic and female clinicians as emotionally aloof. Participants were exposed to anywhere from zero to twelve stereotype disrupters and were on average exposed to 4 stereotype disrupters comments.For our baseline analyses, we first measured comments that defied the gender stereotype (henceforth referred to as the "stereotype disrupter" variable) as a continuous variable: more specifically, as the proportion of all available comments that conveyed experiences that ran counter to the gender stereotype.As we define the gender stereotype as female doctors being more caring and having better bedside manner skills than male doctors, thus the stereotype disrupter is when male doctors are described as particularly warm and caring or female doctors as not so.The coefficient conveys the marginal impact of exposure to an additional disruptive comment on the probability of selecting a female physician from the choice set of twelve primary care clinicians. In the final regression specification, we represented exposure to stereotype disrupter comments based on the proportion of comments (these were clustered to increase the stability of the estimates) that disrupted gender stereotypes.This allows us to identify whether there are non-linear effects in the ways that narrative comments disrupt stereotypes and to identify the threshold at which disruptive comments become consequential for choosing a physician. Statistical analysis To assess whether simple exposure to comments (with their random distribution of valence across genders) altered the propensity to select clinicians who were female, we initially estimate a multivariate logistical regression model.The outcome of interest is the interaction term between being exposed to patient comments (Treatment Group) and caring about bedside manner (Bedside Manner).This tests for the difference in the effect of exposure to patient comments on choice of a female physician, for participants who value bedside manner, relative to those who do not have that preference OR who were not exposed to the narratives. Next, using a second multivariate logistical regression model, we tested the effect of stereotype disrupters on choice of physician.The outcome of interest is stereotype disrupter defined as a continuous variable.Thus, we are able to test for the difference in the effect of being exposed to stereotype disrupter patient comments on choice of female physician. Results The analyses included 1,052 participants.Compared to the general US public (as per the 2019 American Community Survey [53]), our sample was slightly older, more White, and less likely to be in in excellent health (Table 3).In addition, participants did not differ across the treatment and control group in terms of any health or demographic characteristic, with the exception of the proportion of individuals in the Age Group from 30-44 (S1 Table in S1 File).Since there were no differences in most of these characteristics between treatment and control group, they were not included as covariates in the models presented, which the exception of Age, which was included as a covariate.Overall, 51% of participants chose a female physician. Because the model focuses on the choices of those who gave priority to bedside manner, it behooves us to consider how much this priority was affected by exposure to the website.Therefore, we compare preferences for bedside manner before and after the experiment.Roughly a quarter of the sample shifted their reported priority for this attribute.But an equal number of respondents increased the priority given to bedside manner as did those who decreased their priority.Because our primary interest is in assessing the impact of patient comments on the use of stereotypes for a given set of preferences, our baseline models use the prechoice preference ranking.(Later in the paper we replicate these models using the post-choice preferences, allowing us to assess whether exposure to patient comments also alters preference rankings). Effect of patient comments on choice of physician To assess the initial effect of exposure patient comments on choice of physician, the first model estimates the probability of choosing a female physician comparing participants who saw patient comments and reported caring about Bedside Manner to those who did not see any patient comments or who did not care about bedside manner.Those who saw comments and cared about the relational aspects of care had a 67% higher odds of choosing a female physician than participants who did not see a patient comments (p = 0.083).The results do not change when controlling for age (Table 4). Effect of stereotype disruptive comments on choice of physician Measuring stereotype disrupters as a continuous variable shows that for every additional stereotype disrupting comment, there is a 6.8% lower odds (p = 0.016) of choosing a female physician for participants exposed to comments.Here again, the results do not change when controlling for age (Table 5, left column).Fig 4 graphically represents these findings.It illustrated the negative relationship between the odds of choosing a female physician and the number of stereotype disrupter comments on the website.But it also suggests that the relationship may incorporate a threshold effect. To test for these threshold effects, we re-estimated the model with the proportion of disruptive comments clustered into five strata.The results suggest that when exposed to comments wherein at least 40% of patient narratives are disrupting gendered stereotypes, participants were significantly less likely (odds-ratio 0.605, p = 0.035) to select a female physician than those participants who saw fewer or no disruptive comments (Table 5, right column).These findings suggest there is in fact a threshold for disrupting stereotypes.But that threshold is relatively low.Even when consumers are exposed to less than a majority of comments that run counter to stereotypes, there may still be a sufficient accumulation of disruptive comments to measurably reduce the odds of choosing based on gendered stereotyping. Sensitivity analyses To ensure that our estimates of the impact of stereotypes are not unduly sensitive to model specification, we tested a variety of alternative specifications which are presented in the S1 File.First, because past research suggests that exposure to patient narratives can cause some consumers to become distracted from quantifiable (star-rated) performance rankings [27], we wanted to ensure that the apparent reduction in stereotyped selection was not a byproduct of consumers who had become overloaded with the complexity of the choices we faced.To do this, we limited the sample to those whose final selection avoided doctors who scored below average on any of the three star-rated dimensions.Using this restricted sample, we then reestimated the regressions.None of these alternative specifications significantly altered the results presented above (S2 Table in S1 File). In our baseline specifications presented above, we treated as equivalent disruptive comments conveying positive bedside manner for male physicians and those that portrayed the bedside manner of female clinicians in negative terms.But there is no reason to presume that these forms of disruption will have equal impact.In subsequent model specifications, we assessed stereotype disrupters, stratified by physician gender, allowing for stereotype disrupters to have potentially different effects for male and female physicians (S3 Table in S1 File).Here again, we observe no significant differences from the findings reported for the baseline models. Since we know from our findings reported above that exposure to the website led a quarter of our participants to alter their pre-experiment preference of valuing bedside manner, we wanted to assess whether this might alter the apparent impact of comments on stereotyping.This involved re-estimating all the models, but in this case the group for which stereotypes were deemed to be salient was identified based on their expressed preference for bedside manner preference post-experiment.Here again, this revised specification did not significantly alter any of our findings (S4-S6 Tables in S1 File).Our results were also robust to non-linear effects of stereotype disrupters (S7 Table in S1 File). Discussion The findings from this study provide insights into some ways in which stereotyping shapes consumers' choices among clinicians.First and foremost, stereotypes clearly seem to affect the selection of a primary care physicians.The impact of stereotyping on choice becomes more pronounced when consumers encounter reports of other patients' experiences.In this context, for those participants who reported valuing a "warm and caring" physician, being exposed to patient comments increased their chances of choosing a female physician, holding constant the content of these comments.Second, the impact of stereotyping can be altered by the content of other patients' narratives.For those participants that value bedside manner, being exposed to a sufficient proportion of comments that run counter to stereotypes decreased the chance of choosing a female clinician.This aligns with the Activated Stereotype Hypothesis, since exposure to patient narratives led to a greater influence of stereotypes in choosing a physician.Disruption of stereotypes is evident even if there is only moderate exposure to narratives that run counter to the stereotype.Although these findings need to be interpreted in the context of certain methodological limitations discussed below, they hold some potentially important implications for both the understanding of stereotyping in medical settings as well as for clarifying how health policies might address (and potentially mitigate) that impact. Better understanding how stereotyping affects medical consumerism Simple exposure to patient narratives increased the likelihood of choosing a female clinician, consistent with our Activated Stereotype Hypothesis.This pattern also comports with findings from the literature on priming effects related to other complex health choices, [54] as well as those from studies of the impact of stereotyping on voter selection among political candidates [54].But it may initially seem counterintuitive, when the content of so many of the narratives presented on the SelectMD website are inconsistent with gendered stereotyping of clinicians. The explanation rests in the complicated calculus of choosing a physician and the oftenchallenging task of translating narrative accounts into choice preferences.Patients value many skills and attributes in clinicians.Of the dozen attributes that respondents were asked about on the pre-experiment survey, the modal respondent gave high priority to eight of the attributes.That is a lot of attributes to balance when selecting a preferred provider. These are precisely the circumstances in which narrative priming can be most consequential.Roughly 40% of real-life narratives about doctors involve what we have labeled here as "bedside manner" [36].Consumers who casually peruse these comments are reminded that this aspect of medical care matters to the experiences of many other patients.Even if they don't attend closely enough to the content of the comments to try to discern which doctors perform best in terms of these relational aspects of care, the salience of "bedside manner" rises for their selection of a primary care clinician. It is clear that a certain proportion of consumers will not probe narratives that closely.15.7% of the participants in the choice experiment indicated that it was hard for them to discern from the narratives which clinicians were performing best.Those consumers will favor the simpler, star-rated assessments of clinician practices, precisely because those can be readily rank-ordered.If consumers don't pay attention to the narrative content that disrupts stereotypes, the heightened salience of bedside manner activates those stereotypes and makes gender more influential in choice of a physician.Thus, the subset of individuals who value bedside manner within the 15.7% of participants who did not necessarily use patient narratives to choose their physician, are more likely to rely on physician gender stereotypes to find a physician that they believe can fulfill their bedside manner needs. However, when consumers do pay attention to the content of patient comments and these comments frequently run counter to stereotypes, gendered expectations can be disrupted.That requires that at least a third of comments are contrary to stereotypes.In this context, consumers do not appear to strongly "defend" their preconceived gender stereotypes (as some theories of motivated reasoning might predict), since they appear willing to alter their choices away from stereotyped preconceptions, even if a majority of comments remain consistent with stereotypes. How might these insights extend to stereotyping based on other observable attributes such as clinicians' race or age?Clinicians age and race may induce different types of stereotypes than does gender [55,56].Whether these stereotypes would be triggered in the absence of narratives will depend on other information that is available on real-world websites (but not presented on our experimental site), including photo images of the clinician or the year in which they graduated from medical school.To whatever extent stereotypes are triggered by realworld websites designed to support clinician choice, it seems plausible that the stereotype dampening effects of narratives would likely extend to these additional clinician attributes, however this should be tested with further research.Patients' preference for clinicians who seem "similar" to them is associated with their assessed threat of unfair treatment and their hopes that matching will ease communication, comfort, and trust [57].However, though impaired communication and perceived mistreatment are reported less frequently in racially and ethnically concordant pairings [58,59], communication failures and perceived discrimination are sufficiently common in these contexts that the stereotypes supporting racial matching are a rather unreliable predictor of high-quality interactions between patients and providers [60,61]. Might matching be enhanced if consumers had greater access to patients' narrative accounts of their interactions with providers?For some aspects of care-and some motivations for seeking out racially concordant pairings-patients' comments could signal otherwise unmeasured sources of communication breakdowns, lack of empathy, or untrustworthy practices, since all these aspects of care are regularly reported in patients' narratives about their treatment [36].However, to the extent that provider selection is motivated by concerns about racially-targeted discrimination, comments seem a less reliable remedy.Patients less often report on "unfair" treatment in ways that explicate the sources of discriminatory practices.And when they do so, their own race or ethnicity may be difficult to discern from the comment, making it difficult for consumers to determine which patients are at greatest risk for discrimination. Conversely, patient narratives may offer a more reliable way to address flawed stereotypes related to age.Many patients are predisposed toward younger physicians [61], presuming that more recently trained clinicians will have better technical, emotional, and explanatory skills than older doctors [62].Because these broad age-related stereotypes are often inaccurate characterizations for particular clinicians and because these domains of clinician-patient interaction are widely reported-on in patient narratives, there seems to be considerable opportunity for patient comments to erode age-related biases in the same way that they disrupted gendered stereotypes. Stereotyping and the opportunities for policy intervention The implications of these findings suggest that when individuals are given information derived from patient accounts inconsistent with their preconceived notions, they adjust their beliefs using the new "data" presented to them.Whether these lead to better choices, however, depends on our capacity to elicit narratives from a broad cross-section of patients and to do so in ways that encourages narratives that provide a high-fidelity representation of how it feels to get care from a particular clinician.In recent years, the capacity to rigorously elicit patient narratives has improved [63].But one cannot presume that the comments that are currently available on websites are necessarily either representative or complete, leading to other potential biases in consumer choice. A potentially more constructive stance for health policy would involve treating the overall supply of patient comments as a form of public good.This characterization is easy to recognize in countries that have a unified health care delivery and financing system, like the National Health Services in the U.K [64].But it is more difficult to comprehend-and infinitely more difficult to implement-in an American context that remains committed to promoting competition among providers.Over the past decade, there has been a dramatic proliferation of healthcare systems collecting patient narratives about their affiliated clinicians and posting those narratives on-line [6,12].However, since these narratives are being deployed, at least in part, for strategic advantage, there are ample incentives for systems to suppress feedback that offers a more negative assessment of their patients' experiences. Under these circumstances, there are two potential pathways toward preserving a more reliable and robust reservoir of patient comments that can be made freely accessible to consumers.The first approach would involve creating and enforcing norms of good practice for narrative reporting by each individual healthcare system.A second approach would be to assign government (at state and/or federal levels) the responsibility for eliciting and posting patient narratives [12].This could involve collecting comments from patients of all ages, covered by all forms of insurance.Or it could be more focused on the substantial portions of the American public who already have their healthcare financed through Medicare or Medicaid.If the latter approach was taken, it would then be straightforward to take comments collected from Medicare and Medicaid beneficiaries and make them freely available to the general public, much as all Americans can already access the Medicare Compare website to review comparative performance data on many types of health care providers and health plans [65]. Generalizations and the need for additional research The analyses presented above examined consumers' choices among primary care clinicians.But the findings may also have implications for the medical specialties.Past research suggests that stereotypes about physician gender lead to patients preferring a female physician for dermatology, pediatrics, obstetrics/gynecology, and family medicine [66].Likewise, patients prefer male physicians for surgical specialties such as orthopedic surgeons [57].To the extent that actual clinical behavior fails to match these stereotypes for one or more of these specialties, a shortage of comments addressing relational aspects of care may lead to consistent mismatching of patient preferences and provider practices.The capacity for patient comments to disrupt stereotypes in the short-run thus portends the longer-term potential to alter consumer choices in ways that reshape the ways in which aspiring clinicians (a.k.a.medical students and residents) select into particular specialties. These study findings need to be interpreted in the context of some methodological limitations.First, this was a hypothetical choice experiment.Thus, participants may engage with the experiment with less consistent or probing attention than they would if the choices had actual consequences for their medical care.This may imply that our results are an underestimate of the effect that learning from narratives can have on stereotyped choices, as participants in real life would be more likely to carefully engage with the website material (and thus process more fully narrative content which is more difficult to interpret than counting stars) if this was a real-life decision to be made with more consequential stakes.Second, the choice set in this experiment was structured to be carefully balanced according to gender in terms of quantitative performance metrics.In a real-world context, where there will be less balance in other performance metrics, the impact of stereotypes may skew access to other desirable clinician attributes. In addition, stereotyping may also shape how patients interpret their interactions with clinicians themselves, so stereotypes can have a more profound and lasting impact on the physician-patient relationship.As a result, the impact of stereotypes may extend to the interpretation of subsequent patient experiences, in that seeing stereotype reinforcing patient comments may lead to a patient to interpret an interaction with a physician as also stereotype reinforcing, which they themselves will report.Also, the presumption that female physicians will be more caring and warmer than their male counterparts is only one of several gender stereotypes.Future research could help to more carefully parse the multiple ways in which current and potential patients interpret and anticipate outcomes based on clinician gender.In addition, in order to fully assess the impact of gender stereotypes, further analyses are needed, studying other gender stereotypes of clinicians, including the common assumption that male physicians are more technically equipped than female physicians [39,58,67,68]. Conclusion Most patients want to select their primary care clinician with minimal effort and attention, especially those who see themselves in relatively good health [9,35].These are precisely the circumstance in which stereotyping is likely to have the most pronounced influence on choices.Online patient narratives can provide access to information about aspects of clinician skills and behavior that are not reliably measured by other means, helping consumers to choose based on some evidence, rather than relying entirely on stereotypes.How much this might improve matching of patient preferences and clinicians' skills will depend on the availability of narratives that reflect a broad range of patient experiences and convey those experiences in reliable ways.That can and should become a focus for policymakers intending to promote consumer choices as a way to improve health system performance. Table 2 . Summary of the seven experimental arms of the SelectMD 2.0 experiment. When viewing patient comments, respondents could see how commentators rated the positive or negative nature of their own comments and the distribution of positive-to-negative comments (much like how comments are displayed on the Amazon Website).	2023-12-09T05:09:57.073Z	2023-12-07T00:00:00.000	{ "year": 2023, "sha1": "1c9940c0343c1b0dfa8b657993e9184e18fe2319", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "1c9940c0343c1b0dfa8b657993e9184e18fe2319", "s2fieldsofstudy": [ "Medicine", "Psychology", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
252092397	pes2o/s2orc	v3-fos-license	Effects of CYP2C19 and CYP2D6 gene variants on escitalopram and aripiprazole treatment outcome and serum levels: results from the CAN-BIND 1 study Cytochrome P450 drug-metabolizing enzymes may contribute to interindividual differences in antidepressant outcomes. We investigated the effects of CYP2C19 and CYP2D6 gene variants on response, tolerability, and serum concentrations. Patients (N = 178) were treated with escitalopram (ESC) from weeks 0–8 (Phase I), and at week 8, either continued ESC if they were responders or were augmented with aripiprazole (ARI) if they were non-responders (<50% reduction in Montgomery–Åsberg Depression Rating Scale from baseline) for weeks 8–16 (Phase II). Our results showed that amongst patients on ESC-Only, CYP2C19 intermediate and poor metabolizers (IM + PMs), with reduced or null enzyme function, trended towards significantly lower symptom improvement during Phase II compared to normal metabolizers (NMs), which was not observed in ESC + ARI. We further showed that CYP2D6 NMs and IM + PMs had a higher likelihood of reporting a treatment-related central nervous system side effect in ESC-Only and ESC + ARI, respectively. The differences in the findings between ESC-Only and ESC + ARI may be due to the altered pharmacokinetics of ESC by ARI coadministration in ESC + ARI. We provided evidence for this postulation when we showed that in ESC-Only, CYP2C19 and CYP2D6 IM + PMs demonstrated significantly higher ESC concentrations at Weeks 10 and 16 compared to NMs. In contrast, ESC + ARI showed an association with CYP2C19 but not with CYP2D6 metabolizer group. Instead, ESC + ARI showed an association between CYP2D6 metabolizer group and ARI metabolite-to-drug ratio suggesting potential competition between ESC and ARI for CYP2D6. Our findings suggest that dosing based on CYP2C19 and CYP2D6 genotyping could improve safety and outcome in patients on ESC monotherapy. INTRODUCTION Polymorphisms in genes encoding cytochrome P450 (CYP) enzymes, which mediate the Phase I metabolism of many antidepressants, result in variability in enzyme activity and contribute to large interindividual differences in drug metabolism [1,2]. Hence, CYP450 genotyping has the potential to improve the efficacy and tolerability of antidepressants for the treatment of the major depressive disorder (MDD) by guiding medication selection and dosage adjustments according to the genetically predicted rate of drug metabolism of individual patients [3]. Escitalopram (ESC), the S-enantiomer of racemic citalopram, is one of the most effective and well-tolerated selective-serotonin reuptake inhibitors (SSRIs) prescribed for the treatment of MDD [4,5]. ESC is biotransformed to its primary metabolite, Sdesmethylcitalopram (S-DCT), by CYP2C19 and CYP3A4, with a minor role of CYP2D6 [6][7][8]. For patients who do not show symptom improvement with first-line antidepressant monotherapy, augmentation strategies with atypical antipsychotics have been reported to improve outcomes [9]. Aripiprazole (ARI), a second-generation antipsychotic, is an effective augmentation option with standard antidepressant therapy for MDD treatment [10]. ARI is predominantly metabolized by CYP2D6 into its active metabolite, dehydroaripiprazole (DHA). CYP2CD6 and CYP2C19 genes are highly polymorphic with multiple allelic variants associated with altered enzymatic capacity, while variations in the CYP3A4 gene are less common and have little impact on its enzymatic activity. The Clinical Pharmacogenetics Implementation Consortium (CPIC) has published guidelines on CYP2D6 and CYP2C19 phenotype prediction from genotypes [11]. Individuals can be classified into phenotypic subgroups based on inherited alleles associated with differing rates of drug metabolism, including normal metabolizers (NMs), intermediate metabolizers (IMs), poor metabolizers (PMs), and ultra-rapid metabolizers (UMs) [11]. For CYP2C19, there are three additional phenotypic subgroups according to CPIC, including rapid metabolizers (RMs), as well as "likely IM" and "likely PM" for decreased function CYP2C19 alleles with limited data to characterize function [12,13]. Using data from the well-characterized Canadian Biomarker Integration Network in Depression-Study 1 (CAN-BIND-1) in which patients with MDD were treated with ESC monotherapy or ESC with adjunctive ARI, we had three main objectives: 1. to examine the relationships between CYP2C19 and CYP2D6 metabolizer phenotypes, treatment response, and tolerability; 2. to replicate previous findings showing that CYP2C19 and CYP2D6 metabolizer phenotypes influence serum concentrations of ESC and ARI and their metabolite-to-drug ratio; and 3. to explore whether there is a relationship between serum concentrations with the response and side effects of treatment. We hypothesized that, compared to the non-NM metabolizer groups, CYP2C19 and CYP2D6 NMs would (1) be predominant among responders, show greater symptom improvement, and experience less treatment-related side effects, as well as (2) demonstrate lower parent drug concentrations and higher drugto-metabolite ratio. We further hypothesized that (3) differences in serum concentrations would be correlated with variability in treatment response and tolerability. METHODS A detailed description of the protocol for the Canadian Biomarker Integration Network for Depression (CAN-BIND-1) clinical trial has been published elsewhere [10,27]. Treatment protocol The 16-week study protocol consisted of two phases following screening and baseline visits. During Phase I (Weeks 0-8), participants were treated with open-label ESC (10-20 mg/day, flexible dosage) for 8 weeks. At Week 8, participants were classified as "responders" or "non-responders" if they demonstrated Montgomery-Åsberg Depression Rating Scale (MADRS) reductions of ≥50% or <50% from baseline, respectively. During Phase II (Weeks 8-16), responders continued ESC, whereas non-responders to ESC were augmented with ARI (2-10 mg/d, flexible dosage) for the second eight weeks. Blood samples were collected on Weeks 2, 10, and 16 to measure medication levels and on Week 4 for pharmacogenetic analyses. The methods used for the quantification of serum levels of drug and metabolite concentrations is described in the Supplementary Methods. Clinical sample The study sample consisted of 211 participants diagnosed with MDD according to the Diagnostic and Statistical Manual for Mental Disorders IV (DSM-IV-TR; American Psychiatric Association, 2000) and confirmed using the Mini International Neuropsychiatric Interview (MINI) with age ranging from 18 to 61 years. Participants were free of psychotropic medications for at least five half-lives prior to the start of the trial, had a depressive episode duration of ⩾3 months, a total score of ⩾24 in the Montgomery-Åsberg depression rating scale (MADRS) at the time of screening, and fluency in English to complete self-report questionnaires. The exclusion criteria included any other psychiatric diagnosis as the primary diagnosis. including Bipolar I or II, significant neurological disorders or head trauma, high suicidal risk, psychosis in the current episode, or substance dependence or abuse in the past six months. Measures Montgomery-Åsberg depression rating scale (MADRS). Depressive symptoms were assessed using MADRS every 2 weeks from Week 0 to 16. The primary outcomes for response were: (1) response status (responder versus non-responder) on the last visit of Phases I and II (i.e., Week 8 and 16), and (2) the percentage of symptom improvement across visits during Phases I and II. Remission status (remitter versus non-remitter) at the end of Phases I and II was a secondary outcome for a response. These response outcomes are described in the Supplementary Methods. Toronto side effects scale (TSES). The Toronto side effects scale (TSES), administered on Weeks 2, 4, 10, 12, and 16, is a clinical instrument designed to assess the frequency and severity of treatment-related side effects on 5-point Likert scales. The "intensity" score is derived by multiplying the frequency and severity of each side effect [28,29]. The items assessed can be broadly categorized into the central nervous system (CNS), gastrointestinal (GI), and sexual side effects, as well as weight gain (Table S1) [28]. The primary outcomes for side effects were both dichotomous and continuous: (1) absence or presence of side effects within the four categories on the last visit of Phases I and II and (2) the intensity of each category of side effect across visits during each Phase. DNA isolation and genotyping SNPs and haplotypes for CYP2C19 and CYP2D6 that are associated with altered metabolism and are common in the reference population (consisting of Europeans, African Americans, and East Asian ancestry) were included for genotyping. These SNPs cover >95% of the common alleles associated with altered metabolism. Genomic DNA was extracted from venous blood samples using a modified version of the FlexiGene DNA kit (QIAGEN, Hilden, Germany) and sent for genotyping at the CAMH Biobank and Molecular Core Facility (Centre for Addiction and Mental Health, Toronto, Canada). Genotyping was performed using standard TaqMan® Assays (Thermo Fisher Scientific, ON, Canada) according to the manufacturer's protocol to assess alleles and copy number variants (CNVs) in CYP2D6 (1, 2, 3, 4, 5, 6, 9, 10, 17, 29, 36, 41) and in CYP2C19 (1, 2, 3, 17) [30]. CNVs, including deletion (5) and multiplications of CYP2D6, were assessed using a copy-number assay and CopyCaller Version 1.0 (Applied Biosystems, Burlington, ON, Canada). The overall phenotype for CYP2D6 duplications was determined using the results from the SNP and CNV assays (e.g. genotype is reported as 1/3 (xN) if SNP assays revealed 1 and 3 and CNV assay showed more than two copies of the CYP2D6 gene for the same participant). Genotyping results were reviewed by two laboratory staff blind to the clinical data. Predictions of CYP2D6 and CYP2C19 metabolizer phenotypes were based on the expected enzyme activity of the alleles as reported in CPIC guidelines for CYP450 genes (https://cpicpgx.org/guidelines/guideline-forselective-serotonin-reuptake-inhibitors-and-cyp2d6-and-cyp2c19/). Table S2 summarizes the expected enzymatic activity for CYP2C19 and CYP2D6 alleles. The predicted metabolizer phenotype based on CYP2C19 and CYP2D6 genotype is reported in Table S3. Ten percent of the sample was regenotyped for quality control. Subjects for whom genotype was ambiguous were retyped, and if this result remained ambiguous, data from the participants were excluded from further analyses. A genotype is determined to be ambiguous if the sample failed to amplify or it does not clearly cluster with one of the three genotype clusters visualized in the ABI ViiA7 RUO (Applied Biosystems) software post-amplification. Statistical analysis All analyses were conducted using R Version 4. statistics for demographic and clinical characteristics by CYP2C19 (NMs vs. IM + PMs vs. RM + UMs) and CYP2D6 (NMs vs. IM + PMs) metabolizer groups were generated using the chi-squared or Fisher's exact test for categorical variables and the Mann-Whitney U or Kruskal-Wallis tests for continuous variables, as appropriate. There were only two CYP2D6 UMs (two in ESC-Only and zero in ESC + ARI) precluding the creation of a separate group, and therefore they were excluded from analyses. Given the different metabolic pathways of ESC and ARI, ESC-Only and ESC + ARI treatment arms are analyzed separately for Phase II. For analyses involving ESC, CYP2C19 and CYP2D6 metabolizer groups were included as fixed effect independent variables, since both enzymes are involved in the metabolism of ESC [6]. All analyses were also adjusted for age, sex, ancestry, and recruitment site, with the latter, included as a random effect factor in the linear mixed-effects models. Post-hoc comparisons for trends between ungrouped metabolizer phenotypes (NMs, IMs, and PMs for CYP2D6, as well as RMs and UMs for CYP2C19) were conducted for significant associations. Linear models were checked for assumptions of normality and nonlinearity, and log transformation was applied for skewed, non-normally distributed data, where applicable. The effect size for linear models was calculated using Cohen's is the set of all other predictors, R 2 AB is the variance explained for a multiple regression model with all of the predictors, and R 2 A is the variance explained for a model without the predictor (B) for which we want to calculate a "local" effect size [31,32]. The effect is considered small at 0.02, medium at 0.15, and large at 0.35 [33]. Due to heterogeneity in ancestry among study participants, all analyses were repeated in the largest ancestral group, Europeans, to control for population stratification [34]. False discovery rate (FDR) approach was used to control for multiple comparisons in the analysis of each subsample (i.e. total sample for Phase I and treatment arms for Phase II) with a significance threshold of q < 0.05 (two-tailed) [35,36]. For post-hoc comparisons, p < 0.05 was considered significant. Association of CYP2C19 and CYP2D6 metabolizer status with outcome measures. We assessed the dichotomous measures of response (responder vs. non-responder and remitter vs. non-remitter) and side effects (present vs. absent) for the last timepoint in Phases I and II using logistic regression models with total MADRS score at baseline included as a covariate. Given the availability of biweekly MADRS scores and multiple time points for TSES, continuous measures of response (percentage of symptom improvement) and side effects (intensity of each category of side effects) were assessed using linear mixed-effects models that included interactions of CYP2C19 and CYP2D6 metabolizer groups with timepoint, and recruitment site and individual as random effects variables. Association of CYP2C19 and CYP2D6 metabolizer status with measures of drug exposure. Using linear regression models, we examined the effects of CYP2C19 and CYP2D6 metabolizer groups on ESC exposure using three serum measures: concentrations of ESC, its primary metabolite, S-DCT, and the S-DCT/ESC ratio. Serum concentrations were adjusted for dosage (i.e., ng/mL/ mg). Sampling time (i.e., hours since the last dose) was entered as a covariate in the regression models to account for when the medication was taken. In the ESC + ARI treatment arm during Phase II, we also examined the effects of CYP2D6 metabolizer group on dose-adjusted serum concentrations of ARI, its primary metabolite, DHA, and the DHA/ARI ratio using linear regression models, as described above. The CYP2C19 metabolizer group was not included as a fixed effect covariate, as CYP2C19 is not known to be involved in the metabolism of ARI. Associations of drug exposure with outcome measures. Using Spearman's rank correlation, we explored whether measures of unadjusted serum concentrations were associated with symptom improvement and the intensity of side effects during Phase I and II. Sample demographics Participant flow is detailed in Fig. 1. We excluded 31 participants who dropped out prior to Week 8 and therefore lacked MADRS scores and drug serum levels for Phases I and II [27]. Amongst these dropouts, chi-square goodness-of-fit tests show that there were 10 NMs, 13 IM + PMs (11 IM, 2 PM), and 8 RM + UMs (7 RM, 1 UM) for CYP2C19 (χ 2 (2) = 1.23, p = 0.542), and 18 NMs and 12 IM + PMs (6 IM, 0 PM) for CYP2D6 (χ 2 (1) = 1.20, p = 0.273), with one lacking genotyping data. Thus, it does not appear that dropouts were overrepresented in any of the CYP2C19 or CYP2D6 metabolizer groups. For the dropouts for whom MADRS scores and serum levels at Week 2 were available, there were no significant differences in symptom improvement or ESC adj serum concentrations between CYP2C19 or CYP2D6 metabolizer phenotypes (Fig. S1). Two participants who did not have genotyping data were also excluded. Therefore, 178 participants were included in the study (211 recruited−31 dropouts−2 lacking genotyping data = 178). For CYP2D6, the effect of genotype on enzymatic function was unclear for five participants, and genotyping was consistently unsuccessful for one participant. For CYP2C19, there was one participant with a poor-quality sample. Therefore, analyses were conducted on 177 participants for CYP2C19 and 170 participants for CYP2D6 after the exclusion of UMs (N = 2). The distribution of metabolizer phenotypes of CYP2C19 and CYP2D6 did not differ significantly (χ 2 (12) = 6.26, p = 0.902). All participants were adherent to treatment during Phase I based on drug serum levels at Week 2. During Phase II, seven participants were suspected of non-adherence determined by a lack of treatment medication detected in serum at both Weeks 10 and 16, therefore they were not included in Phase II analyses. Nonadherence was not associated with CYP2C19 (χ 2 (2) = 0.29, p = 0.867) or CYP2D6 (χ 2 (1) = 1.29, p = 0.257) metabolizer groups. For demographic characteristics stratified by metabolizer group for the study sample and the European subset (see Tables 1 and S4), respectively. Also, see Supplementary Results for a description of the study sample. A summary of metabolizer status and genotypic frequencies of CYP2C19 and CYP2D6 can be found in Table S3. Association of CYP2C19 and CYP2D6 metabolizer groups with antidepressant response The overall response rates at the end of Phases I and II were 46.62% (83/178) and 68.54% (122/178), respectively. There was no significant impact of either CYP2C19 or CYP2D6 metabolizer groups on response or remission status at the end of Phase I or II (Table S5). During Phase I (Weeks 0-8), symptom improvement across timepoints was not significantly influenced by CYP2C19 or CYP2D6 metabolizer groups ( Fig. 2A, B). During Phase II (Weeks 8-16), in the ESC-Only treatment arm (N = 283 observations), there was a trend for an influence of CYP2C19 metabolizer group on symptom improvement across timepoints (F (2,207) = 3.99, p = 0.020, q = 0.068, f 2 = 0.05). The linear mixed-effects model indicated that the average percentage change in MADRS from baseline was lower in IM + PMs versus NMs (B = −2.34, 95% CI: [−3.62, −0.37]) (Fig. 2C, D). Simple effects analysis revealed that the average percentage change in MADRS from baseline was 2.54% (95% CI: [−4.33, −0.74], p = 0.006) lower in IMs than in NMs with every two-week assessment, while PMs were not statistically different from NMs (Fig. S2C). Overall, the cumulative difference in symptom improvement from baseline between CYP2C19 NMs and IMs was 11.52% (W = 381, p = 0.050, r = 0.28) at trial end in ESC-Only. Follow-up mediation analyses revealed that about 45% of the effect of CYP2C19 IM+PM phenotype on symptom improvement during Phase II in ESC-Only may be mediated by ESC adj serum concentrations (see Supplementary Results). For ESC + ARI (N = 325 observations), the percentage of symptom improvement was uninfluenced by either CYP2C19 or CYP2D6 metabolizer groups (Table S7). Associations between symptom improvement and CYP2C19 and CYP2D6 metabolizer groups were not observed in the European subset following multiple testing corrections (Table S8). Associations of CYP2C19 and CYP2D6 metabolizer groups with antidepressant side effects Analyses of the associations of CYP2C19 and CYP2D6 metabolizer groups with the absence or presence of CNS, GI, sexual side effects and treatment-related weight gain are presented in Table S9. Differences in sample sizes between weeks or side effect categories are accounted for by missed visits or failure to respond to the corresponding item on the TSES. No associations between the intensity of side effects across timepoints with either CYP2C19 or CYP2D6 metabolizer groups were observed (Figs. S10-13). Further, when these tests were repeated in the European subset, no associations were observed (Tables S13 and S14). In ESC + ARI, we observed that there was an association between CYP2C19 metabolizer group and ESC adj concentrations at Weeks 10 (F ( (Fig. S14C). Of note, there was no association between ESC adj serum concentrations and CYP2D6 metabolizer group for ESC + ARI (Fig. 4D). CYP2C19 and CYP2D6 metabolizer groups were not significantly associated with ESC adj concentrations in the European subset (Table S16). Serum S-DCT concentrations. At Weeks 2, 10, and 16, serum levels of S-DCT were available for 172, 152, and 150 participants, respectively. There was no significant association between S-DCT adj serum concentrations and CYP2C19 or CYP2D6 metabolizer groups (Fig. S16). Serum S-DCT/ESC ratio. There were significant effects of CYP2C19 and CYP2D6 metabolizer status on S-DCT adj /ESC adj serum ratio at Week 2 (CYP2C19: F (2,144) = 0.66, p < 0.001, q = 0.003, f 2 = 0.10; Fig. 2 Symptom improvement over time for Phase I and II by CYP2C19 and CYP2D6 metabolizer group. During Phase I, A CYP2C19 or B CYP2D6 metabolizer groups did not have a significant influence on symptom improvement over time. During Phase II, for the ESC-Only treatment arm, the average symptom improvement from baseline for every two-week assessment trended towards being lower in C CYP2C19 IM + PMs compared to NMs, which was not observed in the ESC + ARI group. There were no associations between symptom improvement over the course of Phase II and D CYP2D6 metabolizer groups for any of the treatment arms. All linear mixed effects analyses were adjusted for age, ancestry, sex, and interaction between time and CYP2C19 and CYP2D6 metabolizer groups as fixed effects, and recruitment site and subject as random effects variables. Error bars represent standard error. ARI aripiprazole, ESC escitalopram, IM intermediate metabolizer, NM normal metabolizer, PM poor metabolizer, RM rapid metabolizer, UM ultra-rapid Metabolizer. # indicates trend with q between 0.050 and 0.070. For Phase II, in ESC-Only, S-DCT adj /ESC adj ratio showed an association with CYP2C19 and CYP2D6 metabolizer groups at (Table S15) (Table S16). Association of ESC and ARI exposure with antidepressant response and side effects Results of the Spearman correlation indicated that serum levels of ESC, S-DCT, and their ratio were not associated with symptom improvement, CNS or gastrointestinal side effects, or treatmentrelated weight gain during Phase I and II. In ESC-Only, there was a significant negative association between the intensity of sexual side effects and serum ESC concentrations at Week 10 (r s (67) = −0.36, p = 0.002, q = 0.035), while ESC + ARI did not show this effect (Table S19). The lower the serum levels of ESC levels, the greater the intensity of sexual side effects that are reported. Posthoc analyses revealed this effect was driven by a significant correlation between concentrations of ESC and intensity of anorgasmia (r s (67) = −0.32, p = 0.007) and decreased libido (r s (67) = −0.25, p = 0.038) (Fig. S20). Serum ARI and DHA levels were correlated with symptom improvement for the ESC + ARI treatment arm (r s (83) = −0.36, p = 0.001, q = 0.048; r s (83) = −0.35, p = 0.002, q = 0.048, respectively). The higher the concentrations of ARI and DHA, the lower the percent change in MADRS from baseline at Week 16 (Fig. S21). DISCUSSION To the best of our knowledge, the current study is the first to assess the relationship of CYP2C19 and CYP2D6 metabolizer groups with the response, tolerability, and serum concentrations in individuals on ARI augmentation of ESC for the treatment of MDD, which represents a commonly used practice. Our results showed that CYP2C19 IM + PMs demonstrated a trend towards lower symptom improvement (i.e., the percentage change in MADRS from baseline) during Phase II than NMs amongst patients on ESC monotherapy. Post-hoc comparisons revealed that there was a significant difference specifically between CYP2C19 NMs and IMs, demonstrating a cumulative difference of 11.5% in percentage MADRS change by Week 16, whereas NMs and PMs did not differ, likely due to the small number of PMs in the sample. However, when the clinical relevance of this effect was evaluated using response and remission status, we found that the proportion of responders and non-responders, as well as remitters and non-remitters, did not differ by CYP2C19 metabolizer group. Therefore, although we observed that symptom improvement trended towards being Fig. 4 Dose-adjusted ESC concentrations in serum for Phase I and II by CYP2C19 and CYP2D6 metabolizer groups. During Phase I, A CYP2C19 IM + PMs showed higher mean ESC adj concentrations relative to NMs, whereas there was no significant difference in ESC adj concentrations between NMs and RM + UMs. B A significant difference in ESC adj concentrations was not observed between CYP2D6 metabolizer group. During Phase II, for the ESC-Only treatment arm, C CYP2C19 and D CYP2D6 IM + PMs compared to NMs had higher ESC levels in serum. In the ESC + ARI treatment arm, ESC adj serum levels were associated with only C CYP2C19, but not D CYP2D6, with higher ESC adj concentrations in CYP2C19 IM + PMs relative to NMs. All linear regression analyses were adjusted for age, ancestry, sex, recruitment site, time since last dose, CYP2C19 and CYP2D6 metabolizer groups. Error bars represent standard error. ARI aripiprazole, ESC escitalopram, IM intermediate metabolizer, NM normal metabolizer, PM poor metabolizer, RM rapid metabolizer, UM ultra-rapid metabolizer. q < 0.05; q < 0.01; q < 0.001; # indicates trend with q between 0.050 and 0.070. different between CYP2C19 IM + PMs versus NMs in patients on ESC monotherapy, the size of this effect was small (f 2 = 0.05) and of limited clinical utility. Therefore, our findings warrant further validation in a larger, independent sample. Further, we showed that ESC serum concentrations are influenced by CYP2C19 metabolizer group. CYP2C19 IM + PMs demonstrated a trend towards higher ESC adj serum concentrations and lower S-DCT adj /ESC adj ratio in comparison to NMs during Phase I and in both treatment arms during Phase II, which replicates findings from previous studies [14,15,19,37]. The mediation analysis showed that about 45% of the effect of CYP2C19 IM + PM phenotype on symptom improvement may be mediated by ESC adj serum levels in ESC-Only. These findings taken together suggest that for slower CYP2C19 metabolizers, there are higher concentrations of ESC in serum possibly above the therapeutic range, which negatively impacts symptom improvement over time in individuals on ESC monotherapy (Fig. S23). Therefore, individuals who are CYP2C19 IM and PM may benefit from ESC dose reductions to achieve greater improvements in depressive symptomology. The association between CYP2D6 metabolizer group and serum measures of study medications is more complex due to ESC and its metabolite, S-DCT, being weak inhibitors of CYP2D6 in vitro [8,38]. The underlying mechanisms and the extent to which CYP2D6 activity is affected by ESC inhibition remain to be elucidated. It has been reported that IMs may be more susceptible to phenoconversion by concomitant use of weak to moderate CYP2D6 inhibitors compared to PMs, NMs, and UMs [39]. In this study, CYP2D6 IMs demonstrated elevated ESC concentrations similar to concentrations in PMs, suggesting possible phenoconversion of CYP2D6 IMs by ESC into a lower metabolizer phenotype (Fig. S14). In contrast, ESC is not a known inhibitor of CYP2C19, thus CYP2C19 NMs, IMs, and PMs showed differences in ESC adj concentrations consistent with their predicted enzymatic capacity. Although both treatment arms are affected by possible phenoconversion of CYP2D6 by ESC and its metabolite, a significant association between CYP2D6 metabolizer group and ESC adj serum concentrations was observed in ESC-Only, but not in ESC + ARI (Fig. 4D). This suggests that there may be a difference in the pharmacokinetics of ESC between the two treatment arms, with ESC + ARI being affected by ARI coadministration. We postulated that the differential effect of CYP2D6 metabolizer group on ESC adj serum levels between treatment arms is due to the competition for CYP2D6 by both ESC (a CYP2D6 weak inhibitor) and ARI (a CYP2D6 substrate) in the ESC + ARI treatment arm. This postulation is supported by the observation that, during Phase II, CYP2D6 NMs in ESC + ARI consistently demonstrated higher ESC adj serum levels compared to CYP2D6 NMs in ESC-Only. This is possibly due to more unmetabolized ESC in serum in ESC + ARI as a result of competition with ARI for CYP2D6, which is not present in ESC-Only (Fig. S15). This competition for CYP2D6 by both ESC and ARI shifts the metabolism of ESC to be more dependent on CYP2C19, hence resulting in a lack of a significant association between ESC adj concentrations and CYP2D6 metabolizer group in ESC + ARI. In ESC-Only, where there is no competition for CYP2D6 by Fig. 5 Dose-adjusted serum S-DCT/ESC adj ratio for Phase I and II by CYP2C19 and CYP2D6 metabolizer groups. During Phase I, A CYP2C19 and B CYP2D6 IM + PMs showed lower mean S-DCT ad /ESC adj ratio relative to NMs. Likewise, during Phase II, in the ESC-Only and ESC + ARI treatment arms, C CYP2C19 IM + PMs compared to NMs had lower mean S-DCT ad /ESC adj ratio in serum. D For CYP2D6, IM + PMs displayed lower S-DCT ad /ESC adj ratio in ESC-Only, whereas S-DCT ad /ESC adj ratio was not associated with CYP2D6 metabolizer group in the ESC + ARI treatment arm. All linear regression analyses were adjusted for age, ancestry, sex, site, time since last dose, CYP2C19 and CYP2D6 metabolizer groups. Error bars represent standard error. ARI aripiprazole, ESC escitalopram, IM intermediate metabolizer, NM normal metabolizer, PM poor metabolizer, RM rapid metabolizer, UM ultra-rapid metabolizer. q < 0.05; q < 0.01; *q < 0.001. ARI, the metabolism of ESC is dependent on both CYP2C19 and CYP2D6, resulting in a significant association of ESC adj concentrations with both enzymes. For treatment-related side effects, CYP2D6 NMs and IM + PMs had a higher likelihood of reporting a CNS side effect in ESC-Only and ESC + ARI, respectively. The mechanism underlying this difference between treatment arms in the direction of association between CYP2D6 metabolizer group and the presence of CNS side effects is unclear. CYP2D6 is reported to be expressed centrally, where it may be enzymatically active and involved in the metabolism of endogenous compounds, including neuronal amines (e.g. tyramine to dopamine), as well as peripherally administered drugs [40,41]. Like hepatic CYP2D6, brain CYP2D6 can also be induced and inhibited by medications; therefore, it is possible that the direction of the effect was different between treatment arms due to the phenoconversion of brain CYP2D6 by ESC and competition for CYP2D6 by both ESC and ARI in ESC + ARI, affecting the metabolism and functioning of endogenous systems, including serotonin and dopamine [42]. Our analyses included both CYP2C19 and CYP2D6 metabolizer groups as fixed effects in the same model. This approach was previously shown to be a better predictor of ESC blood levels than when CYP2C19 and CYP2D6 were evaluated individually [43]. A further strength of our study included the repeated measures approach of assessing symptom improvement and intensity of side effects over time which increased statistical power. The limitations of this study include the relatively small sample size when stratifying by treatment arm during Phase II, which limits statistical power to detect differences in response and side effects by CYP450 metabolizer groups. However, our sample was adequately powered to investigate differences in serum levels by CYP450 metabolizer groups during both Phases. Further, we were limited by the low representation of the less common PMs and UMs; therefore, because of power considerations, we employed grouping together slower and faster metabolizer phenotypes. As a result, we conducted post-hoc comparisons for significant results using ungrouped metabolizer phenotypes to distinguish which specific pairs of phenotypes are different. Another study limitation is that comedication was not recorded which might have modulated CYP2C19 or CYP2D6 activity and resulted in phenoconversion [39,44]. Similarly, one subject reported a liver condition and three subjects reported alcohol misuse, which might have affected medication metabolism. Finally, heterogeneity in ancestry within the study sample raises the issue of population stratification resulting in false positive associations. To ameliorate this issue, we conducted the same analyses in only Europeans and identified discrepancies in the results between the total sample and the European subsample which need to be explored in future studies with a larger sample. In summary, our results shed some light on the pharmacokinetics of ESC in vivo. We provided support for the phenoconversion of CYP2D6 by ESC and S-DCT inhibition, which was previously shown in vitro. In ESC + ARI, our results revealed altered pharmacokinetics of ESC with ARI coadministration, both of which may be competing for CYP2D6. Of particular interest is our finding showing that CYP2C19 IM + PMs demonstrated higher ESC adj concentrations and trended towards lower symptom improvement relative to NMs amongst those on ESC monotherapy. These results suggest preemptive CYP2C19 genotyping may be useful to identify patients who are CYP2C19 IM or PM, so that they may be treated with a reduced dose to optimize their treatment outcome. These results are in line with the recommendations provided by CPIC, confirming that "a 50% reduction of recommended starting dose" should be considered for CYP2C19 PMs. Based on these results, dose reductions may also be considered for CYP2C19 IMs. Further, we found an association between CYP2D6 metabolizer group with CNS and sexual side effects, differentially by treatment arm, indicating that CYP2D6 genotyping may also preemptively identify patients susceptible to these side effects.	2022-09-07T13:32:05.267Z	2022-09-06T00:00:00.000	{ "year": 2022, "sha1": "bd3f11b15b1f455d2adb85a79e4fb1ab198053b7", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Springer", "pdf_hash": "eaf6bcfed48d33e602291c90b520a3fca9539561", "s2fieldsofstudy": [ "Biology", "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
246931716	pes2o/s2orc	v3-fos-license	SoK: Function-As-A-Service: From An Application Developer’s Perspective In the past few years, FaaS has gained significant popularity and became a go-to choice for deploying cloud applications and micro-services. FaaS with its unique ‘pay as you go’ pricing model and key performance benefits over other cloud services, offers an easy and intuitive programming model to build cloud applications. In this model, a developer focuses on writing the code of the application while infrastructure management is left to the cloud provider who is responsible for the underlying resources, security, isolation, and scaling of the application. Recently, a number of commercial and open-source FaaS platforms have emerged, offering a wide range of features to application developers. In this paper, first, we present measurement studies demystifying various features and performance of commercial and open-source FaaS platforms that can help developers with deploying and configuring their serverless applications. Second, we discuss the distinct performance and cost benefits of FaaS and interesting use cases that leverage the performance, cost, or both aspects of FaaS. Lastly, we discuss challenges a developer may face while developing or deploying a serverless application. We also discuss state of the art solutions and open problems. Introduction Function-as-a-Service (FaaS) has emerged as a new paradigm that makes the cloud-based application development model simple and hassle-free. In the FaaS model, an application developer focuses on writing code and producing new features without worrying about infrastructure management, which is left to the cloud provider. 1 FaaS was first introduced by Amazon in 2014 as AWS Lambda [3], and since then, other commercial cloud providers have introduced their serverless platforms, i.e. Google Cloud Function (GCF) [22] from Google, Azure Function [14] from Microsoft, and IBM Cloud Function [23] from IBM. There are also several open-source projects like Apache OpenWhisk, Knative, OpenLambda, Fission, and others. At the time of the inception of the Internet, applications were built and deployed using dedicated hardware acting as servers, which needed a high degree of maintenance and often lead to under-utilization of resources [60,61]. Moreover, adding/removing physical resources to scale to varying demand, and debugging an application, was a cumbersome task. Under-utilization of resources and higher cost of maintenance led to the invention of new technologies like virtualization and container-based approaches. These approaches not only increased resource utilization but also made it easy to develop, deploy, and manage applications. Many tools [60,61,79,119] were built to help users orchestrate resources and manage the application. Although virtualization and container-based approaches lead to higher utilization of resources and ease of building applications, developers still have to manage and scale the underlying infrastructure of an application, i.e. virtual machines (VMs) or containers, despite the availability of a number of approaches that would perform reactive or predictive scaling [47,69,92,98,105,128]. To abstract away the complexities of infrastructure management and application scaling, serverless computing emerged as a new paradigm to build, deploy, and manage cloud applications. The serverless computing model allows a developer to focus on writing code in a high-level language (as shown in Table 1) and producing new features of the application, while leaving various logistical aspects like the server configuration, management, and maintenance to the FaaS platform [122]. Even though FaaS has been around for only a few years, this field has produced a significant volume of research. This research addresses various aspects of FaaS from benchmarking/improving the performance of various FaaS platforms/applications, porting new applications into a serverless model, to suggesting altogether new serverless platforms. As serverless computing is still an evolving field, there is a significant need for systematization of the knowledge (SoK) particularly from the perspective of an application developer. We believe that for an application developer, an ideal SoK paper should address three main aspects: 1) current state of FaaS platforms, e.g. performance and features, 2) what makes serverless computing ideal for certain classes of applications, and 3) and future research directions for helping a developer leverage the full potential of FaaS with her limited control over the FaaS platform. Previous SoK papers are generally written from the perspective of the service provider. Castro et al. [56] present an overview of serverless computing and discuss the serverless architecture, development, and deployment model. Hellerstein et al. [77], Jonas et al. [82] and Baldini et al. [52] also provide an overview of serverless computing, and dis-cuss potential challenges that a serverless provider should address for the popularization of serverless computing. Similarly, in [110], challenges and potential research directions for serverless computing are discussed. Eismann et al. [65] perform a systematic review of serverless applications and provide useful insights into the current usage of serverless platforms. Eyk et al. [118] give perspectives on how serverless computing can evolve and identify adoption challenges. Lynn et al. [90] give an overview of various features provided by popular serverless platforms. The aforementioned works generally take the perspective of a service provider and discuss the challenges and optimizations that it should introduce to improve and popularize the FaaS platform and have limited to no discussion from an application developer's perspective. In this paper, we take a closer look at the three aforementioned aspects of FaaS from an application developer's perspective. We assess previous work related to measurements, performance improvement, and porting of applications into the FaaS computing model, and augment this with our own experimental results and insights. While we mainly take an application developer's perspective viewing the FaaS platform as a closed-loop feedback control system, we also discuss improvements and optimizations that a provider can introduce (as a developer can be a provider too in the case of opensource FaaS platforms) to provide a more holistic view to the reader. In this paper, we make the following contributions to the SoK: • We categorize the decisions that an application developer can make during one life cycle of an application into two categories: one-time decisions and online decisions, and discuss their performance and cost implications. • We show that the quick provisioning time, on-demand scaling, and true "pay as you go" pricing model are key factors for FaaS adoption for various classes of applications and discuss potential challenges. • In Section 7, we discuss the challenges and open issues that a developer may face while employing FaaS for her cloud applications. We discuss building tools and strategies for FaaSification and decomposition of legacy applications to better suit the FaaS computing model, optimizing code, tuning resources for serverless applications, and usage of FaaS in conjunction with other cloud services for cost savings. The rest of the paper is organized as follows. We first describe the FaaS computing model and its important features (Section 2). We then present a developer's view of a FaaS platform as a closed-loop feedback control system (Section 3). Next, we look at various measurement studies that investigate different aspects of commercial and open-source FaaS platforms (Section 4). Then we present an economic model of FaaS (Section 5) and compare it with traditional Infrastructureas-a-Service (IaaS), and identify suitable classes of applications that can leverage serverless computing for its performance/cost (Section 6). Lastly, we discuss future challenges and research directions to make FaaS adoption efficient and easy (Section 7). Background Serverless computing was initially introduced to handle less frequent and background tasks, such as triggering an action when an infrequent update happens to a database. However, the ease of development, deployment, and management of an application and the evolution of commercial and opensource FaaS platforms have intrigued the research community to study the feasibility of the serverless computing model for a variety of applications [72,94,128,129]. Moreover, there are systems whose aim is to help developers port their applications to a serverless programming model [62,114]. In a serverless computing model, a developer implements the application logic in the form of stateless functions (henceforth referred to as serverless functions) in the higher-level language. We show various runtimes supported by popular FaaS platforms in Table 1. The code is then packaged together with its dependencies and submitted to the serverless platform. A developer can associate different triggers with each function, so that a trigger would cause the execution of the function in a sandbox environment (mostly containers) with specified resources, i.e. memory, CPU-power, etc. The setup time of the sandbox environment is referred to as cold start. In a typical case, the output of the serverless function is then returned as the response to the trigger. As serverless functions are stateless, a developer has to rely on external storage (like S3 from AWS), messages (HTTP requests) or platform API [32] to persist any data or share state across function instances 2 . The serverless computing model is different from traditional dedicated servers or VMs in a way that these functions are launched only when the trigger is activated, while in the traditional model, the application is always running (hence the term "serverless"). Serverless computing abstracts away the complexities of server management in two ways. First, a developer, only writes the logic of an application in a high-level language, without worrying about the underlying resources or having to configure servers. Second, in case the demand for an application increases, a serverless platform scales up the instances of the application without any additional configuration or cost and has the ability to scale back to zero (discussed in Section 4.4). While FaaS platforms provide typical CPU and memory power to serverless applications, it is their ability to scale quickly (in orders of milliseconds) that gives them a performance advantage over other cloud services. On the contrary, in IaaS, an application developer not only has to specify the additional scaling policies but there can be an additional cost for deploying such autoscaling services and it can take up to minutes to scale up. In Table 1, we show some of the key features provided by popular commercial FaaS platforms 3 . While providing similar services, specific features can vary significantly from one platform to another. Generally, these platforms only allow memory as a configurable resource for the sandbox environment with the exception of GCF which also allows a developer to specify the CPU power. AWS Lambda allocates CPU in proportion to the memory allocated [6]. IBM Cloud Function seems to have a constant allocation of the CPU share regardless of the memory allocation as increasing memory does not improve runtime significantly [93]. Azure Function does not allow any configurable resource and charges the user based on the execution time and memory consumption [11]. While these platforms initially supported applications written in specific languages, they currently support more languages and custom runtimes, making it possible to run any application using FaaS. An important feature of the serverless computing model is that serverless platforms follow the "pay as you go" pricing model. This means a user will only pay for the time a serverless function is running. This model charges a user for the execution time of the serverless function based on the resources configured for the function. A user will not be charged for deploying the function or for idle times. Even though all of the cloud providers follow a similar pricing model, the price for the unit time (Billing Interval) of execution can vary significantly from one cloud provider to another. In the serverless computing model, the abstraction of infrastructure management comes at the cost of little to no control over the execution environment (and underlying infrastructure) of the serverless functions. Depending on the platform, a user can control limited configurable parameters, such as memory size, CPU power, and location to get the desired performance. Since the introduction of serverless platforms, there has been a large body of research work that aims to demystify the underlying infrastructure, resource provisioning, and eviction policies for commercial serverless platforms. Besides, these works have also looked at different aspects of performance, namely cold-starts, concurrency, elasticity, network, and I/O bandwidth shares. These research studies are helpful for the research and developer community to find a suitable serverless platform for their application and also inspire future research. 3 Developer's View of FaaS Serverless platforms are largely black-boxes for application developers, who submit the code of their application (with a few configurations) and in turn, the code gets executed upon the specified triggers. A user has little to no control over the execution environment, underlying resource provisioning policies, hardware, and isolation. A user has control over limited configurations through which they can control the performance of their serverless application. In what follows we categorize the decisions a developer can make for their serverless applications to get the desired performance or optimize their cost. One-Time Decisions: These are the decisions that a developer can make before developing and deploying an application and include selecting the serverless platform, programming language, and location of deployment. These decisions can be dictated by the features that a serverless platform offers such as underlying infrastructure, pricing model, elasticity, or performance metrics -for example, certain languages may have lower cold-start latency or the location of deployment can affect the latency to access the application. We believe changing any of these aspects would incur significant development and deployment cost, hence a developer can make such a decision only once in the life cycle of the application. Online Decisions: A developer has more freedom to change other parameters without a serious effort, including resources (memory, CPU) and concurrency limit. As we show later in this section, these parameters can affect the performance and cost of a serverless application. A developer can employ a more proactive technique to configure her serverless function based on the desired performance metric. Configuring these parameters is also important as serverless platforms provide no Service-Level Objective (SLO), i.e. guarantee on the performance of the serverless function, and a developer's only recourse to get the desired performance is through the careful configuration of these parameters. Later in Section 7, we discuss the challenges of designing proactive approaches by employing feedback control systems. These systems would continually monitor the performance of a serverless application and make these online decisions for the application, as shown in Figure 1. There have been several measurement studies conducted by academic researchers and independent developers that have attempted to demystify different aspects of commercial and open-source serverless platforms. These studies help a developer make one time decisions by identifying the underlying resources, i.e. operating system, CPUs, virtualization technique, and by benchmarking various performance aspects of serverless platforms. Moreover, these studies also look at the effect of configurable parameters (online decisions) on the performance and cost of serverless functions establishing the need to configure these parameters carefully. Measurement Studies In Table 2, we present a classification of previous measurement studies. In this classification, we correlate the decisions (both one time and online) that a developer or a researcher can make in terms of picking the serverless platform, scripting language, and configurations, with different performance aspects, such as cold-start delay, runtime, cost, etc. Every cell in the table indicates the peer-reviewed studies that have looked at the relationship between the controlled variable (decision) and the dependent parameters (performance). In what follows, we describe in greater detail the findings of these measurement studies 4 and explain the effect of choices on different performance aspects. As our main focus in this paper is the main observations and insights of these measurement studies, we do not discuss other aspects such as the reproducibility of these studies. To this end, Scheuner and Leitner [106] present a comprehensive analysis of various measurement studies and discuss the flaws and the gaps in the FaaS measurement and benchmarking literature. Cold Starts This is perhaps the most studied aspect of serverless platforms. There have been several peer-reviewed studies attempting to quantify and remedy the effect of cold starts. The cold start comes from the fact that if a function is not being invoked recently for an amount of time set by the platform (called instance recycling time, and discussed later in this section), the platform destroys the sandbox environment (i.e. container) to free up the resources. On a subsequent new request, the platform will (re-)initialize the sandbox environment and execute the function, hence an extra delay would be incurred. Studies have found that cold starts can be affected by various online and one-time decisions. • Choice of language: These studies show that usually, interpreted languages (Python, Ruby, Javascript) have significantly less (100x) cold-start delays as compared to compiled runtimes (Java, .NET, etc.) [17, 95,122]. This can be due to the fact that a compiled runtime, as in Java, requires the initiation of a compute-intensive JVM, which can incur significant delay [123]. Another aspect to consider is that although interpreted languages may have less of an initial cold start, they suffer from lower execution performance compared to compiled runtimes [33]. • Serverless provider: Studies have shown that different providers can have different cold-start delays depending on their underlying infrastructure or resource provisioning strategy [87,95,97,122]. For example, AWS Lambda tends to place function instances from a user on the same underlying VM [97,122], hence causing contention and increasing the cold start. Similarly, Azure instances are always assigned 1.5GB of memory, possibly increasing the cold-start time. • Resources: Cold start is also impacted by the resources [85,122,123] x [44,122,123] x Network throughput [85,122,123] x [122] x Instance Lifetime [88,120,122] x [122] x Underlying Infrastructure [87,122] x [88] x 95,122]. This can be because of the fact that more resources lead to a faster setup of the execution environment [122]. • Code Package: Studies [17, 123] have shown that code package size, i.e. code and the libraries it uses, can affect the cold-start latency. This is due to the fact that the bigger the code package size, the longer it will take to load into memory [78]. The above insights can help a user develop an application in a particular language, and also configure resources based on the application's needs. If an application is latency-sensitive, a developer may choose to use a scripting language and configure more resources for the serverless function. One has to be careful with configuring more resources for the serverless function to remedy cold start, as it can increase the cost of running the serverless function as explained later in Section 4.3. Based on the finding reported in [122] on commercial serverless platforms, AWS Lambda has the least cold-start delays. Approaches to circumvent the cold start can be divided into two categories: 1) For serverless platforms: Serverless platforms can improve the cold-start latency by having fast sandboxing techniques, efficient function instance 5 placement/scheduling and by keeping the sandbox instances warm for a longer time. While the last approach can be significantly expensive for the platform as it can potentially lead to resource underutilization (discussed in more detail in Section 4.2), there has been a significant body of research focused on improving the cold-start latency through the first two. Advanced containermanagement/sandboxing techniques [45,54,63,97,100,113] employ container reuse, loose isolation between function instances, and memory snapshotting and restoring to achieve a cold-start latency that is as low as 10ms or less [113]. Other approaches suggest optimized routing schemes [43], packageaware scheduling [49], efficient capacity planning [74] and reuse of resources [115] to reduce the cold-start latencies. 5 Function instance refers to the sandbox environment executing the code of a serverless function. 2) For the developers: The aforementioned fast sandboxing approaches will only work if a developer has complete control over the serverless platform. In case a developer is using a commercial serverless platform, their approach to mitigate cold start will be different. In addition to carefully selecting the language and serverless platform to develop and deploy their application based on previous findings, they can also control cold start through carefully configuring resources for the application. There are several articles published [2,18,30,40,86], which suggest certain design changes in the application to avoid unnecessary cold starts such as sending dummy requests to the serverless function that perform early exit without performing any computation. While these approaches may keep the function warm, they can also introduce extra cost (discussed in Section 4.3) as there is a fixed cost charged for each request and some FaaS platforms round up the execution time to the nearest 100ms, so even if the function performs early exit, the user would be charged some cost. A recent feature from FaaS platforms, such as AWS Lambda [36] and Azure Function [10], allows their user to specify a minimum number of function instances to be kept warm all the time to avoid unnecessary cold starts but a user is charged for enabling this feature. Summary: Cold start can be impacted by the virtualization techniques and function eviction policies employed by the serverless platform. From a developer's perspective, the impact of cold start can be controlled through the configurable resources and careful choice of the programming language. Instance Recycling Time and Lifetime When a serverless function is first executed, the serverless platform creates the sandbox environment, loads the function's code in it, and executes the code. After the code has been executed, the sandbox environment is kept in a warm state for a certain amount of time (called instance-recyclingtime) to serve any subsequent request for the same function. If during that time, no subsequent request arrives, the sandbox environment is terminated so as to reuse the resources. A serverless platform may decide to terminate the sandbox environment after it has been in use for a certain period regardless of the usage. This time is called instance-lifetime. Both instance recycling time and instance lifetime are very critical values to configure for not only the serverless platform but also for the users. A low value for these variables would mean that a serverless platform can free the resources quickly and re-purpose them for other applications while increasing the utilization of underlying resources, but for users, it can be devastating as the serverless functions would experience unnecessary cold starts hence degrading the performance of their serverless application. For a commercial serverless platform, it can lead to potential revenue loss by losing customers. While from the user's perspective, longer values would be ideal as their application would always find their serverless functions warm, hence reducing the latencies, but this may end up reducing the utilization of the underlying resource for the serverless platform 6 . For open-source serverless platforms [26,111], a user can configure these values on their own and there have been studies suggesting using popularity analysis to configure these values on a per-application basis [73,111]. But in commercial serverless platforms, these values are decided by the platform and a user has no control over the instance-recycling-time and instance-lifetime. There have been several peer-reviewed studies that looked at this aspect of commercial serverless platforms. Most of these studies followed a similar technique to infer the values for instance-recycling-time and instancelifetime. Commercial serverless platforms allow a serverless function to use a limited amount of persistent storage for the time a sandbox environment is in use. Previous studies [87,120,122] use this storage to store an identifier for the serverless function when the function is invoked for the first time. Later they invoke the same function again and check if the identifier is still present; if it is not, then the sandbox environment was destroyed and the latter execution was done in a new environment. They show that different serverless platforms have different instance-recycling times, with Google Cloud Function having the longest of all (more than 120 minutes). AWS Lambda's recycling time is reported to be around 26 minutes. The authors could not find a consistent value for Azure Functions. Another recent study [17] claims this value to be 20-30 min for Azure Function, 5-7 min for AWS Lambda, and 15 min for Google Cloud Function. Hence, if a serverless function stays inactive for this instance-recyclingtime, the subsequent request would incur an extra delay equal to a cold start. In an independent study [37], the authors established a relation between instance-recycling-time and resources (i.e. memory) configured for the serverless function on AWS Lambda. 6 Remember a user does not pay for idle times in serverless computing, hence this is a lose-lose situation for the serverless platform or cloud provider. They found that a large value of memory configured for the serverless function tends to give it a small instance-recyclingtime. Regarding instance lifetime, in [122], using a similar technique, the authors found that Azure Function has the longest instance-lifetime as compared to AWS Lambda and Google Cloud Function. They also found that in the case of Google Cloud Function, the lifetime of an instance can be affected by the resources configured for the function. It is reported that instance-lifetime of an instance with 128 MB and 2,048 MB memory is 3-31 minutes and 19-580 minutes, respectively. Summary: For a serverless function, instance-recyclingtime is decided by the serverless platform. A serverless platform can employ more pro-active approaches to configure instance-recycling-time based on the application's popularity, as suggested in [111]. For an application developer, a low value for instance-recycling-time would affect performance by incurring extra cold-start delays. A developer can reduce the effect of cold starts by carefully choosing the language of the application and configurable resources. Cost and Performance The cost of cloud usage for one execution of a serverless function on a commercial serverless platform p can be calculated as follows: where T (m) is the run time of the serverless function given resources m and C(p, m) is cost per unit time of resources m from the platform p. G(p) denotes the fixed cost such as API gateway for AWS Lambda; if there is no fixed cost, G(p) can be considered zero. Equation (1) shows that the cost of cloud usage directly depends on the run time of the serverless function and the price per unit time for resources m [7,11,21,25]. Hence all the factors that can impact the run time of a function will also impact the cost of cloud usage. To observe resources runtime resources cost Figure 2: Performance and cost on AWS Lambda the effect of configurable resources on the performance of a serverless function, we deployed various (I/O-intensive, memory-intensive, and CPU-intensive) functions on AWS Lambda and invoked them with varying resource configurations e.g. memory (more details in [44]). We show the observed trends in the performance and cost with respect to the resources in Figure 2, across all function types. It can be seen that more resources lead to faster execution of the serverless function but the performance gain is limited after a certain point. Note that the performance of I/O-intensive and CPU-intensive functions also improves with more memory allocation. It is because of the fact that AWS Lambda allocated CPU share in proportion to the memory allocated [4]. This observation also confirms previous findings made in [44,64,93], which report a similar effect of resources on the performance. Other factors that can affect the performance are summarized below: Cold Starts: A serverless platform may decide to terminate the sandbox environment if it has been inactive for a certain amount of time, as explained in Section 4.2. Hence, serverless functions with less frequent invocations may incur the extra latency of cold start. Concurrency: Previous studies [85,87,88,122] looked at the effect of concurrency on the performance of serverless functions and found that the performance can be negatively impacted by a higher concurrency level. This is due to the particular resource provisioning policies of the serverless platforms as reported in [122]. In particular, AWS Lambda and Azure Function try to co-locate the function instances, hence causing more contention for resources. Recent work [108] shows that concurrency configurations can also impact the performance of serverless functions running on the opensource serverless platform Knative [27]. Co-location: Previous studies [44,122] show that colocation of serverless functions on the same underlying resource can also result in significant performance degradation. Our preliminary experiment on OpenWhisk also confirms these findings. This is due to the fact that multiple function instances hosted on the same VM compete for resources such as disk I/O and network bandwidth, and this competition can adversely affect performance. Underlying Infrastructure and Policies: As discussed in Section 4.6, the underlying infrastructure of commercial serverless platforms consists of diverse resources, and in addition to that, resource provisioning policies for the execution of a serverless function can also vary significantly from one platform to the other [122]. Hence, these aspects can also introduce significant uncertainty in performance. Keeping in mind the tightly coupled nature of performance and cost of serverless functions, it is really important to find the "best" configuration of parameters (online decisions), e.g. memory, CPU, concurrency, such that they not only meet performance expectations but also optimize the cost of cloud usage. Previous approaches [44,64,68,108] use various machine learning and statistical learning approaches to configure parameters, e.g. memory, CPU, concurrency and location, for serverless applications deployed on commercial and opensource serverless platforms. We discuss these approaches in more detail in Section 7.3. Summary: The performance of a serverless function can be impacted by its configurable resources, choice of programming language, and the choice of serverless platform. The usage cost is calculated based on the configurable resources, the execution time, and the unittime cost specified by the serverless platform. Concurrency or Elasticity Concurrency is the number of function instances serving requests for the serverless function at a given time. On-demand scaling by the serverless platforms -i.e. in case the demand for the serverless application increases, the serverless platform initializes more function instances to serve these requests concurrently -is one of the distinct features of the serverless computing model. Unlike IaaS, a user does not have to specify the scaling policies, rather the serverless platforms provision more function instances of the serverless function to cater to increasing demand. Most serverless platforms can scale up to a certain limit and en-queue any subsequent requests until one of the previous requests finishes execution and resources are freed. A platform's ability to scale quickly, and the maximum concurrency level that it can achieve, can be very critical to applications with fluctuating demand. To observe the maximum concurrency level that a commercial platform can support, Wang et al. [122] performed a comprehensive measurement study on three major cloud providers: AWS Lambda, GCF, and Azure Function. They found that out of all three, AWS Lambda was the best, achieving a maximum concurrency level of 200 7 , while GCF and Azure Functions were unable to achieve advertised concurrency levels. FaaSdom [93], a recent benchmarking suite for serverless platforms, also found that AWS Lambda achieves the best latency in the face of an increased request rate for a serverless application -demonstrating its ability to quickly scale out. They also found that one time decisions, such as language and underlying operating system, can also affect the scalability of a serverless application. Another study [85] found that AWS Lambda and GCF perform better for varying demand when compared to IBM Cloud Function and Azure Function. We believe a platform's inability to scale well can come from the fact that scale-out is decided based on measured CPU load, a queue length, or the age of a queued message, which can take time to be logged. On the other hand, AWS Lambda launches a new function instance for a new request if current function instances are busy processing requests, as reported in [12,85]. Using this proactive approach, AWS Lambda can scale out quickly without relying on any other measured indicator. As elasticity is one of the most advertised features of serverless computing, commercial serverless platforms are striving to improve their service by offering higher concurrency limits. AWS Lambda's recent documentation indicates that concurrency limits have increased significantly (>3000) and a user can request further increase [31]. Serverless platforms, such as Apache Openwhisk and Knative from Kubernetes, allow a user to configure a containerlevel concurrency limit, i.e. number of requests that a function instance can serve in parallel (where each request runs as a separate thread) [28,35]. On the other hand, Azure Function allows a user to configure a maximum number of function instances that can be launched on a single VM to avoid the possibility of running out of underlying VM resources [13]. Schuler et al. [96] show that the container-level concurrency limit can affect the application's performance. They also suggest an AI-based (reinforcement learning) technique to configure the concurrency limit for Knative. The fact that a user can configure this particular concurrency limit on the fly also makes this limit an online decision. A user should be careful with configuring the container-level concurrency limit, as function instances running prior to making the configuration change will keep running with the old configuration (until terminated by the platform based on its settings), and only the new instances will assume the new concurrency limit. A user should wait for the system to be stable with the new configuration (i.e., all function instances with the old configuration are terminated) before making any further changes. Summary: Serverless applications can elastically scale without any additional configurations. The maximum number of function instances that can run in parallel is determined by the serverless platform and can vary based on the cloud provider. Studies have found that among commercial serverless platforms, AWS Lambda scales best in terms of throughput. CPU, Network and I/O While using FaaS, a user can only configure certain parameters, e.g. memory, CPU-power, location, and concurrency, other resources such as CPU-, network-and I/O-share are decided by the serverless platform. In [122], the authors find that in case there is no contention, empirical results show that AWS Lambda puts an upper bound on the CPU share for a function with memory m of 2m/3328, while in the case of co-location, function instances share the CPU fairly and each instance's share becomes slightly less than, but still close to the upper bound. Similarly, Google also allocates the CPU share according to the memory allocated to the function. CPU allocation in proportion to memory assigned to a function is also specified in AWS Lambda and GCF's documentation [4]. Contrary to GCF and AWS Lambda, IBM Function does not allocate the CPU share in proportion to memory allocated to the function, as reported in [93], rather it keeps it constant as an increase in memory does not affect the performance of the function. On the other hand, with Azure Function, the CPU share allocated to a function was found to be variable with the serverless function getting the highest CPU share when placed on 4-vCPU VMs. Note placement of function instances on VMs can be random from a user's perspective. In the case of co-location, the CPU share of co-located instances can drop. Similar to CPU share, disk I/O and network performance can also be affected by the resources configured for the serverless function and co-location, as reported in [44,85,122]. The performance usually improves when function instances are allocated more resources [123]. Measuring the network performance of FaaS platforms can be a challenging task considering the constantly changing network conditions and the multiple geographical regions that a developer can choose from, to deploy her serverless applications. A developer should opt for a geographical region that is closer to the intended users to reduce the access latency [123]. Our preliminary experiments also confirm this for the I/O performance, where the performance of I/O-intensive serverless functions improves when allocated more memory, as illustrated in Figure 2. Summary: The CPU, Network, and I/O bandwidth of a serverless function can be impacted by the co-location of multiple functions on the same underlying resource (VM) and the instance placement policies of the serverless platform. An application developer can run various benchmarks (or consult measurement studies) to find the most suitable provider for her application. Underlying Infrastructure In a serverless computing model, a user only focuses on writing the code, and it is the serverless platform's responsibility to execute this code on any infrastructure/hardware. A user has no control over the underlying resources (types of VM where the application code would be executed). A developer may be interested in knowing the underlying infrastructure where their serverless application would be running to optimize the performance of their applications or to make other assumptions about the running environment of their application. There have been several studies that tried to demystify the underlying virtual infrastructure for commercial serverless platforms. Lloyd et al. [87] discovered that serverless functions have access to the "/proc" file system of underlying VMs running the Linux operating system. By inspecting "/proc/cpuinfo", the authors discovered that the underlying VMs run Amazon Linux [1] and use CPUs that are similar to those of EC2 instances. Wang et al. [122] went one step further and using a similar approach, the authors conducted a wide study on all the big commercial serverless platforms, i.e. AWS Lambda, Google Cloud Function, and Azure Functions. They found that Google Cloud Function successfully hides the underlying resources and the only information they could obtain was that there are four unique types of underlying resources. By inspecting "/proc/cpuinfo" and "/proc/meminfo", they found that AWS Lambda uses five different types of VMs having different vCPUs and memory configurations, mostly 2 vCPUs and 3.75GB physical RAM, which is the same as c4.large instances from EC2. The authors also noticed that Azure Function has the most diverse underlying infrastructure. While inspecting the contents of "/proc/*", they came across VMs with 1, 2, or 4 vCPUs, and the vCPU is either Intel or AMD model. Knowing the underlying infrastructure can be helpful for developers to identify various performance-related issues. One example of that could be, a serverless function, running on Azure Function, placed on a VM with 4 vCPUs, can have more CPU share as compared to when placed on other types of VMs. Also, knowing the diversity of the underlying infrastructure can help the researcher explain the variability in performance for a given serverless platform. Summary: Serverless platforms have diverse underlying infrastructure and this can introduce significant variability in the performance of a serverless function even when executed with the same configurable resources. Careful selection of the serverless platform by the application developer, and the usage of more pro-active approaches such as COSE [44] to dynamically configure resources for serverless functions, can mitigate this variability in performance. Serverless Economic Model As discussed earlier, FaaS platforms provide two main features: 1) the ability to scale quickly in the order of milliseconds without any additional configuration, and 2) a unique "pay as you go" pricing model, i.e., a user only pays for the time the code is executing and not the idle time. The first feature helps with catering to bursty demands, i.e. when the demand suddenly increases for a short duration of time. In this section, we will have a detailed look at the latter feature and compare it with the traditional IaaS pricing model. IaaS services like Amazon's EC2 and Google's VM, have pricing models that not only charge based on minutes and seconds of usage but also have a different price per unit time as compared to their FaaS counterparts. In addition to the price factor, these VMs take extra labor to configure and maintain. Previous works [44,64,68,109] use various statistical, machine learning, and measurement-based methods to build the per-execution performance and cost model of serverless applications for a given platform. In this paper, we explain a more general analytical cost model, which in addition to perrequest cost, considers the overall cost of deployment taking into account the demand. Given the execution model of a serverless application for a certain serverless platform, pricing model, and demand (request per second), one can estimate the cost of deploying a serverless application on a commercial FaaS platform. Similarly, a user can calculate the cost of deploying a cloud application by renting VMs from a commercial cloud provider. In [16,103], the authors present an economic model of deploying an application on commercial serverless platforms (FaaS), such as AWS Lambda, and compare it with the economic model when only IaaS resources (VMs) are used to deploy the application. Specifically, the cost of FaaS based deployment can be described as: where COS T FaaS is the total cost (per second) of running an application on a serverless platform. This cost depends on the rate of function invocations (N) and cost per execution from Equation (1), for platform p and resources m allocated to each request. Similarly, the cost for IaaS based deployment (COS T IaaS ) can be calculated as follows: where N is the request arrival rate, V M r_max is the maximum number of requests that a VM can accommodate without violating SLO, and C V M (p) is the cost of renting a particular virtual machine V M from platform p. Note, in the above cost models, we do not consider the free tiers provided by the cloud provider. For example, AWS Lambda provides 1 million requests per month and 400,000 GB-seconds of compute time per month for free [7]. The key takeaways from the studies in [16,103], following the cost models given by (2) and (3), are: • FaaS platforms are cost-effective to deploy an application when the demand (request arrival rate) is below a certain threshold, referred to as Break-Even Point (BEP). Beyond BEP, IaaS resources are cheaper to use for their relatively lower cost per unit time. • The authors also consider the different execution times and resources allocated to each request for the application on both IaaS and FaaS, and show that resources allocated for the execution of each request can also affect the value of BEP. Previous studies such as [44,46,64,68] address the issue of finding the optimal resources for an application in the FaaS and the IaaS model. The cost effectiveness of FaaS for low-rate and bursty computations has been observed and reported by various studies [42,66,67] and has been leveraged by frameworks like LI-BRA [103] and Spock [76]. This unique FaaS economic model has revenue and performance implications for both the cloud provider and the application developer. From the cloud provider perspective, serverless applications are stateless, run for shorter times and a user only pays for the actual utilization of resources. This is in contrast to traditional cloud applications which are generally stateful, run for longer times and a user pays for the lease duration irrespective of the usage. To maximize its revenue, a cloud provider may host multiple serverless applications from users on the same infrastructure and periodically evict applications based on its policies, as discussed in Section 4.2. Moreover, the particular pricing model allows a cloud provider to rent out infrastructure for as little as 1ms. FaaS offering also provides greater flexibility in terms of selecting/managing the underlying infrastructure, e.g., operating system, communication protocols within the datacenter, security measures, and hardware types [67]. From an application developer's perspective, there can be multiple implications of using FaaS. First, there is an extra cost of building an application for FaaS as a traditional application may not work in this computing model. Moreover, depending on the provider, the development of serverless applications can differ significantly, i.e. code/dependency packaging is based on the runtime offered. So, a developer can suffer from vendor lock-in. Second, not only FaaS can be expensive if the demand stays high for a longer period of time, some platforms round up the execution time to, say, the nearest 100ms for cost calculation and a developer may end up paying for unused compute time, for example, if the function only runs for 50ms. Also, because of the provider's eviction policies, an application may experience frequent cold-starts, which in turn affect performance and cost. Lastly, FaaS platforms provide no strict SLO and an application can suffer from variable performance and outages [42,107]. So, a developer should carefully consider all the performance and cost implications before opting for FaaS. Summary: Serverless is more economical/efficient for applications with a low invocation rate and bursty demand. A developer should carefully anticipate the demand for her application and project the cost to decide whether FaaS is a cost-effective option for her application. FaaS Usage Even though serverless computing is a relatively new paradigm and still evolving, it has become a popular choice to deploy cloud applications. We believe that the following distinct features of serverless computing are the main reasons for its adoption and increasing popularity. F1 Development Model: FaaS allows developers to build cloud applications in high-level languages and provides API/CLIs [29,38] to package code, along with dependencies, and to deploy it on the platform, thereby facilitating CI/CD (continuous integration and continuous delivery). Most platforms also provide integrated logging systems [5,9,19,24] for debugging and monitoring. F2 No Back-end Maintenance: The serverless computing model offloads all back-end management from the application developer to the FaaS platform, which is responsible for the set-up and maintenance of underlying resources as well as scalability. F3 Pricing Model: As mentioned earlier, FaaS platforms offer a unique "pay as you go" pricing model. A user does not pay for deploying their application or for idle times. On the other hand, in an IaaS model, if a user has rented a VM, she pays regardless of the usage. F4 On-Demand Scalability: Unlike IaaS, where a developer has to configure scaling policies, serverless platforms assume the responsibility of scaling an application in case there is an increase in demand. F5 Quick Provisioning: Serverless platforms use advanced virtualization techniques, such as containers, to provision new instances of the application, which can be provisioned in the order of 10s of milliseconds [45,54,63,100,113,122]. This feature allows a serverless application to scale out, in case of increasing demand, without suffering from performance degradation. Interesting Use Cases Considering the above development, cost, performance, and management advantages, FaaS is becoming a popular service to deploy cloud applications. Developers have employed FaaS to deploy rather simple DevOps to full production scale applications [20,65,82,110]. We only present here some of the interesting use cases of serverless computing/FaaS. Malawski et al. [94] show that AWS Lambda and GCF can be used to run scientific workflows. Serverless computing can also be employed to solve various mathematical and optimization problems [50,112,124]. Moreover, on-demand computation and scalability provided by serverless computing can be leveraged by biomedical applications [80,83,84]. MArk [128], Spock [76], Cirrus [55] and others [81,117] explore deploying various machine learning applications using FaaS platforms. The authors in [70,121] leverage the higher level of parallelism offered by serverless platforms to train machine learning models. FaaS for its on-demand, cost-effective computation power and elasticity has also been explored to deploy stream processing applications [39,89]. Video processing is one such example, where a user may want to extract useful information from an incoming video stream (video frames), where for each new incoming frame a serverless function can be spawned. Sprocket, ExCamera and others [48,71,129] describe the implementation of video processing frameworks using serverless functions. Authors in [59,101,102] explore the possibility of using serverless computing for IoT applications and services. Yan et al. [125] use serverless computing to build chatbots. Aditya et al. [41] present a set of general requirements that a cloud computing service must satisfy to effectively host SDN-and NFV-based services. Chaudhry et al. [58] present an approach to improve QoS on the edge by employing virtual network functions using serverless computing. Next, we explain the development, deployment, and management challenges of a FaaS application using ML inference modeling as an example application. ML Inference Models using FaaS Since the advent of serverless computing, there have been several efforts exploring the possibility of using this computing model to deploy machine learning applications. ML inference models are one such application, where an application developer can deploy a pre-trained ML model. When a user submits her query through an API, the inference model runs in a cloud service and the result is returned to the user. For QoS purposes, the inference model should return the results within a certain amount of time hence the model execution has an SLO [75]. Traditionally, developers have employed IaaS and other specific services, e.g., Azure MLaaS [34], and AWS Sagemaker [8], to deploy such models but recently, approaches such as MArk [128], Spock [76] and others [81,117] show that FaaS can also be leveraged for such applications for its quick provisioning time and pricing model (pay per request). To deploy such an application, a developer implements the model in a high-level language such as Python and submits this code to the FaaS platform along with any dependencies/libraries [F1]. Usually, these models are Neural Networks (NN) and pre-trained models are either packaged with the code or placed in the external storage (S3 in case of AWS). When a user submits the query, the code along with the dependencies is loaded into a sandbox, and in case of cold start, it only takes few milliseconds [F5]. A developer does not have to worry about the underlying resources where the sandbox is placed as its managed by the FaaS platform [F2]. Queries can be submitted via HTTP request or other methods such as storage event, CLI, SDK, and triggers allowed by the platform. This code then downloads the model from the storage, processes the query in a serverless function, and the results are returned to the user or trigger event. A developer does not pay for the idle time in case the demand goes to zero and precisely pays for the time the model is serving queries [F3]. Moreover, when demand increases, the FaaS platform increases the number of sandbox instances, and scales back when demand decreases [F4]. During the development cycle, a developer has to make decisions related to the language as it can affect performance (cold-start and elasticity as mentioned earlier). A developer can also optimize the code by removing the extra dependencies and library code that is not being used by the application to improve the cold-start latency. Also, the queries have performance requirements (SLO), the amount of resources, such as memory and CPU, configured for the sandbox environment is crucial to get the desired performance. Lastly, based on the query arrival rate, after a certain point, FaaS may not be the most economical option to process these queries, hence using an alternate IaaS based deployment can save substantial costs for a developer. We discuss these challenges and their possible solutions in the next section. Summary: The main driving factors for serverless adoption are simple development/deployment model, quickprovisioning time, on-demand scaling, abstraction of back-end management, and true "pay as you go" pricing model. While serverless adoption is increasing, there are certain challenges that need to be addressed. Developer's Challenges In the previous section, we discussed the suitability of the serverless computing model for various classes of cloud applications. In this section, we will take a closer look at the challenges that a developer may face while importing their application into the FaaS model, and optimizations that can leverage the serverless computing model efficiently. We will particularly focus our discussion on the challenges that a developer can address with limited control (one-time and online decisions). We will also discuss the state of the art solutions suggested to tackle these challenges and what remains unsolved. The discussion and insights presented in this section can help both the developer and researcher to optimize the FaaS usage and build new tools. FaaSification and Decomposition FaaS development and computing models significantly differ from traditional IaaS models. Hence, to deploy a legacy application using FaaS, a developer has to translate the application into this unique model. Recently, there have been approaches such as [62,104,114] that aim to automate this process for applications written in various languages. As pointed out by Yao et al. [126], these approaches either work for selected parts of the application or fail to leverage some of the key performance benefits offered by FaaS. In particular, these approaches replace a selected part of an application with a Remote Procedure Call (RPC) and deploy the selected part as a serverless function. While helpful to quickly deploy legacy applications using FaaS, these approaches miss taking advantage of the elasticity feature offered by FaaS. We believe that an ideal FaaSification tool should not only consider producing the FaaS counterpart of the application but also leverage the elasticity offered by FaaS platforms. For example, through static/dynamic code analysis, the tool should identify the parts of an application that can be parallelized and generate corresponding serverless functions. Now, given a FaaSified version of an application, how can a developer leverage multiple FaaS platforms to optimize performance and cost? FaaS platforms offer diverse features, e.g., elasticity limits, supported languages, configurable parameters, pricing models, etc. Moreover, as we have seen in Section 4, these platforms have varying underlying infrastructure and resource provisioning policies [122]. As a result, the performance and cost of the same application can vary significantly across different serverless platforms. In [51], the authors show that serverless functions with different bottlenecks, such as memory and computation, may have an ideal serverless platform on which they perform the best. This shows that serverless platforms are not one-for-all. Considering an application, which comprises multiple serverless functions with varying compute, memory, I/O bottlenecks, one platform may not suit all of the individual functions. We suggest investigating this idea further, where automated tools may help developers decompose their application into multiple serverless functions and then find the ideal serverless platform for each serverless function. This may require a sophisticated tool to perform code analysis [53] and measurement tools [93,127] which can benchmark serverless platforms for different kinds of workload/computations. Moreover, serverless platforms allow users to configure resources for each component of an application (if deployed as separate serverless functions), which may not be possible for a monolithic application deployed over a VM. In [127], the authors show that decomposing a monolithic application into multiple micro-services, instead of deploying the whole application as one unit, can lead to significant performance and cost gains. The authors also show an example application where decomposition leads to better performance and less cost. We also believe that decomposing an application would allow developers to cost-effectively fine-tune resources for various parts of the application. To the best of our knowledge, we did not come across any previous work that suggests decomposing monolithic serverless applications across multiple providers to optimize the cost or performance. Costless [68] is the closest approach that suggests deploying a serverless application split across two platforms (edge and core) but it assumes that the application is already decomposed into multiple serverless functions. Code Pruning Another optimization that a developer can introduce is to optimize the code of the serverless application and remove any extra dependencies or unused library code. Usually, developers package a whole library along with the code even when the application is utilizing a small portion of it. This can adversely affect performance -as the application code is loaded into the sandbox environment at run time, studies [15,78] have shown that the code package size can affect the cold start. Moreover, because the sandbox environment has limited resources, pruning extra code can also improve the performance of a serverless application. Nimbus [57] is one such framework that performs code optimization for serverless applications written in Java. We believe that this approach can be extended to other more popular languages used for serverless applications such as Python and JavaScript. Parameter Tuning On commercial serverless platforms, a user can only specify limited configurable parameters, such as memory, CPU, and location, for a serverless function. In Section 4, we discussed that measurement studies show that these configurable parameters can affect the cost of cloud-usage and the performance of serverless functions. As serverless platforms do not provide any guarantee (SLO) on the performance of serverless functions, configuring the parameters becomes even more crucial to get the desired performance of an application and optimize cost. There have been a number of proposals suggesting various offline and online techniques to configure these parameters. Costless [68], given a workflow consisting of multiple functions, proposes a technique to efficiently distribute these functions across the edge-and core-cloud while reducing the cost of cloud usage and meeting the performance requirement. This approach relies on (one time) profiling of the performance of a serverless function in the workflow under possible memory configurations. It suggests suitable configurable parameters (memory) based on the profiling results, however, it fails to capture the dynamicity of the execution model. In [108], the authors show that the per-container concurrency limit in Knative can affect the throughput and latency of serverless functions. They suggest a reinforcement learning-based approach to find the optimal concurrency limit for a given deployment of the application. Even though this approach is adaptive, it only targets configuring the concurrency limit, but as discussed earlier, other parameters such as memory, CPU, and location can also impact performance. Moreover, we noticed that the authors did not address the feedback delay issue, which for Knative, in our experience, can be up to several minutes depending on the configuration. Sizeless [64] uses resource-consumption data from thousands of synthetic serverless functions to build a representative performance model. Then, using the performance model and performance logs of the target function, it suggests the best memory configuration. This approach may incur significant cost overhead for running thousands of synthetic functions to get the required data to build the performance model. This approach also requires changes in the serverless application to collect the performance logs and only targets configuring memory for a function written in Node.js and deployed over AWS Lambda. We believe that an ideal configuration finder should be a feedback control system, as illustrated in Figure 1, It should continually monitor the performance of serverless applications and configure these parameters on the fly if needed. There are a number of challenges for designing such systems: 1) serverless platforms have varying underlying infrastructure, resource provisioning policies, sandboxing techniques, and every time a serverless function is invoked, even with the same configurable parameters, performance can vary based on the co-location of functions and underlying resources. This makes it hard to predict the performance of the serverless function; 2) Our experiences with GCF and Kubernetes Knative, show that there can be a significant delay in the feedback loop, i.e. after the configuration is changed and until the new configuration takes effect (up to minutes as mentioned in Section 4.4). This excessive feedback delay can lead to performance instability as the state of the system might change during that time; 3) The impact of the changes in allocated resources on the performance of a serverless function can vary depending on the underlying serverless platform. In our experiments, we noticed that while an increase in allocated memory/CPU improves the performance of a serverless function on AWS Lambda and GCF, it did not significantly affect the performance on Apache OpenWhisk and IBM Function. Maissen et al. [93] make a similar observation about IBM Cloud Functions. COSE [44] is an online statistical learning technique to configure various configurable parameters for delay-bounded chains of serverless functions or single functions. COSE not only achieves the desired performance for a serverless application but also reduces the cost of cloud usage. It can capture the dynamic changes in the execution model stemming from co-location and variable underlying infrastructure. COSE can be easily adapted for other parameters and platforms because it works as a stand-alone system that requires no changes to the serverless application. While COSE addresses most challenges of parameter configuration, it considers similar input sizes across multiple function invocations. COSE may not perform well if the input of the serverless application has large variations as the input size can affect the execution time. Multi-Cloud Usage Serverless functions are executed in light-weight sandbox environments, which can be launched in as few as 10s of milliseconds. So, in case an application experiences a sudden increase in demand, it can seamlessly scale out to cater to the increasing demand. This is a feature of serverless comput-ing that has been leveraged by previous approaches, such as MArk [128], Spock [76], and FEAT [99], to hide the SLO violations for cloud applications deployed using traditional cloud services such as VMs. These approaches redirect a portion of the demand to the serverless counterpart of the application while scaling up traditional cloud resources which can take up to minutes to start up. These approaches may improve the performance of an application by reducing the number of SLO violations during scaling, at the expense of introducing a substantial development cost for a developer to build the serverless counterpart of the application. To reduce the development cost, a developer can employ an automated approach to build the serverless version of the application, similar to the approach suggested in [62,114]. Another limitation of these approaches is that they suggest a one time configuration of resources for the serverless version of the application, which can lead to variations in the performance as explained in Section 7.3. As the goal of such approaches is to reduce the SLO violations, this variation in performance can adversely affect the application. We believe that in addition to performance, serverless computing also offers a unique pricing model, and as discussed in Section 5, serverless computing can be cost-effective for certain demand. For applications with large variations in demand, deploying them on VMs for the periods when demand is low can lead to sub-optimal cost. We propose to build a hybrid framework (Load Balancer, as illustrated in Figure 3) that leverages both aforementioned features of serverless computing, i.e. performance and economic model, by using serverless computing as: (1) an alternative to traditional cloud resources for a certain portion of the demand (consistently instead of only while scaling up VM resources), and (2) a fallback to the serverless counterpart of the application when demand is below BEP. To address the performance uncertainty in the serverless platform, we suggest that in addition to the multi-cloud framework, the developer should also employ more pro-active approaches, similar to COSE [44], to configure resources for the serverless counterpart of the application. COSE suggests the configuration for a serverless application that not only reduces the cost of cloud usage but also meets the specified SLO. We also believe that serverless functions can be used as an alternative to VMs to offload the lightweight computations in a distributed application such as scientific workflows [94], where small tasks requiring more concurrency and elasticity can be implemented as serverless functions while keeping the tasks with longer computation time and requiring more resources on VMs. One can leverage the "utilization" of the computation, i.e. how long the computation is and how often it needs to be executed, to decide whether the computation should be directed (and executed) over a dedicated VM or a serverless platform. The problem is how to optimally distribute computations to minimize the total cost. This is a challenging problem given the inherent performance-cost tradeoffs: VMs are cheaper for high-utilization (long-running and frequent) computations, on the other hand, serverless platforms are cheaper for low-utilization (short-running and less frequent) computations and have the advantage of elasticity. Finally, developers have indeed started to leverage services from different cloud providers. A case study is presented in [20], where an invoicing application is built using various best-in-class services from different commercial cloud providers. The application is built using Google's AI and image recognition services along with two of Amazon's services (Lambda and API Gateway). Summary: During the life cycle of a serverless application, a developer has to address various challenges starting from developing/porting the application in/to a relatively new programming model. To further optimize, a developer can also perform application decomposition and code pruning. She can also rely on various online/offline techniques to configure resources for her application to get the desired performance and optimize cost. Finally, depending on the usage, FaaS may not be the most economical option to run the cloud application, hence a multi-cloud scenario can help applications with fluctuating demand, without compromising on cost and performance. Conclusion Serverless computing has gained significant popularity in recent years. It offers an easy development model, back-end management, along with key performance benefits and a "pay as you go" pricing model. There is a significant amount of research articles addressing various aspects of serverless computing such as benchmarking/improving performance of commercial and open-source serverless platforms, new virtualization techniques for the execution environment, and studying the feasibility of serverless computing for a variety of cloud applications. In this paper, we look at these studies from an application developer's perspective and discuss how these studies can help her make informed decisions regarding her serverless application. We argue that serverless computing is becoming a popular choice to deploy various cloud applications for its distinct cost and performance benefits. While serverless adoption is pacing up, there are still a number of challenges that need to be addressed. We identify potential challenges and open issues that must be addressed to make serverless computing a viable option to deploy cloud applications. We argue that pro-active approaches to configure resources for serverless functions can address the performance uncertainty issue, while frameworks to decompose serverless applications and to leverage various cloud services at the same time can reduce the operational cost as well as enhance the performance of cloud applications.	2022-02-18T16:14:19.076Z	2021-09-22T00:00:00.000	{ "year": 2021, "sha1": "ebbd18615d74ff625a09ea96108e65b83be86b78", "oa_license": "CCBYNC", "oa_url": "https://escholarship.org/content/qt1wg7h0qf/qt1wg7h0qf.pdf?t=r7eku8", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "b10a25016fc3b5c81a7cb68499fc56d7a3d6845c", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [] }
12465854	pes2o/s2orc	v3-fos-license	Intestinal Ascending Colon Morphometrics in Rats Submitted to Severe Protein Malnutrition This paper aims to verify the effects of severe protein malnutrition over the intestinal ascending colon morphometrics in adult rats. 12 rats (90 days old) were divided into 2 groups: control (n = 5) and malnutritioned (n = 7). In the following 90 days, the rats of the control group received a 24% protein chow as the malnourished group received 4% protein chow. The animals were submitted to euthanasia according to the anesthetic protocol. Colon segments were collected and submitted to routine histological processing. The cuts were stained with HE and histochemical techniques for mucines. The morphometric analyses showed the sustenance of the whole wall and muscle tunic thickness, as well as the reduction of the thickness of the mucosa tunic, the amount of goblet cells, the depth of the crypt and the height of the enterocytes as well as their nucleus on malnutritioned animals. The data suggest that protein malnutrition causes alterations on adult rat ascending colon intestinal morphometrics, especially in tissues which present a high level of cell turnover such as the mucosa tunic and consequently their structures such as the enterocytes, goblet cells, and crypts. INTRODUCTION The implications of malnutrition on different organs and body systems may be different according to the kind of organ and age.In the gut, protein malnutrition causes gastric and intestinal mucosa tunic atrophy, villous and microvillous decrease, of the intestinal traffic which may cause constipation, and diarrhea, in cases of compromising of the immunologic system (Waterlow, 1996). Protein malnutrition experimental studies on different-age rat showed that the small intestine presents alterations demonstrated by the reduction of the intestinal wall layer thickness (Natali et al., 1995(Natali et al., , 2000(Natali et al., , 2005;;Torrejais et al., 1995) as well as the reduction of the thickness of the mucosa tunic and enterocyte height (Brandão, 1998). There are a few studies reporting the effects of protein malnutrition on the large intestine which despite of its minor nutrient absorption function, plays an important part on digestion, and most of all, absorption of water and electrolytes, fecal matter traffic, and production of fat-soluble vitamins.Firmansyah et al. (1989) demonstrated that malnutrition also compromises the development of the colon mucosa tunic and its crypts on animals during fetal development. This study analyzed the effects of severe protein malnutrition on adult rat ascending intestinal wall colon morphometrics. MATERIAL AND METHOD The experimental protocol was previously approved by the Animal Research Ethics Committee of the Universidade Paranaense (UNIPAR). All animals had their ascending colon extracted, measured, weighed, then washed in saline solution, and fixed in a 10% buffered formalin in phosphate buffer (pH 7.3).The ascending colon segments were dehydrated in an ascending series of alcohol, diaphanized in xylol and paraffin-embedded for further 3 and 5µm transversal cuts stained with hematoxilin-eosin, Alcian Blue (AB, pH 2.5), and periodic acid Schiff (PAS). The intestinal wall morphometric analysis was carried out from images captured using a Motic B5 optical microscope and digital camera (Moticam 2000, 2.0 M Pixel). Images covering all intestinal circumferences were captured.80 measurements were carried out over all intestinal circumferences of each rat, as 40 measurements were carried out over the crypts.Images captured with 4x objective lense were used to measure the total thickness of the intestinal wall.The images captured with 20x objective lense were used to measure the muscle tunic thickness, the thickness of the mucosa tunic, and the depth of the crypts.HE stained cuts were used for these measurements.Images from ABstained cuts were captured with 100x lenses in order to measure the nucleus largest diameter, as well as, the height of 960 enterocytes (only the ones presenting nucleolus).All measurements were carried out with Motic Images Plus 2.0. Besides the measurements above, the goblet cells in 0.2 mm 2 of the mucosa tunic of every animal were quantified.This quantification was carried out through stained cuts by HE, AB (pH 2.5), and PAS.The account was carried out from images captured using a 40x objective lense. All data was first submitted to the Kolmogorov-Smirnov Test in order to verify the distribution type.Data showing normal distribution are expressed as mean±SD, the others are expressed as median (P25; P75).We used t-Test (normal distribution data) and Mann-Whitney (others distributions) in order to compare the control group and the malnourished group.P values less than 0.05 was considered statistically significant in both cases. RESULTS The control group weighed 332.17±55.87g,as the malnourish group weighed 200.36±28.92gby the end of the experiment.A significant reduction of 34.23% for the animal weight of the malnourish group was observed (p<0.0001). The ascending colon for the malnourish group was smaller in relation to the control group, as presented in Table I. The morphometric analysis did not present any significant difference occurrences for either the total wall thickness or the muscle tunic thickness for both groups, as well as a reduction of the thickness of the mucosa tunic, depth of the crypts, height of enterocytes and their nucleus height for the malnourished animals (Table II). The quantification of the goblet cells by the HE, AB, and PAS techniques presented significant differences while comparing the control group and the malnourish group as shown in Table III. DISCUSSION The animals in this experiment, which were fed with 4% protein chow, presented less body weight gain as observed by several authors using different protein restriction models in young rats (Takano, 1964;Firmansyah & Suharyono, 1969;Torrejais et al.;Natali & Miranda-Neto, 1996;Sant'Ana et al., 1997;Brandão;Meilus et al., 1998;Fiorine et al., 1999;Natali et al., 2000;Zanim et al., 2003) thus demonstrating that the diet protein levels (for either quantity or quality) are essential for body weight gain during development.However, studies on old rats fed with a 8% protein diet did not present any body weight reduction, possibly because such animals present lower metabolic level and less protein intake would be enough for their sustenance (Natali et al., 2005;Schoffen et al., 2005). The gut contributes for only 3 -6% of the total body weight; however, it is the organ responsible for around 20 -35% of the protein turnover as well as the energy expenditure of the entire body (Lobley et al., 1980;McNurlan & Garlick, 1980).Therefore, it is natural to have observed that the colon weight, length, width, and area in malnourished rats were significantly low what also was observed in protein-restricted experiments on the small intestine (Firmansyah & Suharyono;Younoszai & Ranshaw, 1973;Firmansyah et al.;Torrejais et al.;Natali & Miranda-Neto;Meilus et al.;Natali et al., 2005) and rat colons (Firmansyah et al.;Sant'Ana et al., 1997 and2001;Araújo et al., 2003).Deo (1978) states that malnutrition compromises the organ development, and Natali et al. (2005) considered that a smaller organ may be interpreted as an adaptive response to a smaller food intake.Table II.Total wall, muscle tunic and thickness of mucosa tunic, depths of the crypts, height of enterocytes and theirs nucleus for enterocyte of the ascending colons of rats from the control group and the malnourished group. The total wall, muscle tunic, height of enterocytes and their nucleus are expressed as mean±sd.The thickness of the mucosa tunic and the depth of the crypts are expressed as median (P25; P75).p<0.01;*p<0.0001.While comparing other colon-related studies, we verified that the colon was 40% smaller in our control group, as it was 31.5% smaller in Sant' Ana et al. (1997).Such difference can be explained because the amount of protein (8%) used in this study was doubled in relation to ours. The ascending colon wall morphometric analysis showed muscle tunic thickness preservation in animals fed with 4% protein.This result is similar to Schoffen et al. who reduced the proteic level to 8% for old rats.Other authors observed muscle tunic thickness reduction (Torrejais et al.;Natali et al., 2000;2005;Brandão et al., 2003), what may be assigned to not having proteic material accumulated on the cells of the muscle tunic (Schoffen et al.) since they were still growing animals.Natali et al. (1995), Torrejais et al. and Brandão (1998) malnourished rat duodenal wall during pregnancy and lactation, which are critic periods for rat intestine development.Our results, as in Schoffen et al., showed no significant differences for that parameter, possibly, because in nutrient privation cases, the organism is forced to reorganize itself metabolically by selecting priorities for the distribution of the insufficient nutrients available for tissues and organs.For a possible recovering -aimed by any organism under privation -the tissues somehow injured did not heal homogeneously and synchronically, however, because of some tissue singularities, it is notorious that some tissues will reestablish quickly, such as, the connective and epithelial tissue whereas others slowly such as the muscle and nerve tissues.This may explain the muscle tunic sustenance in spite of the malnourished rat mucosa tunic observed in this study. The mucosa tunic of the colon is substantially thicker due to the larger intestinal gland length in relation to the small intestine.Since there are not any villous, the surface of the mucosa tunic is smooth presenting an increasing number of goblet cells (Dellmann & Brown, 1982).The reduction of the thickness of the mucosa tunic results from recurrent investigations analyzing the effects of the nutritional deficiencies over the intestinal morphometrics (Viteri & Schneider, 1974;Rodrigues et al., 1985;Torrejais et al.;Brandão, 1998;Natali et al., 2000;Schoffen et al.).This might be explained by the fact that the mucosa tunic presents high regenerative capacity of its own tissues; therefore, less aminoacid intake would be assigned to low regenerative tissues in order to prevent permanent damage and to avoid the nitrogenic balance to become negative. The depth of the crypts was smaller in malnourished animals in relation to the Control Group (p<0.05), as observed on the colon of rats by Firmansyah et al., and on the small intestine of rats by Brandão (1998).This decrease may possibly be related to the mucous reduction as the crypt is in the intestinal layer.As the crypts present basal cells capable of dividing several times by mitosis and differentiate amongst the number of intestine epithelium, our results suggest that the regeneration of such tissues was damaged. The height of the enterocytes and their nucleus largest diameter was measured.The enterocyte is the epithelial cell of the intestinal mucosa tunic, which carries out the absorption, thus, its number will determine the epithelium absorption capacity (Brandão, 1998).Malnutrition may result in intestinal absorption decrease due to the enterocyte number and height decrease.The experimental malnourishment carried out in this study resulted in the reduction of the enterocyte height and their nucleus, as observed in rats feed with a protein-restricted diet by Brandão (1998).Pires et al. (2003) did not find significant differences while analyzing the enterocyte height of children's small intestines; however, they pointed out to have found a disposition to the height decrease of such cells according to the increase of malnourishment levels, what is close to our findings since the diet proteic levels were 85% less than those for the control group in our experiment.As the colon is responsible for electrolyte and water absorption, malnutrition may be damaging this physiological function in the animals in this study, however, diarrhea was not observed. Because of enterocyte height reduction, microorganism at the lumen may easily invade the organism through the transcellular pathway.Thus, reinforcement for the adhesion of these cells in order to prevent the attack through the paracellular pathway would be natural.Therefore, these cells would be strongly attached to each other through the cellular adhesion molecules.An immunohistochemical study with intra-uterine retardedgrowth rats submitted to protein malnourishment during pregnancy demonstrated an cadherin positive epithelial cell increase (probably enterocytes) in villous (Dalçik et al., 2003).These results may indicate more care regarding positioning, recognition, and epithelial cell unit coating the intestine of protein-deprived animals during pregnancy as a mechanism against adverse conditions. When the goblet cells were quantified -in spite of the chemical nature of their content (HE stained cuts) -we verified they significantly decreased in the control group (around 42.34%).Thus, we infer that the volume of the secreted by these cells (mucines) was smaller for the malnourished group, what may be understood as a way of endogen protein loss contention as well as the assignment of less aminoacid intake for cells, tissues, organs, and metabolic pathways essential for animal survival. Mucines are related to both physiology and gastrointestinal health (Forstner & Forstner, 1994).The proteic part of such molecules is mainly constituted by serine and presents binding sites for oligosaccharides, such as Nacetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acids.According to the carbohydrate composition, the mucines are classified in (1) neutral and the acid subtypes: sulphated and non-sulphated (3).The proportion amongst these classes may be influenced by the diet (Montagne et al., 2004). The impact of protein in the diet over the composition of the mucines has not been largely studied.In this study, the number of PAS positive cells was smaller (37.9%) as well as the AB (pH 2.5) positive cells (32.27%) in the malnourished group.On the other hand, the proportion between acid and neutral mucous was 3.95 for the control group, whereas it was 4.24 for the malnourished group.This probably means that the ascending colon mucous in the animals of the malnourished group was less dense what might have influenced a higher interaction amongst lumen and microvillus molecules thus favoring nutrient absorption.On the other hand, this kind of mucous committees the mucous protection by favoring eruptions, as well as the increase the risk of lesions, ulcerations, and inflammatory diseases, what is important to be considered in relation to the intestinal lumen -an unfriendly environment to the colon.Turck et al. (1993) reported that the composition of the mucine in the colon of 21-day-old shoats milked naturally and artificially.The mucine from the shoats milked by female pigs presented more fucose and glycosamine and less sulphate, thus considered more mature than the mucines from animals artificially milked.It is worth to remark that quantitative and qualitative alterations concerning secretion, composition, and removing of intestinal lumen mucines may modify the defense barrier of the mucosa tunic as well as influence the colon's fermentation capacity what may result in important physiological implications (Smith & Podolsky, 1986;Rhodes, 1989). Intestinal growth and crypt increasing imply in the intestinal epithelial cell increasing and further absorption increase (Natali, 1993); therefore, our results demonstrated that the smaller growth for the organ with reduction of the mucosa area may result in the intestinal absorption damaging.The decrease of the crypt depth and the number of goblet cells has probably interfered in the normal functioning of the mucosa, its regeneration, and nutrient absorption, demonstrating that severe malnutrition reflects harmfully for the large intestine in adult animals. Table I . Length, width, area and weight of the ascending colon from rats of the control group and malnourished group. Table III . Number of goblet cells in a 0.2mm 2 area of the ascending colon from rats of the control and malnourished groups evidenced by hematoxilin-eosine (HE), Alcian Blue (AB, pH 2.5), and periodic acid Schiff (PAS). HERMES, C.; AZEVEDO, J. F.; ARAÚJO, E. J. A. & SANT'ANA D. M. G. demonstrated that the decreasing of the Intestinal ascending colon morphometrics in rats submitted to severe protein malnutrition.Int.	2017-09-26T18:03:30.610Z	2008-03-01T00:00:00.000	{ "year": 2008, "sha1": "ec6c9602c1e2b295452528b88b6c1a60f2834897", "oa_license": "CCBYNC", "oa_url": "http://www.scielo.cl/pdf/ijmorphol/v26n1/art01.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "ec6c9602c1e2b295452528b88b6c1a60f2834897", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Medicine", "Biology" ] }
67861338	pes2o/s2orc	v3-fos-license	HIV-1 Escape from Small-Molecule Antagonism of Vif Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation. permissive cells express a dominant inhibitor of HIV-1 replication that is absent in permissive cells (6,7). Shortly thereafter, CEM15 was isolated from nonpermissive CEM cells, shown to be sufficient to confer the nonpermissive phenotype, and identified as APOBEC3G (A3G), a single-stranded DNA cytidine deaminase (8). HIV-1 has acquired Vif to counteract the highly potent, intrinsic antiviral activities of APOBEC3 proteins, and A3G in particular, that are expressed in the natural cellular targets of HIV-1 infection (reviewed in reference 9). In the absence of Vif, A3G is packaged into virions and deaminates cytidines in the viral minus-strand DNA formed during reverse transcription, resulting in lethal G-to-A hypermutation (10)(11)(12). There is also evidence that A3G packaged into newly formed virions inhibits reverse transcription through a deamination-independent mechanism (13,14). To counteract APOBEC3 inhibition, Vif recruits an E3 ubiquitin ligase complex to APOBEC3 proteins to promote their polyubiquitination and subsequent proteasomal degradation (15,16). Because of the absolute dependence on Vif for viral replication in the host, Vif remains an attractive and yet elusive antiviral target. Small molecules that uncouple the binding of Vif to APOBEC, or that disrupt the formation of the E3 ubiquitin ligase complex, have the potential to act as novel antivirals by promoting the stability of APOBECs. Thus, agents targeting the biological activity of Vif would activate a potent natural defense that could expand clinical treatment options and possibly lead to better strategies aimed at eradication of viral reservoirs. We previously described a small-molecule antagonist of Vif (RN18) that was identified in a cell-based assay aimed at identifying compounds that stabilize A3G in the presence of HIV-1 Vif (17). Initial screening was based on cotransfection of 293T cells with a yellow fluorescent protein fused to A3G and wild-type (wt) or Vif-deleted vectors. Using this system, small molecules with inhibitory activity were identified through increased yellow fluorescent protein (YFP) signal and nonspecific effects could be excluded based on comparison to the fluorescent signal in matched cotransfections using Vif-deficient vector. RN18 exhibited specific, A3G-dependent antiviral activity that was manifest only in nonpermissive cells (expressing A3G) and not in permissive cells devoid of A3G expression (17). On the basis of the structural scaffold of RN18, additional compounds have been synthesized and screened to identify analogs of RN18 with improved antiviral activity. These compounds have been used to generate inhibitor escape mutants that provide evidence for target validation and delineate mechanistic aspects of Vif antagonist resistance. RESULTS To enhance the potency and metabolic stability of RN18, we investigated the isosteric replacement of the amide functionality in RN18. We designed a series of conformationally restricted, biocompatible, and metabolically stable isosteric heterocyclic systems. We synthesized four test molecules by substituting the amide functionality in the lead molecule with isosteric heterocyclic systems such as 1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,4-disubstituted-1,2,3-triazole, and 1,5-disubstituted-1,2,3-triazole. Synthesis of RN18 analogs containing isosteric heterocycles resulted in the discovery of a 1,2,3-triazole as a preferred scaffold for inhibition of HIV-1 Vif function and restoration of A3G levels in nonpermissive H9 cells (18). Further structure-activity relationship (SAR) studies were performed by synthesizing libraries of compounds with preferred scaffolds to identify a number of analogs with the desired antiviral activity profiles, i.e., antiviral activity in nonpermissive (H9) but not in permissive (MT4) cells (18). Three compounds, IMC15, IMC89, and IMB63-II, were prioritized because of their antiviral specificity and potency as assessed by spreading infections in a cell-based assay. Chemical modifications to RN18 leading to increased potency are presented in Fig. 1. HIV-1 replication was potently inhibited in a dose-dependent manner in nonpermissive cells (Fig. 2a to c), but no inhibition was observed in permissive cells (Fig. 2d to f). Half-maximal inhibitor concentrations (IC 50 s) were determined for the compounds and relative to those of RN18 (5.7 M), each of the analogs exhibited increased potency (IMC15, 2.61 M; IMB63-II, 2.15 M; IMC89, 0.62 M). To validate the mechanism of action, each of the Vif antagonists were used to generate inhibitor escape mutants. Wild-type HIV-1LAI (GenBank accession number K02013) was passaged in nonpermissive H9 cells in stepwise increasing concentrations of the analogs to generate variants with the capacity to replicate in high concentrations of analogs. Previous experiments using inhibitor concentrations ranging from 1 to 50 M showed dose-dependent reductions in wild-type virus replication and substantial inhibition at the lower concentrations used (Fig. 2). On the basis of these data, passage 1 H9 cells infected with wild-type HIV-1 were maintained in culture medium containing inhibitors at a 1 M final concentration. Increases in inhibitor concentrations were gradual and dependent on the ability of the virus to overcome inhibition at a given concentration. Similar cultures were maintained in parallel without addition of the inhibitor to assess changes in the virus occurring spontaneously during prolonged H9 cell passage. Transition from one passage to the next was initiated when the level of virus production from treated cells was like that observed for cultures not maintained under conditions of selective pressure. The durations of the passages differed depending on the inhibitor concentration used but generally fell within a range of 2 to 4 weeks. New passages were initiated by exposing uninfected H9 cells to filtered culture supernatants from the previous passage with the addition of elevated concentrations of drugs. After 16 passages (ϳ11 months), viruses had the capacity to replicate efficiently in H9 cell cultures containing 100 M IMC15, IMC89, or IMB63-II. Replication profiles were similar for all analog-resistant viruses, and representative data utilizing IMC15 are presented for nonpermissive (Fig. 3a) and permissive (Fig. 3b) cell infections. While virus passaged in the presence of analogs exhibited resistance to those analogs, the analog-resistant viruses remained sensitive to RN18. The IC 50 values for wild-type and V142I (IMC15-resistant) viruses were 2.6 M and Ͼ25 M, respectively. In contrast, The V142I mutation did not affect the IC 50 for RN18, which was ϳ5.5 M for both the wild type and the V142I variants. For comparison, the IC 50 values for the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) efavirenz was Ͻ1 nM for the wild-type, RN18 analog-resistant (RAR), and V142I variants. Since we expected resistance to a Vif antagonist to develop because of changes in Vif, the vif gene was sequenced throughout the culture period. At alternating passages, the vif gene was amplified from cell cultures containing inhibitors and sequenced to detect changes that might confer resistance. Purified viral RNA was amplified by RT-PCR using primers that flank Vif, and amplification products were purified and submitted for Sanger sequencing by Genewiz (South Plainfield, NJ). Sequence trace file peak heights were used to estimate the relative amounts of wild-type and mutant virus present in the cultures at the alternating passages. For example, representative results showed that wild-type V142 (codon GTA) transitioned to V142I (codon ATA) in the presence of increasing amounts of IMC15 during long-term passage (see Fig. S1 in the supplemental material). As summarized in Fig. 4a, exposure to each of the RN18 analogs resulted in the rapid selection of isoleucine for valine at position 142 in Vif. Interestingly, this valine is part of a hydrophobic interaction domain immediately adjacent to the Vif SOCS box that promotes binding to EloC (15,16). There is a binding pocket for V142 in EloC, and alteration at that site might influence the binding of Vif to EloC. To further explore the binding of Vif to EloC, structural modeling was used to define antagonist binding with and without the altered side chain at Vif amino acid 142. To visualize the interactions of IMC15 with the Vif-CBF-␤-CUL5-EloB-EloC complex, the compound was docked to a crystal structure of the complex (PDB identifier [ID] 4N9F) (19) using the molecular modeling program Schrödinger (20)(21)(22). The docking grid was defined as a 25-Å cube centered on coordinates x ϭ 25.19, y ϭ Ϫ14.1, and z ϭ Ϫ96.93, near V142 of Vif. As shown in Fig. 5, IMC15 is predicted to bind to the complex primarily through interactions with Vif, although some Van der Waals interactions with loop 5 of EloC are also observed. IMC15 is primarily stabilized through -cation interactions with Arg 167 of Vif. To understand how IMC15 might disrupt binding of Vif to A3G, the molecular modeling program Coot (23) was used to model the Vif-APOBEC interface, aligning residues from each protein known to be critical for binding (PDB ID 4N9F and 5K81) (19,24,25). Fig. 5a shows the modeled Vif-CBF-␤-CUL5-EloB-EloC complex with A3G, where IMC15 clearly interrupts binding interactions between Vif and A3G. Further detailed analyses revealed that primary Van der Waals interactions were stabilized with Val 142 and -cation interactions with Arg 167 (Fig. 5b). To understand how the V142I mutation would affect binding of IMC15 to Vif, the mutation was generated using the Prime module of Schrodinger and IMC15 was again docked to the protein using a 25-cubic-Å grid centered on the coordinates x ϭ 25.19, y ϭ Ϫ14.1, and z ϭ Ϫ96.93. In this instance, no favorable binding interactions were observed, suggesting that IMC15 is unable to bind to the site with that mutation. Loss of inhibitor binding would restore the ability of Vif to mediate the degradation of A3G, contributing to the observed resistance to the Vif antagonists. Similarly, RN18 was docked against the Vif-CBF-␤-CUL5-ELOB-ELOC complex to compare its predicted binding pose with that of IMC15. As shown in Fig. S2, the predicted binding pose for RN18 showed only weak Van der Waals interactions with Vif and was stabilized through interactions with EloC, namely, electrostatic interactions with Glu 92 and a weak hydrogen bond with the backbone of Ile 95. Modeling with the V142I Vif mutant complex also indicated little change in the expected binding mode of RN18. The Van der Waals contacts observed between RN18 and EloC in the wt Vif complex are maintained with the V142I mutant complex, with only a slight change in orientation of the anisole ring in RN18 observed. No change in contacts was expected HIV-1 Vif Inhibitor Resistance ® between V142 and the V142I mutation. Therefore, it is likely that RN18 binds to a site that is different from that mediating IMC-15 binding on the Vif-CBF-␤-CUL5-ELOB-ELOC complex. To identify additional changes that might contribute to inhibitor escape, subgenomic regions of viral RNA from several passages were amplified and sequenced. Sequencing revealed polymorphisms that were not present in the control virus that could potentially contribute to the resistance phenotype. A single nucleotide change in the long terminal repeat (LTR) converted the last cysteine residue in Nef to a stop codon. In addition to changing Nef, the C-to-A transversion at genomic position 9007 LAI potentially modifies an enhancer element important for transcriptional activation (26,27). Treatment with each of the inhibitors resulted in the selection for this mutation, although with delayed kinetics relative to the V142I substitution in Vif (Fig. 4b). The polymorphism maps to the binding site for lymphocyte enhancing factor-1 (LEF-1; Los Alamos HIV sequence database) and may function to increase the transcriptional activity of the virus (28). Vpr was also inactivated during passage of virus in our H9 cell cultures containing each of the analogs. However, it is unclear whether this contributes to the resistance phenotype since Vpr was also lost during passage of virus in the absence of antagonist (Fig. 6). Vpr is cytostatic, FIG 5 In silico model of IMC15 bound to Vif in complex with the E3 ligase and A3G. IMC15 is rendered in stick form, with surface density represented in gray (a). Vif is rendered as a dark blue ribbon, with EloB in orange, EloC in light green, CBF-␤ in light blue, Cullin 5 in red, and A3G in dark green. IMC15 binds Vif in proximity to the APOBEC binding region, destabilizing Vif-APOBEC interactions. IMC15 makes primary stabilizing Van der Waals interactions with V142 and -cation interactions with Arg 167 (b). IMC15 is rendered in stick form, with surface density shown in gray. Vif is rendered as a dark blue ribbon, with CBF-␤ as a light blue ribbon. Sharkey et al. ® and inactivation of vpr during long-term culture of HIV-1 is a well-recognized phenomenon (29). Additional mutations were noted at some passages but appeared to be stochastic. The V142I Vif and C9007A and Vpr null changes emerged early and were maintained throughout the culture period ( Fig. 4 and 6). Since all three antagonists drove the development of the same mutations, a single full-length virus clone was generated and sequenced. Virus collected at passage 22 in H9 cell culture containing 100 M IMB63-II was used to infect fresh H9 cells. Total DNA was purified from cells 10 days postinfection, and two halves of the proviral DNA were PCR amplified and recombined to generate a full-length virus clone. In addition to the key mutations, several other changes were found within the cloned genome; a complete list is presented in Table 1. We noted that the replication kinetics of the cloned analog-resistant virus appeared to be accelerated relative to that of wild-type virus (Fig. 3a). This difference is likely to have been A3G dependent, since the replication kinetics of wild-type and analogresistant viruses were identical in permissive cells (Fig. 3b). This was surprising since wild-type virus should completely nullify A3G and, as such, should have the highest degree of fitness in nonpermissive cells. To investigate further, we measured the relative fitness levels of the parental and analog-resistant variants in pairwise competition experiments in nonpermissive H9 and permissive MT4 cells. In addition to the antagonist-resistant clone, a Vif V142I point mutant virus was included in the analysis. Cells were also infected with virus stocks singly to demonstrate replicative competence. As in the previous experiments, all viruses replicated similarly in permissive MT4 cells (Fig. 7c), while virus production levels varied in nonpermissive H9 cells such that analog-resistant virus cloned from late-passage culture exhibited enhanced replication kinetics relative to the wild-type virus (Fig. 7b). For dual infections, virions from culture supernatants of H9 cells were collected at 2-day intervals and the relative amounts of wild-type and antagonist-resistant viruses were measured by specific, quantitative PCR (qPCR) of cDNAs using primers that discriminated between the different alleles (Fig. 7a). The Vif V142I point mutant was modestly more fit than the wild-type virus in H9 cells (Fig. 7d), but the analog-resistant virus rapidly predominated in H9 cell coinfections (Fig. 7e). DISCUSSION In this study, we identified and evaluated inhibitors of HIV-1 Vif function in vitro and delineated the mechanism of action of these novel antiretroviral compounds involving the binding of Vif to EloC. To our knowledge, this is the first report describing HIV-1 escape from small-molecule inhibition of Vif activity. Each of the Vif antagonists used in this study drove the rapid selection for the same polymorphism within the Vif SOCS box, while the no-treatment control virus was unchanged for the culture duration. On the basis of crystallographic data, one would predict that changes at Vif V142 could impact binding to EloC (15). Vif targets A3G for polyubiquitination by mimicking cellular SOCS box-containing proteins via its BC box that binds to EloB and EloC. Residues in the C-terminal region of Vif, including V142, make side chain interactions with helix 3 and helix 4 of EloC, and the protein surface of EloC shows a binding pocket for V142 (15). This is consistent with our structural modeling, which places IMC15 at this Vif-EloC binding interface, and the modeling suggests that inhibitor binding may be disrupted when valine is replaced by isoleucine at position 142. In further support of this, a small molecule (VEC5) identified by virtual screening and validated by subsequent biochemical analysis binds at the same site and exhibits antiviral activity by disrupting the Vif-EloC interaction (30,31). The complete genome of the cloned inhibitor-resistant virus was sequenced, and several mutations, in addition to the Vif V142I, were identified. Considering the location of the mutations in the context of previously published work, the Vpr null mutation and the C9007A transversion may contribute to high-level resistance to the Vif antagonists used here. Hache et al. reported that Vif-deleted HIV-1 evolved to replicate in nonpermissive cells due to Vpr inactivation and a single mutation in the LTR promoter that increased transcription levels (32). It was postulated that Vpr inactivation was required to eliminate a Vpr-interacting factor that facilitates restriction by A3G, since Vpr knockout was not observed in control cells not expressing A3G (32). In contrast, Vpr was mutated in our system in each of the cultures containing Vif antagonists as well as in the no-treatment culture. In general, T cells infected in vitro with an HIV-1 isolate that contains an intact Vpr gene fail to divide due to the cell cycle arrest properties of Vpr and eventually die (29,33,34). Although establishing cell lines chronically infected with HIV-1 is possible, most produce virus particles with mutations in Vpr that render the protein nonfunctional (29,35). Interestingly, the same Vpr nonsense mutation that emerged in our cultures had previously been detected at high frequency in HIV-1 serially passaged in vitro and in peripheral blood mononuclear cells (PBMC) from chronically infected individuals and may represent a "hot spot" for Vpr inactivation (35). Further study will be required to elucidate whether inactivation of Vpr in our system contributes to the observed inhibitor-resistant phenotype. The C9007A transversion in the distal region of the LTR promoter suggests that the mutation could elevate proviral transcriptional activity. It maps to the binding site for LEF-1, a regulatory protein that shares homology with high-mobility group protein-1, which was previously shown to potently activate the T cell receptor ␣ enhancer in T lymphocytes (36). C9007A also results in a one-amino-acid truncation of Nef, but an exhaustive search of the literature failed to find a description of an important biological property for the terminal cysteine in Nef or to indicate that its loss would have an impact on Nef function. The mutation emerged in each of the Vif antagonist cultures but not in virus replicating in the untreated control cells. On the basis of sequencing data, selection for the enhancer mutation was delayed relative to that of the Vif mutation and likely represents a secondary modification that may elevate virus production to further overcome the restriction by A3G, which was previously reported to be a mechanism by which Vif-deleted HIV-1 can evolve to replicate in nonpermissive cells expressing A3G (32). Results of efforts to directly compare the levels of transcriptional activity of wild-type and variant promoters using reporter plasmid constructs were uninformative. Measuring the relative levels of promoter activities may require a nucleosome-assembled in vitro approach, since LEF-1 transactivation is a chromatindependent process whereby LEF-1 functions to effectively counteract nucleosomal repression of HIV-1 transcription and similar effects are not observed using transfected naked plasmid DNA (28). Although attempts to measure relative levels of promoter activities were unsuccessful, the cloned virus exhibiting the resistance phenotype clearly possessed a growth advantage in infected cells relative to the wild-type virus. This suggests that, in the context of wild-type virus, Vif does not completely subjugate A3G and that even wild-type HIV-1 is subject to the antiviral effects of A3G. This is supported by studies demonstrating the presence of proviruses with evidence of APOBEC-mediated editing in HIV-1-infected individuals (37,38). An inability of wild-type HIV-1 to completely neutralize A3G might be advantageous to viral replication in vivo since it may contribute to sequence evolution and adaptation to environmental pressures such as antiviral immunity. In summary, we have identified and evaluated new HIV-1 Vif antagonists on the basis of the structure of a lead compound (RN18) that possesses increased solubility and elevated potency. Mutations contributing to HIV-1 escape from these Vif antagonists localize to a motif that is essential for Vif function, demonstrating that the inhibitors specifically target the Vif-APOBEC axis. Structural modeling strongly supports these conclusions and suggests that the loss of inhibitor binding contributes to the development of resistance. Further optimization based on structure-function relationships, especially in the context of the Vif-EloC interaction, has the potential to identify novel Vif inhibitors with clinical efficacy. MATERIALS AND METHODS Reverse transcriptase assay for HIV-1 replication. H9 and MT4 cells were maintained in RPMI 1640 medium (Invitrogen) containing 25 mM HEPES and L-glutamine with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen) at 37°C in a humidified incubator (5% CO 2 ). Cells were seeded at 2 ϫ 10 5 per well in a 24-well plate, treated overnight with antiviral agents, and infected with HIV-1 (X4-tropic HIV-1LAI) at 2 ϫ 10 5 cpm reverse transcriptase units per well. Viral replication was monitored every day or every 2 days by measuring reverse transcriptase activity in culture supernatants. Measurement of relative viral fitness levels. Cells were maintained as described in the section on the reverse transcriptase assay procedure and were infected using 2 ϫ 10 5 cpm reverse transcriptase units per well either with single-virus stock or a combination of wild-type and variant viruses (V142I Vif mutant virus or cloned RN18 analog-resistant virus). Cells were pelleted and washed 4 h after infection to remove residual inoculum. Viral replication was monitored every day (MT4 cell infections) or every 2 days (H9 cell infections) by reverse transcriptase assay. At each time point, total RNA was purified from 100 l culture supernatant. RNA was converted to cDNA using random hexamers, and the relative amounts of wild-type and variant viral genomes were quantitated by allele-specific qPCR using HiDi Taq polymerase (myPOLS, Constance, Germany), which efficiently discriminates between single-base mismatches at the 3= end of primers during annealing and extension. Forward primers are listed in Fig. 7a and started at nucleotide 5022 of HIV-1LAI (GenBank accession number K02013). Sequence of the common reverse primer is LAI 5393 GAGTAACGCCTATTCTGCTATGTCGA and that of the fluorogenic reporter probe is LAI 5236 56-FAM (6-carboxyfluorescein)-ACATTTTCC/ZEN/TAGGATTTGGCTCCATGGCTTAG3 IABkFQ (Integrated DNA Technologies). Docking with Schrödinger. In silico docking of IMC15 to the Vif-CBF-␤-CUL5-EloB-EloC complex with APOBEC3G was performed using the Glide docking module of the Schrödinger 11.5 modeling software suite. The Vif-CBF-␤-CUL5-EloB-EloC complex and APOBEC3G structures were first refined using Prime. Missing side chains and hydrogen atoms were resolved before docking, and the Optimized Potentials for Liquid Simulations All-Atom (OPLS) force field and the Surface Generalized Born (SGB) continuum solution model was used to optimize and minimize the crystal structures. Ligprep was used to generate a minimized three-dimensional (3D) structure for IMC15 using the OPLS 2001 force field. Docking was performed with Glide XP. The docking grid was defined as a 25-Å cube centered on the coordinates x ϭ 25.19, y ϭ Ϫ14.1, and z ϭ Ϫ96.93), near V142 of Vif. Sequencing. For each passage presented in Fig. 4 and 6, viral RNA was purified from 200 l of culture supernatant and an aliquot was converted to cDNA by random hexamer priming. High-fidelity Phusion II polymerase (Thermo Fisher Scientific) was used for PCR amplification of the cDNA with primers specific for the LTR, Vif, and Vpr. Products were purified with silica columns (Qiagen), quantified, and used as templates for Sanger DNA sequencing (Genewiz). Sequencing data were analyzed using MacVector software, and sequence variants were identified by ClustalW alignments and visual evaluation of sequence trace files. For the full-length RN18 analog-resistant (RAR) viral clone, multiple overlapping sequence files were generated and assembled into a single contiguous file using Sequencher (Gene Codes Corporation). Data accessibility. Alignments performed with the parental virus sequence (GenBank accession number K02013) guided annotation of the inhibitor-resistant virus sequence prior to submission to GenBank (accession number MH843935). ACKNOWLEDGMENTS We dedicate this work to the memory of Eugene Charov, who diligently provided technical support throughout the project. We acknowledge grant support from the National Institutes of Health (MH100942, MH093306, AI096109) to M. Stevenson. We also acknowledge core facility support from the Miami Center for AIDS Research (P30AI073961).	2019-03-11T17:22:21.228Z	2019-02-26T00:00:00.000	{ "year": 2019, "sha1": "b240904017af0c124e84becb2327fdd401013cb5", "oa_license": "CCBY", "oa_url": "https://mbio.asm.org/content/mbio/10/1/e00144-19.full.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b240904017af0c124e84becb2327fdd401013cb5", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
231705919	pes2o/s2orc	v3-fos-license	The effects of the COVID-19 lockdown and alcohol restriction on trauma-related emergency department cases in a South African regional hospital The objective of this study was to compare the effect of the Covid-19 lockdown and the alcohol restriction on the number of cases that presented to the Emergency Department (ED) with the same time period two years prior. The method used was a retrospective review of medical records, directly comparing the types and numbers of trauma cases as well as non-trauma cases that presented to the ED in March and April 2020 with the same period two years prior. Our results showed a reduction during both months of lockdown compared to the same time period in 2018 with trauma cases in March 2020 down 33.14% and April 2020 down 57.93%. The non-trauma ED cases were down 2.52% in March and 37.43% in April compared to two years prior. When comparing only the last 6 days of March, a significant percentage decrease is visible as trauma cases fell from 20.79% in 2018 to just 8.58% of the total cases in 2020. In conclusion, our data showed a significant reduction in almost all types of ED cases during the lockdown period, but most significantly in trauma-related cases which was likely due, inter alia, to the prohibition of alcohol sales, gatherings and unnecessary travel. Introduction The Coronavirus disease of 2019 (COVID-19) and the resultant pandemic has been an unprecedented event in recent history, with its effects being felt across all spheres of society. The respiratory virus, which is highly contagious, has spread rapidly across the world leading to widespread infection, morbidity and even mortality. A multitude of strategies have been employed in order to mitigate, not only the spread of the virus, but also the concomitant demand for health resources, which has had an effect on the economy and on general daily activities. Some countries have implemented stringent COVID-19-related regulations while others have opted for a more relaxed approach to dealing with the virus [1,2]. South Africa, in particular, has implemented a relatively "strict" approach to this pandemic, which includes a risk-determined, levelbased system of lockdown. This system includes various level-based restrictions which, amongst other things, prevents the sale of goods deemed as "non-essential". The highest levels, namely level 5 and 4, included a prohibition on the sale of alcohol and tobacco products, as well as the restriction of mass gatherings and travel, except for essential workers and essential activities in terms of the regulations promulgated under the State of Disaster Act. The lockdown officially began on 26 March 2020 and is still ongoing at present [2]. South Africa's public healthcare system is severely under-resourced and hospitals have struggled to manage the ordinary trauma burden, even prior to the COVID-19 pandemic [9]. While the primary aim of these regulations was to reduce the rate of new infections and slow down the spread of the virus, one of the other objectives was also to "manage" the demand on already strained healthcare system. This particular study was conducted with the aim of comparing the number of trauma-and non-trauma-related cases seen at the Pholosong Regional Hospital Emergency Department during the lockdown period in March and April 2020 when compared to the same period in 2018. Pholosong Regional Hospital Emergency department is a 24 hour facility that sees on average 3246 patients per month during the initial quadrimesters of the years 2018, 2019 and 2020, with the maximum number of monthly patients seen during that period being 4506 (April 2019) and the minimum being 2195 (April 2020). Of this total, on average 16.1% are trauma-related cases. Method This is a single-centre, observational study that involved a retrospective review of medical records. We directly compared the numbers and variety of trauma-related patients seen during the lockdown period in March and April 2020, with the same time period two years prior. No added criteria or exclusions were utilised and the data is a direct comparison therefore preventing bias. Non-trauma ED patients were also studied and used as a comparison but patients whose primary complaints were not injury-related were excluded from trauma section of the study. A significant limitation to our study was the lack of adequate trauma records from the year 2019. Another setback was that during data population, records department does not separate patients according to gender or age and thus we were not able to add these demographics. Ideally, these would have been included in our study to make it more robust and to compare the trend over the same period in three different years. Results As illustrated in Fig. 1, the majority of trauma-related injuries, with the exception of burns and crush injuries, reduced during the lockdown period. The most significant reduction is seen in the month of April as cases dropped from 592 in 2018 to 249 cases in 2020, down by 57.93%. Trauma cases in March were also reduced by 33.14% from 673 cases in 2018 to 450 in 2020 (partial lockdown). When we take into account only the days in which the lockdown was active, namely the last 6 days of March, we notice that 2020 only had a total of 53 trauma cases compared to 163 cases in 2018. This is a 67.48% reduction. Trauma cases in March 2020 (partial lockdown) made up 12.01% of the total patients seen in the ED, down from 17.51% in March 2018. The month of April 2020 also saw a reduction in trauma cases, as just 11.34% of the total patients seen in the ED were trauma-related, which was down from 16.88% in April 2018. In the period of 26-31 March 2018, trauma cases made up a significant 20.79% as opposed to just the 8.58% of cases during the same period in 2020. The total number of patients seen in the ED, illustrated in Table 1, was down significantly in April 2020 to just 2195 from 3508 in the same month in 2018. This is a reduction of 37.43%. March 2020 (partial lockdown) only saw a 2.52% reduction in total patient numbers when compared to March 2018. But again if we take into account just the 6 days of March that fell into the lockdown period, we notice that this results in a 21.07% reduction in 2020 compared to the same period in 2018. Discussion The implementation of the lockdown regulations has changed the way people use and access healthcare facilities. While most hospitals and clinics around the world are still functioning and treating patients as per normal, or with slightly reduced capacities, the amount of people visiting these facilities for non-COVID-19-related reasons has reduced significantly [3] ( Table 2). One of the most significant reductions that has been noted is in the number of trauma patients presenting to the Pholosong Regional Hospital, in particular, the Emergency Department (ED). This 570 bed hospital is a moderately sized, regional hospital in the East Rand area of the Gauteng Province, South Africa. The hospital serves a population of more than 900,000 people in its catchment area and, as such, regularly deals with a variety of trauma and alcohol-related cases [4]. Emergency departments around the world see significant alcoholrelated visits on a regular basis, with these cases placing a huge burden on these facilities. This burden is not only limited to human or financial resources, but can also lead to psychological and even physical strain on healthcare workers [5]. In addition to the above, patients who present with alcohol-related injuries generally have an increased length of stay in the ED, when compared to non-alcohol related patients. Time is a precious resource in emergency medicine and a few minutes can mean the difference between life and death, especially in a resuscitation situation [6]. Alcohol-related trauma in particular, is also a significant challenge to pre-hospital healthcare service providers. McNicholl et al., showed that up to 29% of cases that present to EDs on weekends are in some way alcohol-related, with a large number of patients making use of ambulance services to transport them to hospitals and other healthcare facilities. The study was also able to illustrate that these patients are more likely to leave the hospital, against medical advice in some cases, even before being seen by a doctor. This is not only a waste of time and valuable resources, but it also places these patients at risk of being incorrectly diagnosed or of not receiving the necessary treatment for their condition, which will undoubtedly have a negative impact on their morbidity and mortality [7]. Data collected from emergency departments also show that patients with alcohol-related traumatic injuries tend to have more severe injuries than non-intoxicated controls, in particular when it comes to injuries that involve the head and face [8]. When comparing the number of trauma cases seen for each respective month during the different years, one can appreciate that the month of April had a far greater decline than the month of March during the lockdown period. One can also see that the total patient numbers seen in the ED were also reduced, and as with the trauma cases, more significantly during the month of April 2020. However, the month of March 2020 only saw a 2.52% reduction when compared to the same month in 2018. But if we take into account just the 6 days of March that fell into the lockdown period, we notice that this results in a 21.07% reduction in 2020 compared to the same period in 2018. In April, the total patient numbers were reduced by 37.43%, yet the total trauma patients were reduced by 57.93%. Thus trauma cases were more significantly reduced when compared to non-trauma cases. There are multiple factors that may have contributed to this pattern. One reason for this variability is that the lockdown only commenced towards the end of March 2020 and, as such, the month was only partially affected by the lockdown regulations. Furthermore, aside from the banning of alcohol and gatherings, there was progressive public awareness of the dangers associated with COVID-19 as significant public education efforts were made by governments around the world, including South Africa. As such, many people became more adherent to the lockdown regulations in the subsequent weeks than was the case initially. This was also compounded by stricter enforcement of lockdown regulations by security forces, including the police as well as the army who were deployed by the national government to maintain order [10,11]. April 2020 saw the most significant reduction in trauma, in particular with regard to soft tissue injuries caused by motor vehicle accidents (MVA), which declined by 89.3% and head injuries which declined by 89.6%. Both of these injury types have significant impacts on morbidity and mortality. An interesting finding is that March 2018 had two public holidays compared to only one in 2020 and April 2018 had two public holidays compared to the three in April 2020. This means that even with an additional holiday, the trauma cases still decreased [6,7,9]. Another interesting finding is that crush injuries actually increased during the study period from 0 cases in April 2018 to 5 cases in April 2020. This may be due to the socio-economic strain caused by the lockdown regulations, as crush injuries are commonly related to mob justice assaults in communities [13]. Further investigation is needed in order to ascertain the true reason for this increase, which falls outside of the scope of this paper. One limitation of this study is that it was not possible to separate alcohol-related trauma from non-alcohol related trauma and, as such, determine the direct correlation of the alcohol ban on trauma cases. Other shortcomings include not being able to compare the demographics of the trauma, in particular age and gender, as well as not being able to separate paediatric trauma from adult trauma ED cases due to the lack of data. Conclusions Our data, although limited, shows a significant reduction in almost all types of ED cases during the lockdown period, but most significantly in trauma-related cases during the lockdown period as compared to the same period in 2018 just two years prior. The reduction in numbers is most likely attributable to the COVID-19 lockdown regulations which included, inter alia, a prohibition on alcohol sales, gatherings and unnecessary travel. The Presidency announced that as of 1 June 2020, South Africa would move into level 3 of the lockdown which, amongst other things, allowed for the sale of alcohol for private consumption, as well as the opening of places of worship with a limited number of congregants [12]. It remains to be seen if this will result in an increase in the trauma cases that present to our ED and, as such, a follow up study will be required to assess the outcome. While the world continues to battle the COVID-19 pandemic and the effects thereof, it is reassuring to know that there have been some "positive" outcomes which, even though inadvertent, have resulted in the saving of lives, the targeted use of resources and has assisted in the battle against the high rates of traumatic injury that plague our country. It is our hope that this data will be of benefit to South Africa as well as to other countries, not only in Africa but around the globe, when planning Table 2 Pholosong Regional Hospital Emergency Department patient statistics (added more data to give better representation of trauma cases out of total ED cases as well as total patient numbers). and forming regulations to deal with both the current as well future pandemics. Dissemination of results The above findings were shared with management at the Pholosong hospital and were published in issue 8 of the Pholosong Hospital newsletter which was published on 12/06/2020.	2021-01-13T14:08:29.909Z	2021-01-09T00:00:00.000	{ "year": 2021, "sha1": "c1114b1ed42d0c87693a6e81dd50f3958f481033", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.afjem.2020.12.001", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "3377eba3dd439fe728e2122d4d21a49098d49a83", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
209167258	pes2o/s2orc	v3-fos-license	Gallic Acid and Quercetin as Intelligent and Active Ingredients in Poly(vinyl alcohol) Films for Food Packaging Gallic acid (GA) and quercetin (QC) were used as active ingredients in poly(vinyl alcohol) (PVA) film formulations obtained by solvent casting process. The effect of two different percentages (5 and 10 % wt.) on morphological behavior, thermal stability, optical, mechanical, and release properties of PVA were investigated, while migration with food stimulants and antioxidant properties were tested taking into account the final application as food packaging systems. The results showed how different dispersability in PVA water solutions gave different results in term of deformability (mean value of ε PVA/5GA = 280% and ε PVA/5QC = 255%, with 190% for neat PVA), comparable values for antioxidant activity at the high contents (Radical Scavenging Activity, RSA(%) PVA/10GA = 95 and RSA(%) PVA/10QC = 91) and different coloring attitude of the polymeric films. It was proved that GA, even if it represents the best antioxidant ingredient to be used with PVA and can be easily dispersed in water, it gives more rigid films in comparison to QC, that indeed was more efficient in tuning the deformability of the PVA films, due the presence of sole hydroxyl groups carrying agent. The deviation of the film coloring towards greenish tones for GA films and redness for QC films after 7 and within 21 days in the simulated conditions confirmed the possibility of using easy processable PVA films as active and intelligent films in food packaging. Introduction In the last few years, the use of pure phenolic compounds and phenolic extracts in active polymeric packaging has attracted a particular interest, since these compounds show potent antimicrobial and antioxidant activity in food systems and their intake can make a contribution to human health. Being the phenolic compounds able to interact with the environment and the product to extend shelf life, their addition to the package reduces the need to use synthetic antioxidants in the plastic, limiting the risk of potential toxicity by migration, and protects at the same time the package content. Equally, the extra protection of the food provided by the slow release of antioxidants from the package can make it possible to reduce the direct addition of chemicals to the food. Polyphenols belong to a wide category of plant derived natural compounds exhibiting many biological properties [1], including anticancer [2,3], anti-inflammatory [4,5], antimicrobial and antioxidant activity [6]. Among these compounds, gallic acid (3,4,5-trihydroxybenzoic acid, GA) and quercetin (3,3 ,4 ,5,7-pentahydroxyflavone, QC) are two potent antioxidants, due both to their redox properties and structural features (Figure 1), that impart high antioxidant activity: the three hydroxyl groups for GA [7] and the four hydroxyl groups on the A Limiting the analysis to their use in poly(vinyl alcohol) (PVA) based films, the literature reports the incorporation of tannic acid [10], hydroxytyrosol and its derivatives [11][12][13], tea plant [14][15][16] and rosemary extracts, to produce antioxidant films potentially useful to increase the shelf life of food [17,18]. The same approach was used in PVA blends and nanocomposites, as in the case reported by Wu et al. [19] and Matos de Carvalho et al. [20], where the authors respectively demonstrated that PVAstarch films impregnated with 500-1000 ppm of catechins and PVA cast film with solid lipid nanoparticles entrapping α-tocopherol showed relevant antioxidant capacity and controlled release of the active ingredients, confirming the possibility of their use in food preservation. It is also well known in the literature that dyes containing phenolic or conjugated substances, are useful for colorimetric determination of pH in an intelligent manner. Thus, presence of polyphenols in polymeric films can act as pH indicators and this use has been extensively investigated. In the specific case of PVA based films, Ma et al. [21] developed a pH indicator which consisted of polyvinyl alcohol (PVA)-chitosan nanoparticles and mulberry extract and they found that change in color indicator from red to green was correlated with the presence of volatile nitrogenous compounds, which is characteristic of fish spoilage. Anthocyanins that were extracted from roselle immobilized onto starch-PVA-based film were also proposed by Zhai et al. [22] as visual colorimetric film for volatile nitrogenous compounds released in fish spoilage. In addition, Liu et al. [23] developed an intelligent starch/poly-vinyl alcohol (PVA) capable of monitoring pH changes and inhibiting undesired microbial growth in pasteurized milk. The results suggested that the intelligent films reported here show good capability for both alerting and inhibiting food spoilage. A recent example of QC loaded PVA can be found in He at al. [24], where the authors prepared, via physically mixing QC aggregation-induced emission luminogens (AIEgens) as fluorescence sensors for detecting biogenic amines formed during the spoilage of seafood. Again, Ma et al. [25] prepared pH sensing film from tara gum-cellulose nanocrystal incorporated with natural dye (grape skin extract) to evaluate the pH changes of the milk at ambient temperature for 48 h. During the test, the color of indicator clearly changed from bright red (acidic) to dark green (alkaline), which can be correlated with microbial contamination and pH decrease. The authors suggested that the developed pH sensing film could be used as a visible color indicator and changes in color of the film provide information to monitor the packaged food freshness. With the same aim, in parallel with morphological, thermal, and mechanical behavior of produced films, the antioxidant activity of PVA containing GA and QC at two different percentage (5 and 10% wt.) before and after release in food simulated solutions and their change in color were here investigated. The variations in terms of color for films was then correlated to swelling behavior Limiting the analysis to their use in poly(vinyl alcohol) (PVA) based films, the literature reports the incorporation of tannic acid [10], hydroxytyrosol and its derivatives [11][12][13], tea plant [14][15][16] and rosemary extracts, to produce antioxidant films potentially useful to increase the shelf life of food [17,18]. The same approach was used in PVA blends and nanocomposites, as in the case reported by Wu et al. [19] and Matos de Carvalho et al. [20], where the authors respectively demonstrated that PVA-starch films impregnated with 500-1000 ppm of catechins and PVA cast film with solid lipid nanoparticles entrapping α-tocopherol showed relevant antioxidant capacity and controlled release of the active ingredients, confirming the possibility of their use in food preservation. It is also well known in the literature that dyes containing phenolic or conjugated substances, are useful for colorimetric determination of pH in an intelligent manner. Thus, presence of polyphenols in polymeric films can act as pH indicators and this use has been extensively investigated. In the specific case of PVA based films, Ma et al. [21] developed a pH indicator which consisted of polyvinyl alcohol (PVA)-chitosan nanoparticles and mulberry extract and they found that change in color indicator from red to green was correlated with the presence of volatile nitrogenous compounds, which is characteristic of fish spoilage. Anthocyanins that were extracted from roselle immobilized onto starch-PVA-based film were also proposed by Zhai et al. [22] as visual colorimetric film for volatile nitrogenous compounds released in fish spoilage. In addition, Liu et al. [23] developed an intelligent starch/poly-vinyl alcohol (PVA) capable of monitoring pH changes and inhibiting undesired microbial growth in pasteurized milk. The results suggested that the intelligent films reported here show good capability for both alerting and inhibiting food spoilage. A recent example of QC loaded PVA can be found in He at al. [24], where the authors prepared, via physically mixing QC aggregation-induced emission luminogens (AIEgens) as fluorescence sensors for detecting biogenic amines formed during the spoilage of seafood. Again, Ma et al. [25] prepared pH sensing film from tara gum-cellulose nanocrystal incorporated with natural dye (grape skin extract) to evaluate the pH changes of the milk at ambient temperature for 48 h. During the test, the color of indicator clearly changed from bright red (acidic) to dark green (alkaline), which can be correlated with microbial contamination and pH decrease. The authors suggested that the developed pH sensing film could be used as a visible color indicator and changes in color of the film provide information to monitor the packaged food freshness. With the same aim, in parallel with morphological, thermal, and mechanical behavior of produced films, the antioxidant activity of PVA containing GA and QC at two different percentage (5 and 10% wt.) before and after release in food simulated solutions and their change in color were here investigated. The variations in terms of color for films was then correlated to swelling behavior of polymeric films in Polymers 2019, 11,1999 3 of 20 presence of the active ingredients, having in mind to use the produced films as indicators for safe food packaging applications. Preparation of PVA Based Formulations PVA based formulations were prepared by solvent casting technique. The concentrations of active GA and QC were fixed at 5 and 10% wt., as previously reported [26]. Firstly, 0.5 g of PVA was dissolved in 10 mL of distilled water under magnetic stirring for 2 h at 80 • C. The polymeric solution was kept under magnetic stirring to reach room temperature (RT). PVA based formulations were obtained mixing, under magnetic stirring, the polymeric solution with a specific amount of GA or QC, previously dispersed in distilled water (0.1 g of active ingredient in 10 mL of distilled water) for 1 h at RT. Uniform dispersion of QC or GA in aqueous solution was obtained applying a magnetic stirring and a sonication bath, both at RT for 1 h (Scheme 1). Polymers 2019, 11,1999 3 of 20 of polymeric films in presence of the active ingredients, having in mind to use the produced films as indicators for safe food packaging applications. Preparation of PVA Based Formulations PVA based formulations were prepared by solvent casting technique. The concentrations of active GA and QC were fixed at 5 and 10% wt., as previously reported [26]. Firstly, 0.5 g of PVA was dissolved in 10 mL of distilled water under magnetic stirring for 2 h at 80 °C. The polymeric solution was kept under magnetic stirring to reach room temperature (RT). PVA based formulations were obtained mixing, under magnetic stirring, the polymeric solution with a specific amount of GA or QC, previously dispersed in distilled water (0.1 g of active ingredient in 10 mL of distilled water) for 1 h at RT. Uniform dispersion of QC or GA in aqueous solution was obtained applying a magnetic stirring and a sonication bath, both at RT for 1 h (Scheme 1). The polymeric solutions of neat film and binary films were cast in a Teflon® mold and evaporated at RT. Films having thicknesses in the range 30-60 μm were obtained and equilibrated for 7 days at 53% RH (an oversaturated magnesium nitrate solution was used to control the RH) in desiccators, by using magnesium nitrate-6-hydrate oversaturated solution, after the processing and before the characterizations, in accordance to the literature [12,27]. Characterization of PVA Based Formulations Field emission scanning electron microscopy (FESEM, Supra 25-Zeiss) was used to investigate morphologies of both GA and QC and fractured surface (surface obtained after the tensile test) morphology of PVA based formulations. The surfaces were gold sputtered in order to offer electric conductivity and the samples were observed using an accelerating voltage of 2.5 kV. FTIR measurements for QC, GA, and PVA formulations were performed by using a Jasco FT-IR 615 spectrometer (Jasco Inc, Easton, MD, USA) in the 400-4000 cm −1 range in attenuated total reflection (ATR) mode. Thermal characterization of active ingredients and PVA based formulations was carried out by both differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA). DSC (DSC-TA Instrument, Q200) measurements were performed in the temperature range from −25 to 240 °C, at a heating rate of 10 °C min −1 . Three samples were used to characterize each material. The crystallinity degree (melting crystallinity degree (Xm) and crystallinity degree at cooling scan (Xc)) was calculated according to the following Equation (1): The polymeric solutions of neat film and binary films were cast in a Teflon®mold and evaporated at RT. Films having thicknesses in the range 30-60 µm were obtained and equilibrated for 7 days at 53% RH (an oversaturated magnesium nitrate solution was used to control the RH) in desiccators, by using magnesium nitrate-6-hydrate oversaturated solution, after the processing and before the characterizations, in accordance to the literature [12,27]. Characterization of PVA Based Formulations Field emission scanning electron microscopy (FESEM, Supra 25-Zeiss) was used to investigate morphologies of both GA and QC and fractured surface (surface obtained after the tensile test) morphology of PVA based formulations. The surfaces were gold sputtered in order to offer electric conductivity and the samples were observed using an accelerating voltage of 2.5 kV. FTIR measurements for QC, GA, and PVA formulations were performed by using a Jasco FT-IR 615 spectrometer (Jasco Inc., Easton, MD, USA) in the 400-4000 cm −1 range in attenuated total reflection (ATR) mode. Thermal characterization of active ingredients and PVA based formulations was carried out by both differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA). DSC (DSC-TA Instrument, Q200) measurements were performed in the temperature range from −25 to 240 • C, at a heating rate of 10 • C min −1 . Three samples were used to characterize each material. The glass transition temperature (T g ), melting temperature (T m ), crystallization temperature (T c ), and relative enthalpies (∆H m and ∆H c ) were evaluated. The crystallinity degree (melting crystallinity degree (X m ) and crystallinity degree at cooling scan (X c )) was calculated according to the following Equation (1): where ∆H is the enthalpy for melting or crystallization; ∆H 0 is enthalpy of melting for a 100% crystalline PVA sample, taken as 161.6 J g −1 [28,29] and (1 − m f ) is the weight fraction of PVA in the sample. TGA (Seiko Exstar 6300) experiments were performed for each film from 30 to 600 • C at 10 • C min −1 under a nitrogen atmosphere (250 mL min −1 ) in order to evaluate the effect active ingredient (AI) addition on the thermal stability of PVA matrix. The mechanical properties (tensile tests) of neat PVA and PVA/AI based systems were performed on rectangular probes as indicated in the EN ISO 527-5 standard, with a crosshead speed 5 mm min −1 , a load cell 500 N by using a digital Lloyd testing machine (Lloyd Instrument LR 30K Segens worth West, Foreham, UK). Average tensile strength (σ B ), elongation at break (ε B ), and Young's modulus (E) were calculated from the resulting stress-strain curves. The measurements were done at room temperature and at least five samples for each formulation were tested. The Double-sided, total immersion migration tests were carried out with 12 cm 2 of films and 20 mL of simulant (area-to-volume ratio around 6 dm 2 L −1 ) in triplicate at 40 • C in an oven (J. P. Selecta, Barcelona, Spain). Samples were taken at 1, 3, 7, 10, 21 days in triplicate; a blank test for the simulant was also included. After the migration tests, each solution was recovered and stored at −4 • C before analysis. The color variations of PVA films were investigated by means of a spectrophotometer (CM-2300d Konica Minolta, Japan). Data were acquired by using the SCI 10/D65 method whereas CIELAB color variables, as defined by the Commission Internationale de 1 Éclairage (CIE 1995), were used. Film specimens were placed on a white standard plate and L, a, and bparameters were determined. L value ranges from 0 (black) to 100 (white); a* value ranges from −80 (green) to 100 (red); and b* value ranges from −80 (blue) to 70 (yellow). Samples were analyzed in triplicate, and three measurements were taken at random locations on each of the studied films. The total color difference ∆E* and gloss values were calculated as indicated in Equation (2): GA and QC Release Studies in Food Simulant (Specific Migration) GA and QC released by PVA based formulations in food simulant for every contact time (1, 3, 7, 10, and 21 days) were analyzed by UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). Absorbance values were measured at 270 nm and at 375 nm for GA and QC, respectively. Standard curves for GA (range 4.0-20 µg mL −1 ) and QC (range 2.0-14 µg mL −1 ) have been used. Samples were analyzed in Polymers 2019, 11, 1999 5 of 20 triplicate and results are the mean ± standard deviation (SD) and expressed as µg of antioxidant per mL of food simulant. Antioxidant Activity of Films Radical scavenging activity of PVA based films at initial and after the contact with food simulant (simulant D, 50 v/v% of ethanol) at different times (1, 3, 7, 10, and 21 days) was determined by using a spectroscopic method according to the procedure proposed in literature by Byun et al. [30]. The different PVA systems (0.1 g) were cut into small pieces and immersed in 2 mL of methanol for 24 h at RT. An aliquot of methanol extract (1 mL) was mixed with 1 mL of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) in methanol (50 mg L −1 ). The mixture was allowed to stand at RT in the dark for 60 min. The absorbance was measured at 517 nm using a UV spectrometer (Lambda 35). The DPPH mixture solution of methanol extracted from neat PVA was used as control. DPPH radical scavenging activity (RSA) was measured according to the Equation (3): where A sample is the absorbance of sample and A control is the absorbance of the control. Antioxidant Activity of Food Simulant Solutions The antioxidant activity of the food simulant solutions was determined using the DPPH assay according to the literature already reported by us [11,12]. Briefly, 1.5 mL of DPPH solution (50 mg L −1 ) was mixed with 1.0 mL of samples and the optical density was recorded at 517 nm after 60 min of reaction in the dark. The DPPH radical scavenging activity was expressed as RSA% according to the Equation (3). The DPPH mixture solution of simulant from neat PVA films was used as control. Statistical Analysis The experimental results were expressed as mean ±SD of three replicates. Statistical differences were calculated using one-way analysis of variance (ANOVA), followed by Tukey's honest significant difference (HSD) post hoc test. GA and QC Characterization The thermal stability of QC and GA in inert atmosphere was determined by TGA tests, with a run from 30 to 700 • C under nitrogen at 10 • C/min. Figure 2a shows TG/DTG profiles for GA and QC. In the case of QC, that it is proved to be highly hygroscopic, the first loss was observed at 103 • C, due to the loss of H 2 O molecules from the crystal lattice of its hydrate form [31,32]. The dehydration is complete at 160 • C, no further mass loss was observed until 300 • C, which marks the onset of its thermal degradation [33]. After that, a sharp exothermic peak was observed in QC at 340 • C, due to decomposition of organic matter: we found that a significant amount of complex (38.6%) remains after heating the polymer to 700 • C, and this is attributed to the formation of new catechol-catechol bonds [34]. In the case of GA, the first low intensity degradation peak centered at 87 • C is due to the degradation of water and volatile compounds with low molecular weight, while a main peak centered at ca. 264 • C, that corresponds to the main chain scission, was detected [35]. Finally, minor and progressive degradation phenomena of lower intensity were observed at temperatures above 400 • C, which are related to the residual decomposition of GA at high temperature [36]. The FTIR spectra for QC and GA are shown in Figure 2b. In the case of QC, OH groups stretching were detected at 3403, 3321 (not shown), 1448, and 1014 cm −1 (the large presence of OH groups accounts for the solubility of the polymer in mix solvents such as ethanol/water), whereas OH bending of the phenol function was detectable at 1381 cm −1 . The C=O aryl stretching absorption was evident at 1662 cm −1 . Aromatic ring stretch bands for C=C were detected at 1610, 1561, and 1520 cm −1 . The signal for ν(C-O) can be found at 1462 cm −1 [37], other bands associated with the vibrations of C-O bonds appear also at 1132 cm −1 (C-OH stretching). The in-plane bending band of C-H in aromatic hydrocarbon was detectable at 1318 cm −1 , and out-of-plane bending bands were evident at 930, 822, 678, and 603 cm −1 . Bands at 1262, 1198, and 1168 cm −1 are related, respectively, to the C-O stretching in the aryl ether ring, the C-O stretching in phenol, and the C-CO-C stretch and bending in ketone [38,39], other signals for C-OH deformation and -C-OH stretch vibration were found at 1214 cm −1 and 1091 cm −1 . Minor signals attributable to substituted benzene can be found at 941, 864, 795, and 700 cm -1 [40]. FTIR spectrum for GA powder exhibits characteristic peaks at 3482 cm −1 and 3281 cm −1 , which are due to stretching of the O-H groups (not shown), a sharp absorption band of carboxylic group -COOH at 1695 cm −1 , absorption bands of -OH at 866 cm −1 , as well as -C-OH at 1320 cm −1 , 1375 cm −1 (Phenol (or) tertiary alcohol, OH bend), 1244 cm −1 (C-O stretch) and peak at 766, 1021, 1100, and 1614 cm −1 , a C=C stretching vibration can be observed at 1539 cm −1 [41]. The peaks located at 1614, 1539, 1446 cm −1 represented the aromatic ring C=C stretching vibrations [42]. There are several peaks in 1300-1000 cm −1 region (1336, 1306, 1203 cm −1 ) that could be assigned to the stretching vibration of C-O bond and bending vibration of O-H [43]. Minor signals related to substituted benzene can be found at 985, 790, and 700 cm −1 . GA powder (appearance and chemical formula in Figure 3a) was characterized by crystals of small size and regular shape with an apparently smooth surface [44] (Figure 3c) while, after water dispersion/drying, appeared individualized with a fibrous structure, reduced diameters, and increased lengths. In details, FESEM image ( Figure 3e) shows that GA was organized as long (10-100 μm) filaments with diameter of 500-550 nm. In the case of poorly water soluble QC (appearance and chemical formula in Figure 3b), it has been found that, in its powdered form, it consists of irregular rod structures [45,46] (Figure 3d) while, after dispersion in water, reduced particle size was noted (Figure 3f): particle size uniformity was obtained and lack of larger needle-type crystalline structures was found [47,48], in analogy with a previous study where QC morphology in relationship to the interaction with water under room temperature conditions was analyzed [49]. The FTIR spectra for QC and GA are shown in Figure 2b. In the case of QC, OH groups stretching were detected at 3403, 3321 (not shown), 1448, and 1014 cm −1 (the large presence of OH groups accounts for the solubility of the polymer in mix solvents such as ethanol/water), whereas OH bending of the phenol function was detectable at 1381 cm −1 . The C=O aryl stretching absorption was evident at 1662 cm −1 . Aromatic ring stretch bands for C=C were detected at 1610, 1561, and 1520 cm −1 . The signal for ν(C-O) can be found at 1462 cm −1 [37], other bands associated with the vibrations of C-O bonds appear also at 1132 cm −1 (C-OH stretching). The in-plane bending band of C-H in aromatic hydrocarbon was detectable at 1318 cm −1 , and out-of-plane bending bands were evident at 930, 822, 678, and 603 cm −1 . Bands at 1262, 1198, and 1168 cm −1 are related, respectively, to the C-O stretching in the aryl ether ring, the C-O stretching in phenol, and the C-CO-C stretch and bending in ketone [38,39], other signals for C-OH deformation and -C-OH stretch vibration were found at 1214 cm −1 and 1091 cm −1 . Minor signals attributable to substituted benzene can be found at 941, 864, 795, and 700 cm -1 [40]. FTIR spectrum for GA powder exhibits characteristic peaks at 3482 cm −1 and 3281 cm −1 , which are due to stretching of the O-H groups (not shown), a sharp absorption band of carboxylic group -COOH at 1695 cm −1 , absorption bands of -OH at 866 cm −1 , as well as -C-OH at 1320 cm −1 , 1375 cm −1 (Phenol (or) tertiary alcohol, OH bend), 1244 cm −1 (C-O stretch) and peak at 766, 1021, 1100, and 1614 cm −1 , a C=C stretching vibration can be observed at 1539 cm −1 [41]. The peaks located at 1614, 1539, 1446 cm −1 represented the aromatic ring C=C stretching vibrations [42]. There are several peaks in 1300-1000 cm −1 region (1336, 1306, 1203 cm −1 ) that could be assigned to the stretching vibration of C-O bond and bending vibration of O-H [43]. Minor signals related to substituted benzene can be found at 985, 790, and 700 cm −1 . GA powder (appearance and chemical formula in Figure 3a) was characterized by crystals of small size and regular shape with an apparently smooth surface [44] (Figure 3c) while, after water dispersion/drying, appeared individualized with a fibrous structure, reduced diameters, and increased lengths. In details, FESEM image (Figure 3e) shows that GA was organized as long (10-100 µm) filaments with diameter of 500-550 nm. In the case of poorly water soluble QC (appearance and chemical formula in Figure 3b), it has been found that, in its powdered form, it consists of irregular rod structures [45,46] (Figure 3d) while, after dispersion in water, reduced particle size was noted (Figure 3f): particle size uniformity was obtained and lack of larger needle-type crystalline structures was found [47,48], in analogy with a previous study where QC morphology in relationship to the interaction with water under room temperature conditions was analyzed [49]. Figure 4, (Panel A) shows the microstructure of the fractured sections of PVA based formulations. PVA film is characterized by a homogeneous, uniform, and smooth fractured surface (Figure 4a) [29,50]. This behavior underlines the good processability and film-forming ability of PVA during solvent casting process. The addition of GA and QC affects the homogeneity and the uniformity of PVA films at both concentrations (5 and 10 wt. %). GA in polymer matrix processed by solvent casting results well dispersed, due to its high hydrophilicity and solubility in water solution [51]. The PVA/GA based films show a porous stretched fractured surface, due to the GA rearrangement during the evaporation phenomenon (Figure 4b,d). The FESEM images of PVA/QC based films (Figure 4c,e) show the presence of QC not properly solubilized in aqueous solution (see the arrows). As already observed by FESEM analysis for QC, after dispersion in water solution reduced particle size was noted (Figure 4d) [47]. In Figure 4, Panel B, images for surfaces of the same samples are reported: the results confirmed the presence of a flat and uniform morphology for the different materials, that was substantially unaffected by the presence of the active ingredients (only a limited roughness was found in the case of QC containing films). Figure 4, (Panel A) shows the microstructure of the fractured sections of PVA based formulations. PVA film is characterized by a homogeneous, uniform, and smooth fractured surface (Figure 4a) [29,50]. This behavior underlines the good processability and film-forming ability of PVA during solvent casting process. The addition of GA and QC affects the homogeneity and the uniformity of PVA films at both concentrations (5 and 10 wt. %). GA in polymer matrix processed by solvent casting results well dispersed, due to its high hydrophilicity and solubility in water solution [51]. The PVA/GA based films show a porous stretched fractured surface, due to the GA rearrangement during the evaporation phenomenon (Figure 4b,d). The FESEM images of PVA/QC based films (Figure 4c,e) show the presence of QC not properly solubilized in aqueous solution (see the arrows). As already observed by FESEM analysis for QC, after dispersion in water solution reduced particle size was noted (Figure 4d) [47]. In Figure 4, Panel B, images for surfaces of the same samples are reported: the results confirmed the presence of a flat and uniform morphology for the different materials, that was substantially unaffected by the presence of the active ingredients (only a limited roughness was found in the case of QC containing films). Thermal Analysis Thermal properties of PVA and PVA based films were evaluated by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) under nitrogen atmosphere, with the goal of assessing the effect of both GA and QC at different weight contents on thermal stability of PVA matrix. Table 1 summarizes the values of parameters related to glass transition, crystallization and melting phenomena of PVA and PVA based films, measured during the cooling and the second heating scans. In the cooling scan, a decrease in the Tg with the increase of active ingredients amount was noted, indicating that the mobility of the PVA chains, probably due to electrostatic interactions between the GA (or QC) and the PVA, diminished [52]. During the cooling, when the content was increased from 0 to 5% for both GA and QC, the crystallization enthalpy increased, manifesting that the crystallinity also increased. However, PVA/10GA and PVA/10QC showed a reduced crystallinity value: apparently, higher contents of active ingredients can hinder the chains packing and inhibit crystals growth, as already observed by Peng et al. [53]. Moreover, the analogous trend was found for the crystallization temperature Tc, that gradually decreased with the addition of GA and QC from 5 to 10% wt. [10]. During the second heating scan, neat PVA glass transition temperature occurred at 77 °C. With the addition of both GA and QC at the two different amounts, Tg gradually increased to a mean value of 85 °C when 10% wt. of both active ingredients was incorporated, whereas a notable enhancement of Tm values during the second heating scan, due to reduced mobility of PVA chains, was also registered [15]. In addition, PVA/GA and PVA/QC films exhibited narrower endothermic peaks than PVA alone (data not shown). Zhu et al. also observed that when polyphenol catechins are mixed with poly(ε-caprolactone) or poly(3-hydroxypropionate), only one Tg exists for all component ranges, and this value increases as the content of polyphenol in the system increases [54]. Thermal Analysis Thermal properties of PVA and PVA based films were evaluated by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) under nitrogen atmosphere, with the goal of assessing the effect of both GA and QC at different weight contents on thermal stability of PVA matrix. Table 1 summarizes the values of parameters related to glass transition, crystallization and melting phenomena of PVA and PVA based films, measured during the cooling and the second heating scans. In the cooling scan, a decrease in the T g with the increase of active ingredients amount was noted, indicating that the mobility of the PVA chains, probably due to electrostatic interactions between the GA (or QC) and the PVA, diminished [52]. During the cooling, when the content was increased from 0 to 5% for both GA and QC, the crystallization enthalpy increased, manifesting that the crystallinity also increased. However, PVA/10GA and PVA/10QC showed a reduced crystallinity value: apparently, higher contents of active ingredients can hinder the chains packing and inhibit crystals growth, as already observed by Peng et al. [53]. Moreover, the analogous trend was found for the crystallization temperature Tc, that gradually decreased with the addition of GA and QC from 5 to 10% wt. [10]. During the second heating scan, neat PVA glass transition temperature occurred at 77 • C. With the addition of both GA and QC at the two different amounts, T g gradually increased to a mean value of 85 • C when 10% wt. of both active ingredients was incorporated, whereas a notable enhancement of T m values during the second heating scan, due to reduced mobility of PVA chains, was also registered [15]. In addition, PVA/GA and PVA/QC films exhibited narrower endothermic peaks than PVA alone (data not shown). Zhu et al. also observed that when polyphenol catechins are mixed with poly(ε-caprolactone) or poly(3-hydroxypropionate), only one T g exists for all component ranges, and this value increases as the content of polyphenol in the system increases [54]. T g -glass transition temperature; T m -melting temperature; T c -crystallization temperature; ∆H m -melting enthalpy; ∆H c -crystallization enthalpy; X m -melting crystallinity degree (X m ); and X c -crystallinity degree at cooling scan. (a-c) Different superscripts within the same column indicate significant differences among formulations regarding the same scan (p < 0.05). The effect of QC and GA on degradative behavior of PVA matrix was also investigated by TGA, mass loss and derivative weight loss curves are reported in Figure 5a. All the studied formulations were characterized by the presence of a multi-step degradation behavior: the first weight loss, at low temperature centered at around 80-110 • C, corresponds to the removal of weakly bound water or dehydration. The second and the third degradation temperatures correspond to the degradation of neat PVA [55]. In particular, the second/main degradation step of PVA film corresponds to the chain scissoring, with removal of residual acetate groups, due to the incomplete hydrolysis of PVA that remains in the chains, while the third degradation step is due to the cyclization reaction and continual elimination of residual acetate groups [13]. GA containing films displayed enhanced T max and decrease in the rate of degradation (indicated by the broadening of DTG curve), with the shift of the second degradation peak from 247 • C for neat PVA to 264 and 267 • C, respectively for PVA/5GA and PVA/10GA. It is considered that interactions between the terminal -OH groups of the biopolymer chain with -OH, -CO, and/or -COOH chemical groups of the GA components via hydrogen bonding are responsible for the observed thermal stability improvement [56,57]. QC containing films showed prominent effect on the thermal properties with huge improvement in the T max values, with the shift of the second degradation peak to 276 and 344 • C, respectively for PVA/5QC and PVA/10QC, confirming the observation of Dhand et al. [58], where it was found that strong covalent or non-covalent interactions between the −OH groups of PVA with −OH moieties of heteroaromatic catechol structures conferred a thermal shift, that was more evident in the case of complex polyphenols. Mechanical Response The mechanical response of the active ingredients based PVA films was here investigated taking into account the final application in the industrial sector for which the mechanical characteristics are critical issues. The performed tensile tests permitted to evaluate the influence of GA and QC on the PVA mechanical properties, experimental results are reported in Figure 5b and Table 2. The elongation at break of PVA neat film was measured at 195 ± 45%, underlining the ductile nature of the selected polymer matrix, as already previously observed in the literature [12]. The addition of different amounts of GA and QC to PVA induced a modification in the plastic response of PVA matrix. In particular, the deformation at break for PVA/5GA increased to about 280% as a result of a plasticizing effect [59], while the value was maintained similar to unfilled system by adding 10% wt. of GA in the PVA matrix. It is reasonable to argue that the presence of phenolic compounds having hydrophilic properties might have contributed to increase the inter-chain interactions, reducing the flexibility of the PVA films [57,60]. In comparison with neat PVA, a limited decrease of strength values was also observed, that can be explained by the change of intramolecular bonding with the addition of GA and QC. PVA/5QC films showed tensile strength values higher than PVA/5GC, and this result can be related to the inter-molecular interactions between hydrophilic groups in PVA and polyhydroxyl groups of quercetin [61]. On the other hand, tensile strength of PVA/10QC was weaker than that of PVA. This was because the incorporation of excessive amount of hydrophobic quercetin made the inner structure of PVA film become discontinue [62]. In analogy with results from Yoon et al. [63], that reported the variation in flexibility and strength of PVA films containing additives having both hydroxyl and carboxyl groups (glycerol and succinic acid) and glycerol (only hydroxyl groups), in our case we observed that, when GA having both hydroxyl and carboxyl groups is added to PVA films, more rigid films were found than those with only hydroxyl group-containing agent (QC). 50 ± 7 c 60 ± 10 a 170 ± 10 a 520 ± 110 a,b (a-c) Different superscripts within the same column indicate significant differences among formulations (p < 0.05). Overall Migration, Antioxidant Activity of PVA Formulations and Simulant Solutions at Different Times Overall migration test is used to determine the limit value at which the substances that compose the polymeric base of specific films for food packaging applications can migrate into foods during the different phases, in particular during the transport and commercialization of foodstuffs [64]. In this research work, the analysis was performed by using one food simulant, ethanol 50% (v/v) (D1), utilized to established the behavior of polymeric films in contact with fatty foods. Table 3 summarizes the migrated levels for all the systems, all the values were lower than the migration limit for foodstuffs contact materials (60 mg kg −1 simulant). The obtained results demonstrate the potential applicability of the realized polymeric systems in contact with the food. Generally, the migrated value increases with increase of QC (or GA) content in the matrix [26,65]. The presence of GA in PVA films induces a slight increase of migration levels in comparison with PVA/QC based films (PVA/5GA = (6.8 ± 0.1) mg kg −1 and PVA/10GA = (9.9 ± 0.5) mg kg −1 for GA, PVA/5QC = (6.6 ± 0.4) mg kg −1 and PVA/10QC = (9.5 ± 0.4) mg kg −1 for QC). This behavior can be related to the tendency of GA to better solubilize in aqueous solutions with respect of QC [66] (Table 3). Additionally, the oxidation of foodstuffs during transportation and storage leads to a series of negative changes in food, such as the sensory characteristics of the product (e.g., changes in aesthetic aspect and color, rancidity, and smell) [67,68]. According to this, the efficiency of the produced PVA/GA and PVA/QC based formulations as antioxidant packaging systems was also verified, by testing the reduction of stable free radical DPPH, the obtained data were expressed as RSA (%) and reported in Figure 5c and Table 4. The high antioxidant activity of GA and QC is related to the number and position of the free phenolic hydroxyl groups in the QC and GA molecule [69,70], that intercept free radical chain, inducing the formation of a stable end product, which does not initiate or propagate oxidation of lipids [70]. In Table 4, RSA values for PVA/GA and PVA/QC films evaluated at different times was also presented: no significant differences among the different formulations and among the same formulation at different times were noted (except for the time 0 that shows the lowest value of RSA). The radical scavenging activity reached a maximum reduction of DPPH radicals at 7 days in contact of food simulant (Table 4) Furthermore, the residual antioxidant activity resulting from specific migration tests of GA and QC containing PVA films was determined (Figure 6a . Indeed, all samples showed an antioxidant activity in line with the release studies, that will be further discussed. Release Tests in Food Simulant Release tests were done according to the European Standard EN 13130-200522 and European Commission Regulation 10/2011 [71], to define the release profile of GA and QC incorporated into the PVA matrix. Thus, PVA/GA and PVA/QC systems were immersed into the simulant used for fatty foods (ethanol 50% v/v) for 21 days. All samples were examined at 1, 3, 7, 10, and 21 days, measuring released GA and QC, by UV-vis spectrophotometry (Figure 6b). In all formulations, measured values for released QC and GA are correlated with their concentrations in the films. Indeed, the migration levels from PVA/10GA are increased respect to PVA/5GA, which have been measured, respectively, as 701.54 µg/mL (day 21) and 296.76 µg/mL (day 21). PVA/QC followed the same trend with 495.96 µg/mL (day 21) for PVA/10QC and 231.11 µg/mL (day 21) for PVA/5QC, respectively released from the PVA matrix. In similar migration experiments, it has been reported that only 1.15% of the QC had been released after 48 h in 95% EtOH and that no more QC was released when the test time was extended to 72 h [24]. FTIR characterization for PVA films after the different release times was also performed, with the main aim of correlating the RSA/release tests information with the physical state of the PVA films containing GA and QC at the two different contents. In Figure 7a, the spectra of the PVA films at the different times are reported. It can be commented that, in case of neat PVA, no substantial variations were observed, with exception of the peak at 1322 cm −1 (due to -CH bending), 1142 cm −1 (C-O stretching band), and 915 cm −1 (assigned to the CH 2 rocking vibration) that were more intense, as reported by Zuo et al. [72], PVA membrane after immersion in aqueous ethanol solution showed variation in contact angle values for fully hydrolyzed PVA, attributed to the reorientation of hydroxyl groups at the surface of the membrane. In our case, when the PVA surface contacts the ethanol/water feed mixture, polar −OH groups reoriented at the surface, creating a more hydrophilic conformation (confirmed even by the more intense signal, for our PVA films, of -OH band centered at 3310 cm −1 from 0 to 21 days, levelled after 10 days, confirming the results of PVA 99% water contact angle already observed in [72], where ethanol molecules adsorb from solution onto a PVA film surface in an ordered and cooperative way governed by H-bonding, resulting in a more hydrophobic surface, when the hydrolysis degree of PVA is higher than 96%. These interesting packaging materials indeed exhibit a coloration due to the addition of the two ingredients: color of such materials can be considered definitely useful in many applications, such as in the labeling of packaged food, since color of solutions/films can be not only tuned by using natural phytocompounds, but they could be employed as indicators of polymers ageing time when a pH variation is observed. According to this, color parameters for the produced films at time 0 and after different release times were measured. The initial deviation (time 0) of b value from 0.19 ± 0.02 to 5.66 ± 0.02 and increased value of ΔE (6.36 instead of 0.56) showed, for PVA/5GA sample, the tendency towards yellowing and total color changes of films compared to the control (PVA film), essentially due to the presence of well dispersed polyphenolic compounds. When higher amounts of GA was introduced, reduced tendency to yellowness was measured (b = 3.00 ± 0.02), probably related to a limited dispersion of the active ingredient, as already confirmed by mechanical and morphological analysis of PVA/10GA films. In the case of GA and QC containing films (Figure 7b), spectra analysis evidenced a relative intensity variation for the peaks at 1091 and 1142 cm −1 , assigned to stretching vibrations of C=O in PVA. More intense peaks at 1375 cm −1 and 1237 cm −1 , related to vibration of phenol alcohol in GA, were noted at 7 and 21 days, while a signal at 1040 cm −1 (C-O stretching) resulted less intense: these results are in line with the detected amount of GA in the ethanolic solutions at the same times. In the case of QC, the modified intensity of bands for aromatic C=C at 1611 and 1522 cm −1 , in-plane bending band of C-H in aromatic hydrocarbon at 1320 cm −1 , C-O and C-CO-C stretch stretching in the aryl ether ring at 1263 and 1168 cm −1 confirmed the evident release of the QC ingredient. On the other hand, it was observed, during the release tests, that the overall swelling degree of GA added films was higher than those of QC added films at 7 and 21 days, and it has been confirmed by analysis of the morphology for swelled fractured surfaces at time 7 and 21 days (Figure 8a): due to the more hydrophilic character of hydroxyl and carboxyl groups containing additive, GA containing films resulted more porous than both neat PVA and PVA/QC films, justifying the increased released amount in case of GA. Nevertheless, as previously observed by Luzi et al. in PVA/Hydroxytyrosol systems [12], the addition of an hydrophilic active ingredient generally enhances the water absorption, which in turn can be also correlated to the lower values of crystallinity degree measured for formulation at 10% wt. of GA, as reported in Table 1. ± 0.02 and increased value of ΔE (6.36 instead of 0.56) showed, for PVA/5GA sample, the tendency towards yellowing and total color changes of films compared to the control (PVA film), essentially due to the presence of well dispersed polyphenolic compounds. When higher amounts of GA was introduced, reduced tendency to yellowness was measured (b = 3.00 ± 0.02), probably related to a limited dispersion of the active ingredient, as already confirmed by mechanical and morphological analysis of PVA/10GA films. These interesting packaging materials indeed exhibit a coloration due to the addition of the two ingredients: color of such materials can be considered definitely useful in many applications, such as in the labeling of packaged food, since color of solutions/films can be not only tuned by using natural phytocompounds, but they could be employed as indicators of polymers ageing time when a pH variation is observed. According to this, color parameters for the produced films at time 0 and after different release times were measured. The initial deviation (time 0) of b value from 0.19 ± 0.02 to 5.66 ± 0.02 and increased value of ∆E (6.36 instead of 0.56) showed, for PVA/5GA sample, the tendency towards yellowing and total color changes of films compared to the control (PVA film), essentially due to the presence of well dispersed polyphenolic compounds. When higher amounts of GA was introduced, reduced tendency to yellowness was measured (b = 3.00 ± 0.02), probably related to a limited dispersion of the active ingredient, as already confirmed by mechanical and morphological analysis of PVA/10GA films. After the release tests (values for 7 and 21 days are also included in Table 5) and immersion in the simulant solution, only the color of the PVA/5GA films deviated towards green (a = −3.64 ± 0.08 when compared with neat PVA (a = −0.17 ± 0.01)), while the a values turned to be less negative (a = −0.65 ± 0.09) in PVA/10GA, and the b remained substantially unvaried at 21 days. In the case of QC containing films, even the a value was greatly modified towards red, due to the initial color of the filler itself, with yellow basic color shifted to orange-like hues (Figure 8b). Since color change in intelligent films is largely due to the generation of organic acids during anaerobic respiration of anaerobic bacteria or facultative anaerobic bacteria under hypoxic or anaerobic condition, higher acidity values could indicate a time evolving inferior freshness [73]. According to these results, it could be reasonable to use these films as intelligent labels to detect acidity variations in the packages during relatively short food life. Conclusions The results of this study suggest that GA and QC as active ingredients in PVA films can be used to tune not only the antioxidant behavior, but also the deformability of the produced films. It was proved that high content of active ingredients (10% wt.) can be easily released in simulated ethanolic solution and can express high antioxidant activity. At the same time, the active ingredients can hinder the chains packing and inhibit the crystals growth, giving more rigid films, as detected in GA containing films. Nevertheless, QC, that was less dispersible in PVA solutions at the same conditions and showed reduced RSA activity in comparison with GA, can be indeed more efficient in tuning the deformability of the water cast PVA films, due the presence of sole hydroxyl groups carrying agent. The capability of both polyphenols of altering the color of the films after release at different specific times also confirmed the possibility of using these easy processable PVA films as active and intelligent films in food packaging when a specific shelf life (3-21 days) is required.	2019-12-05T09:07:20.796Z	2019-12-01T00:00:00.000	{ "year": 2019, "sha1": "ebe3f51876c9f9ac950f8e52cc87f7baaec0b04f", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4360/11/12/1999/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "445e85ad1d4e1eeaeba6e613c4280bf653121d71", "s2fieldsofstudy": [ "Materials Science", "Agricultural and Food Sciences" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
210699975	pes2o/s2orc	v3-fos-license	DMCNN: A Deep Multiscale Convolutional Neural Network Model for Medical Image Segmentation Medical image segmentation is one of the hot issues in the related area of image processing. Precise segmentation for medical images is a vital guarantee for follow-up treatment. At present, however, low gray contrast and blurred tissue boundaries are common in medical images, and the segmentation accuracy of medical images cannot be effectively improved. Especially, deep learning methods need more training samples, which lead to time-consuming process. Therefore, we propose a novelty model for medical image segmentation based on deep multiscale convolutional neural network (CNN) in this article. First, we extract the region of interest from the raw medical images. Then, data augmentation is operated to acquire more training datasets. Our proposed method contains three models: encoder, U-net, and decoder. Encoder is mainly responsible for feature extraction of 2D image slice. The U-net cascades the features of each block of the encoder with those obtained by deconvolution in the decoder under different scales. The decoding is mainly responsible for the upsampling of the feature graph after feature extraction of each group. Simulation results show that the new method can boost the segmentation accuracy. And, it has strong robustness compared with other segmentation methods. Introduction Medical imaging makes a critical difference in clinical diagnosis [1][2][3]. Recently, with the progress of medical imaging technology and the continuous development of artificial intelligence image processing, medical image processing technology has gradually developed into a key research field. It is vital in clinical application. The aim of medical image segmentation technology is to segment the interested part by some deep automatic segmentation algorithms and make the segmentation results as close as possible to the original structure of the region [4]. Segmentation of medical image has big significance in clinical diagnosis and pathological diagnosis. Measuring lesion volume with segmented images can assist doctors to determine the disease and make treatment plans [5]. Medical imaging segmentation (MIS) is an indispensable stage in ROI (region of interest) extraction, quantitative analysis, and 2D reconstruction. The images will be segmented with the same or similar features (such as intensity, color, and texture) into separated areas, particularly to extract the lesion areas with special meanings or other regions of interest (ROI) from the complex background, so as to provide basis for clinical analysis [6]. Magnetic Resonance Imaging (MRI) uses the principle of nuclear magnetic resonance, which not only has high soft tissue resolution but also provides rich and high-resolution three-dimensional brain tissue information. Therefore, how to segment the medical images accurately in MRI images is becoming a challenging task in medical image research [7]. Through the analysis of research status, we summarize three kinds of traditional MIS methods: (1) manual segmentation method, which is tedious, excessive labor, subjective, prone to error, and not suitable for large-scale research [8]; (2) semiautomatic segmentation method, which requires accurate control of prior parameters and consumes much time in the process of parameter tuning [9]; (3) the traditional segmentation methods such as graphbased, deformation model, and active appearance model [10][11][12], which is based on simple registration method. However, due to the differences between the hippocampus, in terms of the segmentation efficiency and accuracy, a simple registration method is still not ideal. Currently, deep learning has attracted more attention, and the model based on deep CNN and its variants have been diffusely used in various fields of medical image processing and also achieved better results [13][14][15]. For example, Cha [16] presented MR brain image automatic segmentation method using a CNN network. The method was independent on explicit features only requiring a single MR (magnetic resonance) image. Lu et al. [17] used twodimensional convolutional neural network to evaluate the segmentation of electron microscope images. Zhang et al. [18] adopted deep CNN to evaluate the segmentation of multimodal brain images. Although the models based on CNN have obtained better performance, these methods have a common problem, namely, all networks take image block as input, due to a large amount of overlapping image blocks, the redundant computation will increase the time cost for testing the network, and the image block size will influence the capability of the trained network. To solve the problem of image segmentation, many researchers have come up with many approaches based on the fully convolutional network (FCN) model to remedy limitations of image segmentation. FCN can take the entire image as the input of the network and generate the corresponding output of the whole image, thus avoiding the problems caused by the use of image blocks. However, it had low efficiency. So, a deep multiscale CNN model is put forward for medical image segmentation. The major contributions are illustrated as follows: (1) First, we extract the regions of interest from the raw medical images. Then, the data augmentation is operated to obtain more training dataset. The encoder, U-net, and decoder models are used for constructing our proposed segment framework. (2) Encoder is mainly responsible for feature extraction of 2D image slice. (3) In different scales of the decoder, it will acquire the features of each encoder's block by deconvolution operation, and then the U-net joins them together. (4) The decoding is mainly responsible for the upsampling of the feature graph after feature extraction of each group. This paper is originated as follows. In Section 2, related works are introduced for the image segmentation. Section 3 describes the proposed DMCNN in detail. In Section 4, we conduct experiments and give analysis. Section 5 concludes the work. Inception Model. In order to make the convolutional neural network have better learning ability, the most direct and effective method is to make the network layer deeper. However, there are some disadvantages in this operation: (1) if the training number and dataset are limited, more parameters will easily lead to overfitting; (2) if the network is larger, it is hard to utilize due to the greater endless computation; (3) the deeper the network is, the gradient will disappear, which leads to the diffusion of gradient. Under this situation, it is difficult to optimize the network model. Inception v1 is proposed in 2014 [19], the convolution layer of 1 × 1, 3 × 3, and 5 × 5, and pooling layer of 3 × 3 are stacked together, which increases the width of the network and also enhances the adaptability of the network in terms of the scale. This operation can extract features from different scales. An important improvement in Inception v31 is decomposition, the two-dimensional convolution of N × N is divided into two-dimensional convolutions of 1 × N and N × 1. The advantage of this method is that it can not only accelerate the computation but also increase the nonlinearity of the network. Batch Normalization. BN aims to add a standardized processing for the input data of each layer in the training process of neural network, which also belongs to the network layer. Previously, we mentioned that in addition to the output layer of the network, the parameters of the lower layer of the network are updated during the training, which caused the change of the distribution of the input data in the latter layer. In each layer, it is better to add a preprocessing operation. For example, the data in the third layer of network are normalized. Then, it inputs the third layer for calculation, so that we can solve the problem of "Internal Covariate Shift" [20][21][22]. By introducing batch standardization method, the network's convergence speed will be greatly increased. The overfitting can also be controlled. The dropout and regularization operation will be realized with little utilization. End-To-End Models and Jump Connections. Compared to the traditional image block-based convolutional neural network model, the end-to-end model utilizes the entire image as input. The entire image will be generated as output [23,24]. The image block-based model needs to foretell each size of pixel in the slice separately. Therefore, the end-to-end model adopted in this paper can evidently decrease the time consumption when segmenting images. Generally, end-to-end models primarily include fully CNN and faster CNN. It combines the feature maps of different levels. Unlike FCN, the U-net model adopts jump connection to join the feature information obtained from the shrinking coding path and the deconvolution operation in the expanded path together which is favourable for obtaining multiscale feature information to strengthen the network's feature extraction ability. Data Preprocessing. For the acquired medical images, the region of interest should be extracted and preprocessed to serve as the samples for training and testing the network. After that, gray level regularization is carried out on all acquired ROIs. And, the mean and SD (standard deviation) are measured. The gray level regularization is assessed by subtracting mean value and dividing by SD. In the subsequent training and testing process, the extracted images are transported into the network model as samples, and the extracted region is shown in Figure 1. For example, the size of the raw image is 256 × 256, and after ROI extraction, we get the 128 × 128 patch and input it into network for training. Data Extension. Since labeled public medical image datasets are little online and they are inconvenient to use, training a deep CNN model is troublesome [25]. In our proposed model, we first adopt some newest data argumentation methods to expand the raw data in order to increase the number of available training samples. In this paper, five data expansion methods are adopted including vertical direction reversal, random angle rotation, random translation, horizontal direction reversal, and image deformation. Figure 2 shows an example of the image expansion. Proposed Network Model. To raise the accuracy of medical image segmentation, spatial information of images and relevant information between 2D slices are effectively utilized. The proposed deep multiscale convolutional neural network model contains three parts: encoder, U-net, and decoder as shown in Figure 3. Encoder. It is mainly responsible for feature extraction of 2D slices, whose network structure is shown in Figure 4. Small convolution kernel in convolutional network is conducive to capturing local information, while large convolution kernel is conducive to capturing global information. However, ROIs are different in 2D slices, and it is difficult to select an accurate and universal convolution kernel. For this purpose, we use three different convolution layers (1 × 1, 3 × 3, 5 × 5) to extract information of multiple scales in Inception V1, which can extract more features. Additionally, to reduce the amount of computation, asymmetric convolution kernel is used in this experiment to decompose the N × N two-dimensional convolution into two one-dimensional convolutions with 1 × N and N × 1. Meanwhile, to expand the receptive field of convolution and perfectly obtain multiscale information without increasing the size of parameters, this paper adds dilated convolutions with expansion coefficients of 2 and 4, respectively. For an ordinary convolution layer of 3 × 3, the receptive field of its convolution kernel is 3 × 3. In this paper, after the employment of dilated convolution (DC), the size of parameters remains unchanged, but the receptive field of the convolution kernel becomes 7 × 7 and 15 × 15. It can be seen that DC greatly increases the receptive field of the convolution layer without increasing parameters number. As shown in Figure 4, there are three kinds of dilated convolution with expansion coefficients of 1, 2, and 4, respectively, in the encoded part, and the receptive fields of the corresponding convolution kernel are 3 × 3, 7 × 7 and 15 × 15, respectively. After using the three receptive fields with different sizes, it is not only conducive to feature extraction but also conducive to better capturing the pathological area features. Then, after cascading the feature graphs extracted from five different convolution layers, we utilize two ordinary 3 × 3 convolution layers in feature extraction process. Finally, to reduce the size of the feature map, it connects a maximum pooling layer. In Figure 4, the number of channels in each convolution layer is 16. Considering the convergence of networks, batch normalization is added behind the convolution layer, and ReLu layer is used as the activation function. Figure 5 shows the proposed U-net network structure employed. The U-net network structure contains two parts as follows: The U-net model in this paper is displayed as Figure 5. U-Net Model. The proposed entire U-net network consists of twentyeight convolution layers. In here, twenty-four convolution layers are spread over four convolution blocks and four deconvolution blocks. The contraction encoder in deep Figure 4. Each convolutional block contains two convolutional layers (conv). Each convolutional layer utilizes a 3 × 3 convolutional kernel to carry out the convolution. The step size is 1. Synchronously, each convolutional layer follows a BN layer and a ReLu layer to modify the network performance. ReLu activation function has sparse ability, and it can better learn the relatively sparse features from the effective data dimension and play the role of feature automatic decoupling. In each convolution block, its first convolution layer can double feature graphs. The number of feature graphs will be increased to 64. After four convolution layers, the number of feature graphs is increased from 64 to 1024. Between each convolution block, the sampling method of traditional U-net adopts maximum pooling. In this paper, we use the 2 × 2 convolution kernel with step length 2 for down-conv operation to achieve a convolution block feature on the image of the sampling operation. Through this down- Conv + BN + ReLu 4 convolution blocks 3 up-convolution blocks Conv + BN + ReLu Conv + sigmoid conv operation, the size of feature graph is reduced by half with iterative deepening, so that the size of input original image is decreased from 128 × 128 layer by layer to 8 × 8. The expansion decoder of the U-net model contains three deconvolution blocks as shown in the right of Figure 6. The deconvolution up-conv operation adopts the 3 × 3 convolution kernel. The step size is 2, and the size of the feature graph is increased by twice the original size through the deconvolution operation. This process can recover the feature graph as the raw input image in the last deconvolution block. Meanwhile, the number of feature graphs is halved after each deconvolution operation. The feature graphs obtained by deconvolution are cascaded with the corresponding feature graphs in the convolution block as the feature input of the deconvolution block. Two convolution layers are in each deconvolution block. The 3 × 3 convolution kernel is utilized (the step size is 1). The first convolution layer will reduce the number of feature graphs by half after every cascading. Not exactly the same as the original U-net structure, the presented U-net structure is filled with zero filler in each convolution layer. According to formula (1), the output size of the deep multiscale CNN model can be guaranteed to be consistent with the input image data size by using zero padding: where I input and I output are on behalf of the input and output images' size in DMCNN, respectively. F represents the convolution kernel with size 3 × 3. P denotes the fill size with 1 × 1. S � 1 stands for step size in this paper. At the end of the proposed U-net model in this paper, we skillfully adopt a convolution layer (whose size is 1 × 1) to lessen the number of feature graphs to 1. The final output will be disposed by the Sigmoid function. We can obtain the value of each pixel between 0 and 1. The lesion area is a probability distribution. Through the above processing, the final image is considered as the probability graph of DMCNN. The value corresponding to each pixel indicates the probability that the point belongs to the lesion. Decoding Part. It is mainly responsible for upsampling the feature graph after extracting each group feature, and the structure is shown in Figure 7. The decoder section contains one deconvolution layer and one convolution layer. Both deconvolution and convolution have batch-normalization and ReLu. After the upsampling, the feature graph becomes the same resolution as the input image. Finally, the final segmentation result is obtained by softmax classifier to analyze the end-to-end segmentation. Loss Function. Different from the commonly used pixel point-based softmax loss function [26,27], Dice loss function is based on region loss function. In medical image segmentation, Dice index is often used to measure the overlap rate between the object and the detection area. If the Dice value is larger, then the overlap degree is higher, and the segmentation effect is better. However, Dice index cannot be directly used as a loss function, so we use the improved Dice function. Dice function is a function that gives feedback to network parameters after independent evaluation for each area [28]. The calculation form and process of Dice function are in good agreement with medical image segmentation. Therefore, Dice loss function used in this paper is defined as follows: where g stands for the ground truth. p is the predicted value. v is the number of pixels in each image block. Dice always is used as a loss function, when comparing the probability graph with the labeled. The background part whose labeled value is 0 will not be calculated into the loss to avoid the situation of unbalanced category and accelerate the convergence of the network and improve the segmentation accuracy. Dataset and Evaluation Index. The dataset is from ADNI (Alzheimer's Disease Neuroimaging Initiative: adni.loni.usc. edu) [29,30]. In this advanced researches, 100 groups of brain MRI images and segmented hippocampal tags are obtained from ADNI library. From this group, 80 groups are randomly selected for cross-validation, and the remaining 20 groups are for testing. To improve the segmentation veracity, this study preprocesses the data with three steps. First, consider that the hippocampus only accounts for a small part of the whole brain MRI image and other parts are invalid areas. The pixel values in brain MRI are statistically analyzed, and the images are cropped into 80 × 80 × 40 including the hippocampus and the blank area around it. In this way, invalid background information can be reduced without any influence on the integrity of valid information. Second, to accelerate the convergence of the network and consider the inconsistency of the pixel values of MRI images in ADNI, the mean and SD methods are utilized to normalize the images. Thirdly, making allowances for the small number of samples in the dataset, we enhance the obtained MRI images by left rotation and right rotation and finally obtain 400 MRI images. To accurately reflect the performance differences between algorithms, we use uniform platform. The hardware environment of the experiment is NVIDIA GTX1060Ti, Intel Corei7 processor, and the software environment was Keras2.2.4. In the experiment, glorot normal distribution method is used to initialize the weight. The image size is 300 × 300 pixel used in this section. Execution environment is GPU and Geforce GTX 1060. The parameters used in DMCNN are given in Table 1. To quantitatively evaluate the performance of the new approach, dice similarity coefficient (DSC), sensitivity (SEN), and predictive positivity value (PPV) are selected as the evaluation indexes for the medical image segmentation result. They are defined as follows: where P denotes the lesion region segmented by the presented algorithm. T expresses the region of Ground truth. P∩T represents the pixel region of the intersection between the algorithm's segmentation region and the true segmentation region. Comparative Analysis of Segmentation. In this paper, 100 images before augmentation and 400 images after augmentation are segmented by the proposed method. The evaluation indexes in above section are used for evaluation. The comparison results of DSC, SEN, and PPV are given in Table 2. From Table 2, it reveals that data augmentation can greatly improve the segmentation accuracy. This also fully proves the importance of datasets in the deep learning model construction process. The size of the datasets can directly affect the learning capability of the model. To verify that DMCNN can effectively capture information between slices, we conduct the comparison between multiscale convolutional neural network and single-scale CNN in this paper. Other conditions remain unchanged. The effectiveness of the proposed model can be observed from Table 3. We can see that the segmentation accuracy of DMCNN is significantly higher than that of single-scale CNN, which further verifies that DMCNN can better learn more feature information between slice sequences than single-scale CNN. Meanwhile, we study the effect of different network models on experiment results. Two representative segmentation methods including U-net and 2D U-net network model are compared with our deep multiscale U-net given in Table 4. The segmentation accuracy obtained by the DMCNN method is higher than that of the other two methods, indicating that it can extract features more efficiently and improve the segmentation accuracy. Compared with the multiple groups of up-and downsampling layers in U-net and 2D U-net networks, the proposed network model only contains one up-and downsampling layer, which greatly reduces the size of the parameters. The number of parameters of the encoding part and decoding part is below 5000, which significantly reduces the computation time in this article. We also conduct comparison experiment with state-ofthe-art segment methods including TLWK [31], MNF [32], and SUSAN [33] on our medical data. The results are given in Table 5. TLWK adopted the traditional random forest regression method, and MNF adopted the multiscale method. They are all automatic segmentation methods, due to the large gap between different individuals, and the accuracy and efficiency of segmentation are often not ideal, which is not as high as the precision of the automatic segmentation method in this paper. SUSAN simply improves 2D U-net, so the results are not very good. In general, the proposed method combining convolution neural network and multiscale U-net model is superior to other current methods for medical image segmentation. And, the time consumption is shorter than other methods too. Figures 8-10 are the segmentation comparison results in terms of hippocampus, retinal blood vessel, sarcoma, and meningioma. Given the segmented image, the IoU measure gives the similarity between the predicted region and the ground truth region for an object and is defined by following equation: where TP, FP, and FN denote the true positive, false positive, and false negative counts, respectively. The results are given in Table 6. We can see that the proposed segment method has the better result. Conclusions This paper proposes a medical image segmentation method based on multiscale convolutional neural network. This method can realize automatic segmentation of medical images and has high accuracy of segmentation. The CNN model in this paper not only reduces the amount of computation but also effectively captures multiscale information. In addition, the use of U-net fully mines the relevant information between slice sequences. Taking relevant medical image segmentation as an example, the experimental results on ADNI database show that the segmentation method in this paper is superior to other methods. The proposed method can perform segmentation tasks more easily and accurately. In the future, studying on deeply deep learning methods to segment images and applying them into different types of images and different practical engineering are warranted. Data Availability The data used to support the findings of this study are available from the corresponding author upon request.	2020-01-02T21:45:59.677Z	2019-12-26T00:00:00.000	{ "year": 2019, "sha1": "79ec996d53d400953c9647e78d12bec4681b1e48", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1155/2019/8597606", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "28def63fc4b2f0c59a91aa6b2cc03f09686ce3b5", "s2fieldsofstudy": [ "Medicine", "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Medicine" ] }
219924142	pes2o/s2orc	v3-fos-license	The complete chloroplast genome sequence of Populus davidiana, and a comparative analysis with other Populus species Abstract Populus davidiana plays an important ecological role in boreal and temperate forests, serving as wildlife habitats and watersheds. The complete chloroplast genome sequence of P. davidiana was characterized from Illumina pair-end sequencing. The chloroplast genome of P. davidiana was 155,325 bp in length, containing a large single-copy region (LSC) of 84,679 bp, a small single-copy region (SSC) of 16,862 bp, and two inverted repeat (IR) regions of 26,892 bp. The overall GC content is 36.80%, while the corresponding values of the LSC, SSC, and IR regions are 34.5%, 30.5%, and 42.5%, respectively. The genome contains 131 complete genes, including 86 protein-coding genes (62 protein-coding gene species), 37 tRNA genes (29 tRNA species), and 8 rRNA genes (4 rRNA species). The neighbour-joining phylogenetic analysis showed that P. davidiana and P. hopeiensis clustered together as sisters to other Populus species. Introduction Populus davidiana occurs in northern and central parts of China, plus Mongolia, Korea, and the Far East of Russia (Zheng et al. 2017;Hou et al. 2018). Populus davidiana is widely distributed in the Northern Hemisphere and plays an important ecological role in boreal and temperate forests, serving as wildlife habitats and watersheds; they can dominate riparian forests, but are ecologically adaptable. Populus davidiana has wide geographic distribution, high intraspecific polymorphism, adaptability to different environments, combined with a relatively small genome size. Consequently, P. davidiana represents an excellent model for understanding how different evolutionary forces have sculpted the variation patterns in the genome during the process of population differentiation and ecological speciation (Neale and Antoine 2011). Moreover, we can develop conservation strategies easily when we understand the genetic information of P. davidiana. In the present research, we constructed the whole chloroplast genome of P. davidiana and understood many genome variation information about the species, which will provide beneficial help for population genetics studies of P. davidiana. The fresh leaves of P. davidiana were collected from Lijiang city (100 23 0 N, 26 88 0 E). Fresh leaves were silica-dried and taken to the laboratory until DNA extraction. The voucher specimen (SY002) was laid in the Herbarium of Chongqing University of Arts and Sciences and the extracted DNA was stored in the -80 C refrigerator of the Key Laboratory of College of Landscape Architecture and Life Science. We extracted total genomic DNA from 25 mg silicagel-dried leaf using a modified CTAB method (Doyle 1987 (Nurk et al. 2017) to assemble chloroplast genomes. We used P. tremula (GenBank: NC_027425) as a reference genome. We annotated the chloroplast genome with the software DOGMA (Wyman et al. 2004), and then corrected the results using Geneious 8.0.2 (Campos et al., 2016) and Sequin 15.50 (http://www.ncbi.nlm.nih.gov/Sequin/). The complete chloroplast genome of P. davidiana (National Genomics Data Center accession number GWHAMJQ01000000) was characterized from Illumina pairend sequencing. The complete chloroplast genome sequence of P. davidiana was characterized from Illumina pair-end sequencing. The chloroplast genome of P. davidiana was 155,325 bp in length, containing a large single-copy region (LSC) of 84,679 bp, a small single-copy region (SSC) of 16,862 bp, and two inverted repeat (IR) regions of 26,892 bp. To confirm the phylogenetic location of P. davidiana within the family of Populus, we used the complete chloroplast genomes sequence of P. davidiana and 21 other related species of Populus and Salix babylonica and Salix arbutifolia as outgroup to construct phylogenetic tree. The 22 chloroplast genome sequences were aligned with MAFFT (Katoh and Standley 2013), and then the neighbour-joining tree was constructed by MEGA 7.0 (Kumar et al. 2016). The results confirmed that P. davidiana was clustered with P. hopeiensis (Figure 1). Disclosure statement No potential conflict of interest was reported by the author(s). Funding Financial support for this research was provided by the Science and Technology Research Project of Chongqing Education Commission [KJQN201801336]. Data availability statement The data that support the findings of this study are openly available National Genomics Data Center at https://bigd.big.ac.cn/ search?dbId=gwh&q=GWHAMJQ01000000, accession number GWHAMJQ01000000.	2020-06-11T09:10:53.270Z	2020-06-04T00:00:00.000	{ "year": 2020, "sha1": "579afad4c3927a63d68913194415886d093ddf3e", "oa_license": "CCBY", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/23802359.2020.1773344?needAccess=true", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "6f7c8d5b150f9d12519b7b5351ae15e325be9d82", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
14676933	pes2o/s2orc	v3-fos-license	Drug Target Identification and Elucidation of Natural Inhibitors for Bordetella petrii: An In Silico Study Environmental microbes like Bordetella petrii has been established as a causative agent for various infectious diseases in human. Again, development of drug resistance in B. petrii challenged to combat against the infection. Identification of potential drug target and proposing a novel lead compound against the pathogen has a great aid and value. In this study, bioinformatics tools and technology have been applied to suggest a potential drug target by screening the proteome information of B. petrii DSM 12804 (accession No. PRJNA28135) from genome database of National Centre for Biotechnology information. In this regards, the inhibitory effect of nine natural compounds like ajoene (Allium sativum), allicin (A. sativum), cinnamaldehyde (Cinnamomum cassia), curcumin (Curcuma longa), gallotannin (active component of green tea and red wine), isoorientin (Anthopterus wardii), isovitexin (A. wardii), neral (Melissa officinalis), and vitexin (A. wardii) have been acknowledged with anti-bacterial properties and hence tested against identified drug target of B. petrii by implicating computational approach. The in silico studies revealed the hypothesis that lpxD could be a potential drug target and with recommendation of a strong inhibitory effect of selected natural compounds against infection caused due to B. petrii, would be further validated through in vitro experiments. Introduction Infectious diseases are major dreadful threat to human life, spread through many causative agents like bacteria, fungus, virus, etc. Therefore, development of potential drug is highly essential to fight against infectious diseases. Generally, methods of traditional drug design are tedious, time consuming, and cost intensive. Hence, several multidisciplinary approaches have gained research attention to reduce the time and cost during drug development process. In silico drug design is one of such approach, provides remarkable opportunity for identification of novel lead compounds as comparison to other conventional methods. Drug target identification is an important step involved in computer aided drug design process. In this context, the present study is planned to identify a potential drug target against infectious diseases caused by Bordetella petrii using various computational tools and techniques. B. petrii is a gram-negative coccobacilli of the phylum Proteobacteria, has been found exclusively in close association with humans and various warm-blooded animals due to its independent existence as an environmental facultative anaerobe [1]. B. petrii exhibits the host associated properties, and its strains have been detected from environmental samples like microbial consortia degrading aromatic compounds, marine sponges, polluted soil and a grass root consortium [2][3][4][5][6]. The genome of B. petrii contains 5,287,950 base pairs, and has been observed with greatest numbers of expressed virulence factors [7]. Infectious diseases like mandibular osteomyelitis [8], suppurative mastoiditis [9], chronic pulmonary diseases [10], respiratory infection in case of bronchiectasis and cystic fibrosis [11,12] have been reported due to B. petrii infection. Again, exposure to soil or water containing B. petrii may lead to local colonization and eventually serious nosocomial infections SN Rath, et al. Natural Inhibitors for Drug Target of Bordetella petrii [13,14]. Some other bacteria like Escherichia coli [15], Pseudomonas aeruginosa [16], Chlamydophila pneumoniae [17], Staphylococcus aureus [18], and Haemophilus influenzae [19] have also been identified as a cause for such types of infections. Many Food and Drug Administration (FDA) approved drugs have been used against these pathogens, but those drugs have less effect on B. petrii due to development of substantial resistance [10], generated an opportunity to identify a potential drug target as well as to discover a novel lead compound against B. petrii using computational techniques. Again, due to adverse effect of most of the anti-infectious drugs of synthetic origin, natural compounds with anti-infectious properties have been tested in both in vivo and in vitro to be an alternative approach towards the treatment. In this context, some of the natural compounds such as ajoene (Allium sativum), allicin (A. sativum), cinnamaldehyde (Cinnamomum cassia), curcumin (Curcuma longa), gallotannin (active component of green tea and red wine), isoorientin (Anthopterus wardii), isovitexin (A. wardii), neral (Melissa officinalis), and vitexin (A. wardii) have been acknowledged with anti-bacterial properties [20][21][22][23][24][25]. Curcumin is a naturally occurring polyphenolic compound, a common component of Indian spice turmeric, having anti-tumor, anti-oxidant, and anti-inflammatory properties. It has been reported as an agent to reduce bacterial colony formation and inhibit lung cancer progression in high-risk chronic obstructive pulmonary disease (COPD) patients [20]. Similarly, gallotannin is an active component of green tea and red wine has been observed as a potent inhibitor of calcium-activated Clchannel, may be used for the treatment of cystic fibrosis [21]. Again, in vitro study of three flavone C-glycosides such as isoorientin, vitexin, and isovitexin isolated from A. wardii have been reported as potent inhibitors for interleukin-8, and matrix metalloproteinase-1, an inflammatory marker in case of COPD [22]. Moreover, most of the bacterial infection caused due to formation of bio film in the host. In this regard, an active compound cinnamaldehyde of C. cassia have been identified as a strong inhibitor against quorum sensing (QS) biofilm formation due to E. coli, and Vibrio harveyi [23] infection. Similarly, two extracts of A. sativum namely allicin and ajoene have been accounted for inhibitory effect against P. aeruginosa and E. coli [24]. Likewise, neral is an active component of M. officinalis, has been reported as anti-bacterial, anti-fungal, and anti-oxidant properties against P. aeruginosa, Klebsiella pneumoniae, S. aureus, and Citrobacter koseri infection [25], and may be applied for several infectious diseases including nosocomial infection. As, all of these nine natural compounds have been established with anti-bacterial properties, may be useful for treatment of B. petrii infection, encouraged authors to elucidate their inhibitory effect against the putative drug target of B. petrii through in silico approach. Subtractive proteomics The complete protein datasets of B. petrii (whole genome project accession No. PRJNA28135) were retrieved from Genome database of National Centre for Biotechnology Information (NCBI) web server (http://www.ncbi.nlm.nih. gov/genome/). The essentiality search [26] was carried out through the Database of Essential Gene (DEG) for screening out the essential proteins responsible for the survival of B. petrii in the host. The bit score parameter was considered as greater or equal to 100 for homology search using Basic Local Alignment Search Tool (BLAST) while accessing DEG (http://tubic.tju.edu.cn/deg/blast.php) database, to select bacterial essential proteins. The non-redundancy search was performed through CD-HIT (http://weizhong-lab.ucsd.edu/ cdhit_suite/cgi-bin/index.cgi) web server to remove the redundant proteins among selected essential proteins of B. petrii. Further, by considering the resulted proteins of B. petrii, the sequence similarity search was carried out against human [27][28][29] followed by human gut flora microorganisms [30,31] using BLASTP algorithm to eliminate proteins homologous with human and human gut flora microorganisms respectively. During similarity search, the threshold for expected value (e-value) was fixed as greater or equal to 0.001. Furthermore, the protein sequences which are putative, unknown, hypothetical, probable, conserved hypothetical and unnamed protein were removed manually. The study of sub cellular localization was performed to identify the cytoplasm proteins among the selected proteins [32] using CELLO (http://cello.life.nctu. edu.tw/). Generally, proteins are annotated and ranked as per their evidence level in UniProt database which is also a factor to be considered while selecting putative drug targets. Afterwards, the Virulence Factor Database (VFDB) was explored, and the virulence proteins were screened out (http://www.mgc.ac.cn/VFs/blast/blast.html) among the selected targets of B. petrii through VFDB BLASTP search against VFDB core data sets with consideration of threshold e-value as greater than 0.0001 [31]. The above methodology is described through a flow diagram (Fig. 1A). Pathway analysis The participation of virulent proteins in any unique pathways of pathogen could be ensured and analyzed through the exploration of databases, is a common procedure to be followed while predicting putative drug targets. In this study, the pathway analysis was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/). Here, the exploration of KEGG database for pathway study was conducted twice. In the first case, the search was carried out for all obtained virulent proteins against existing pathways of B. petrii using KEGG BLAST algorithm. Secondly, unique pathways of B. petrii (pathways present in B. petrii, not in human) were sort listed from the KEGG database by comparing all pathways between B. petrii and human. Further, selection of common unique pathways [26,32,33] was made between the observed pathways of selected targets and obtained unique pathways of B. petrii. Furthermore, the selection pathways which are essential for the survival of the pathogen were made after detail inspection of obtained pathways. Finally, the key proteins regulating those pathways of B. petrii were considered as putative drug targets (Fig. 1B). Protein-protein network study The putative drug targets of B. petrii obtained after pathway analysis were subjected for protein-protein network analysis using STRING (http://string-db.org/) web server. Here, the interaction between the drugs targets were quantified on the basis [34] of confidence value (0.007) and the level of interaction (maximum as 50). The drug targets observed as having higher numbers of interaction with other drug targets among the chosen data sets were considered for further analysis (Fig. 1B). Similarity search and domain prediction Similarly search was carried out for selected proteins using BLASTP algorithm against Protein Data Bank (PDB) database (http://www.rcsb.org/pdb/). The closest homolog for each selected target protein was considered as template based on lowest e-value and highest percentage of similarity. The conserved domain region of potential drug target was predicted using conserved domain database (CDD) of NCBI web server (http://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi) with default parameters (Fig. 1B). Structure prediction and evaluation Phyre2 (Protein Homology/Analogy Recognition Engine) server was used for modelling of three dimensional structure of putative drug target of B. petrii. The algorithm of Phyre2 server predict three dimensional structure of a protein sequence using the principles and techniques of homology and/or fold recognition, as structural folding pattern is more conserved in evolution than its amino acid sequence. Hence, a protein of interest (drug target) can be modelled with reasonable accuracy basing on a distantly related protein with known (template) structure [35]. The energetically stable structure was obtained through structural refinement and energy minimization using ModRefiner algorithm [36] of Zhang Lab web server (http://zhanglab.ccmb.med.umich.edu/ ModRefiner/). The structural superimposition of refined model with template model was performed using Discovery Studio 3.5 suite. The structural quality and reliability of refined model were evaluated using RAMPAGE [37], ProSA-web [38], Protein Quality Predictor (ProQ) [39], and ERRAT [40] web tools. Molecular docking Molecular docking between the predicted model of potential drug target with each of the nine natural compounds was conducted using AutoDock-4.2 algorithm (http://autodock.scripps.edu/), by considering the protein structure as rigid whereas ligands were kept as flexible. AutoDock algorithm requires pre calculated grid maps, one for each atom type present in the flexible molecules being docked, and it stores the potential energy arising from the interaction with rigid macro molecules [41]. Ligands were docked around the identified conserved region within the functional domain of the selected drug target. The favorable poses were selected on the basis of lowest binding energy calculated by AutoDock which indicates for most favorable binding conformation between receptor and ligands. Docking procedure was carried out as per normal metho-dology for protein-ligand docking [42,43]. The visualization and analysis of interaction between docked complexes were made using Discovery Studio 3.5 suites. Screening of putative drug targets Availability of whole genome information of B. petrii DSM 12804 (accession No. PRJNA28135) in Genome database of NCBI web server encouraged authors to retrieve the entire set of 5,031 protein sequences in order to identify a potential drug target against it. Primarily, screening was made through identification of essential genes of B. petrii, considered as primitive strategy [26] for target identification. Here, out of total 5,031 proteins, only 1,841 were obtained as essential proteins and hence suspected as primary targets of B. petrii. To remove redundancy [29,30] among the putative drug targets, a non-redundancy search was carried out which resulted 1,802 proteins as non-redundant. Search for sequence homology with human proteins is an essential step to be followed for identification of proteins present only in pathogen but not in human [27][28][29]. Again, due to symbiotic relationship among human and human gut micro biota, homology search between proteins of pathogen and human gut flora microorganisms is also a crucial step [30,31] to be performed to eliminate the target proteins which are homologous to human gut flora microorganism. The resulted 799 numbers of proteins of B. petrii were not shown any significant similarity with human proteome as well as human gut flora proteome, hence were selected for downstream analysis. Furthermore, the reduction in protein dataset was done by selecting proteins reported as active and as well as functional. Out of 799 proteins, only 509 numbers of functionally characterized proteins were selected using the information available at UniProt database. As suggested by the literature, only cytoplasm proteins are suitable drug targets whereas membrane bound proteins are generally considered for vaccine development [32]. Among them only cytoplasm proteins (334 numbers) were considered for further study. Furthermore, screening was carried out on the basis of evidence report. As of UniProt database report, the evidence which supports the existence of proteins are classified as: (1) experimental evidence at protein level; (2) experimental evidence at transcript level; (3) protein inferred from homology; (4) protein predicted; and (5) protein uncertain. Out of these evidences, evidence at protein level is the best choice for protein existence, as it indicates the existence of the protein experimentally. But in the current investigation, not a single protein has been observed satisfying this criterion. Only 135 proteins which were inferred from homology subjected to virtual study. Identification of pathogenic proteins having virulence factor is also obligatory [31] while screening a drug target against any pathogenic microorganism. Therefore, homology search was carried out between 135 numbers of proteins and the core virulence proteins of B. petrii using VFDB BLAST. The result suggested, out of 135 selected proteins only 26 number of proteins found with virulence factor, which strongly supported for further study to predict potential drug target against B. petrii ( Figs. 1 and 2). Analysis of metabolic pathways The pathway analysis was conducted to screen out the putative drug targets and their involvement in important and unique pathways of the pathogen B. petrii which are essential for the survival of the pathogen. The preliminary search in KEGG database suggested for involvement of 22 proteins in different important pathways of B. petrii, and therefore the dataset of putative drug target reduced from 26 to 22. Further, exploration of all available pathways for both human and pathogen was conducted and compared, resulted 38 unique pathways present in B. petrii but absent in human. Furthermore, the comparison was made between the identified important pathways of B. petrii for 22 drug targets and 38 unique pathways identified in B. petrii, resulted 12 putative drug targets (Table 1) contributing towards the 3 common and unique pathways of B. petrii namely, lipopolysaccharide biosynthesis, two component system, and bacterial chemotaxis. As per literature suggest, lipopolysaccharide biosynthesis is an essential pathway [44] for the survival of B. petrii, a major component of the outer membrane of gram negative bacteria and referred as endotoxin. It is comprised of a hydrophilic polysaccharide and a hydrophobic component referred as lipid A which is responsible for major bio reactivity of endotoxin [44], also serves as a physical barrier providing protection towards bacteria from its surroundings [45]. Intense inspection on lipopolysaccharide biosynthesis pathway (Fig. 3) suggested, out of 12 selected putative drug targets (Table 1) only eight numbers (lpxH, kdsB, lpxK, lpxD, lpxB, kdsA, lpxC, and Bpet0453) were as key regulatory proteins [46]. Again, involvement of two components namely, amino acid and sugar metabolism and pentose phosphate pathway were found as regulating the complete pathway. lpxC, lpxD, lpxH, lpxB, and lpxK proteins are involved in amino acid and sugar metabolism, and among them lpxH was discarded as the synthesis of lpxB could alternatively possible by lpxD. Further, lpxB and lpxK were also rejected as potential drug target as the regulation of kdtA and waaA through lpxB and lpxK were otherwise possible by the proteins involved in pentose phosphate pathway. Furthermore, Bpet0453 (5.3.1.13), (Fig. 3), and regulating synthesis of kdtA and waaA, but the synthesis of same proteins was also control by proteins involved in amino acid and sugar metabolism to carried out the whole lipopolysaccharide biosynthesis. So the key proteins kdsA, kdsB, and Bpet0453 of pentose phosphate pathway were discarded. Finally, two proteins namely lpxD and lpxC were selected on the basis of their highly involvement in the regulation of lipopolysaccharide biosynthesis pathway than any other proteins (Figs. 2B and 3). Protein network analysis Protein network analysis is generally used for selection of drug targets having interaction with maximum numbers of other drug targets, an efficient technique to discover a potent drug target against pathogens [34]. Here, the biological interaction was studied for 2 obtained drug targets of B. petrii, and the assessment supported for both lpxD (with lpxC, lpxB, kdsA, kdsB, lpxH, lpxK, and bpet0453) and lpxC (with lpxD, lpxB, kdsA, kdsB, lpxK, and bpet0453) proteins showing interaction with other drug targets among the chosen data set (Table 2, Fig. 4). Therefore, lpxD and lpxC proteins were strongly recommended to be potential drug targets against the pathogen B. petrii. Identification of potent drug target and domain prediction Further, template structure identification for lpxD and lpxC proteins were undergone through similarity search against PDB. The template protein structure (PDB ID: 3PMO) from P. aeruginosa was selected for the drug target lpxD of B. petrii with 91% query coverage and 49% of identity where as template structure (PDB ID: 4FW3) from P. aeruginosa for lpxC protein of B. petrii with 88% query coverage and 56% of identity (Table 3). Though, it has been already established that lpxC catalyzes initially during the biosynthesis of the lipid A component of lipopolysaccharide and may be a potent drug target to block the lipopolysaccharide pathway of pathogens [47], the current investigation suggested, the status of lpxC protein B. petrii as un reviewed (Table 3) and have shown less number of interaction with other drug targets in comparison to lpxD (Table 2). Again, as per literature report, lpxD has a key role in biosynthesis of the lipid A component of lipopolysaccharide, and its inhibitors are also potentially attractive as antibacterial agents, as there is no close similarity found with human proteins [48]. Also, lpxD has a vital role in bio film formation which is causing nosocomial infection [16] and the status obtained from UniProt web server as reviewed (Table 3). This seems lpxD could be a potential drug target against B. petrii infection. Therefore, lpxD was proposed as a potent drug target subjected for further in silico study. Through BLAST search and inspection in CDD database, the conserved amino acid resides within the left-handed parallel beta helix (LbH) domain of lpxD were identified (Fig. 5A) Structure prediction and refinement of proposed drug target Due to absence of experimental structure the tertiary model of lpxD (UniProt ID: A9INS9) protein was predicted by the single highest scoring template i.e., lpxD protein of P. [35] web server with 100% confidence. Protein attains the lowest free energy during the native state; therefore the stability of the predicted model was improved through energy minimization [36] procedure, supported by the Root Mean Square Deviation score 0.612 obtained through the structural superimposition of lpxD models both before and after energy minimization (Fig. 5B). The refined lpxD protein model is publically available in Protein Model Database with PMDB ID (PM0080538). Model evaluation of proposed drug target The correctness and quality of predicted lpxD protein model of B. petrii was verified through RAMPAGE [37], ProSA-web [38], ProQ [39], and ERRAT [40] online server. The RAMPAGE analysis revealed 99.5% residues were folded properly suggesting good backbone folding pattern ( Fig. 5C). Further, Z-score for lpxD model calculated by ProSA-web to be -6.84 (Fig. 5D) which is well within the range of native conformation of experimental structures. Furthermore, ProQ computed a Levitt-Gerstein (LG) score of 2.785 and Max sub score to be 0.225 for the predicted model. A ProQ LG score ＞2.5 is necessary for suggesting a model is of good quality [39], implying high accuracy level of predicted structure. Also, the overall quality factor for lpxD model of B. petrii was predicted as 73.23% (Fig. 5E) by ERRAT web server. All of these evaluations recommended the reliability of the predicted lpxD model of B. petrii. Molecular docking analysis The results of molecular docking were intended for energetically favorable binding poses for each nine natural compounds within the active pocket (LbH domain) of proposed drug target of B. petrii (Fig. 5A) as lowest binding energy and inhibition constant are generally preferred for favorable binding mode while docking small compounds with target protein molecule [42,43]. Again, among them isoorientin (-5.66 kcal/mol), curcumin (-5.38 kcal/mol), isovitexin (-5.22 kcal/mol) found with lower binding energy and inhibition constants i.e., 71.35 μm, 114.67 μm, and 149.85 μm, suggesting as strong inhibitors of lpxD protein. Further, the binding stability was proved through existence of hydrogen bonding pattern between the protein-ligand complexes ( Table 5). Presence of hydrophobic amino acid residues within the binding site highly contributes towards the stability during molecular interaction between protein and small compound. Here, the resulted hydrophobic interaction between the docked complexes also supported for a strong binding affinity of natural compounds towards the proposed drug target (lpxD) of B. petrii (Table 6, Fig. 7). Also, the presence of some positively and negatively charged amino acid residues of lpxD within 4 Å distance from the ligands suggested for electrostatic force of interaction between docked complexes ( Table 6, Fig. 7). Again, among nine active natural compounds, isoorientin (-5.66 kcal/mol) was observed as the most suitable inhibitor with lowest binding energy and inhibition constant (71.35 μm), and formed hydrogen bond with amino acid residues like Ala 186, Glu 149, and Gly 227 of lpxD ( 165, Ala 186, and Leu 226 within 4 Å distance from the ligand ( Table 6, Fig. 7F). All of these above observation established isoorientin to be a strong inhibitor against lpxD of B. petrii. In conclusions, Identification of potential drug target has a vital role in designing a novel drug against any infectious disease. B. petrii strains have been reported as causing infections in humans and various warm blooded animals. In the present investigation, authors attempted to narrow down the complete data set of known 5,031 protein sequences of B. petrii DSM 12804 (accession No. PRJNA28135) in order to identify a potential drug target computationally. In silico study revealed lpxD protein of B. petrii as a potential drug target, is an essential protein of the pathogen and involved in the biosynthesis of the lipid A component of lipopolysaccharide. Again, the observations made through computational study also strongly supported lpxD to be a potential drug target due to the absence of lpxD protein in any human essential pathways as well as without any significant percentage of sequence homology with human and human gut flora micro biota. Again, previous literature reports also established the involvement of lpxD in nosocomial infections caused due to pathogens and hence, could be used as a potential drug target [15,16] against B. petrii. Furthermore, due to adverse effect of synthetic drugs, the uses of active natural compounds have gained an increasing attention towards the treatment or prevention of infectious diseases. Moreover, some of the phytochemicals like ajoene (garlic), allicin (ginger), cinnamaldehyde (Chinese cinnamon), curcumin (turmeric), gallotannin (green tea and red wine), isoorientin (blueberry relative found in Central and South America), isovitexin (blueberry relative found in Central and South America), neral (lemon balm), and vitexin (blueberry relative found in Central and South America) have been reported as having anti-oxidant, anti-inflammatory, and anti-bacterial properties, might be used against COPD, cystic fibrosis, QS biofilm formation, nosocomial infection, and other types of bacterial infection [20][21][22][23][24][25]. Therefore, authors excited to apply molecular docking approach to check the inhibitory effects of above mentioned nine natural compounds (Table 4, Fig. 6) against the proposed drug target (lpxD) of B. petrii. The docking study results also supported Genomics & Informatics Vol. 14, No. 4, 2016 for the confirmation of the mechanism of blocking of lipid A biosynthesis in B. petrii which is essential for the survival of the bacterium. Again, isoorientin was noticed with highest inhibitory effect among nine natural compounds supported by lowest binding energy and inhibition constant (Table 5). Binding stability was also confirmed through observation of strong hydrophobic, electrostatic and hydrogen bonding interactions between protein-ligand complexes (Tables 5 and 6, Fig. 7F). Hence, isoorientin could be suggested as most suitable natural compounds against the proposed drug target (lpxD) of B. petrii. The overall hypothesis generated through implementation of in silico approach would be a great aid and value towards designing a novel drug against B. petrii infection and which would be further validated through in vitro experiments.	2018-04-03T05:48:51.338Z	2016-12-01T00:00:00.000	{ "year": 2016, "sha1": "aa97f3276f03ef0c3577f5ae6920b46c861d156f", "oa_license": "CCBYNC", "oa_url": "http://genominfo.org/upload/pdf/gni-14-241.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "aa97f3276f03ef0c3577f5ae6920b46c861d156f", "s2fieldsofstudy": [ "Biology", "Chemistry", "Environmental Science", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
11731738	pes2o/s2orc	v3-fos-license	Investigation of Free-Standing Plasmonic Mesoporous Ag/CMK-8-Nafion Composite Membrane for the Removal of Organic Pollutants with 254-nm UV Irradiation “Carbon-based material” has demonstrated a great potential on water purification due to its strong physical adsorption to organic pollutants in the water. Three-dimensional cubic ordered mesoporous carbon (CMK-8), one of the well-known ordered mesoporous carbons, was prepared by using nanocasting method with mesoporous silica (KIT-6) as the template. In this study, CMK-8 blended with Nafion polymer to form a free-standing mesoporous CMK-8-Nafion composite membrane. The synthesis of high crystallinity CMK-8 was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). More than 80% methyl orange (MO) removal efficiency was observed under 254-nm UV irradiation after 120 min. Ninety-two percent recycling performance was remained after four recycling tests, which indicated a reliable servicing lifetime for the water purification. Furthermore, an additional layer of plasmonic silver nanoparticles (Ag NPs) was integrated into this CMK-8-Nafion membrane for higher pollutant removal efficiency, attributing from the generation of plasmon-resonance hot electrons from Ag NPs. A 4-in. CMK-8-Nafion composite membrane was also fabricated for the demonstration of potential large-scale utilization. Background Maintaining a constant supply of clean water has become a vital issue in this decade because of the increasing number of contaminants produced from industrial wastes worldwide. Various organic contaminants such as dioxin, ethylbenzene, and polycyclic aromatic hydrocarbons are frequently found in wastewater and are substantially harmful to human health and ecological security [1,2]. Therefore, development of efficient methods for decontamination and disinfection of water, particularly of drinking water sources, is urgently required. Several approaches have been adopted for removing organic pollutants from water. For example, reverse osmosis [3], ion exchange process [4], biochemical processes [5], and physical adsorption [6,7] are generally used for water purification. Of these technologies, physical adsorption is the most commonly used because of its low cost and easy operation. Carbon-based materials [8,9] such as activated carbon and carbon nanotubes are promising candidates for water purification because of their exceptional capabilities of adsorbing various organic contaminants through numerous bonding types, such as electrostatic interactions, π-π bonding, hydrogen bonding, and hydrophobic interactions [10,11]. Organic water pollutants can also be photocatalytically decomposed with semiconductor materials that exploit the electron-hole pairs (excitons) from the conversion of incident photons. These high-energy electrons and holes react with aqueous solutions at solid and solution interfaces to generate •OH and O 2 •− , triggering the decomposition of organic pollutants in wastewater [12]. Scheme 1 illustrates a possible explanation for the enhanced mechanism and reaction route in an Ag/ CMK-8-Nafion system. Under photon irradiation at 254 nm, hot electrons and holes are excited from the surfaces of silver nanoparticles (NPs) to produce superoxide radical anions (O 2 •− ) and hydroxyl radicals (•OH), respectively; the primary oxidizing species correspond to photocatalytic oxidation processes [13][14][15]. In addition to activated carbon and carbon nanotubes, ordered mesoporous carbon is another effective material used for removing pollutants from wastewater [16][17][18][19]. Ordered mesoporous carbon has been receiving much attention because of its high surface area, high conductivity, and highly uniform and regular pore sizes, which facilitate mass transport [20][21][22][23]. Moreover, ordered mesoporous carbon has been successfully employed in energy storage devices such as fuel cells [24][25][26] and supercapacitors [27]. Different structures, sizes, and shapes of ordered mesoporous carbon can be explicitly synthesized by varying fabrication parameters and surfactant concentrations [28,29]. In this study, we propose the application of a free-standing CMK-8-Nafion composite membrane in the photo-induced decomposition of methyl orange (MO). Mesoporous carbon CMK-8 can not only adsorb MO [30] but can also effectively absorb photons due to their blackbody property [31,32], contributing to the additional photo-induced decomposition of MO [16]. This unique dual mechanism consisting of physical adsorption and photocatalytic decomposition is discussed under different experimental conditions. Finally, we also layered silver plasmonic NPs [33,34] onto this free-standing mesoporous CMK-8-Nafion composite membrane to further enhance the removal of organic water pollutants. Methods High-quality samples of mesoporous silica KIT-6 and the corresponding mesoporous carbon CMK-8 were prepared using a process similar to that used in previous studies [35]. CMK-8 has a reversed cubic structure, which was then replicated using KIT-6 as a hard template. A dilute H 2 SO 4(aq) solution was added to sucrose solution with weight ratios of 1 g KIT-6/1.25 g sucrose/ 5 g H 2 O/0.14 g H 2 SO 4 . The colloid mixture was dried at 333 K for 6 h and dehydrated at 433 K for 6 h. The aforementioned steps were repeated again with a mixture of 0.8 g sucrose/3.2 g H 2 O/0.09 g H 2 SO 4 . The resultant dark brown powders were carbonized under argon atmosphere at 1173 K for 1 h. The silica template was removed with 1 M hydrofluoric acid in a solution of 50% ethanol and 50% H 2 O, and CMK-8 was finally collected. For the fabrication of CMK-8-Nafion composite membranes, designated amounts of CMK-8 were mixed with Nafion solution at a solid-content ratio of 30%, and this CMK-8-Nafion precursor was ultrasonically agitated for 10 min before the casting step. Each mesoporous CMK-8-Nafion membrane was formed by pouring CMK-8-Nafion precursor solution in a 4-in. petri dish and was then solidified at 323 K for 40 min. In addition, for the deposition of the silver NP layer, a precursor composed of silver acetylacetonate weighing 0.035 g [Ag(acac); 98%, Acros] was dissolved in 30 mL deionized water mixed with 5 mL 99.5% alcohol and the prepared mesoporous CMK-8-Nafion membrane was then immersed in the solution for 15 h [36]. After examination of several samples, the average thickness of the Ag/CMK-8-Nafion membrane was 0.3-0.4 mm. The microstructures and morphologies of KIT-6 and CMK-8 were examined using a scanning electron microscope (SEM; Scheme 1 Enhanced MO decomposition mechanism and reaction route in an Ag/CMK-8-Nafion system FE-SEM; HITACHI S-4800) and a transmission electron microscope (JEOL JEM-2100F). The pore sizes and specific surface areas were analyzed using N 2 adsorption/desorption analysis under 77 K (Micromeritics; ASAP2020). The mesostructures of KIT-6 and CMK-8 were confirmed by small-angle (2θ of 0.5°-8°) powdered X-ray diffraction (XRD) by using Cu Kα radiation (λ = 0.154 nm; scan rate of 1°/min). The chemical states of silver NPs were examined using an X-ray photoelectron spectroscope (XPS; ULVAC-PHI Versa-probe) with Al Kα X-rays and a 45°photoelectron takeoff angle. A 1-eV flooding electron source and 7-eV Ar + was applied for charge compensation during spectrum acquisition. Finally, a UV-Vis integrating sphere was used to evaluate the performance of organic pollutant decomposition by the free-standing mesoporous CMK-8-Nafion membranes under UV irradiation at 254 nm. Figure 1a, b presents high-resolution TEM images of mesoporous microstructures of silica KIT-6, and the corresponding three-dimensional (3D) structures of ordered mesoporous carbon CMK-8 exhibited a wellordered honeycomb structure with a uniform pore size. As depicted in Fig. 1c, d, long-range ordering porosity of KIT-6 and CMK-8 can be observed from XRD patterns in the low-angle range; this finding is in agreement with the result of HRTEM. Because of the high surface area of 3D cubic CMK-8, fast mass transfer kinetics become possible, which is not ably beneficial for organic molecule adsorption. The N 2 adsorption/desorption isotherms (77 K) were measured for examining the specific surface area of CMK-8. The N 2 adsorption/ desorption isotherms (77 K) of CMK-8 exhibited an essentially type-IV isotherm (according to the IUPAC classification) with a broad hysteresis loop, which had the typical characteristics of capillary condensation in mesoporous channels (Fig. 1e). In addition, according to the Brunauer-Emmett-Teller method, CMK-8 possessed a specific surface area of 840.67 m 2 g −1 . The Barrett-Joyner-Halenda analysis of the desorption branch Fig. 1 Characterization of KIT-6 and corresponding CMK-8. a, b High-magnification TEM images. c, d Small-angle XRD patterns. e N 2 adsorption/ desorption isotherms (77 K) of CMK-8. f, g SEM image and corresponding EDX elemental mappings of C, Ag, and Ag/CMK-8-Nafion membranes of the isotherm indicated that the pores had an average diameter of approximately 4 nm. CMK-8 is expected to provide a substantial number of active sites for physical adsorption of organic pollutants because of its high specific surface area and porous nature. To fabricate an Ag/CMK-8-Nafion membrane, these CMK-8 powders were mixed with Nafion solution, followed by a chemical reduction process for the deposition of Ag NPs. The morphology of the Ag/CMK-8-Nafion membrane was examined using an SEM, and the results are presented in Fig. 1f. The corresponding energy-dispersive X-ray spectroscopy mapping of C and Ag elements was also performed; the Ag NP distributions on the surfaces of CMK-8-Nafion membranes were clearly observable (Fig. 1g). Results and Discussion To obtain substantial hot electron generation from a plasmonic resonance process, it is essential to preserve the neutrality of silver NPs because neutral silver NPs provide a more severe plasmon resonant condition than oxidized silver NPs do; therefore, characterizing the surface chemical state of silver NPs is crucial. XPS spectroscopy was used for examining the Ag/CMK-8-Nafion system. The elemental survey spectrum of the Ag/CMK-8-Nafion membrane was measured, and the four main elements, namely Ag, C, F, and O, were identified (Fig. 2a). On the basis of this typical survey, the atomic percentage of Ag was estimated to be approximately 5.0%. The high-resolution spectrum of C1s was obtained and is presented in Fig. 2b. The nonsymmetrical peak shape indicated that multiple chemical states of carbon were present in the Ag/CMK-8-Nafion sample; hence, deconvolution was performed to identify each component. The most pronounced peak, located at 284.3 eV, was attributed to the C-C graphitic bonding of CMK-8, indicating that no chemical reactions occurred between silver NPs and CMK-8. The peak at 285.9 eV can be assigned to the carbon bonded to C * H 2 CFH n , and the peak at 288.0 eV can be attributed to CH 2 C * FH n . Another broad peak at 290.8 eV can be considered as the superposition of signals from -CF 2 -, -OCF-, and -OCF 2groups. As presented in Fig. 2c, a symmetrical Ag3d5 peak was obtained in the high-resolution scan, indicating that Ag NPs were successfully reduced on the surface of the CMK-8-Nafion membrane through a physical adsorption approach without any other chemical bonding. Furthermore, because of the binding energy of Ag, Ag oxides and Ag fluorides differed by only a few tenths of an eV. Thus, determining the oxidation of Ag only by using the Ag3d5 peak position is difficult. For more accurate characterization, Ag MNN auger electrons were also examined. A cross-comparison of simultaneous measurements of the Ag3d binding energy and Ag MNN kinetic energy (KE) peak can determine the chemical state and prevent the confusion of shifts. The Ag MNNKE is given as follows: According to the Al Kα X-ray photon energy (1486.6 eV), the AgM 5 N 5 N 5 binding energy was calculated as 1133.8 eV. As presented in Fig. 2d, the KE of AgMNN was 358.8 eV (6.0 eV added to the KE data on M 5 N 5 N 5 to obtain the KE [37,38]. The auger interpretation of the binding energy (367.39 eV; Fig. 2c) and the KE of AgMNN (358.8 eV) indicate the existence of metallic silver NPs in our sample, suggesting that the sample has active plasmon resonance and substantial hot electron generation. To investigate the efficiency and mechanism of MO decomposition, we performed three experimental setups for testing the removal rates of MO: (i) UV irradiation at 254 nm with no membrane, (ii) a CMK-8-Nafion membrane only in darkness, and (iii) a CMK-8-Nafion under UV irradiation at 254 nm. Figure 2a-c illustrates the evolution of the UV-Vis spectra of the MO solution under these three decomposition conditions. The absorption peak of MO at 463 nm was obtained from the conjugated structure constructed through an azo bond. A decrease in the peak intensity indicated the decomposition of MO and decoloration of the solution. MO demonstrated a very slight self-degradation under UV irradiation at 254 nm after 120 min (Fig. 3a). When the CMK-8-Nafion membrane was examined in darkness, it exhibited a strong physical adsorption ability for MO even without UV irradiation. A noticeable decrease at an absorption peak of 463 nm was observed with the passage of time (Fig. 3b). Photo-induced decomposition of MO was examined by placing a CMK-8-Nafion membrane under irradiation at 254 nm (Fig. 3c). The MO decomposition efficiency of this photo-enhanced process was nine times higher than that of CMK-8 in darkness, suggesting that the photo-induced decomposition process was achieved by incident photons with preadsorbed MO molecules on the CMK-8-Nafion surface. In addition, we examined the recycling stability of the CMK-8-Nafion membrane (Fig. 3d). The results demonstrated that the CMK-8-Nafion membrane still retained 92% of its original efficiency after four consecutive 120-min decomposition cycles. We fabricated a 4-in. free-standing CMK-8-Nafion membrane to demonstrate the potential practical use of these mechanisms. A similar evaluation process was performed on this 4-in. CMK-8-Nafion membrane. Figure 4a presents the UV-Vis spectrum evolution of MO decomposition under UV irradiation at 254 nm. After 150 min of irradiation, more than 80% of organic MO was successfully removed from the solution. Figure 4b depicts the corresponding photographs of the decoloration of the MO solution with an increase in irradiation time. The 4-in. CMK-8-Nafion membrane had a robust framework of CMK-8 and Nafion, which did not leave any unnecessary legacy products in the cleaned water even after several recycling tests, eliminating the additional effort of removing photocatalytic filter detritus. To further improve the MO decomposition process, we introduced a layer of plasmonic silver NPs onto the surface of the CMK-8-Nafion membrane to achieve additional MO decomposition efficiency by the generation of hot electrons from plasmon-resonance NPs. Silver NPs were prepared using a previously reported chemical reduction process. Figure 5a presents the absorption spectrum of CMK-8-Nafion and Ag/CMK-8-Nafion membranes. The CMK-8-Nafion membrane exhibited a typical broad photon absorption from 350 to 800 nm because of the blackbody characteristics of CMK-8. In the Ag/CMK-8-Nafion membrane sample, an additional pronounced silver plasmonic absorption peak [39] was observed at approximately 310 nm, presenting a slight blueshift caused by the low dielectric constant of CMK-8. With the integration of silver NPs, more than 98% MO decomposition was achieved within 120 min under UV irradiation at 254 nm (Fig. 5b). This decomposition enhancement of approximately 20% is attributable to hot electrons generated on the surfaces of silver NPs by the plasmon decay process, substantially raising the population of active radicals in the solution and providing an additional reaction route for the MO decomposition process. Notably, because the excellent molecule adsorption ability of the CMK-8 also provides a perfect reaction ground for these active oxidizing species with preadsorbed MO molecules, the whole decomposition reaction proceeds with the positive feedback of an avalanche. Finally, we compared the decomposition efficiency of CMK-8-Nafion in darkness and under UV Fig. 5 a Absorption spectra of CMK-8-Nafion with and without silver NPs. b UV-Vis spectrum evolution of MO decomposition with the Ag/CMK-8-Nafion membrane. c A comparison of MO decomposition efficiency levels with different experimental conditions irradiation and the efficiency of Ag/CMK-8-Nafion under UV irradiation (Fig. 5c). As expected, the Ag/CMK-8-Nafion sample exhibited the highest MO decomposition efficiency because of additional hot electrons and holes generated from silver NPs. Conclusions Free-standing CMK-8-Nafion membranes were fabricated for improving MO decomposition in wastewater. The basic membrane removed pollutants with an efficiency level of more than 80% after 120 min of UV irradiation at 254 nm. A reliability test indicated that the basic CMK-8-Nafion membrane still retained 92% of its original efficiency after four consecutive MO decomposition processes. Furthermore, with the integration of a silver NP layer, 98% MO decomposition efficiency was achieved, which was approximately 20% higher than that of the basic CMK-8-Nafion membrane. Finally, we demonstrated the feasibility of fabricating a 4-in. free-standing CMK-8-Nafion membrane for high-throughput wastewater treatment.	2018-04-03T01:30:20.047Z	2017-05-19T00:00:00.000	{ "year": 2017, "sha1": "22a6f380e54e6ec4d0bc2a81fde0537cf6686f87", "oa_license": "CCBY", "oa_url": "https://nanoscalereslett.springeropen.com/track/pdf/10.1186/s11671-017-2124-7", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "22a6f380e54e6ec4d0bc2a81fde0537cf6686f87", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
248009725	pes2o/s2orc	v3-fos-license	Duodenal Metastasis of Breast Cancer: A Case Report and Review of the Literature Rationale: The usual metastases of breast cancer are mainly represented by the following organs: lymph nodes, bone, liver, lung, pleura and brain. While gastrointestinal involvement remains rare. Patient Concerns: A 67-year-old postmenopausal woman presented with abdominal pain and obstructive symptoms, she was diagnosed with duodenal metastasis of breast cancer with infiltration of the duodenal mucosa by lobular breast carcinoma. The patient had undergone the right breast mastectomy for infiltrating lobular carcinoma 6 years previously. Diagnosis: Metastatic infiltration of the duodenal mucosa by mammary lobular carcinoma was proven histologically and immunohistochemically. Lessons: The importance of thinking about metastases of the gastrointestinal tract in the presence of non-specific and vague symptoms and of clearly differentiating in this context these metastases from primary gastrointestinal tumors. INTRODUCTION Breast cancer is the most common malignant tumor in women and the second most common cause of cancer death in them. The usual metastases of breast cancer are mainly represented by the following organs: lymph nodes, bones, liver, lungs, pleura and brain. While gastrointestinal and retroperitoneal organ involvement remains rare. Although ductal carcinoma infiltrant (DCI) is the predominant type of breast cancer, lobular carcinoma infiltrant (LCI) is the least common histological type but is responsible for almost all metastasis to the gastrointestinal tract 1, 2. We present a case of duodenal metastasis in a woman with a personal history of infiltrating lobular carcinoma of the breast diagnosed 6 years ago. CASE REPORT In February 2021, a 67-year-old menopausal Caucasian woman was admitted to the emergency room for intense liver colic of acute onset. Our patient had a personal history of a lobular infiltrating breast carcinoma RH + HER2 -Ki67 at 50%, metastatic to bone, the patient had benefited from several therapeutic lines (an initial right mastectomy.... ), currently under treatment with Palbociclib and Flvestrant , however she had no notable family history. The history of her illness dates back to two days after her admission with the sudden onset of abdominal pain in the right hypochondrium in a hemi-girdle towards the right scapula, permanent, unbearable, rated at 9/10 on the pain scale, without any aggravating or calming factors, so it was a hepatic colic type of pain associated with early postprandial food vomiting, all evolving in a context of asthenia, anorexia. The physical examination found a conscious patient well oriented in time and space, eupneic, tachycardia at 114 beats per minute, normotensive, the body mass index (BMI) was equal to 14 kg/m2, loss of autonomy (WHO at 3-4) and in whom the abdominal examination objectified a sensitivity in the right hypochondrium and gastric lapping In front of this symptomatology an abdominal ultrasound was carried out which objectified a vesicular lithiasis with dilatation of the intrahepatic bile ducts and a borderline choledochus. This was completed by an endoscopic ultrasound with the following steps: descent by the endoscope under visual control to the bulb. Doubt on a duodenal stenosis. By trans-bulbar approach the choledochus was slightly dilated to 8 mm, with the presence of a stone. The gallbladder is lithiasis. A perihepatic effusion was noted and a suspicious subcentimetric lesion in the left liver. It was decided to repeat the examination with a gastroscope which confirmed the presence of a stenosis at the D1-D2 junction impassable by the gastroscope, opacification objective a long stenosis, realization of multiple biopsies at the level of the stenosis. Immunohistochemical analysis showed infiltration of the chorion of the duodenal mucosa by a carcinomatous proliferation, arranged in sheets and trabeculae. The tumor cells are medium-sized, with eosinophilic cytoplasm, and not very cohesive. The nuclei are enlarged, hyperchromatic, with irregular outlines. The residual duodenal glands are not dysplastic. On immunohistochemistry, the tumor cells express CK7, estrogen (50%) and progesterone (25%) receptors. They do not express Cadherin, CDX2 or CK20. In conclusion, it was an infiltration of the duodenal mucosa by a mammary lobular carcinoma. The thoracic-abdominal-pelvic CT scan showed dilatation of the intrahepatic bile ducts without identifiable choledocholithic obstruction; D1-D2 stenosis without identifiable pancreatic tumor; contrast enhancement of the second sigmoid loop with thickening of the wall; absence of identifiable digestive artery occlusion and peritoneal effusion, particularly perihepatic and perisplenic. The positron emission tomography examination showed a hepatic hypermetabolic lesion (segments VI and IV) extending over 15 mm and 8 mm of major axis in the axial plane respectively: to be confronted with a hepatic MRI for characterization; a diffuse hypermetabolism of the first and third duodenal segments, without detectable scannographic lesion; an intense and circumferential sigmoidal hypermetabolism (27 × 15 mm) and a probable hypermetabolic centimetric adenopathy, located near the pancreatic head. For the exploration of the sigmoidal hyper metabolism, a rectosigmoidoscopy only objectified a diverticulosis without any other associated lesion. DISCUSSION Duodenal metastases from breast cancer are rare 3. The clinical manifestations of duodenal metastases are nonspecific, with clinical symptoms of abdominal pain, nausea, vomiting, or upper GI bleeding 4, 5. In some cases duodenal metastases of breast cancer may present as a picture of upper bowel obstruction 6. All these clinical symptoms are not specific for metastatic origin, hence the interest to complete the investigations by paraclinical examinations, in particular by upper GI endoscopy with multiple biopsies, as immunohistochemical study is the most reliable way to differentiate metastatic duodenal tumors from primary duodenal tumors. In the majority of cases, duodenal metastases appear years after the diagnosis of breast cancer, which is the case of our patient because the diagnosis of duodenal metastasis was made 6 years after the diagnosis of breast cancer. 127 Nakamura et al., showed from autopsy studies that the incidence of breast cancer metastasis to the gastrointestinal tract was 8.9% 7. In Annals of Surgical Oncology, McLemore showed in a large series of patients followed for metastatic breast cancer that only 73 patients had gastrointestinal metastases out of a total of 12001 patients and that small bowel metastases accounted for 19% of breast metastases to the gastrointestinal tract and confirmed that lobular breast carcinoma was responsible for almost all cases, almost 80% of gastrointestinal metastases 8. Duodenal metastasis of breast cancer has a poor prognosis and requires prompt and effective diagnostic and therapeutic management. This immediate management is strongly associated with improved quality of life and short and long-term prognosis 8. CONCLUSION Duodenal involvement as a late site of distant breast cancer metastasis is relatively rare. The exact incidence of duodenal metastases is difficult to estimate, as in most cases patients remain asymptomatic or minimally symptomatic and the symptoms present are nonspecific to the site of metastasis, making early diagnosis difficult. Duodenal metastases usually appear within 2 to 13 years after diagnosis of breast cancer. The immunohistochemical study remains the gold standard for the diagnosis of duodenal breast cancer metastases.	2022-04-01T11:54:24.422Z	2022-03-17T00:00:00.000	{ "year": 2022, "sha1": "2e24b8242e16d46d66aec9357b4d6672e1c5b862", "oa_license": null, "oa_url": "https://doi.org/10.36348/sjpm.2022.v07i03.008", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "2e24b8242e16d46d66aec9357b4d6672e1c5b862", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
2396191	pes2o/s2orc	v3-fos-license	Sugarcoated isolation: evidence that social avoidance is linked to higher basal glucose levels and higher consumption of glucose Objective: The human brain adjusts its level of effort in coping with various life stressors as a partial function of perceived access to social resources. We examined whether people who avoid social ties maintain a higher fasting basal level of glucose in their bloodstream and consume more sugar-rich food, reflecting strategies to draw more on personal resources when threatened. Methods: In Study 1 (N = 60), we obtained fasting blood glucose and adult attachment orientations data. In Study 2 (N = 285), we collected measures of fasting blood glucose and adult attachment orientations from older adults of mixed gender, using a measure of attachment style different from Study 1. In Study 3 (N = 108), we examined the link between trait-like attachment avoidance, manipulation of an asocial state, and consumption of sugar-rich food. In Study 4 (N = 115), we examined whether manipulating the social network will moderate the effect of attachment avoidance on consumption of sugar-rich food. Results: In Study 1, fasting blood glucose levels corresponded with higher attachment avoidance scores after statistically adjusting for time of assessment and interpersonal anxiety. For Study 2, fasting blood glucose continued to correspond with higher adult attachment avoidance even after statistically adjusting for interpersonal anxiety, stress indices, age, gender, social support and body mass. In Study 3, people high in attachment avoidance consume more sugar-rich food, especially when reminded of asocial tendencies. Study 4 indicated that after facing a stressful task in the presence of others, avoidant people gather more sugar-rich food than more socially oriented people. Conclusion: Results are consistent with the suggestion that socially avoidant individuals upwardly adjust their basal glucose levels and consume more glucose-rich food with the expectation of increased personal effort because of limited access to social resources. Further investigation of this link is warranted. Introduction When God looked upon man, he or she contended that, "It is not good for the man to be alone. " (Genesis,2:18). Research indeed suggests that humans draw strength from combined efforts in coping with situational demands. Social bonding serves security provision functions (Mikulincer and Shaver, 2007) and regulates stress, negative affect and arousal (Bowlby, 1973;Beckes and Coan, 2011). Some have suggested that highly social individuals can manage demands on their energy more efficiently (Coan, 2008;Beckes and Coan, 2011). For example, other people can help care for offspring (Ehrenberg et al., 2001), assist when ill or injured (Townsend and Franks, 1995), share resources (Roger and De Boer, 2001), and contribute vigilance for potential threats (Davis, 2010;Ein-Dor et al., 2011b). Nevertheless, some individuals tend to distrust others' goodwill, strive to maintain independence, and often distance themselves from other people when dealing with threats and negative emotions. These people, often called "avoidantly attached" (Mikulincer and Shaver, 2007), tend to cope with threats by deemphasizing distress and vulnerability and by attempting to cope independently, without seeking others' help (Fraley and Shaver, 1997). Because they do not share the cost of many of life's metabolically expensive activities, they are likely to employ a traitlike strategy of increased preparedness for individual decision making, problem solving, threat vigilance, and even the regulation threat vigilance-a strategy that increases their personal "budget" for rapidly accessible metabolic resources (Beckes and Coan, 2011;Gross and Proffitt, 2013). Recently, Bales (2012) have found indications for such a strategy among male titi monkeys, and Henriksen et al. (2014) have found such a strategy among less socially connected people (i.e., those who score high on measures of loneliness). In the present research, we examined this premise by testing (1) whether people who tend to avoid social ties-high scorers on attachment avoidance-maintain in their blood a higher basal concentration of circulating glucose, the most easily accessible metabolic fuel of the human body and brain ( Vannucci and Vannucci, 2000); and (2) whether attachment avoidance corresponds with greater consumption of sugar-containing food such as carbohydrates. Attachment theory (Bowlby, 1973(Bowlby, , 1980(Bowlby, , 1982 proposes that human beings possess an innate psychobiological system (the attachment behavioral system) that motivates them to seek proximity to significant others (attachment figures) when they need protection from threats. Throughout a history of interactions with attachment figures, people develop trait-like attachment orientations that are relatively stable over time (Ainsworth et al., 1978;Mikulincer and Shaver, 2007). The pioneering work by Ainsworth et al. (1978) has indicated that when attachment figures regularly respond sensitively to a person's needs, he or she develops a sense of attachment security while acquiring constructive strategies for coping with threats and regulating negative emotions. "Secure" people generally cope with threats either by relying on internal resources developed with the help of security-enhancing attachment figures or by effectively seeking support from others or collaborating with them (Shaver and Mikulincer, 2002). Secure individuals generally have high self-esteem, trust other people, and perceive the world as a relatively safe place (Mikulincer and Shaver, 2007). These socially oriented tendencies might also help them to reduce energy expenditure relative to energy consumption (Coan, 2008;Gross and Proffitt, 2013). For example, their vigilance-related processing might decrease because they can rely on the combined efforts of others to detect threats (Roberts, 1996). Indeed, secure individuals are less attentive to signs of danger, and show the longest delays in warning others about dangers they do detect (Ein-Dor et al., 2011a,b;Ein-Dor and Orgad, 2012). Secure people may also reduce their energy expenditure by depending more on others to identify and acquire resources, assist with health-related needs, and help nurture offspring (Coan, 2008;Gross and Proffitt, 2013). Attachment theory suggests that when attachment figures are unavailable, unreliable, or rejecting of bids for support, a person may become chronically wary of depending on others (Bowlby, 1982;Mikulincer and Shaver, 2007). A common manifestation of insecure attachment is avoidance, which is marked by extreme independence, lack of intimacy and self-disclosure, and emotionregulation strategies characterized by social distancing. Citing the brain's tendency to act as a Bayesian predictor of future outcomes, Beckes and Coan (2011) contend that "a history of finding others to be relatively unhelpful and unreliable may lead to priors that predict low levels of social support, biasing individuals to make "bets" that social resources are unlikely to obtain, and leading to an increased recruitment of personal resources in the presence of various life challenges. " (p. 981). Because people who avoid social ties need to recruit their own resources more often than their secure counterparts, they might need more rapid access to freely available metabolic resources-a budgeting strategy that could, in theory, lead to greater basal levels of glucose in the bloodstream, and to greater consumption of sugar-containing food. Recently, Dickinson et al. (2014) have supported the notion that glucose intake relates to Bayesian-based decision making and not to heuristic-based ones. In the present research, we examine this possibility by relating attachment avoidance disposition with glucose budgeting strategies. Recently, Bales (2012) have revealed several indications for an equivalent mechanism among male titi monkeys. In the wild, male titi monkeys leave their family and travel for long distances looking for a mate. The researchers have employed a laboratory setting and discovered that after the male titi monkeys detach from their kin, they show an increase in blood glucose concentration, which decreases once they reattach with a mate. Regarding glucose intake, Henriksen et al. (2014) have indicated that perceived loneliness was linked with elevated intake of sugar-rich beverages, whereas indices of social connectedness, such as relationship satisfaction, being married, having supporting friends, and having a sense of togetherness at work, was related to lower intake of sugar-rich beverages. These tendencies were significant even after adjusting for body mass index, self-image, physical activity and mental health. Jaremka et al. (2015) also linked loneliness with higher hunger among women with low body mess. Glucose is the predominant organic fuel for all animal species, including humans (Kety, 1950). This is equally true for all of the body's activities, including cerebral metabolism (Heininger, 2002). Moreover, glucose tends to be efficiently utilized to produce the maximal amount of chemical energy possible (Vannucci and Vannucci, 2000). Importantly, circulating blood glucose levels are sensitive to negative emotions (Skaff et al., 2009), and can be conditioned to prevailing social circumstances over longer periods of time (Woods and Kuskosky, 1976;Brummett et al., 2005;Henriksen et al., 2014) via several central mechanisms (Anthony et al., 2006). Lower activity in at least one such mechanism, the ventral striatum, is associated with greater insulin resistance and higher circulating blood glucose (Ryan et al., 2012). Moreover, the ventral striatum tends to be less active among people who avoid social attachments (Vrtička et al., 2008). If people who tend to avoid social ties place their own personal resources under greater demand-including demands that can be unpredictable and urgent-then maintaining higher basal glucose levels via these and other central mechanisms could provide them with the metabolic resources to more rapidly and independently overcome life's many challenges. Research also suggests that various psychological processes influence the intake of sugar-containing food (e.g., Henriksen et al., 2014) and that depletion of body-energy elicits specific motivations and behaviors that allow the acquisition and replenish of metabolic resources Gailliot et al., 2007;Wang and Dvorak, 2010;Aarøe and Petersen, 2013). For example, Wang and Dvorak (2010) have shown that increasing blood glucose levels by drinking sugar-rich beverage, led participants to place more value in future reward over present goods. Such shifts in motivations and behaviors may be interpretable as adaptive strategies to maintain optimal level of glucose in the blood (Aarøe and Petersen, 2013). Social baseline theory (Beckes and Coan, 2011;Gross and Proffitt, 2013;Coan and Sbarra, 2015), suggests that a person's optimal level of glucose in the bloodstream may be set by the expected quality and breadth of his or her social network. People with asocial tendencies, such as those high in attachment avoidance, may need set this optimal level higher, because they expect others either cannot or will not aid them when in need. Along with other centrally mediated mechanisms, the maintenance of higher glucose levels may entail eating more sugar-rich food. In the present research, we designed four studies that examined the link between attachment avoidance, basal glucose level, and consumption of sugar-rich food. In Study 1, participants simply completed a self-report measure of attachment avoidance and had their finger pricked to measure blood glucose concentration. We predicted that the higher the attachment avoidance, the greater the basal glucose level. In Study 2, we examined whether the association between avoidance from social ties and basal glucose would generalize to (a) a different culture (Israel as compared with United States); (b) women and men from a different age group (adults and late adults as compared with young adults); and (c) a different measure of attachment avoidance. In Study 2, we also adjusted the analysis for the possible contributions of body mass index, gender, age, social support, time of assessment and three robust indices of stress-self-report ratings on anxiety-related symptoms, physicians' diagnosis of hypertension symptoms, and fasting basal ratio of cortisol to adrenal androgen dehydroepiandrosterone (DHEA). In Study 3, we examined the link between trait-like attachment avoidance, manipulation of an asocial state, and consumption of sugar-rich food. Study 3 allowed us to examine the causal link between asocial tendencies and glucose-related correlates. We predicted that the higher the attachment avoidance, the greater the consumption of sugar-rich food. We also predicted that under a state of asocial expectations, people would consume more sugar-rich food than under a control condition. In Study 4, we examined whether manipulating the social network will moderate the effect of attachment avoidance on consumption of sugar-rich food. Specifically, participants underwent a challenging task either alone or in pairs, and then ate cereals high in sugar for breakfast. We predicted that attachment avoidance will be linked with greater consumption of sugar-rich food specifically in the paired condition, because asocial people do not expect to benefit from the aid of others. Study 1 In Study 1, we examined whether attachment avoidance predicts fasting basal levels of glucose in the blood. To this end, participants completed self-report measure of attachment avoidance, and gave a blood sample to assess their blood glucose concentration. Materials and Methods Participants As part of a larger study of social support, 60 undergraduate women ranging in age from 18 to 21 years were recruited in pairs from the University of Virginia's Department of Psychology Participant Pool. Participants were instructed to avoid all eating and drinking (except water) for 3 h prior to their participation, and were informed that participation involved, among other things, finger pricks for the purpose of measuring blood glucose concentration. All participants consented to the study, and were either given course credit or compensated financially for their participation. Study 1 was approved by the University of Virginia's institutional review board (IRB; granted to JAC and MAW; HSR # 14240). Materials and Procedure Results reported here are drawn from a separate set of studies designed to investigate associations between emotion regulation and blood glucose concentration. Participants were separated upon arrival, and, after consenting to the study, gave a history of everything they had orally consumed for the past 12 h. If participants had adhered to the fasting procedure, they then gave a baseline blood glucose measure before engaging in additional study procedures not reported here. Blood glucose levels were measured with a Hemocue 201 Glucose tester (in mg/dL). At the end of the session, participants filled out a variety of questionnaires. Attachment orientations were assessed with the experiences in close relationships-revised scales (ECR-R; Fraley et al., 2000). Participants rated the extent to which each item was descriptive of their feelings in close relationships on a 7-point scale ranging from not at all (1) to very much (7). Eighteen items assessed attachment anxiety (e.g., "I am afraid that I will lose my partner's love") and 18 assessed avoidance (e.g., "I prefer not to show a partner how I feel deep down"). The reliability and validity of these scales have been repeatedly demonstrated (Fraley et al., 2000;Mikulincer and Shaver, 2007). In the present study, Cronbach αs were 0.89 for the anxiety items and 0.94 for the avoidance items, and the two scores were significantly correlated, FIGURE 1 \| The scatterplot depicts the association between attachment avoidance and fasting basal glucose level. Consistent with our hypothesis, the higher the participants' attachment avoidance score, the higher their fasting basal glucose level. Also, as the attachment avoidance score increased, the association between attachment avoidance and fasting basal glucose level increased in its relative magnitude. Note that the area within the light dashed lines represents the normal range of fasting basal glucose levels. By contrast, the heavy dashed line near the top depicts the level at which fasting basal glucose levels become clinically significant. r(58) = 0.37, p < 0.01. After completing these questionnaires, participants completed a socio-demographic questionnaire and were debriefed and thanked. Results and Discussion Participants' fasting basal glucose level was examined using a curve estimation regression analysis (estimating linear and quadratic relations), in which participants' attachment avoidance score served as the predictor, and their fasting basal glucose level served as the outcome measure. Estimating a linear association between attachment avoidance and fasting basal glucose level, we observed that the higher the participants' attachment avoidance score, the greater their fasting basal glucose level, F(1, 58) = 12.89, β = 0.43, R 2 = 0.18, p < 0.001. Adding the quadratic estimation yield marginally significant increment in the association, t(57) = 1.96, p = 0.055, boosting the β to 0.48 and the R 2 to 0.23 (see Figure 1). When time of testing was included in the regression model, there was no effect of time of testing, t(57) = 1.24, p = 0.22, and the model estimating linear and quadratic effects of attachment avoidance on fasting basal glucose remained significant, F (3,55) = 6.28, p = 0.001, R 2 = 0.21. No similar associations were observed between fasting basal glucose and attachment anxiety. In line with our prediction, women who tend to avoid depending on others for support had greater fasting basal levels of glucose in their blood than their more socially oriented counterparts. The higher levels of basal blood glucose found in highly avoidant individuals may serve as a metabolic reservoir that provides people high in attachment avoidance with the needed energy for rapid, independent responses to unpredictable contextual demands. Study 1, however, cannot rule out an alternative explanation that social avoidance promotes elevated levels of tension and stress and thus, in turn, higher levels of fasting basal blood glucose. In fact, among humans as well as many other animals, elevated basal blood glucose is a reliable marker of stress (Armario et al., 1996). Moreover, these results do not speak to whether the association between attachment avoidance and fasting basal blood glucose generalizes to men. We designed Study 2 to address these questions. Study 2 In study 2, we collected data on people's attachment orientations (avoidance and anxiety), fasting basal blood glucose level, three independent indicators of tension and stress, and the following control measures: age, gender, social support, BMI, and time of assessment. Next, we examined whether attachment avoidance was positively related to fasting basal glucose levels independent of our tension and stress measures. We selected multiple methods to measure tension and stress, including self-report ratings on anxiety-related symptoms, physicians' diagnosis of hypertension symptoms, and fasting basal ratio of cortisol to adrenal androgen DHEA. Elevated cortisol/DEHA ratios are associated with tension, stress, and related psychopathology (Goodyer et al., 2001). Following Wang and Dvorak (2010), we also adjusted the analysis for the possible effects of body mass index, gender, age, social support, and time of assessment. Also, to address the generalizability of the present research, we sampled 285 Israeli adults (49.82% men) who were significantly older than the participants in Study 1, and used a different measure of attachment orientation. Thus, we were able to examine whether the results of Study 1 could be replicated in a sample that was (a) from a different culture, (b) mixed by gender, (c) older, and (d) assessed using a different measure of attachment avoidance. We hypothesized that attachment avoidance would be associated with higher fasting basal levels of glucose, and that the indicators of tension and stress would not account for that association. Materials and Methods Participants Study 2 was part of ongoing longitudinal research conducted at Ruppin Academic Center (Cloninger and Zohar, 2011). Twohundred-eighty-five Israeli participants (143 women and 142 men), ranging in age from 42 to 90 years (Mdn = 58), volunteered to participate in the study, which included a free medical examination at a well-known medical facility (Mor Institute for Medical Data Ltd). Study 2 was approved by the Hillel Yaffe Medical Center's Helsinki committee (granted to RC; HSR # 42\2007). Measures and Procedure The study spanned two sessions. In the first session, participants, who were recruited by a series of public lectures, mailbox pamphlets, and word of mouth, were individually invited to Ruppin Academic Center for a morning of interview, self-report, and cognitive testing. Attachment orientations were assessed with a Hebrew-language questionnaire developed by (Mikulincer et al., 1990;Mikulincer and Erev, 1991). In completing this questionnaire, participants rated the extent to which each item was descriptive of their ECR on a 7-point scale ranging from not at all (1) to very much (7). Eight items assessed avoidant attachment (e.g., "I am uncomfortable when other people get too close to me") and 7 assessed anxious attachment (e.g., "I worry about being abandoned"). In the present study, Cronbach αs were 0.67 for the anxiety items and 0.76 for the avoidance items. Previous research has shown high concordance between this brief measure and the 36-item ECR measure (Brennan et al., 1998). Mean scores were computed for each scale, and the two scores were significantly correlated, r(280) = 0.55, p < 0.001. General anxiety level was assessed with a Hebrew version of the Brief Symptom Inventory (BSI; Derogatis and Melisaratos, 1983)-a 53-item self-report inventory in which participants rate the extent to which they have been aggravated (0 = "not at all" to 4 = "extremely") in the past week by various symptoms. The BSI anxiety subscale comprised six items (e.g., "Feeling tense or keyed up"), and its reliability and validity have been repeatedly demonstrated (Boulet and Boss, 1991). In the present study, Cronbach α was 0.81, and thus self-rated anxiety was calculated by averaging the item ratings. Social support was assessed with the multidimensional scale of perceived social support (MSPSS; Zimet et al., 1990). It includes items to assess perceived support from friends, family and an intimate partner (e.g., "There is a special person who is around when I am in need"). In the present study, Cronbach α was 0.93, and thus a total score of perceived social support was calculated by averaging the item ratings. Upon completion of the first session, participants were given a referral for a medical examination at the Mor Institute for Medical Data Ltd, and the medical center administrator was given the contact details of the prospective patient. Participants were invited to the medical center in the morning after a fast of 12 h. We controlled for time of awakening, morning activity, caffeine consumption and smoking, factors that can affect morning cortisol levels. All participants were instructed not to exercise before coming to the examination. The medical examination was conducted independently of all other study variables, by medical staff blind to the study goals and hypotheses. Upon participants' arrival, a nurse drew blood samples into serum tubes containing aprotinin (500 kallikrein-inhibiting units, or KIU, per ml of blood). The samples were centrifuged at 1,600 × g for 15 min at 4°C, and then transferred to plastic tubes and stored at -80°C. Cortisol was measured by the TKCO1-Coat-A-Count kit (Diagnostic Products Corporation, Los Angeles, CA, USA), and DHEA was assessed with the DHEA-DSL-9000-Active TM DHEA coated tube radioimmunoassay kit (Diagnostic System Laboratories, Webster, TX, USA). Basal glucose levels were measured with the Roche Diagnostics Serum Work Area Modular Analytics P-800 auto-analyzer (Roche Diagnostics, Basel, Switzerland). After the blood tests, an expert physician examined the participants to establish the occurrence of various medical conditions, including clinical hypertension, diabetes, and obesity. Test results were sent to the last author's (AHZ) research laboratory as well as to the participants. Upon request, the results were also sent to the participants' general practitioner in the community. Results and Discussion Participants' fasting basal glucose level was examined using hierarchical regression 1 . In the first step of the analysis, we introduced participant age as a predictor because age alone can cause systematic variations in fasting basal glucose levels (Shimokata et al., 1991). Following Wang and Dvorak (2010), we also adjusted the analysis for the possible effects of body mass index, gender, age, social support, and time of assessment. In the second step of the analysis, we added participants' attachment avoidance and anxiety scores as predictors. The analysis revealed that older age corresponded with marginally higher fasting basal glucose, b = 0.11, 95% CI for b (−0.01, 0.23), β = 0.11, p = 0.067. The addition of attachment scores in the second step of the analysis significantly increased the amount of variance accounted for, ∆F(2, 257) = 4.76, p = 0.009, ∆R ² = 0.04. Consistent with our hypothesis, the analysis revealed that the higher the participants' attachment avoidance score, the greater their fasting basal glucose level, b = 2.18, 95% CI for b (0.74, 3.62), β = 0.22, p = 0.003, replicating the results of Study 1. Attachment anxiety was not related to participants' fasting basal glucose, b = −0.74, 95% CI for b (−2.19, 0.71), β = −0.07, p = 0.32. Supplementary logistic regression analyses revealed that attachment avoidance was not significantly related to the likelihood of suffering from diabetes, Exp(b) = 1.11, p = 0.72, or obesity, Exp(b) = 0.86, p = 0.37, indicating that attachment avoidance was associated with normal levels of fasting basal glucose. To assess the possibility that avoidance from social ties relates to greater fasting basal glucose level through heightened distress and tension, we conducted a multiple mediation analysis (Preacher and Hayes, 2008), where the association between attachment avoidance and fasting basal glucose level was modeled as being mediated by three indicators of tension and distress: (a) self-report level of anxiety, (b) cortisol/DHEA ratio, and (c) endorsement of clinical hypertension (see Figure 2). Following Wang and Dvorak (2010), we also adjusted the analysis for the possible effects of body mass index, gender, age, social support, and time of assessment. The specified mediation pathways did not account for the association between attachment avoidance and fasting basal glucose level (i.e., bias-corrected bootstrap analyses were not significant, which indicate non-significant mediation pathways). Also, the association between attachment avoidance and basal glucose level remained significant after the inclusion of all three indicators of tension and distress, b = 2.08, p = 0.005. Given that we had sufficient power (i.e., above 80%) to discover weak-to-moderate mediation paths (i.e., mediation paths comprised two βs of 0.20; Fritz and MacKinnon, 2007), these null results are unlikely to stem from insufficient statistical power. In line with our prediction, people high in attachment avoidance tended to maintain higher fasting basal glucose levels than their more secure counterparts. In addition, elevated tension and stress did not account for this association. Studies 1 and 2 have only linked attachment avoidance with higher basal glucose level but not with greater consumption of sugar-rich food. In addition, FIGURE 2 \| The structural model above depicts a multiple mediation model to examine whether tension and stress-related indicators mediate the link between attachment avoidance and fasting basal glucose level. Solid paths represent significant predictions; dotted paths represent non-significant predictions. The coefficient in parenthesis represents the association between attachment avoidance and glucose level after the inclusion of the tension and stress-related indicators. Studies 1 and 2 are correlational in nature and preclude the ability to draw conclusions regarding the possible causal link between avoidance and glucose-related indices. We designed Study 3 to address these limitations. Study 3 In Study 3, we examined the link between trait-like attachment avoidance, manipulation of an asocial state, and consumption of sugar-rich food. We predicted that the higher the attachment avoidance, the greater the consumption of sugar-rich food. We also predicted that under a state of asocial expectations, people would consume more sugar-rich food than under a control condition. Materials and Methods Participants One-hundred-and-eight Israeli participants (59 women and 49 men), ranging in age from 17 to 30 years (Mdn = 22), volunteered to participate in the study. Study 3 was approved by the Interdisciplinary Center (IDC) Herzliya's IRB. Measures and Procedure The participants were a convenience sample recruited in IDC. Participants were approached by a research assistant between 11 and 12 AM, who asked them to participate in a study on storytelling appraisal. Upon their consent, participants were invited to a quiet room, and were asked to complete self-report measures of attachment orientation. Specifically, attachment orientation was assessed with a Hebrew version of the ECR scales (Brennan et al., 1998). Participants rated the extent to which each item was descriptive of their feelings in close relationships on a 7point scale ranging from not at all (1) to very much (7). Eighteen items assessed attachment anxiety (e.g., "I worry about being abandoned") and 18 assessed avoidance (e.g., "I prefer not to show a partner how I feel deep down"). The reliability and validity of these scales have been repeatedly demonstrated (Mikulincer and Shaver, 2007). In our study, Cronbach αs were 0.70 for the anxiety items and 0.92 for the avoidance items. Mean scores were computed for each scale, and the two scores were significantly correlated, r(106) = 0.55, p < 0.001. After completing the questionnaire, each participant was presented with one randomly assigned hypothetical story, out of two possible stories. The stories were used to manipulate a state of asocial expectations. Participants were asked to read and appraise the story by writing about their thoughts and feelings following the story that they have just read. In the asocial condition participants read the following paragraph: "Don doesn't like to come home from college for the holidays. If he could afford to go someplace else, he would. He doesn't have many friends back home but he likes it that way. He is not very open with members of his family, and he has few friends he can depend on and talk to when he is in need of support. He prefers to be self-reliant". In the control condition participants read the following paragraph: "Don, a college student, runs errands for a living. In his regular route, he first goes to the supermarket to buy groceries for his customers for the week. Next, he stops by the mechanic's shop to change the oil for a customer's car. He then goes to pick up tickets to a basketball game for another customer. He gets himself tickets for him and his friends as well. After dropping off all the tickets, he heads over to the game. Then, after the game, he heads back to campus and finishes readings and assignments he has for class the next day. " Participants then completed a short demographic sheet, were thanked, and were offered to eat as much Elite™chocolate chunks as they like as a compensation for their time. The number of chocolate chunks eaten served as the dependent variable. Results and Discussion The number of chocolate chunks eaten was examined using hierarchical regression. In the first step of the analysis, we introduced the measures of attachment avoidance and anxiety, and condition (−1 = control, 1 = asocial) as predictors. We also added gender as a covariate. In the second step of the analysis, we added the interactions between attachment avoidance and condition, and attachment anxiety and condition. To ease interpretation of results and to avoid multicollinearity, we centered the measures of attachment avoidance and anxiety around their grand mean. Consistent with our hypothesis, the analysis revealed that the higher participants' attachment avoidance, the more chocolate chunks they ate, b = 1.22, 95% CI for b (0.01, 2.43), β = 0.23, p = 0.05. Participants in the asocial condition ate more chocolate chunks than participants in the control condition, b = 1.26, 95% CI for b (0.39, 2.13), β = 0.27, p = 0.005. These effects, however, were moderated by the interaction between attachment avoidance and condition, b = 1.42, 95% CI for b (0.15, 2.69), β = 0.27, p = 0.029. Using Hayes's (2013) procedure, which is based on simple slopes test, we found that among participants high in attachment avoidance, the manipulation of asocial expectations generated an increase in the amount of chocolate chunks eaten, b = 2.56, p < 0.001 (see Figure 3). Among people low on attachment avoidance, and, hence, the more secure, the effect of the asocial manipulation was not significant, b = 0.28. Finally, the analysis indicated that attachment anxiety was associated with fewer chocolate chunks eaten, b = −1.45, 95% CI for b (−2.57, −0.33), β = −0.30, p = 0.01. In line with our prediction, people high in attachment avoidance tended to eat more chocolate chunks especially after being primed with asocial sentiments, while secure people, who commonly trust others and work in collaboration with them, did not. Study 4 In Study 3, we have found that manipulating a state of asocial expectations affected the consumption of sugar-rich food among people high in attachment avoidance but not among secure people. We designed Study 4 to examine whether people who score high in attachment avoidance may consume more sugary cereal after being paired with another person under stressful conditions. Participants underwent a challenging task either alone or in pairs, and then ate cereals high in sugar for breakfast. We predicted that attachment avoidance will be linked with greater consumption of sugar-rich food specifically in the paired condition, because asocial people do not expect to benefit from the aid of others as compared with more secure individuals. Materials and Methods Participants One-hundred-and-fifteen Israeli undergraduate students (65 women and 50 men), ranging in age from 18 to 33 years (Mdn = 22), from the IDC, Herzliya participated in the study for course credit. Study 4 was approved by the IDC Herzliya's IRB. Measures and Procedure Participants were invited to IDC's laboratory complex between 11 and 12 AM either alone or in pairs. They were told that in order to save them time and effort, they will be taking part in two unrelated studies. First, participants were told they are going to take part in a study on body temperature and later in another experiment on eating habits. The procedure was a cover story that was used to conceal the true nature of our study. After giving their informed consent, participants were asked to complete two questionnaires. Attachment orientation was assessed with a Hebrew version of the ECR, as in Study 3. In our study, Cronbach αs were 0.76 for the anxiety items and 0.89 for the avoidance items. Mean scores were computed for each scale, and the two scores were significantly correlated, r(113) = 0.41, p < 0.001. Next, to support our cover story, participants completed a bogus body temperature questionnaire, which consisted of rating the frequency of daily habits related to temperature (e.g., "I like showering in very hot water"), on a 7-point scale ranging from disagree strongly (1) to agree strongly (7). After completing these questionnaires, participants were put through a cold presser stress task (Lovallo, 1975). It is an extensively used laboratory stress manipulation, in which participants are put through a cold stimulus for few minutes. In this case, participants were asked to put their hands in ice water up to a minute and a half. Participants in the paired condition each had their own bowl but sat in close contact with each other. Following the task, participants were thanked and asked to take part in our second study, which explored the link between daily habits and appetite. They filled in a bogus habits questionnaire, which consisted of rating different everyday habits. Next, they were given a box of cereal and a bowl, and were asked to pour in the bowl as much cereal as they think they might eat for breakfast. Participants in the paired condition were asked to do this separately, having one participant wait in a second room while the first participant poured their cereal. The bowl was then put aside, and the participants were debriefed and thanked. Once the participants left the bowls were weighed by EatSmart™Precision Pro scale. Results and Discussion The weight of cereals was examined using hierarchical regression. In the first step of the analysis, we introduced the measures of attachment avoidance and anxiety, and condition (−1 = alone, 1 = in pairs) as predictors. We also added gender as a covariate. In the second step of the analysis, we added the interactions between attachment avoidance and condition, and attachment anxiety and condition. To ease interpretation of results and to avoid multicollinearity, we centered the measures of attachment avoidance and anxiety around their grand mean. Consistent with our hypothesis, the analysis revealed a significant interaction between attachment avoidance and condition, b = 7.02, 95% CI for b (0.70, 13.35), β = 0.23, p = 0.03. Hayes's (2013) procedure indicated that only in the paired condition, higher attachment avoidance corresponded with a higher quantity of cereal, b = 12.02, p < 0.001 (see Figure 4). In the alone condition, the link between attachment avoidance and weight of cereals was not significant, b = 2.02. In line with our prediction, attachment avoidance was linked with greater allocation of sugar-rich food specifically in the paired condition. It seems that secure individuals can benefit from the presence of others while performing a stressful task. As a result, FIGURE 4 \| Probing the interaction between attachment avoidance and condition in predicting the weight of cereals. Only in the paired condition, the higher the attachment avoidance, the greater the amount of cereals allocated. they are less exhausted and/or expect to need a lower amount of metabolic resources at the aftermath of the task. Avoidant people share no such expectation, and hence, they may be more exhausted and/or expect to need a greater amount of metabolic resources as compared with secure people. General Discussion Economy of action-a principal of biological systems-posits that all organisms must take in more energy than they expend if they are to survive, grow, and reproduce (Proffitt, 2006;Gross and Proffitt, 2013). In practice, the economy of action principle creates pressure for all organisms to conserve resources whenever possible. Recently, Coan and colleagues (Beckes and Coan, 2011;Coan and Sbarra, 2015) proposed that socially oriented people conserve, on average and in the aggregate, more energy than asocial people, because they construe-in largely implicit ways-social relationships as opportunities to conserve resources by sharing the load of life's myriad situational demands (Coan et al., 2006). In the studies reported here, we have examined the possibility that people who tend to avoid social resources would devote higher levels of a rapidly deployable metabolic resource-glucose-to their bloodstream and to consume more sugar-rich food, in order to increase their all-purpose access to that resource. That is because they are not disposed to share the cost of many of life's metabolically expensive activities, from physical labor to complex decisionmaking, problem solving and vigilance for threats. Specifically, we hypothesized that people high in attachment avoidance would maintain higher basal levels of glucose-the predominant organic fuel for a multitude of biosynthetic processes, including cerebral metabolism (Vannucci and Vannucci, 2000)-and consume more sugary foods. Results supported these hypotheses. In Study 1, we found that women who chronically tend to distance themselves from social resources-those high in attachment avoidance-maintained higher fasting basal glucose levels than more socially oriented women. In Study 2, we replicated the results of Study 1 in a different culture, among women as well as men, from a different age group, and using a different measure of attachment avoidance. Thus, the association between social avoidance and basal glucose level seems robust. Study 2 also showed that although people high in attachment avoidance maintain higher basal levels of glucose, there was no evidence that they are exceptionally prone to glucose-related disorders such as diabetes and obesity. One advantage of Study 2 was that it allowed us to address alternative explanation for the association between the avoidance of social resources and fasting basal glucose levels. For example, research suggests that a small and unsupportive social network, and especially feelings of loneliness, may trigger severe feelings of negativity as well as compromised health (Cacioppo et al., 2003) via mechanisms that include-among other things-elevated basal glucose levels (Armario et al., 1996;Whisman, 2010). Thus, the high amount of basal glucose associated with attachment avoidance may stem merely from the stress of relative social isolation and not because of a "bet" that more personal resources are needed because social resources are unlikely to obtain. Our results indicated, however, that the association between attachment avoidance and fasting basal glucose level remained significant even after statistically adjusting for three sensitive and multimodal indicators of distress: self-reported anxiety, symptoms of hypertension, and the cortisol/DHEA ratio. Thus, the association between attachment avoidance and basal glucose results from something other than current distress in our participants. In addition, the effect of attachment avoidance remained significant after adjusting for various other measures that might affect the pattern of results, including age, gender, body mass, social support, and time of assessment. We posit again that the association between fasting glucose and attachment avoidance reflects a "bet" by more avoidant participants that the challenges they face will require independent solutions, a bet that itself probably stems from a social history of unreliable or deficient access to social resources (Bowlby, 1973;Coan, 2008). Our contention is in line with recent findings linking glucose-related decision making with Bayesian reasoning (Dickinson et al., 2014). In Study 3, we found that people who chronically tend to distance themselves from social resources-those high in attachment avoidance-consume more sugar-rich food, especially when reminded of asocial tendencies. Study 4 extended these findings, suggesting that after facing a stressful task in the presence of others, avoidant people gather more sugar-rich food than more socially oriented people. These results are in keeping with recent research linking loneliness and lack of social network with greater consumption of sugar-rich beverages (Henriksen et al., 2014). Our findings are also in line with recent theory and research regarding the potentially adaptive nature of social avoidance. Specifically, Ein-Dor et al. (2010) contended that although avoidant people are more likely to rely on self-protective fightor-flight responses in times of danger, they might also more rapidly identify and enact protective maneuvers when faced with a threatening situation-an advantage contributing to the relative frequency of the trait. At the individual level, the potential adaptability of this approach is obvious, but this kind of behavior may sometimes save other people's lives as well, by thwarting a threat or identifying an escape route that can benefit the group to which the avoidant person belongs. Indeed, Ein-Dor et al. (2011b) did observe that attachment-related avoidance was associated with speedier escape responses to an experimentally manipulated danger-a room gradually filling with smoke, apparently because of a malfunctioning computer-and therefore with greater group safety. In addition, observed that avoidant individuals were better equipped than their less avoidant peers to succeed and be satisfied with professional singles tennis and computer science because these fields reward self-reliance, independence, and the ability to work without proximal social support from loved ones. Thus, it seems that trait-like attachment avoidance is more of an adaptation to a relatively independent way of life, a view that is somewhat different that the contemporary view of avoidant individuals as more globally deficient (Mikulincer and Shaver, 2007). The results of this research also add to a growing body of evidence for the adaptive nature of individual variation in personality. For instance, Nettle (2006) has argued that such variability can be understood in terms of tradeoffs among fitness costs and benefits: "Behavioral alternatives can be considered as tradeoffs, with a particular trait producing not unalloyed advantage but a mixture of costs and benefits such that the optimal value for fitness may depend on very specific local circumstances" (p. 625). There are, of course, some limitations to our studies. First, we emphasize that the correlational nature of Studies 1 and 2 precludes confident conclusions about the direction of causality in the link between avoidance and fasting basal glucose levels. Theory and research on attachment, however, do suggest that attachment orientations, including attachment avoidance, are formed in early childhood and are moderately stable over periods of years (Mikulincer and Shaver, 2007). Moreover, Coan and colleagues (Coan, 2008(Coan, , 2010Beckes and Coan, 2011;Coan and Sbarra, 2015) have drawn from a large body of animal and human neuroscientific research to specifically predict that relative isolation should cause increased demands on metabolic resources. In addition, Studies 3 and 4 have revealed that manipulating asocial tendencies and/or people's social network induces changes in people's consumption of sugar-rich food. In combination, there is reason to believe that avoidance is driving the association with fasting basal glucose levels and not vice versa. Future studies might also benefit from the inclusion of other personality measures to rule out the possibility that our findings regarding avoidance are attributable to other traits. Of course, by now many attachment studies have included measures of the Big Five personality traits, and attachment measures typically predict theoretically expected variables even when the Big Five traits are statistically adjusted for (Mikulincer and Shaver, 2007). Ultimately, our findings raise the possibility that people who consistently avoid the use of social resources and strive to maintain independence, compensate for these tendencies in part by maintaining a higher basal glucose level in their blood, and possibly by consuming more sugar-rich food-a strategy for rapidly accessing the metabolic fuel that helps them successfully face various life challenges alone.	2016-06-18T02:09:40.205Z	2015-04-23T00:00:00.000	{ "year": 2015, "sha1": "650ca3003f6e7122b9874d5691d99b814ac485a6", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fpsyg.2015.00492/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "97fb67ae54c3fd0b362dba4a3d75243fcf3cc32e", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [ "Psychology", "Medicine" ] }
219530894	pes2o/s2orc	v3-fos-license	Conservative Q-Learning for Offline Reinforcement Learning Effectively leveraging large, previously collected datasets in reinforcement learning (RL) is a key challenge for large-scale real-world applications. Offline RL algorithms promise to learn effective policies from previously-collected, static datasets without further interaction. However, in practice, offline RL presents a major challenge, and standard off-policy RL methods can fail due to overestimation of values induced by the distributional shift between the dataset and the learned policy, especially when training on complex and multi-modal data distributions. In this paper, we propose conservative Q-learning (CQL), which aims to address these limitations by learning a conservative Q-function such that the expected value of a policy under this Q-function lower-bounds its true value. We theoretically show that CQL produces a lower bound on the value of the current policy and that it can be incorporated into a principled policy improvement procedure. In practice, CQL augments the standard Bellman error objective with a simple Q-value regularizer which is straightforward to implement on top of existing deep Q-learning and actor-critic implementations. On both discrete and continuous control domains, we show that CQL substantially outperforms existing offline RL methods, often learning policies that attain 2-5 times higher final return, especially when learning from complex and multi-modal data distributions. Introduction Recent advances in reinforcement learning (RL), especially when combined with expressive deep network function approximators, have produced promising results in domains ranging from robotics [26] to strategy games [3] and recommendation systems [31]. However, applying RL to real-world problems consistently poses practical challenges: in contrast to the kinds of data-driven methods that have been successful in supervised learning [21,9], RL is classically regarded as an active learning process, where each training run requires active interaction with the environment. Interaction with the real world can be costly and dangerous, and the quantities of data that can be gathered online are substantially lower than the offline datasets that are used in supervised learning [8], which only need to be collected once. Offline RL, also known as batch RL, offers an appealing alternative [10,14,27,2,25,46,30]. Offline RL algorithms learn from large, previously collected datasets, without interaction. This in principle can make it possible to leverage large datasets, but in practice fully offline RL methods pose major technical difficulties, stemming from the distributional shift between the policy that collected the data and the learned policy. This has made current results fall short of the full promise of such methods. Directly utilizing existing value-based off-policy RL algorithms in an offline setting generally results in poor performance, due to issues with bootstrapping from out-of-distribution actions [27,14] and overfitting [12,27,2]. This typically manifests as erroneously optimistic value function estimates. If we can instead learn a conservative estimate of the value function, which provides a lower bound on the true values, this overestimation problem could be addressed. In fact, because policy evaluation and improvement typically only use the value of the policy, we can learn a less conservative lower bound Q-function, such that only the expected value of Q-function under the policy is lower-bounded, as opposed to a point-wise lower bound. We propose a novel method for learning such conservative Qfunctions via a simple modification to standard value-based RL algorithms. The key idea behind our method is to minimize values under an appropriately chosen distribution over state-action tuples, and then further tighten this bound by also incorporating a maximization term over the data distribution. Our primary contribution is an algorithmic framework, which we call conservative Q-learning (CQL), for learning conservative, lower-bound estimates of the value function, by regularizing the Q-values during training. Our theoretical analysis of CQL shows that only the expected value of this Q-function under the policy lower-bounds the true policy value, preventing extra under-estimation that can arise with point-wise lower-bounded Q-functions, that have typically been explored in the opposite context in exploration literature [39,24]. We also empirically demonstrate the robustness of our approach to Q-function estimation error. Our practical algorithm uses these conservative estimates for policy evaluation and offline RL. CQL can be implemented with less than 20 lines of code on top of a number of standard, online RL algorithms [18,7], simply by adding the CQL regularization terms to the Q-function update. In our experiments, we demonstrate the efficacy of CQL for offline RL, in domains with complex dataset compositions, where prior methods are typically known to perform poorly [11] and domains with high-dimensional visual inputs [4,2]. CQL outperforms prior methods by as much as 2-5x on many benchmark tasks, and is the only method that can outperform simple behavioral cloning on a number of realistic datasets collected from human interaction. Preliminaries The goal in reinforcement learning is to learn a policy that maximizes the expected cumulative discounted reward in a Markov decision process (MDP), which is defined by a tuple (S, A, T, r, γ). S, A represent state and action spaces, T (s \|s, a) and r(s, a) represent the dynamics and reward function, and γ ∈ (0, 1) represents the discount factor. π β (a\|s) represents the action-conditional in the dataset, D, and d π β (s) is the discounted marginal state-distribution of π β (a\|s). The dataset D is sampled from d π β (s)π β (a\|s). On all states s 0 ∈ D, letπ β (a 0 \|s 0 ) := s,a∈D 1[s=s0,a=s0] s∈D 1[s=s0] denote the empirical behavior policy, at that state. Off-policy RL algorithms based on dynamic programming maintain a parametric Q-function Q θ (s, a) and, optionally, a parametric policy, π φ (a\|s). Q-learning methods train the Q-function by iteratively applying the Bellman optimality operator B * Q(s, a) = r(s, a) + γE s ∼P (s \|s,a) [max a Q(s , a )], and use exact or an approximate maximization scheme, such as CEM [26] to recover the greedy policy. In an actor-critic algorithm, a separate policy is trained to maximize the Q-value. Actorcritic methods alternate between computing Q π via (partial) policy evaluation, by iterating the Bellman operator, B π Q = r + γP π Q, where P π is the transition matrix coupled with the policy: P π Q(s, a) = E s ∼T (s \|s,a),a ∼π(a \|s ) [Q(s , a )] , and improving the policy π(a\|s) by updating it towards actions that maximize the expected Q-value. Since D typically does not contain all possible transitions (s, a, s ), the policy evaluation step actually uses an empirical Bellman operator that only backs up a single sample. We denote this operatorB π . Given a dataset D = {(s, a, s )} of tuples from trajectories collected under a behavior policy π β : Offline RL algorithms based on this basic recipe suffer from action distribution shift [27,48,25,30] during training, because the target values for Bellman backups in policy evaluation use actions sampled from the learned policy, π k , but the Q-function is trained only on actions sampled from the behavior policy that produced the dataset D, π β . We also remark that Q-function training in offline RL does not suffer from state distribution shift, because the Q-function is never queried on out-of-distribution states. Since π is trained to maximize Q-values, it may be biased towards out-of-distribution (OOD) actions with erroneously high Q-values. In standard RL, such errors can be corrected by attempting an action in the environment and observing its actual value. However, the inability to interact with the environment makes it challenging to deal with Q-values for OOD actions in offline RL. Typical offline RL methods [27,25,48,46] mitigate this problem by constraining the learned policy [30] away from OOD actions. The Conservative Q-Learning (CQL) Framework In this section, we develop a conservative Q-learning (CQL) algorithm, such that the expected value of a policy under the learned Q-function lower-bounds its true value. A lower bound on the Q-value prevents the over-estimation that is common in offline RL settings due to OOD actions and function approximation error [30,27]. We use the term CQL to refer broadly to both Q-learning methods and actor-critic methods, though the latter also use an explicit policy. We start by focusing on the policy evaluation step in CQL, which can be used by itself as an off-policy evaluation procedure, or integrated into a complete offline RL algorithm, as we will discuss in Section 3.2. Conservative Off-Policy Evaluation We aim to estimate the value V π (s) of a target policy π given access to a dataset, D, generated by following a behavior policy π β (a\|s). Because we are interested in preventing overestimation of the policy value, we learn a conservative, lower-bound Q-function by additionally minimizing Q-values alongside a standard Bellman error objective. Our choice of penalty is to minimize the expected Qvalue under a particular distribution of state-action pairs, µ(s, a). Since standard Q-function training does not query the Q-function value at unobserved states, but queries the Q-function at unseen actions, we restrict µ to match the state-marginal in the dataset, such that µ(s, a) = d π β (s)µ(a\|s). This gives rise to the iterative update for training the Q-function, as a function of a tradeoff factor α: In Theorem 3.1, we show that the resulting Q-function,Q π := lim k→∞Q k , lower-bounds Q π at all (s, a). However, we can substantially tighten this bound if we are only interested in estimating V π (s). If we only require that the expected value of theQ π under π(a\|s) lower-bound V π , we can improve the bound by introducing an additional Q-value maximization term under the data distribution, π β (a\|s), resulting in the iterative update (changes from Equation 1 in red): In Theorem 3.2, we show that, while the resulting Q-valueQ π may not be a point-wise lowerbound, we have E π(a\|s) [Q π (s, a)] ≤ V π (s) when µ(a\|s) = π(a\|s). Intuitively, since Equation 2 maximizes Q-values under the behavior policyπ β , Q-values for actions that are likely underπ β might be overestimated, and henceQ π may not lower-bound Q π pointwise. While in principle the maximization term can utilize other distributions besidesπ β (a\|s), we prove in Appendix D.2 that the resulting value is not guaranteed to be a lower bound for other distribution besidesπ β (a\|s). Theoretical analysis. We first note that Equations 1 and 2 use the empirical Bellman operator, B π , instead of the actual Bellman operator, B π . Following [40,24,38], we use concentration properties ofB π to control this error. Formally, for all s, a ∈ D, with probability ≥ 1 − δ, , where C r,T,δ is a constant dependent on the concentration properties (variance) of r(s, a) and T (s \|s, a), and δ ∈ (0, 1) (see Appendix D.3 for more details). Now, we show that the conservative Q-function learned by iterating Equation 1 lower-bounds the true Q-function. Proofs can be found in Appendix C. Next, we show that Equation 2 lower-bounds the expected value under the policy π, when µ = π. We also show that Equation 2 does not lower-bound the Q-value estimates pointwise. The value of the policy under the Qfunction from Equation 2,V π (s) = E π(a\|s) [Q π (s, a)], lower-bounds the true value of the policy obtained via exact policy evaluation, V π (s) = E π(a\|s) [Q π (s, a)], when µ = π, according to: The analysis presented above assumes that no function approximation is used in the Q-function, meaning that each iterate can be represented exactly. We can further generalize the result in Theorem 3.2 to the case of both linear function approximators and non-linear neural network function approximators, where the latter builds on the neural tangent kernel (NTK) framework [23]. Due to space constraints, we present these results in Theorem D.1 and Theorem D.2 in Appendix D.1. In summary, we showed that the basic CQL evaluation in Equation 1 learns a Q-function that lower-bounds the true Q-function Q π , and the evaluation in Equation 2 provides a tighter lower bound on the expected Q-value of the policy π. For suitable α, both bounds hold under sampling error and function approximation. Next, we will extend on this result into a complete RL algorithm. Conservative Q-Learning for Offline RL We now present a general approach for offline policy learning, which we refer to as conservative Q-learning (CQL). As discussed in Section 3.1, we can obtain Q-values that lower-bound the value of a policy π by solving Equation 2 with µ = π. How should we utilize this for policy optimization? We could alternate between performing full off-policy evaluation for each policy iterate,π k , and one step of policy improvement. However, this can be computationally expensive. Alternatively, since the policyπ k is typically derived from the Q-function, we could instead choose µ(a\|s) to approximate the policy that would maximize the current Q-function iterate, thus giving rise to an online algorithm. We can formally capture such online algorithms by defining a family of optimization problems over µ(a\|s), presented below, with modifications from Equation 2 marked in red. An instance of this family is denoted by CQL(R) and is characterized by a particular choice of regularizer R(µ): Variants of CQL. To demonstrate the generality of the CQL family of optimization problems, we discuss two specific instances within this family that are of special interest, and we evaluate them empirically in Section 6. If we choose R(µ) to be the KL-divergence against a prior distribution, ρ(a\|s), i.e., R(µ) = −D KL (µ, ρ), then we get µ(a\|s) ∝ ρ(a\|s) · exp(Q(s, a)) (for a derivation, see Appendix A). Frist, if ρ = Unif(a), then the first term in Equation 3 corresponds to a soft-maximum of the Q-values at any state s and gives rise to the following variant of Equation 3, called CQL(H): Second, if ρ(a\|s) is chosen to be the previous policyπ k−1 , the first term in Equation 4 is replaced by an exponential weighted average of Q-values of actions from the chosenπ k−1 (a\|s). Empirically, we find that this variant can be more stable with high-dimensional action spaces (e.g., Table 2) where it is challenging to estimate log a exp via sampling due to high variance. In Appendix A, we discuss an additional variant of CQL, drawing connections to distributionally robust optimization [37]. We will discuss a practical instantiation of a CQL deep RL algorithm in Section 4. CQL can be instantiated as either a Q-learning algorithm (with B * instead of B π in Equations 3, 4) or as an actor-critic algorithm. Theoretical analysis of CQL. Next, we will theoretically analyze CQL to show that the policy updates derived in this way are indeed "conservative", in the sense that each successive policy iterate is optimized against a lower bound on its value. For clarity, we state the results in the absence of finitesample error, in this section, but sampling error can be incorporated in the same way as Theorems 3.1 and 3.2, and we discuss this in Appendix C. Theorem 3.3 shows that any variant of the CQL family learns Q-value estimates that lower-bound the actual Q-function under the action-distribution defined by the policy, π k , under mild regularity conditions (slow updates on the policy). Theorem 3.3 (CQL learns lower-bounded Q-values). Let πQ k (a\|s) ∝ exp(Q k (s, a)) and assume that D TV (π k+1 , πQ k ) ≤ ε (i.e.,π k+1 changes slowly w.r.t toQ k ). Then, the policy value underQ k , lower-bounds the actual policy value, The LHS of this inequality is equal to the amount of conservatism induced in the value,V k+1 in iteration k + 1 of the CQL update, if the learned policy were equal to soft-optimal policy forQ k , i.e., whenπ k+1 = πQ k . However, as the actual policy,π k+1 , may be different, the RHS is the maximal amount of potential overestimation due to this difference. To get a lower bound, we require the amount of underestimation to be higher, which is obtained if ε is small, i.e. the policy changes slowly. Our final result shows that CQL Q-function update is "gap-expanding", by which we mean that the difference in Q-values at in-distribution actions and over-optimistically erroneous out-of-distribution actions is higher than the corresponding difference under the actual Q-function. This implies that the policy π k (a\|s) ∝ exp(Q k (s, a)), is constrained to be closer to the dataset distribution,π β (a\|s), thus the CQL update implicitly prevents the detrimental effects of OOD action and distribution shift, which has been a major concern in offline RL settings [27,30,14]. Theorem 3.4 (CQL is gap-expanding). At any iteration k, CQL expands the difference in expected Q-values under the behavior policy π β (a\|s) and µ k , such that for large enough values of α k , we have that ∀s, When function approximation or sampling error makes OOD actions have higher learned Q-values, CQL backups are expected to be more robust, in that the policy is updated using Q-values that prefer in-distribution actions. As we will empirically show in Appendix B, prior offline RL methods that do not explicitly constrain or regularize the Q-function may not enjoy such robustness properties. To summarize, we showed that the CQL RL algorithm learns lower-bound Q-values with large enough α, meaning that the final policy attains at least the estimated value. We also showed that the Q-function is gap-expanding, meaning that it should only ever over-estimate the gap between in-distribution and out-of-distribution actions, preventing OOD actions. Practical Algorithm and Implementation Details Algorithm 1 Conservative Q-Learning (both variants) 1: Initialize Q-function, Q θ , and optionally a policy, π φ . 2: for step t in {1, . . . , N} do 3: Train the Q-function using GQ gradient steps on objective from Equation 4 θt := θt−1 − ηQ∇ θ CQL(R)(θ) (Use B * for Q-learning, B π φ t for actor-critic) 4: (only with actor-critic) Improve policy π φ via Gπ gradient steps on φ with SAC-style entropy regularization: φt := φt−1 + ηπE s∼D,a∼π φ (·\|s) [Q θ (s, a)−log π φ (a\|s)] 5: end for We now describe two practical offline deep reinforcement learning methods based on CQL: an actor-critic variant and a Q-learning variant. Pseudocode is shown in Algorithm 1, with differences from conventional actorcritic algorithms (e.g., SAC [18]) and deep Q-learning algorithms (e.g., DQN [35]) in red. Our algorithm uses the CQL(H) (or CQL(R) in general) objective from the CQL framework for training the Q-function Q θ , which is parameterized by a neural network with parameters θ. For the actor-critic algorithm, a policy π φ is trained as well. Our algorithm modifies the objective for the Q-function (swaps out Bellman error with CQL(H)) or CQL(ρ) in a standard actor-critic or Q-learning setting, as shown in Line 3. As discussed in Section 3.2, due to the explicit penalty on the Q-function, CQL methods do not use a policy constraint, unlike prior offline RL methods [27,48,46,30]. Hence, we do not require fitting an additional behavior policy estimator, simplifying our method. [7] for the discrete control experiments. The tradeoff factor, α is automatically tuned via Lagrangian dual gradient descent for continuous control, and is fixed at constant values described in Appendix F for discrete control. We use default hyperparameters from SAC, except that the learning rate for the policy is chosen to be 3e-5 (vs 3e-4 or 1e-4 for the Q-function), as dictated by Theorem 3.3. Elaborate details are provided in Appendix F. Related Work We now briefly discuss prior work in offline RL and off-policy evaluation, comparing and contrasting these works with our approach. More technical discussion of related work is provided in Appendix E. Off-policy evaluation (OPE). Several different paradigms have been used to perform off-policy evaluation. Earlier works [43,42,44] used per-action importance sampling on Monte-Carlo returns to obtain an OPE return estimator. Recent approaches [32, 16,36,49] use marginalized importance sampling by directly estimating the state-distribution importance ratios via some form of dynamic programming [30] and typically exhibit less variance than per-action importance sampling at the cost of bias. Because these methods use dynamic programming, they can suffer from OOD actions [30, 16,19,36]. In contrast, the regularizer in CQL explicitly addresses the impact of OOD actions due to its gap-expanding behavior, and obtains conservative value estimates. Offline RL. As discussed in Section 2, offline Q-learning methods suffer from issues pertaining to OOD actions. Prior works have attempted to solve this problem by constraining the learned policy to be "close" to the behavior policy, for example as measured by KL-divergence [25, 48,41,46], Wasserstein distance [48], or MMD [27], and then only using actions sampled from this constrained policy in the Bellman backup or applying a value penalty. Most of these methods require a separately estimated model to the behavior policy, π β (a\|s) [14,27,48,25,46], and are thus limited by their ability to accurately estimate the unknown behavior policy, which might be especially complex in settings where the data is collected from multiple sources [30]. In contrast, CQL does not require estimating the behavior policy. Prior work has explored some forms of Q-function penalties [22,47], but only in the standard online RL setting augmented with demonstrations. Luo et al. [34] learn a conservatively-extrapolated value function, V (s) by enforcing a linear extrapolation property over the state-space, and use it alongside a learned dynamics model to obtain policies for goal-reaching tasks. Alternate prior approaches to offline RL estimate some sort of uncertainty to determine the trustworthiness of a Q-value prediction [27, 2, 30], typically using uncertainty estimation techniques from exploration in online RL [40,24,39,6]. These methods have not been generally performant in offline RL [14,27,30] due to the high-fidelity requirements of uncertainty estimates in offline RL [30]. The gap-expanding property of CQL backups, shown in Theorem 3.4, is related to how gap-increasing Bellman backup operators [5,33] are more robust to estimation error in online RL. Experimental Evaluation We compare CQL to prior offline RL methods on a range of domains and dataset compositions, including continuous and discrete action spaces, state observations of varying dimensionality, and high-dimensional image inputs. We first evaluate actor-critic CQL, using CQL(H) from Algorithm 1, on continuous control datasets from the D4RL benchmark [11]. We compare to: prior offline RL methods that use a policy constraint -BEAR [27] and BRAC [48]; SAC [18], an off-policy actor-critic method that we adapt to offline setting; and behavioral cloning (BC). Gym domains. Results for the gym domains are shown in Table 1. The results for BEAR, BRAC, SAC, and BC are based on numbers reported by Fu et al. [11]. On the datasets generated from a single policy, marked as "-random", "-expert" and "-medium", CQL roughly matches or exceeds the best prior methods, but by a small margin. However, on datasets that combine multiple policies ("-mixed", "-medium-expert" and "-random-expert"), that are more likely to be common in practical datasets, CQL outperforms prior methods by large margins, sometimes as much as 2-3x. Adroit tasks. The more complex Adroit [45] tasks (shown on the right) in D4RL require controlling a 24-DoF robotic hand, using limited data from human demonstrations. These tasks are substantially more difficult than the gym tasks in terms of both the dataset composition and high dimensionality. Prior offline RL methods generally struggle to learn meaningful behaviors on these tasks, and the strongest baseline is BC. As shown in Table 2, CQL variants are the only methods that improve over BC, attaining scores that are 2-9x those of the next best offline RL method. CQL(ρ) with ρ =π k−1 (the previous policy) outperforms CQL(H) on a number of these tasks, due Table 1: Performance of CQL(H) and prior methods on gym domains from D4RL, on the normalized return metric, averaged over 4 seeds. Note that CQL performs similarly or better than the best prior method with simple datasets, and greatly outperforms prior methods with complex distributions ("-mixed", "-random-expert", "-medium-expert"). to the higher action dimensionality resulting in higher variance for the CQL(H) importance weights. Both variants outperform prior methods. Table 2: Normalized scores of all methods on AntMaze, Adroit, and kitchen domains from D4RL, averaged across 4 seeds. On the harder mazes, CQL is the only method that attains non-zero returns, and is the only method to outperform simple behavioral cloning on Adroit tasks with human demonstrations. We observed that the CQL(ρ) variant, which avoids importance weights, trains more stably, with no sudden fluctuations in policy performance over the course of training, on the higher-dimensional Adroit tasks. AntMaze. These D4RL tasks require composing parts of suboptimal trajectories to form more optimal policies for reaching goals on a MuJoco Ant robot. Prior methods make some progress on the simpler U-maze, but only CQL is able to make meaningful progress on the much harder medium and large mazes, outperforming prior methods by a very wide margin. Kitchen tasks. Next, we evaluate CQL on the Franka kitchen domain [17] from D4RL [13]. The goal is to control a 9-DoF robot to manipulate multiple objects (microwave, kettle, etc.) sequentially, in a single episode to reach a desired configuration, with only sparse 0-1 completion reward for every object that attains the target configuration. These tasks are especially challenging, since they require composing parts of trajectories, precise long-horizon manipulation, and handling human-provided teleoperation data. As shown in Table 2, CQL outperforms prior methods in this setting, and is the only method that outperforms behavioral cloning, attaining over 40% success rate on all tasks. Offline RL on Atari games. Lastly, we evaluate a discrete-action Q-learning variant of CQL (Algorithm 1) on offline, image-based Atari games [4]. We compare CQL to REM [2] and QR-DQN [7] on the five Atari tasks (Pong, Breakout, Qbert, Seaquest and Asterix) that are evaluated in detail by Agarwal et al. [2], using the dataset released by the authors. provided with only the first 20% of the samples of an online DQN run. Note that CQL is able to learn stably on 3 out of 4 games, and its performance does not degrade as steeply as QR-DQN on Seaquest. Following the evaluation protocol of Agarwal et al. [2], we evaluated on two types of datasets, both of which were generated from the DQN-replay dataset, released by [2]: (1) a dataset consisting of the first 20% of the samples observed by an online DQN agent and (2) datasets consisting of only 1% and 10% of all samples observed by an online DQN agent ( Figures 6 and 7 in [2]). In setting (1), shown in Figure 1, CQL generally achieves similar or better performance throughout as QR-DQN and REM. When only using only 1% or 10% of the data, in setting (2) ( We also present an empirical analysis to show that Theorem 3.4, that CQL is gap-expanding, holds in practice in Appendix B, and present an ablation study on various design choices used in CQL in Appendix G. Discussion We proposed conservative Q-learning (CQL), an algorithmic framework for offline RL that learns a lower bound on the policy value. Empirically, we demonstrate that CQL outperforms prior offline RL methods on a wide range of offline RL benchmark tasks, including complex control tasks and tasks with raw image observations. In many cases, the performance of CQL is substantially better than the best-performing prior methods, exceeding their final returns by 2-5x. The simplicity and efficacy of CQL make it a promising choice for a wide range of real-world offline RL problems. However, a number of challenges remain. While we prove that CQL learns lower bounds on the Q-function in the tabular, linear, and a subset of non-linear function approximation cases, a rigorous theoretical analysis of CQL with deep neural nets, is left for future work. Additionally, offline RL methods are liable to suffer from overfitting in the same way as standard supervised methods, so another important challenge for future work is to devise simple and effective early stopping methods, analogous to validation error in supervised learning. [ Appendices A Discussion of CQL Variants We derive several variants of CQL in Section 3.2. Here, we discuss these variants on more detail and describe their specific properties. We first derive the variants: CQL(H), CQL(ρ), and then present another variant of CQL, which we call CQL(var). This third variant has strong connections to distributionally robust optimization [37]. CQL(H). In order to derive CQL(H), we substitute R = H(µ), and solve the optimization over µ in closed form for a given Q-function. For an optimization problem of the form: the optimal solution is equal to µ * (x) = 1 Z exp(f (x)), where Z is a normalizing factor. Plugging this into Equation 3, we exactly obtain Equation 4. CQL(ρ). In order to derive CQL(ρ), we follow the above derivation, but our regularizer is a KLdivergence regularizer instead of entropy. The optimal solution is given by, where Z is a normalizing factor. Plugging this back into the CQL family (Equation 3), we obtain the following objective for training the Q-function (modulo some normalization terms): CQL(var). Finally, we derive a CQL variant that is inspired from the perspective of distributionally robust optimization (DRO) [37]. This version penalizes the variance in the Q-function across actions at all states s, under some action-conditional distribution of our choice. In order to derive a canonical form of this variant, we invoke an identity from Namkoong and Duchi [37], which helps us simplify Equation 3. To start, we define the notion of "robust expectation": for any function f (x), and any empirical distributionP (x) over a dataset {x 1 , · · · , x N } of N elements, the "robust" expectation defined by: can be approximated using the following upper-bound: where the gap between the two sides of the inequality decays inversely w.r.t. to the dataset size, O(1/N ). By using Equation 6 to simplify Equation 3, we obtain an objective for training the Q-function that penalizes the variance of Q-function predictions under the distributionP . The only remaining decision is the choice ofP , which can be chosen to be the inverse of the empirical action distribution in the dataset,P (a\|s) ∝ 1 D(a\|s) , or even uniform over actions,P (a\|s) = Unif(a), to obtain this variant of variance-regularized CQL. B Discussion of Gap-Expanding Behavior of CQL Backups In this section, we discuss in detail the consequences of the gap-expanding behavior of CQL backups over prior methods based on policy constraints that, as we show in this section, may not exhibit such gap-expanding behavior in practice. To recap, Theorem 3.4 shows that the CQL backup operator increases the difference between expected Q-value at in-distribution (a ∼ π β (a\|s)) and out-ofdistribution (a s.t. µ k (a\|s) π β (a\|s) << 1) actions. We refer to this property as the gap-expanding property of the CQL update operator. Function approximation may give rise to erroneous Q-values at OOD actions. We start by discussing the behavior of prior methods based on policy constraints [27,14,25,48] in the presence of function approximation. To recap, because computing the target value requires E π [Q(s, a)], constraining π to be close to π β will avoid evaluatingQ on OOD actions. These methods typically do not impose any further form of regularization on the learned Q-function. Even with policy constraints, because function approximation used to represent the Q-function, learned Q-values at two distinct state-action pairs are coupled together. As prior work has argued and shown [1,12,28], the "generalization" or the coupling effects of the function approximator may be heavily influenced by the properties of the data distribution [12,28]. For instance, Fu et al. [12] empirically shows that when the dataset distribution is narrow (i.e. state-action marginal entropy, H(d π β (s, a)), is low [12]), the coupling effects of the Q-function approximator can give rise to incorrect Q-values at different states, though this behavior is absent without function approximation, and is not as severe with high-entropy (e.g. Uniform) state-action marginal distributions. In offline RL, we will shortly present empirical evidence on high-dimensional MuJoCo tasks showing that certain dataset distributions, D, may cause the learned Q-value at an OOD action a at a state s, to in fact take on high values than Q-values at in-distribution actions at intermediate iterations of learning. This problem persists even when a large number of samples (e.g. 1M ) are provided for training, and the agent cannot correct these errors due to no active data collection. Since actor-critic methods, including those with policy constraints, use the learned Q-function to train the policy, in an iterative online policy evaluation and policy improvement cycle, as discussed in Section 2, the errneous Q-function may push the policy towards OOD actions, especially when no policy constraints are used. Of course, policy constraints should prevent the policy from choosing OOD actions, however, as we will show that in certain cases, policy constraint methods might also fail to prevent the effects on the policy due to incorrectly high Q-values at OOD actions. How can CQL address this problem? As we show in Theorem 3.4, the difference between expected Q-values at in-distribution actions and out-of-distribution actions is expanded by the CQL update. This property is a direct consequence of the specific nature of the CQL regularizer -that maximizes Q-values under the dataset distribution, and minimizes them otherwise. This difference depends upon the choice of α k , which can directly be controlled, since it is a free parameter. Thus, by effectively controlling α k , CQL can push down the learned Q-value at out-of-distribution actions as much is desired, correcting for the erroneous overestimation error in the process. Empirical evidence on high-dimensional benchmarks with neural networks. We next empirically demonstrate the existence of of such Q-function estimation error on high-dimensional MuJoCo domains when deep neural network function approximators are used with stochastic optimization techniques. In order to measure this error, we plot the difference in expected Q-value under actions sampled from the behavior distribution, a ∼ π β (a\|s), and the maximum Q-value over actions sampled from a uniformly random policy, a ∼ Unif(a\|s). That is, we plot the quantitŷ over the iterations of training, indexed by k. This quantity, intuitively, represents an estimate of the "advantage" of an action a, under the Q-function, with respect to the optimal action max a Q k (s, a ). Since, we cannot perform exact maximization over the learned Q-function in a continuous action space to compute ∆, we estimate it via sampling described in Equation 8. We present these plots in Figure 2 on two datasets: hopper-expert and hopper-medium. The expert dataset is generated from a near-deterministic, expert policy, exhibits a narrow coverage of the state-action space, and limited to only a few directed trajectories. On this dataset, we find that∆ k is always positive for the policy constraint method (Figure 2(a)) and increases during training -note, the continuous rise in∆ k values, in the case of the policy-constraint method, shown in Figure 2(a). This means that even if the dataset is generated from an expert policy, and policy constraints correct target values for OOD actions, incorrect Q-function generalization may make an out-of-distribution action appear promising. For the more stochastic hopper-medium dataset, that consists of a more diverse set of trajectories, shown in Figure 2(b), we still observe that∆ k > 0 for the policy-constraint method, however, the relative magnitude is smaller than hopper-expert. In contrast, Q-functions learned by CQL, generally satisfy∆ k < 0, as is seen and these values are clearly smaller than those for the policy-constraint method. This provides some empirical evidence for Theorem 3.4, in that, the maximum Q-value at a randomly chosen action from the uniform distribution the action space is smaller than the Q-value at in-distribution actions. On the hopper-expert task, as we show in Figure 2(a) (right), we eventually observe an "unlearning" effect, in the policy-constraint method where the policy performance deteriorates after a extra iterations in training. This "unlearning" effect is similar to what has been observed when standard off-policy Q-learning algorithms without any policy constraint are used in the offline regime [27, 30], on the other hand this effect is absent in the case of CQL, even after equally many training steps. The performance in the more-stochastic hopper-medium dataset fluctuates, but does not deteriorate suddenly. To summarize this discussion, we concretely observed the following points via empirical evidence: • CQL backups are gap expanding in practice, as justified by the negative∆ k values in Figure 2. • Policy constraint methods, that do not impose any regularization on the Q-function may observe highly positive∆ k values during training, especially with narrow data distributions, indicating that gap-expansion may be absent. • When∆ k values continuously grow during training, the policy might eventually suffer from an unlearning effect [30], as shown in Figure 2(a). C Theorem Proofs In this section, we provide proofs of the theorems in Sections 3.1 and 3.2. We first redefine notation for clarity and then provide the proofs of the results in the main paper. Notation. Let k ∈ N denote an iteration of policy evaluation (in Section 3.1) or Q-iteration (in Section 3.2). In an iteration k, the objective -Equation 2 or Equation 3 -is optimized using the previous iterate (i.e.Q k−1 ) as the target value in the backup. Q k denotes the true, tabular Q-function iterate in the MDP, without any correction. In an iteration, say k + 1, the current tabular Q-function iterate, Q k+1 is related to the previous tabular Q-function iterate Q k as: Q k+1 = B π Q k (for policy evaluation) or Q k+1 = B π k Q k (for policy learning). LetQ k denote the k-th Q-function iterate obtained from CQL. LetV k denote the value function,V k := E a∼π(a\|s) [Q k (s, a)]. A note on the value of α. Before proving the theorems, we remark that while the statements of Theorems 3.2, 3.1 and D.1 (we discuss this in Appendix D) show that CQL produces lower bounds if α is larger than some threshold, so as to overcome either sampling error (Theorems 3.2 and 3.1) or function approximation error (Theorem D.1). While the optimal α k in some of these cases depends on the current Q-value,Q k , we can always choose a worst-case value of α k by using the inequalitŷ Q k ≤ R max /(1 − γ), still guaranteeing a lower bound. We first prove Theorem 3.1, which shows that policy evaluation using a simplified version of CQL (Equation 1) results in a point-wise lower-bound on the Q-function. Proof of Theorem 3.1. In order to start, we first note that the form of the resulting Q-function iterate, Q k , in the setting without function approximation. By setting the derivative of Equation 1 to 0, we obtain the following expression forQ k+1 in terms ofQ k , ∀ s, a ∈ D, k,Q k+1 (s, a) =B πQk (s, a) − α µ(a\|s) π β (a\|s) . Accounting for sampling error. Note that so far we have only shown that the Q-values are upperbounded by the the "empirical Bellman targets" given by,B πQk . In order to relateQ k to the true Q-value iterate, Q k , we need to relate the empirical Bellman operator,B π to the actual Bellman operator, B π . In Appendix D.3, we show that if the reward function r(s, a) and the transition function, T (s \|s, a) satisfy "concentration" properties, meaning that the difference between the observed reward sample, r (s, a, r, s ) ∈ D) and the actual reward function r(s, a) (and analogously for the transition matrix) is bounded with high probability, then overestimation due to the empirical Backup operator is bounded. Formally, with high probability (w.h.p.) ≥ 1 − δ, δ ∈ (0, 1), Hence, the following can be obtained, w.h.p.: Q k+1 (s, a) = B πQk (s, a) ≤ B πQk (s, a) − α µ(a\|s) π β (a\|s) + C r,T,δ R max 1 − γ . Now we need to reason about the fixed point of the update procedure in Equation 9. The fixed point of Equation 9 is given by: thus proving the relationship in Theorem 3.1. In order to guarantee a lower bound, α can be chosen to cancel any potential overestimation incurred by C r,T ,δ Rmax 1−γ . Note that this choice works, since (I − γP π ) −1 is a matrix with all non-negative entries. The choice of α that guarantees a lower bound is then given by: Of course, we need µ(a\|s) > 0 wheneverπ β (a\|s) > 0, for this to hold, and this is assumed in the theorem statament. Note that since, C r,T ,δ Rmax 1−γ = 0, whenB π = B π , any α ≥ 0 guarantees a lower bound. Next, we prove Theorem 3.3 that shows that the additional term that maximizes the expected Q-value under the dataset distribution, D(s, a), (or d π β (s)π β (a\|s), in the absence of sampling error), results in a lower-bound on only the expected value of the policy at a state, and not a pointwise lower-bound on Q-values at all actions. Proof of Theorem 3.2. We first prove this theorem in the absence of sampling error, and then incorporate sampling error at the end, using a technique similar to the previous proof. In the tabular setting, we can set the derivative of the modified objective in Equation 2, and compute the Q-function update induced in the exact, tabular setting (this assumesB π = B π ) and π β (a\|s) =π β (a\|s)). Note that for state-action pairs, (s, a), such that, µ(a\|s) < π β (a\|s), we are infact adding a positive quantity, 1 − µ(a\|s) π β (a\|s) , to the Q-function obtained, and this we cannot guarantee a point-wise lower bound, i.e . ∃ s, a, s.t.Q k+1 (s, a) ≥ Q k+1 (s, a). To formally prove this, we can construct a counter-example three-state, two-action MDP, and choose a specific behavior policy π(a\|s), such that this is indeed the case. Note that the marked term, is positive since both the numerator and denominator are positive, and this implies that D CQL (s) ≥ 0. Also, note that D CQL (s) = 0, iff π(a\|s) = π β (a\|s). This implies that each value iterate incurs some underestimation,V k+1 (s) ≤ B πV k (s). Now, we can compute the fixed point of the recursion in Equation 12, and this gives us the following estimated policy value: thus showing that in the absence of sampling error, Theorem 3.2 gives a lower bound. It is straightforward to note that this expression is tighter than the expression for policy value in Proposition 3.2, since, we explicitly subtract 1 in the expression of Q-values (in the exact case) from the previous proof. Incorporating sampling error. To extend this result to the setting with sampling error, similar to the previous result, the maximal overestimation at each iteration k, is bounded by The resulting value-function satisfies (w.h.p.), thus proving the theorem statement. In this case, the choice of α, that prevents overestimation w.h.p. is given by: s∈D a∈D π(a\|s) π(a\|s) π β (a\|s)) − 1 −1 . Next we provide a proof for Theorem 3.3. Proof of Theorem 3.3. In order to prove this theorem, we compute the difference induced in the policy value,V k+1 , derived from the Q-value iterate,Q k+1 , with respect to the previous iterate B πQk . If this difference is negative at each iteration, then the resulting Q-values are guaranteed to lower bound the true policy value. If (a) has a larger magnitude than (b), then the learned Q-value induces an underestimation in an iteration k + 1, and hence, by a recursive argument, the learned Q-value underestimates the optimal Q-value. We note that by upper bounding term (b) by D TV (πQ k ,π k+1 ) · max a πQ k (a\|s) π β (a\|s) , and writing out (a) > upper-bound on (b), we obtain the desired result. Finally, we show that under specific choices of α 1 , · · · , α k , the CQL backup is gap-expanding by providing a proof for Theorem 3.4 Proof of Theorem 3.4 (CQL is gap-expanding). For this theorem, we again first present the proof in the absence of sampling error, and then incorporate sampling error into the choice of α. We follow the strategy of observing the Q-value update in one iteration. Recall that the expression for the Q-value iterate at iteration k is given by: Now, the value of the policy µ k (a\|s) underQ k+1 is given by: :=∆ k ,≥0, by proof of Theorem 3.2. Now, we also note that the expected amount of extra underestimation introduced at iteration k under action sampled from the behavior policy π β (a\|s) is 0, as, where the marked quantity is equal to 0 since it is equal since π β (a\|s) in the numerator cancels with the denominator, and the remaining quantity is a sum of difference between two density functions, a µ k (a\|s) − π β (a\|s), which is equal to 0. Thus, we have shown that, , computed under the tabular Q-function iterate, Q k+1 , from the previous equation, we obtain that Now, by choosing α k , such that any positive bias introduced by the quantity (µ k (a\|s) − π β (a\|s)) T (a) is cancelled out, we obtain the following gap-expanding relationship: for, α k satisfying, thus proving the desired result. To avoid the dependency on the true Q-value iterate, Q k , we can upper-bound Q k by Rmax 1−γ , and upperbound (π β (a\|s) − µ k (a\|s)) T B π k Q k (s, ·) by D TV (π β , µ k ) · Rmax 1−γ , and use this in the expression for α k . While this bound may be loose, it still guarantees the gap-expanding property, and we indeed empirically show the existence of this property in practice in Appendix B. To incorporate sampling error, we can follow a similar strategy as previous proofs: the worst case overestimation due to sampling error is given by In this case, we note that, w.h.p., Hence, the presence of sampling error adds D TV (π β , µ k ) · 2·C r,T ,δ Rmax 1−γ to the value of α k , giving rise to the following, sufficient condition on α k for the gap-expanding property: concluding the proof of this theorem. D Additional Theoretical Analysis In this section, we present a theoretical analysis of additional properties of CQL. For ease of presentation, we state and prove theorems in Appendices D.1 and D.2 in the absence of sampling error, but as discussed extensively in Appendix C, we can extend each of these results by adding extra terms induced due to sampling error. D.1 CQL with Linear and Non-Linear Function Approximation Theorem D.1. Assume that the Q-function is represented as a linear function of given state-action feature vectors F, i.e., Q(s, a) = w T F(s, a). Let D = diag (d π β (s)π β (a\|s)) denote the diagonal matrix with data density, and assume that F T DF is invertible. Then, the expected value of the policy under Q-value from Eqn 2 at iteration k + 1, a)], lower-bounds the corresponding tabular value, The choice of α k in Theorem D.1 intuitively amounts to compensating for overestimation in value induced if the true value function cannot be represented in the chosen linear function class (numerator), by the potential decrease in value due to the CQL regularizer (denominator). This implies that if the actual value function can be represented in the linear function class, such that the numerator can be made 0, then any α > 0 is sufficient to obtain a lower bound. We now prove the theorem. Proof. In order to extend the result of Theorem 3.2 to account for function approximation, we follow the similar recipe as before. We obtain the optimal solution to the optimization problem below in the family of linearly expressible Q-functions, i.e. Q := {Fw\|w ∈ R dim(F) }. By re-arranging terms, and converting it to vector notation, defining D = diag(d π β (s)π β (s)), and referring to the parameter w at the k-iteration as w k we obtain: underestimation . Now, our task is to show that the term labelled as "underestimation" is indeed negative in expectation under µ(a\|s) (This is analogous to our result in the tabular setting that shows underestimated values). In order to show this, we write out the expression for the value, under the linear weights w k+1 at state s, after substituting µ = π, Now, we need to reason about the penalty term. Defining, P F := F F T DF −1 F T D as the projection matrix onto the subspace of features F, we need to show that the product that appears as a penalty is positive: π(a\|s) T P F π(a\|s)−π β (a\|s) π β (a\|s) ≥ 0. In order to show this, we compute minimum value of this product optimizing over π. If the minimum value is 0, then we are done. By solving for η (using the condition that a density function sums to 1), we obtain that the minimum value of f (π) occurs at a π * (a\|s), which satisfies the following condition, Using this relation to compute f , we obtain, f (π * ) = 0, indicating that the minimum value of 0 occurs when the projected density ratio matches under the matrix (P F + P T F ) is equal to the projection of a vector of ones, 1. Thus, ∀ π(a\|s), f (π) = π(a\|s) T P F π(a\|s) − π β (a\|s) π β (a\|s) ≥ 0. This means that: ∀ s,V k+1 (s) ≤V k+1 LSTD-Q (s) given identical previousQ k values. This result indicates, that if α k ≥ 0, the resulting CQL value estimate with linear function approximation is guaranteed to lower-bound the value estimate obtained from a least-squares temporal difference Q-learning algorithm (which only minimizes Bellman error assuming a linear Q-function parameterization), such as LSTD-Q [29], since at each iteration, CQL induces a lower-bound with respect to the previous value iterate, whereas this underestimation is absent in LSTD-Q, and an inductive argument is applicable. So far, we have only shown that the learned value iterate,V k+1 (s) lower-bounds the value iterate obtained from LSTD-Q, ∀ s,V k+1 (s) ≤V k+1 LSTD-Q (s). But, our final aim is to prove a stronger result, that the learned value iterate,V k+1 , lower bounds the exact tabular value function iterate, V k+1 , at each iteration. The reason why our current result does not guarantee this is because function approximation may induce overestimation error in the linear approximation of the Q-function. In order to account for this change, we make a simple change: we choose α k such that the resulting penalty nullifes the effect of any over-estimation caused due to the inability to fit the true value function iterate in the linear function class parameterized by F. Formally, this means: choose α k to make this negative And the choice of α k in that case is given by: Finally, we note that since this choice of α k induces under-estimation in the next iterate,V k+1 with respect to the previous iterate,V k , for all k ∈ N, by induction, we can claim that this choice of α 1 , · · · , α k is sufficient to makeV k+1 lower-bound the tabular, exact value-function iterate. V k+1 , for all k, thus completing our proof. We can generalize Theorem D.1 to non-linear function approximation, such as neural networks, under the standard NTK framework [23], assuming that each iteration k is performed by a single step of gradient descent on Equation 2, rather than a complete minimization of this objective. As we show in Theorem D.2, CQL learns lower bounds in this case for an appropriate choice of α k . We will also empirically show in Appendix G that CQL can learn effective conservative Q-functions with multilayer neural networks. Theorem D.2 (Extension to non-linear function approximation). Assume that the Q-function is represented by a general non-linear function approximator parameterized by θ, Q θ (s, a). let D = diag(d π β (s)π β (a\|s)) denote the matrix with the data density on the diagonal, and assume that ∇ θ Q T θ D∇ θ Q θ is invertible. Then, the expected value of the policy under the Q-function obtained by taking a gradient step on Equation 2, at iteration k + 1 lower-bounds the corresponding tabular function iterate if: Proof. Our proof strategy is to reduce the non-linear optimization problem into a linear one, with features F (in Theorem D.1) replaced with features given by the gradient of the current Q-function Q k θ with respect to parameters θ, i.e. ∇ θQ k . To see, this we start by writing down the expression for Q k+1 θ obtained via one step of gradient descent with step size η, on the objective in Equation 2. Using the above equation and making an approximation linearization assumption on the non-linear Q-function, for small learning rates η << 1, as has been commonly used by prior works on the neural tangent kernel (NTK) in deep learning theory [23] in order to explain neural network learning dynamics in the infinite-width limit, we can write out the expression for the next Q-function iterate, Q k+1 θ in terms ofQ k θ as [1,23]: To simplify presentation, we convert into matrix notation, where we define the \|S\|\|A\| × \|S\|\|A\| matrix, ∇ θQ k , as the neural tangent kernel matrix of the Q-function at iteration k. Then, the vectorizedQ k+1 (with µ = π) is given by, Finally, the value of the policy is given by: (15) Term marked (b) in the above equation is similar to the penalty term shown in Equation 14, and by performing a similar analysis, we can show that (b) ≥ 0. Again similar to howV k+1 LSTD-Q appeared in Equation 14, we observe that here we obtain the value function corresponding to a regular gradientstep on the Bellman error objective. Again similar to before, term (a) can introduce overestimation relative to the tabular counterpart, starting atQ k : Q k+1 =Q k − η B πQk −Q k , and we can choose α k to compensate for this potential increase as in the proof of Theorem D.1. As the last step, we can recurse this argument to obtain our final result, for underestimation. D.2 Choice of Distribution to Maximize Expected Q-Value in Equation 2 In Section 3.1, we introduced a term that maximizes Q-values under the dataset d π β (s)π β (a\|s) distribution when modifying Equation 1 to Equation 2. Theorem 3.2 indicates the "sufficiency" of maximizing Q-values under the dataset distribution -this guarantees a lower-bound on value. We now investigate the neccessity of this assumption: We ask the formal question: For which other choices of ν(a\|s) for the maximization term, is the value of the policy under the learned Q-value,Q k+1 ν guaranteed to be a lower bound on the actual value of the policy? To recap and define notation, we restate the objective from Equation 1 below. We define a general family of objectives from Equation 2, parameterized by a distribution ν which is chosen to maximize Q-values as shown below (CQL is a special case, with ν(a\|s) = π β (a\|s)): In order to answer our question, we prove the following result: Theorem D.3 (Necessity of maximizing Q-values under π β (a\|s).). For any policy π(a\|s), any α > 0, and for all k > 0, the value of the policy.,V k+1 ν under Q-function iterates from ν−CQL,Q k+1 ν (s, a) is guaranteed to be a lower bound on the exact value iterate,V k+1 , only if ν(a\|s) = π β (a\|s). Proof. We start by noting the parametric form of the resulting tabular Q-value iterate: The value of the policy under this Q-value iterate, when distribution µ is chosen to be the target policy π(a\|s) i.e. µ(a\|s) = π(a\|s) is given by: We are interested in conditions on ν(a\|s) such that the penalty term in the above equation is positive. It is clear that choosing ν(a\|s) = π β (a\|s) returns a policy that satisfies the requirement, as shown in the proof for Theorem 3.2. In order to obtain other choices of ν(a\|s) that guarantees a lower bound for all possible choices of π(a\|s), we solve the following concave-convex maxmin optimization problem, that computes a ν(a\|s) for which a lower-bound is guaranteed for all choices of µ(a\|s): max ν(a\|s) min π(a\|s) a π(a\|s) · π(a\|s) − ν(a\|s) π β (a\|s) a π(a\|s) = 1, a ν(a\|s) = 1, ν(a\|s) ≥ 0, π(a\|s) ≥ 0. We first solve the inner minimization over π(a\|s) for a fixed ν(a\|s), by writing out the Lagrangian and setting the gradient of the Lagrangian to be 0, we obtain: where ζ(a\|s) is the Lagrange dual variable for the positivity constraints on π(a\|s), and η is the Lagrange dual variable for the normalization constraint on π. If π(a\|s) is full support (for example, when it is chosen to be a Boltzmann policy), KKT conditions imply that, ζ(a\|s) = 0, and computing η by summing up over actions, a, the optimal choice of π for the inner minimization is given by: π * (a\|s) = 1 2 ν(a\|s) + 1 2 π β (a\|s). Now, plugging Equation 20 in the original optimization problem, we obtain the following optimization over only ν(a\|s): Solving this optimization, we find that the optimal distribution, ν(a\|s) is equal to π β (a\|s). and the optimal value of penalty, which is also the objective for the problem above is equal to 0. Since we are maximizing over ν, this indicates for other choices of ν = π β , we can find a π so that the penalty is negative, and hence a lower-bound is not guaranteed. Therefore, we find that with a worst case choice of π(a\|s), a lower bound can only be guaranteed only if ν(a\|s) = π β (a\|s). This justifies the necessity of π β (a\|s) for maximizing Q-values in Equation 2. The above analysis doesn't take into account the effect of function approximation or sampling error. We can, however, generalize this result to those settings, by following a similar strategy of appropriately choosing α k , as previously utilized in Theorem D.1. D.3 CQL with Empirical Dataset Distributions The results in Sections 3.1 and 3.2 account for sampling error due to the finite size of the dataset D. In our practical implementation as well, we optimize a sample-based version of Equation 2, as shown below: whereB π denotes the "empirical" Bellman operator computed using samples in D as follows: where r is the empirical average reward obtained in the dataset when executing an action a at state s, i.e. r = 1 \|D(s,a)\| si,aiinD 1 si=s,ai=a · r(s, a), andT (s \|s, a) is the empirical transition matrix. Note that expectation under π(a\|s) can be computed exactly, since it does not depend on the dataset. The empirical Bellman operator can take higher values as compared to the actual Bellman operator, B π , for instance, in an MDP with stochastic dynamics, where D may not contain transitions to all possible next-states s that can be reached by executing action a at state s, and only contains an optimistic transition. We next show how the CQL lower bound result (Theorem 3.2) can be modified to guarantee a lower bound even in this presence of sampling error. To note this, following prior work [24, 39], we assume concentration properties of the reward function and the transition dynamics: Assumption D.1. ∀ s, a ∈ D, the following relationships hold with high probability, Under this assumption, the difference between the empirical Bellman operator and the actual Bellman operator can be bounded: This gives us an expression to bound the potential overestimation that can occur due to sampling error, as a function of a constant, C r,T,δ . E Extended Related Work and Connections to Prior Methods In this section, we discuss related works to supplement Section 5. Specifically, we discuss the relationships between CQL and uncertainty estimation and policy-constraint methods. Relationship to uncertainty estimation in offline RL. A number of prior approaches to offline RL estimate some sort of epistemic uncertainty to determine the trustworthiness of a Q-value prediction [27, 14, 2, 30]. The policy is then optimized with respect to lower-confidence estimates derived using the uncertainty metric. However, it has been empirically noted that uncertainty-based methods are not sufficient to prevent against OOD actions [14,27] in and of themselves, are often augmented with policy constraints due to the inability to estimate tight and calibrated uncertainty sets. Such loose or uncalibrated uncertainty sets are still effective in providing exploratory behavior in standard, online RL [40,39], where these methods were originally developed. However, offline RL places high demands on the fidelity of such sets [30], making it hard to directly use these methods. How does CQL relate to prior uncertainty estimation methods? Typical uncertainty estimation methods rely on learning a pointwise upper bound on the Q-function that depends on epistemic uncertainty [24,39] and these upper-confidence bound values are then used for exploration. In the context of offline RL, this means learning a pointwise lower-bound on the Q-function. We show in Section 3.1 that, with a naïve choice of regularizer (Equation 1), we can learn a uniform lower-bound on the Q-function, however, we then showed that we can improve this bound since the value of the policy is the primary quantity of interest that needs to be lower-bounded. This implies that CQL strengthens the popular practice of point-wise lower-bounds made by uncertainty estimation methods. Can we make CQL dependent on uncertainty? We can slightly modify CQL to make it be account for epistemic uncertainty under certain statistical concentration assumptions. Typical uncertainty estimation methods in RL [40,24] assume the applicability of concentration inequalities (for example, by making sub-Gaussian assumptions on the reward and dynamics), to obtain upper or lower-confidence bounds and the canonical amount of over-(under-) estimation is usually given by, O where C r and C T are constants, that depend on the concentration properties of the MDP, and by appropriately choosing α, i.e. α = Ω(n(s, a)), such that the learned Q-function still lower-bounds the actual Q-function (by nullifying the possible overestimation that appears due to finite samples), we are still guaranteed a lower bound. F Additional Experimental Setup and Implementation Details In this section, we discuss some additional implementation details related to our method. As discussed in Section 4, CQL can be implemented as either a Q-learning or an actor-critic method. For our experiments on D4RL benchmarks [11], we implemented CQL on top of soft actor-critic (SAC) [18], and for experiments on discrete-action Atari tasks, we implemented CQL on top of QR-DQN [7]. We experimented with two ways of implementing CQL, first with a fixed α, where we chose α = 5.0, and second with a varying α chosen via dual gradient-descent. The latter formulation automates the choice of α by introducing a "budget" parameter, τ , as shown below: Equation 24 implies that if the expected difference in Q-values is less than the specified threshold τ , α will adjust to be close to 0, whereas if the difference in Q-values is higher than the specified threshold, τ , then α is likely to take on high values, and thus more aggressively penalize Q-values. We refer to this version as CQL-Lagrange, and we found that this version outperforms the version with a fixed α on the gym-MuJoCo D4RL benchmarks, and drastically outperforms the fixed α version on the more complex AntMazes. Table 3 (with 10% data), α = 4.0 chosen uniformly for Table 3 (with 1% data), and α = 0.5 for Figure 1. Computing log a exp(Q(s, a). CQL(H) uses log-sum-exp in the objective for training the Qfunction (Equation 4). In discrete action domains, we compute the log-sum-exp exactly by invoking the standard tf.reduce_logsumexp() (or torch.logsumexp()) functions provided by autodiff libraries. In continuous action tasks, CQL(H) uses importance sampling to compute this quantity, where in practice, we sampled 10 action samples each at every state s from a uniform-at-random Unif(a) and the current policy, π(a\|s) and used these alongside importance sampling to compute it as follows using N = 10 action samples: Hyperparameters. For the D4RL tasks, we built CQL on top of the implementation of SAC provided at: https://github.com/vitchyr/rlkit/. Our implementation mimicked the RLkit SAC algorithm implementation, with the exception of a smaller policy learning rate, which was chosen to be 3e-5 or 1e-4 for continuous control tasks. Following the convention set by D4RL [11], we report the normalized, smooth average undiscounted return over 4 seeds for in our results in Section 6. The other hyperparameters we evaluated on during our preliminary experiments, and might be helpful guidelines for using CQL are as follows: • Q-function learning rate. We tried two learning rate values [1e − 4, 3e − 4] for the Qfunction. We didn't observe a significant difference in performance across these values. 3e − 4 which is the SAC default was chosen to be the default for CQL. • Policy learning rate. We evaluated CQL with a policy learning rate in the range of [3e − 5, 1e − 4, 3e − 4. We found 3e − 5 to almost uniformly attain good performance. While 1e − 4 seemed to be better on some of our experiments (such as hopper-medium-v0), but it performed badly with the real-human demonstration datasets, such as the Adroit tasks. We chose 3e − 5 as the default across all environments. • Lagrange threshold τ . We ran our preliminary experiments with three values of threshold, τ = [2.0, 5.0, 10.0]. However, we found that τ = 2.0, led to a huge increase in the value of α (sometimes upto the order of millions), and as a result, highly underestimated Q-functions on all domains (sometimes upto the order of -1e6). On the datasets with human demonstrations -the Franka Kitchen and Adroit domains, we found that τ = 5.0 obtained lower-bounds on Q-values, whereas τ = 10.0 was unable to prevent overestimation in Q-values in a number of cases and Q-values diverged to highly positive values (> 1e+6). For the MuJoCo domains, we observed that τ = 10.0 gave rise to a stable curve of Q-values, and hence this threshold was chosen for these experiments. Note that none of these hyperparameter selections required any notion of onine evaluation, since these choices were made based on the predicted Q-values on dataset state-actions pairs. • Number of gradient steps. We evaluated our method on varying number of gradient steps. Since CQL uses a reduced policy learning rate (3e-5), we trained CQL methods for 1M gradient steps. Due to a lack of a proper valdiation error metric for offline Q-learning methods, deciding the number of gradient steps dynamically has been an open problem in offline RL. [30]. Different prior methods choose the number of steps differently. For our experiments, we used 1M gradient steps for all D4RL domains, and followed the convention from Agarwal et al. [2], to report returns after 5X gradient steps of training for the Atari results in Table 3. Other hyperparameters, were kept identical to SAC on the D4RL tasks, including the twin Q-function trick, soft-target updates, etc. In the Atari domain, we based our implementation of CQL on top of the QR-DQN implementation provided by Agarwal et al. [2]. We did not tune any parameter from the QR-DQN implementation released with the official codebase with [2]. G Ablation Studies In this section. we describe the experimental findings of some ablations for CQL. Specifically we aim to answer the following questions: We start with question (1). On three MuJoCo tasks from D4RL, we evaluate the performance of both CQL(H) and CQL(ρ), as shown in Table 5. We observe that on these tasks, CQL(H) generally performs better than CQL(ρ). However, when a sampled estimate of log-sum-exp of the Q-values becomes inaccurate due to high variance importance weights, especially in large action spaces, such as in the Adroit environments, we observe in Table 2 that CQL(ρ) tends to perform better. Next, we evaluate the answer to question (2). On three MuJoCo tasks from D4RL, as shown in Table 6, we evaluate the performance of CQL(H), with and without the dataset maximization term in Equation 2. We observe that omitting this term generally seems to decrease performance, especially in cases when the dataset distribution is generated from a single policy, for example, hopper-medium. Table 6: Average return obtained by CQL(H) and CQL(H) without the dataset average Q-value maximization term. The latter formulation corresponds to Equation 1, which is void of the dataset Q-value maximization term. We show in Theorem 3.2 that Equation 1 results in a weaker lower-bound. In this experiment, we also observe that this approach is generally outperformed by CQL(H). Next, we answer question (3). Table 7 shows the performance of CQL and CQL-Lagrange on some gym-MuJoCo and AntMaze tasks from D4RL. In all of these tasks, we observe that the Lagrange version (Equation 24, which automates the choice of α using dual gradient descent on the CQL regularizer, performs better than the non-Lagrange version (Equation 4). In some cases, for example, the AntMazes, the difference can be as high as 30% of the maximum achievable performance. On the gym MuJoCo tasks, we did not observe a large benefit of using the Lagrange version, however, there are some clear benefits, for instance in the setting when learning from hopper-mixed dataset. Table 7: Average return obtained by CQL(H) and CQL(H) with automatic tuning for α by using a Lagrange version. Observe that both versions are generally comparable, except in the AntMaze tasks, where an adaptive value of α greatly outperforms a single chosen value of α.	2020-06-09T01:01:10.729Z	2020-06-08T00:00:00.000	{ "year": 2020, "sha1": "28db20a81eec74a50204686c3cf796c42a020d2e", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "fe2ac46ca18e8a33c7232f07861ffa4dbcfa2d70", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
233364374	pes2o/s2orc	v3-fos-license	Management Control System In University Libraries : Challenges and Remedies Management control system is an essential process by which all members of the organisation work as team to achieve a better output and improved service delivery. Despite the importance of management control system, university libraries in South East, Nigeria seem to have issues with its application. This study investigated management control system, issues and prospects in university libraries. The main purpose of this study was to assess the management control system and challenges associated with its application in university libraries in South East, Nigeria. The research design adopted was descriptive survey design. The population of the study was 163 librarians in federal and state university libraries in South-East, Nigeria, and was all used without sampling as the number was considered manageable. The instrument used in gathering data for the study is questionnaire which was analysed using mean and standard deviation. Findings revealed that university libraries employed performance appraisal system, duties rotation system, controlled activities system and regulation of staff attendance. Result also showed that management control system contributes to effective job performance by enabling employees to understand their job expectations and encourages librarians' attitude to work. Challenges associated with utilising management control system include lack of adequate staff development, training and effective communication. Strategies for enhancing management control system in university libraries include adequate financial support to libraries to enable them acquire necessary facilities in order to achieve effective management control system, frequent training and development programme. The researcher recommended that federal and state governments should make funds available to university libraries to enable them adopt effective management control system and procure facilities and resources to enhance job performance. INTRODUCTION University library is a thriving hub in the university where students, lecturers and researchers visit to meet their information needs. The strength of the university library determines how effective research, teaching and learning in the university would be. These intellectual activities are the core essence for which university library is established. The reason is that research, teaching and learning are central to information resources and service that the library provides. University library is a library within the university for educational and research purposes. The University library is meant to support the university's academic programme, which among others include learning, teaching and research. The libraries constitute a very vital component of a vibrant university system. It contributes to the capital human knowledge and resource development. They play essential role in the education and training of staff and student by providing resources for research and scholarly purposes (Igbokwe, 2011). Several authors have conducted a research on management and therefore, come up with different definitions, such as Hartzell (2010), who defines management as a process of organising, using and controlling human activities and other resources towards specific ends; the group of persons responsible for running, organisation or directing human activities. Management involves utilising available resources, whether inform of machine, money or people effectively. In the other hand, management can also be defined as the people responsible for the management of an organisation which is directing, planning, running of its operation, implementation of its policies and attainment of its objectives. Management control is a mechanism used by the management of an organisation with the purpose of creating values and improving the operations of an organisation to help them in achieving their objectives systematically and evaluate the effectiveness of management policies . Management control system is that aspect of management that provides means for achieving effective job performance of the employees. Ben (2008) affirms that management control system for effective job performance is an essential process by which all members of the organisation work as team for achieving a better output and improved service delivery. According to Mohammed (2018), the federal and state university libraries share similar management policies and administrative pattern to an extent that contributes to librarians' outputs. Ben states that the primary objective of management control system in an organisation is to enhance performance through strategic planning processes. Implementation of proper management control system in university libraries will facilitate the performance of the entire library system. The university libraries in south east Nigeria can become the envy of all other academic libraries if they improve on the job performance of the librarians. Proper implementation of management control system in the organisation, helps to align the department, section/unit and individual's goals; identify key priority areas for judicious allocation of scarce resources; bring about improvement in processes and systems in the university library; provide a common sense of direction to all staff; support leadership, motivating and teambuilding processes; facilitate change management initiatives; recognise talents and release potential, and ensure effective service delivery to meet expectations of the library patrons (Asogwa, 2014. P. 50-51) Statement of the Problem It has been observed in literature that management control system enhances job performance of employees. However, discovery based on our experiences with regards to librarians' job performance in university libraries in South East, Nigeria, indicated seemingly, lack of commitment and poor work attitude of librarians Lack of commitment and poor work attitude │ 51 of librarians in this context refers to unserious behaviour of librarians in carrying out their duties. This will adversely affect meeting the information needs of library users thereby hindering progress in research, scholarship and the institutional development. This state of affair may be attributed to lack of effective management control system leading to librarians' inadequate use of their skills in carrying out effective library services. Management control system ensures that librarians' abilities and talents are properly utilised in achieving library objectives. It is empirically unknown if the university in South East, Nigeria employed management control system and the extent to which it is used to improve job performance of librarians. In other words, to the best of researcher's knowledge, it seems no study has been carried out on this topic. In the light of the above undesirable state of affairs, it therefore becomes imperative for a study to examine the management control system in university libraries, challenges and enhancement strategies. Hence, this study attempts to assess management control system, issues and prospects in university libraries in South East Nigeria. Purpose of the Study The general purpose of this study is to assess the management control system, issues and prospects in university libraries in South East, Nigeria. Specifically, the study is intended to: measure in his quest to make efficient and effective use of resources by applying some principles of planning, organising, controlling, commanding and coordinating. Management becomes more indelible in organisations involving human and material resources and certain organisational services. From the foregoing, management gives an organisation direction to meet organisation's goal and objectives. Hitt, Black and Lyman (2008) define management as a process of utilising a set of resources in a manner that enhances accomplishment of tasks in an organisational setting. They further add that management involves a series of activities and operations such as evaluating, planning and deciding. From the definition, management can be seen as a process that involves using a set of resources, bringing together and putting into use a variety of resources as regard to human, material, financial and information to achieve the objective of an organisation. It is also found from the definition by Hitt et al. that management is an action carried out in a goal-directed manner within an organisation by people with different job responsibilities designed to achieve common purposes. Without management control and coordination, university library will tantamount to unruly group of people lacking unit, direction and purpose. Management control system plays significant roles in the organisation. Asogwa (2014) identifies four roles of management control system in an organisation which are as follows: linking individual employee objectives with the organisation's mission and strategic plans. Therefore, the employee has a clear concept on how he contributes to the achievement of the overall administration; focusing on setting clear performance objectives and expectations; defining clear development plans as part of the process and conducting regular discussions throughout the performance cycle which include such things as coaching, monitoring, feedback and assessment. Management control system enables an organisation to attain its goals and objectives which involves a calculative procedure and adequate utilisation of available resources to achieve maximum efficiency of operation . It is not uncommon to identify challenges with management control system either in the area of implementation, maintenance or compliance in the university library. These challenges can lead to poor job performance of library staff. It is therefore, imperative to identify possible challenges to effective management control system in relation to job performance of university library staff. One of the challenges associated with management control system is inadequate staff training development. This has posed serious threats to the management control system of university libraries. According to Raseroka (2010), challenges associated with management control system of university libraries cannot be divorced from lack adequate staff development and training. It appears that the management executives of university libraries in general assume that once librarians have been provided with basic professional training, they are equipped for life. But the assumption is wrong due to changes, innovation and introduction of new technologies. The dynamic information environment, however, demands continual renewal of skills and reinterpretation of operations. Ifidon and Ifidon (2007) assert that the purpose of staff recruitment and evaluation which are integral part of management control is defeated if they are not complemented by staff development and training. Any management control system that does not give staff development and training a priority suffers redundancy. Rosenberg (1997) opines that when there is inadequate development and training of staff, it will result to knowledge gap and skill deficiency which is disastrous to the institution concerned. Poor communication system also hinders the effective management control system of university libraries. Communication policies adopted and practiced can affect the librarians' │ 53 performance either positively or negatively. The communication between the university hierarchy and library management of many university libraries has proven to be very difficult. According to Ngalla (2007), university libraries complain of the uncooperative attitudes of their institutional authorities which make communication very difficult and consequently left many issues unresolved. In spite the roles of management control system in improving job performance, Dutse (2011) states that the relationship between librarians and the university authority has always been difficult, resulting in poor communication and other untold interference in the management control system. Another challenge to effective management control system in university libraries is lack of adequate infrastructures. Management control system does not operate in the vacuum but rather in the library environment. Raseroka (2010), states that Library buildings are essential infrastructures because they provide the single most used facility on any campus in which information related needs are met. If there is no adequate infrastructure upon which management control system dwells, the performance of library staff would be affected. Similarly, Dutse (2011) states that the infrastructural problem which constitute a barrier to effective management control system is lack of maintenance culture especially in the aspect of information and communication system. Some Nigerian university libraries could have acquired personal computers and other necessary software packages for their library operations but unfortunately, there is irregular or non-maintenance and upgrading of such hardware and software. Management control system that is devoid of proper infrastructure and maintenance of information system especially in this era that information communication technology is thriving in libraries is planning for performance drain. Lack of adequate funding poses great challenges to effective management control system in university libraries. Uzomba, Oyebola and Izuchukwu (2015) opine that significant number of libraries, especially in developing countries suffer poor funding. The result of the above fact is that management control system would not be effectively implemented in poor funding situation. Therefore, library requires funds for acquisition of modern facilities for the smooth administration of the library and training of librarians on how to develop and utilise modern facilities for effective job performance. Lack of adequate finance poses a great barrier to implementation of effective management control system. If there should be efficient administration of any library, there is need for adequate funding. Similarly, Ngalla (2007) opines that the prevailing funding problem in universities and their libraries has made the institutions lack necessary equipment. This poses threats to library management control system. Perhaps, adequate funds are important, not only for the purchase of library equipment and information resources, but also for training and developing staff, motivation and other performance management facilities. This, calls for the need for university authorities to review the funding policies of university libraries in Nigeria to enable them meet international standard. Aguolu (2008) reveals various factors that impose serious challenges to effective management control system in any given organisation. These include: Staff collusion: This refers to the compensating relaxation of interrelated checks by staff that operates complementary roles for their own mutual benefits. An example is a situation where the university librarian or acquisition librarian connive with the book vendor to inflate the price of the materials ordered for their mutual advantage. Another example is a situation where university library management agrees with the university human resources committee to include fictitious names or ghost workers on the payroll and they both share the cash. Wittington and Pany (2008) opine that control activities whether manual or automated may be circumvented by collusion among two or more staff or inappropriate management override of management control. │ Management overriding established control: This refers to the management failure or reluctance in enforcing those controls in their personal activities or the activities of their relations and friends. For instance, Aguolu (2008) argues that management is usually under temptation to resist submitting themselves and their relations to personal scrutiny checks or to enforce the required controls in their own affairs. Apart from the reluctance on the part of the management to submit themselves to established controls, Aguolu posits that employees that are charged with the responsibility of enforcing these controls may not be willing to extend these controls to management out of fear respect or intimidation by management. In a similar vein, Messier, Glover and Prawitt (2008) admit that management override established control is a serious threat to effective management control system. They further stress that management control system is only as effective as the personnel who implemented and performed the controls. Abuse of authority: This refers to misuse of authorisation control by the officials in whom such authority is vested. Aguolu (2008) likens abuse of authority to a situation where an official fails to perform an expected control or performs such control to the detriment of the establishment. In the library perspective, the situation is such that the head of a section uses his position to victimise his subordinates. Staff incompetence: Aguolu (2008) states, "Incompetence, lack of care or integrity, fatigue or human error can vitiate the effectiveness of an otherwise good system of control". An example is a situation where the chief cataloguer in the university library, in a hurry fails to check the correctness of bibliographic entries in the cataloguing worksheet before inputting them into OPAC. It could also be a situation where the University Librarian (UL) fails to carry out the necessary checks hence, signing an invalid voucher or a forged cheque. These human errors can indeed, affect librarians performance and also the outcome of management control system. According to Wittington and Pany (2008) human error may be made in the performance of control as a result of a misunderstanding of instructions, mistakes of judgment, carelessness, distraction or fatigue. They also stressed that human errors may also occur in designing, maintaining or monitoring automated controls and these have adverse effects on management control system. Alteration in the system: Aguolu (2008) states that drastic or frequent alteration in the system for whatever reason can lead to a total breakdown in the management control system. The alteration may be a result of frequent staff turnover, introduction of automation or digitalisation of library resources and services. As challenges to effective management control system in university libraries have been discussed, it is imperative to look at the possible strategies that can be used to overcome the challenges and enhance management control system for effective job performance in university libraries. Ifidon and Ifidon (2007) suggest that there should be adequate development and training of the librarians to enable them make effective contribution to the library's service mission and also make them attain satisfaction that goes beyond economic (that is, self -pride, self-respect and achievement of organisation's goal) and prepare the staff for the challenge of complex modern technology, which requires an integration of activities and persons of diverse and specialised competence. They further opine that adequate development and training will enable library workers develop a life of their own outside the library; make staff receptive to change and innovation rather than resistant to them; create a climate where the dignity of employees as human beings, not just as factors contributing to the library's efficiently, is respected; and prepare the individual for a new, different and higher responsibility. Libraries require proper funding in order to run its management control system effectively enough that the job performance of librarians is enhanced effectively. Uzomba, Oyebola and │ 55 Izuchukwu (2015) recommended that libraries should be supported financially to enable them acquire the necessary facilities. With adequate funding, training could be provided for the librarians in order to render quality services to the users. Aguolu (2008) opines that management control requires effective segregation of duties to ensure that no one person carries out three aspects of any transaction such as authorisation, custodian and recording. He states that in the work process, there should be a check on the other person's duties to ensure adherence to the control system. By this arrangement, Aguolu posits that no one person is in a position both to commit fraud and conceal his action by falsifying the record. A case scenario in the library is when an acquisition librarian, after acquiring the books and the proper check is done to ensure materials received are consistent with what were ordered for before stamping and accessioning of the materials, then the materials are passed to the chief cataloguer for proper cataloguing before passing them to circulation librarian for users consumption. Adoption of separation of duties in the libraries has been taken more serious in the contemporary era. Chukwu (2010) emphasises more on the importance of separation of duties between departments and individuals, such that an individual staff or department does not handle a particular activity from the beginning to the end. Aguolu (2008) however, opines that there should be adequate supervisory control. This implies that the job performed by one staff is subject to the approval of a higher official. This can be done by supervising superior officials; scrutinising the job of junior officers or subordinates; authorization from a higher official for a job to be performed; physical controls to ensure that specified corrective measures are carried out especially where an established control breaks down. RESEARCH METHOD Descriptive survey design was adopted in conducting the study. The area of the study is South East geo-political zone of Nigeria. The population of this study comprised total of 163 librarians in ten government university libraries in South East Nigeria which were all studied without sampling as the population is manageable. The instrument used for this study was structured questionnaire which was arranged in 4 clusters with 57 items which was constructed in line with the research questions. The questionnaire was face validated by three experts and their observations and suggestions which include rephrasing some questions, terms, options and formats were used to produce the final draft of the instrument. The reliability of the instrument was trial-tested on 30 Librarians from another university library who were not part of the population of this study. Upon analysis of the responses to the questionnaire, Cronbach Alpha method was used to determine the internal consistency of the items of the instrument. The instrument shows an overall reliability of 0.96. Copies of the questionnaire were administered to librarians in the ten government university libraries in South East, Nigeria. With the aid of five trained research assistants, the questionnaire was administered and completely retrieved from the respondent. The questionnaire was analysed using mean and standard deviation. The mean was interpreted in line with the 4 point rating scale. Consequently, decision was taken based on real limit of numbers as follows: Research Question One What types of management control system are employed for effective job performance in the university libraries? Key, HE=Highly Employed, E= Employed, R=Rank, D= Decision, SD= Standard Deviation Results in table 1 shows the mean ratings and standard deviation of librarians on the types of management control system employed in their university libraries. Using the principle of real limit of numbers, the results showed that performance appraisal system had the highest mean rating of 3.52 followed by duties rotation system with the mean rating of 3.35;controlled activities system, performance management system and penal system/sanctions for breach of official rules and regulation with the mean ratings of 3.28. These mean ratings are within the range of 2.50 -3.59 set as a criterion for employed. The overall mean of 3.26 with a standard deviation of .53 showed that the librarians employ these types of management control system in university libraries. Finally, comparing the two types of ownership of institution used in the study, it was observed that the respondents indicated in federal and state universities that performance appraisal system is mostly employed in university libraries in South East, Nigeria (mean 3.56 and 3.45 respectively). Research Question Two To what extent does management control system contribute to effective job performance in the university libraries? Table 2 above shows the mean ratings of the respondents on the extent to which management control system contributes to effective job performance in university libraries. Using the principle of real limit of numbers, the results of the data analysis revealed that management control system to a great extent contributes to effective job performance in university libraries in the following ways: it enables employees to understand their job expectations, it helps the employees to achieve accuracy and reliability of records, it enables the employees realise the objectives of the library, it inspires employees' to put in their best and enhances the behaviour of employees. Also, the overall mean showed that management control system enables employees to understand their job expectations (mean=3.57) is ranked highest, while management control system improves employees' attitude to work (mean=3.27) is ranked lowest as extent management control system contributes to effective job performance in university libraries. The standard deviation values for the seven evaluation activities ranged from 0.49 to .59 which implied that the respondents were not far from one another in their responses and that their responses were not far from the mean. Research Question Three What are the challenges associated with utilising management control system for effective job performance in the university libraries? Table 3 revealed that, the mean ratings of the responses of the respondents on the fifteen (15) identified items of challenges associated with utilising management control system for effective job performance in library had mean values ranging from 3.16 to 3.59; which means that the respondents agreed that lack of adequate staff development and training, inadequate communication system, lack of adequate infrastructure, lack of appropriate sanctions for breach of official rules and regulation, lack of acknowledgement of staff performance through rewards, promotion and inadequate funding were challenges associated with utilising management control system for effective job performance in library. The standard deviation values for the seven evaluation activities ranged from 0.81 to 1.03 which implied that the respondents were not far from one another in their responses and that their responses were not far from the mean. Research Question Four What are the strategies for enhancing the management control system for effective job performance in the university libraries? Table 4 shows the mean ratings of respondents on the strategies for enhancing the management control system approach for effective job performance in university libraries. Using the principle of real limit of numbers, the results of the data analysis revealed that Libraries should be supported financially to enable them acquire necessary facilities and maintain management control system (mean=3.60), there should be frequent library staff training and development programmes (mean=3.59), there should be periodic performance review to assess the performance of staff over a period of time (mean=3.52) and there should be adequate supervisory control to ensure that job performed by one staff is subject to the approval of a higher official (mean=3.50) are very appropriate strategies for enhancing the management control system approach for effective job performance in university libraries. Other appropriate strategies were remuneration system and working condition should be improved on to encourage staff compliance with the management control system (mean=3.43), there should be effective segregation of duties to ensure that no one staff carries out the whole library functions at a time (mean=3.42), the management control system should be designed to ensure that the personnel operating the system are adequately motivated to carry out the duties assigned to them (mean=3.42) and that there should be improved reward system for librarians who perform exceptionally (mean=3.42). The standard deviation values for the evaluation activities ranged from 0.52 to .70 which implied that the respondents were not far from one another in their responses and that their responses were not far from the mean. │ The discussion of findings was organised in line with the objectives of the study and the research questions. Types of management control system employed in university libraries in South East Nigeria The research revealed that the most employed management control system in university libraries in South-East Nigeria include performance appraisal system, duties rotation system, controlled activities system, performance management system, penal system/sanctions for breach of official rules and regulation, duties segregation system and Regulation of staff attendance. The finding is in line with Howell (2017) who stated that no matter the type of library, some level of management control system must be applied such as performance evaluation; duties rotation systems; benchmarking system; duties segregation system; internal audit system; staff evaluation system; controlled activities system; supervisory control system; internal control system; organisational structure system; internal check system; performance management system and penal system/sanction for breach of official rules and regulations and regulation of attendance register. In consonant with the finding, Ifidon and Ifidon (2007) assert that the purpose of performance appraisal which are integral part of management control in university libraries is defeated if they are not complemented by staff development and training system approach. From the foregoing, it could be seen that the type of management control systems employed in federal and state university libraries are similar. This could be as a result of common objectives of both university libraries and similar characteristics of the librarians working in the university libraries. The extent to which management control system contributes to effective job performance in university libraries Result revealed the extent to which management control system contributes to effective job performance in university libraries. The findings showed that management control system contributes to effective job performance to a great extent by enabling employees to understand their job expectations, helping employees achieve accuracy and reliability of records, enabling the employees realise the objectives of the library, inspiring employees' to put in their best and also enhances the behaviour of employees. In line with the finding, Asogwa (2014) opined that management control system links individual employee objectives with the organisation's mission and strategic plans and enable them achieve high performance rate. Therefore, the employee has a clear concept on how he contributes to the achievement of the overall business objective; focusing on setting clear performance objectives and expectations through the use of results, actions and behaviours; defining clear development plans as part of meeting job expectations. Also in agreement with the findings, Ben (2008) affirms that management control system is an essential process by which all members of the organisation work as partners for achieving a better output and improved service delivery. He further states that the primary contributions of management control system to the organisation is to: enhance performance at all levels; to establish clear links between the institutional development, the delivery of quality services and the development of employees at work; create a common bond of ownership among all employees as well as an environment where all individuals are developed, motivated and inspired to maintain a positive attitude to work and deliver a quality expectation. │ 63 The results revealed the challenges associated with utilising management control system for effective job performance in university library in South East, Nigeria The result shows that lack of adequate staff development and training, inadequate communication system, lack of adequate infrastructure, lack of appropriate sanctions for breach of official rules and regulation, lack of acknowledgement of staff performance through rewards, promotion, etc. and inadequate funding are major factors hindering effective utilisation of management control system in libraries. In line with the findings, Raseroka (2010) stated that lack adequate staff development and training inhibits the effectiveness of utilising management control system in university libraries. Inadequate communication was discovered as another inhibiting factor to effective utilisation of management control system. In agreement with the finding, Dutse (2011) and Ngalla (2007) expressed that the relationship between librarians and the university authority has always been difficult, resulting in poor communication and other untold interference in the management control system. Result also revealed that inadequate funding hinders effective utilisation of management control system in university libraries. In conformity with the finding, Uzomba, Oyebola and Izuchukwu (2015) stated that many libraries; especially in developing countries are poorly funded. The implication of the above exposition is that management control system would not be effectively operational. Therefore, library requires funds needed to acquire modern facilities for the smooth running of the library and training of staff on how best to develop and handle modern facilities needed for effective job performance. This was supported by Ngalla (2007) who posits that the persistent funding problem is a major problem militating against higher institutions in general and the libraries in particular which resulted in university libraries being poorly equipped. Strategies for enhancing management control system for effective job performance in university libraries. It could be seen in table 4 that the mean rating value for all the 14 strategies suggested by the respondents are far above than just the accepted mean of 2.50. This shows that the problems could be addressed if the strategies are followed diligently. The strategies majorly rest on availability of funds, library staff training and development programmes, periodic performance review, duties segregation among all. The suggestions are consistent with Uzomba, Oyebola and Izuchukwu (2015) that libraries should be supported financially to enable them acquire the necessary facilities and also that training be provide for the librarians in order to render quality services to the users. In line with the finding of the study, Wittington and Pany (2008) reinstated that management control system should maintain effective performance review and segregation of duties to enhance and facilitate effective implementation. Furthermore, Aguolu (2008) opines that management control requires effective segregation of duties to ensure that no one person carries out three aspects of any transaction such as authorisation, custodian and recording. From the foregoing, it could be seen that university libraries deserves to be given adequate financial support to enable them acquire necessary management control system facilities and also embark on staff training and development programmes which determines how efficient management control system impacts on job performance. As suggested by the respondents, assessing the performance of staff through periodic performance review positions the attention of the librarians towards application of their talents, skills and knowledge to the actualisation of their job descriptions.	2021-04-23T12:11:40.921Z	2021-02-27T00:00:00.000	{ "year": 2021, "sha1": "330cf2f6943e233ab484a750c22e6a4c41aaac4e", "oa_license": "CCBYNC", "oa_url": "http://jurnal.upnyk.ac.id/index.php/ijcbm/article/download/4358/3339", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "330cf2f6943e233ab484a750c22e6a4c41aaac4e", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [ "Business" ] }
232226711	pes2o/s2orc	v3-fos-license	Development of the systematic observation of COVID-19 mitigation (SOCOM): Assessing face covering and distancing in schools Introduction: During the COVID-19 pandemic, some K-12 schools resumed in-person classes with varying degrees of mitigation plans in the fall 2020. Physical distancing and face coverings can minimize SARS-CoV-2 spread, the virus that causes COVID-19. However, no research has focused on adherence to mitigation strategies during school days. Thus, we sought to develop a systematic observation protocol to capture COVID-19 mitigation strategy adherence in school environments: The Systematic Observation of COVID-19 Mitigation (SOCOM). Methods: We extended previously validated and internationally used tools to develop the SOCOM training and implementation protocols to assess physical-distancing and face-covering behaviors. SOCOM was tested in diverse indoor and outdoor settings (classrooms, lunchrooms, physical education [PE], and recess) among diverse schools (elementary, secondary, and special needs). Results: For the unique metrics of physical-distancing and face-covering behaviors, areas with less activity and a maximum of 10–15 students were more favorable for accurately capturing data. Overall proportion of agreement was high for physical distancing (90.9%), face covering (88.6%), activity type (89.2%), and physical activity level (87.9%). Agreement was lowest during active recess, PE, and observation areas with ≥20 students. Conclusions: Millions of children throughout the USA are likely to return to school in the months ahead. SOCOM is a relatively inexpensive research tool that can be implemented by schools to determine mitigation strategy adherence and to assess protocols that allow students return to school safely and slow the spread of COVID-19. Introduction The impact of the coronavirus disease 2019 , caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), has been widespread, with 30 million cases diagnosed and over 550,000 deaths in the USA as of March 31, 2021. When COVID-19 was declared a pandemic, schools were among the first operations to close to prevent community spread. However, in the fall of 2020, 56 million school-aged children (5-17 years of age) resumed education as some schools opened to in-person classes with varying public health mitigation plans [1]. Children and adolescents comprise asymptomatic, symptomatic, and hospitalized populations of SARS-CoV-2-infected individuals and their capability of spreading disease to school staff and family has yet to be resolved [2][3][4][5]. However, mitigation procedures can minimize SARS-CoV-2 outbreaks even in the close quarters of overnight summer camps [6]. Viral aerosolization can occur due to the general social and physically interactive nature of school-aged children during classroom learning, communal dining, recess, and physical education (PE) classes. Comprehensive return-to-school plans emphasize adherence to accepted SARS-CoV-2 viral mitigation strategies procedures, including physical distancing (staying at least 6 feet from others who are not from the same household) and face cover wearing [7][8][9]. Specifically, new guidelines released by the Centers for Disease Control and Prevention highlight the need to promote behaviors that reduce COVID-19's spread, including maintaining healthy environments and operations, and preparing for when someone gets sick [10]. Recognizing mitigation strategies represent unnatural behaviors for K-12 students, attention has been focused on strategies to implement and operationalize mitigation protocols in schools. Little emphasis has been given to how to quantify adherence. Without adherence metrics, effectiveness of SARS-CoV-2 transmission mitigation procedures cannot be ascertained. Direct observation approaches developed to assess levels of PA in a variety of real-world environments were readily adapted to measure COVID-19 mitigation strategies in K-12 schools. The original System for Observing Play and Leisure Activity in Youth (SOPLAY), designed for PA settings in schools, and the System for Observing Play and Recreation in Communities (SOPARC), designed to include playgrounds and parks, were designed to obtain direct information on PA in open spaces [20,28]. SOPLAY and SOPARC are based on momentary time sampling techniques in which systematic and periodic scans of individuals and contextual factors in PA environments are made and they have been adapted for multiple settings [21]. Given SOPARC's proven ability to obtain reliable observational data, we sought to test a new strategy of using systematic observation to capture COVID-19 mitigation strategy adherence, including physical distancing and face coverings, among grade-school-aged children in diverse school settings: The Systematic Observation of COVID-19 Mitigation (SOCOM). Study Setting Four schools in Orange County, California, were recruited to participate in a Safe School Restart study to begin essential research on COVID-19 transmission in children and adolescents. The aim of Safe School Restart was to determine how effectively state and regional guidelines slowed viral transmission as schools restarted. Private schools across the country have been able to expend the resources necessary to develop and implement effective mitigation strategies. Public schools, particularly Title 1 public schools, have had greater difficulty in accessing necessary resources [29,30]. Consequently, our study includes four representative schools in Orange County, California: 1. Private K-12 school serving predominantly middle-and uppermiddle socioeconomic status (SES) students. 2. K-6 public school serving predominantly Latino, lower SES students. 3. K-8 public charter school located in a predominantly Latino, lower SES neighborhood. 4. K-6 public charter school serving predominantly children with special needs, including down syndrome, autism spectrum disorders, etc. The participating schools had varying levels of COVID-19 plans that were made in effort to meet the CDC guidelines that were in place during the study period. Plans included thoughtful and thorough screening, mask wearing, social distancing, hand hygiene, guest visitations, and testing procedures. All schools had some level of in-person learning with three schools permitting only a small proportion of students to attend in-person. SOCOM was designed to observe during four distinct school day sessions: classroom learning, active recess (regularly scheduled periods for PA and play that was monitored by trained staff or volunteers), instructional PE classes, and communal dining (lunch). Partnerships with Schools Schools were selected for Safe School Restart to reflect the wide range of SES, ethnic diversity, and school layouts and facilities that exist within Orange County, California, allowing for a determination of the viability of the SOCOM tool in different settings ( Table 1). Building on previous relationships, the schools were approached as colleagues and partners, an approach that facilitated approvals for the study by schools and school districts. Great care was taken to plan with school staff, so elements of the study were clear, acceptable, and followed current school policies established in response to the COVID-19 pandemic. SOCOM Development In the summer of 2020, the SOCOM protocol was developed to focus on assessing COVID-19 mitigation procedures (physicaldistancing and face-covering behaviors). Paper and electronic observation forms were developed to capture data (Figs. 1 and 2). The custom electronic data capture system optimized for mobile devices was developed by the Center for Biomedical Informatics, UC Irvine Institute for Clinical and Translational Science. The platform leverages the Bootstrap responsive user interface framework and was programmed in ASP.Net C#. In addition to the SOPARC-based data fields that research coordinators can enter manually based on their field observations, the system is also capable of obtaining local weather information automatically through an application programming interface (API) provided by openweathermap.org. All data captured are stored in a HIPAA-compatible environment hosted at the Enterprise Data Center of the UCI Health. Measures Primary measures included physical-distancing, face-covering behavior, PA levels, and activity type ( Table 2). In addition, observed individuals were categorized into sex/age groups (female students, male students, female adults, and male adults). Data were recorded to identify specific observation target area, date, grades in attendance, time of observation, and weather conditions. Training In preparation for SOCOM, observers (research assistants from the University of California, Irvine's Pediatric Exercise and Genomics Research Center) studied the written protocol and participated in a 4-hour workshop. Original written and video SOPARC training materials were used during the workshop (https:// activelivingresearch.org/soparc-system-observing-play-and-recreationcommunities). Training included reviewing definitions and coding conventions, differentiating among codes, coding practice, and orientation to the observation tools. Observer preparation also included target area mapping strategies. Coding conventions and how to differentiate among various PA levels included video lectures and practice with feedback from videotaped samples through original SOPARC training. Distinguishing between face coverings, physical distancing, and activity type definitions was taught using photographs and videos. Observers Camplain et al. practiced coding and received feedback on their scoring before engaging in field practice. Observation Preparation Maps of school grounds from Google Maps or school administrators were used to precisely identify outdoor target areas, spaces in which activities might occur and were small enough to accurately code people using them. The size, location, and boundaries for both indoor and outdoor target areas were identified and the sequential order for observing them established. As COVID-19 recommendations and restrictions varied among schools and time periods, visiting them was imperative before finalizing school-specific observation protocols and visitation days. Administrative-level school officials escorted observers around the school to note the general layout and major features of the facilities and to specifically identify potential target areas for observation. Physical activity/physical education class One grass location was available for physical activity/physical education classes. 14 K-8 public charter school Classroom learning Students had their own desks with a plastic U-shaped divider in one classroom. 12 Communal dining Lunch was provided in the classroom and students ate at their own desk. 12 Active recess No recess was available. 0 Physical activity/physical education class No physical activity/physical education classes were available. 0 K-6 special needs public charter school Classroom learning Students sat at their own desk placed 6 ft apart with plastic U-shaped dividers in one large classroom (30 × 15 feet). 10 Communal dining Students ate lunch at tables in a room (150 × 75 feet) in which siblings could sit close to one another while other students were on the opposite end of the lunch table from other students. In many cases, students did not sit 6 ft apart. 10 Active recess A large grass area and basketball court (150 × 75 feet) were available 10 Physical activity/physical education classes Classes were provided using online instruction (ZUMBA) inside a classroom. 10 Before official data collection began, all observers attended a field-based training at one of the schools using the developed protocol and data collection form to practice using the protocol and form. Observers used the schedule of observations like that for official observations but were able to discuss protocols and category classifications. Camplain et al. Recording Procedures To establish reliability during visits to target areas, two to three observers simultaneously completed observations either by using paper forms or individual cell phones ( Figs. 1 and 2). Timestamps and weather information were uploaded automatically into the electronic form. After recording the characteristics of a target area, the observers simultaneously completed area scans by making independent "visual sweeps" from left to right using the same pace. Separate scans were conducted for female students, male students, female adults, and male adults, and each characteristic (physical distancing, face covering, PA, and activity type) for a maximum of 16 scans per target area. For example, during the first scan for female students, observers recorded whether each was physically distanced, not physically distanced, or unknown. During the second scan, observers recorded data for each female student's face covering. For PE classes and active recesses, a third scan was performed to record the PA level of each female student as sedentary, walking, or vigorous. Finally, for female students, a scan was made to categorize each as being interactive or individual. This scanning procedure was then completed for male students, female adults, and male adults in the target area (if any). Observers then moved to the next target area. Occasionally (e.g., unusual large numbers of people in the area), target areas were subdivided into smaller sub-areas so more accurate measures could be obtained and data for these sub-areas were later summed to provide an overall measure. During groups of scans, observers also took qualitative notes on contextual information. When needed, observers used paper observation forms and later entered the information into the electronic data format. Weekly meetings were conducted with observers and research team to discuss challenges experienced during observations and to review the study protocol. Planned Data Analysis For physical-distancing, face-covering behavior, PA level, and activity type, counts will be tallied for those engaged in each group in each school and observation area to obtain a summary score for female students, male students, female adults, and male adults. A proportion of individuals in each physical-distancing scenario, face-covering behavior, PA level, and activity type will be calculated by age/sex group. Observations During a school visit, observation times varied by the number of target areas and people present and ranged from 2 min (in classrooms) to 15 min (in dining rooms). Communal areas generally had more people and more target areas and more scans were required. When large numbers of people were present (>20), some observers preferred the paper entry format compared to the electronic form as they could code more easily. Thus, areas with less activity and target areas with a maximum of 10-15 students were more favorable for accurately and confidently capturing data. The presence of observers may have influenced staff and student behavior, especially indoors. Schools had restrictions on visitors due to COVID-19 protocols and teachers sometimes inquired why observers were there. Meanwhile, when students were more physically active (e.g., PE classes) observers typically went unnoticed. Reliability Reliability data for PA, physical-distancing, and face-covering behaviors were collected during all observation periods using two assessors who made simultaneous, independent observations. The overall proportion of agreement from 166 scans was calculated for each variable (face covering, 88.6%; physical distancing, 90.9%; and activity type, 89.2%). When assessed by observation area, agreement was lowest during active recess and PE classes (79.7% to 99.1%). Agreement was lower when there were ≥20 students present (80.2%) compared to <20 students (90.2%). Reliability for PA level calculated for 34 observations (collected during active recess and PE classes) showed the proportion of agreement was 87.9%, similar to previous reliability studies using SOPARC [28]. Preliminary Results The SOCOM activity codes had been used in other observation systems [20,31,32], and with their construct validity being established via heart rate monitoring among 4-to 18-years-old children [31,33] and with accelerometers in schools [34]. Subsequently, we were able to compare PA results to previous studies. During recess in the three schools that provided recess, 25.8% of students were sedentary, 21.5% walked, and 52.8% engaged in vigorous activity. The proportion of students engaged in walking or Partialindividual had face covering on but either his/her nose or mouth was not covered 3. Not onindividual had no face covering on, but one was visible (e.g., mask in hand or hanging from ear) 4. Noneindividual had no visible face covering anywhere on body Physical activity levels 1. Sedentaryindividual was lying down, sitting, or standing in place 2. Walkingindividual was engaged in walking at normal pace 3. Vigorousindividual was engaged in an activity more vigorous than an ordinary walk (e.g., causing increased heart rate, such as when jogging, swinging, doing cartwheels). Activity type 1. Interactiveindividual was engaged in an interactive activity where he/she was using a shared piece of equipment or touched another person 2. Individualindividual was engaged in an independent activity with no shared equipment vigorous activity (74.2%) was higher than those in previous studies (51-68%) [20]. Overall during PE classes, 38.6% of students were sedentary, 25.0% were walking, and 36.4% were engaged in vigorous activity. Meanwhile, the PE class program differed substantially by school as did the resulting activity levels. Students in the K-6 special needs public charter school had PE within their classrooms and subsequently a high proportion of them were sedentary (87.5%) as students were still in the classroom. In the private and public schools providing formal PE classes, 34% of students were sedentary. Additional preliminary data on mitigation using SOCOM linked to SARS-CoV-2 infection in the four schools are also available [35]. Discussion Systematic observation has been widely used in collecting information on children's and adolescents' behaviors [20,21,28,31]. Therefore, we sought to test a new strategy of using systematic observation to capture COVID-19 mitigation strategy adherence, including physical distancing and face coverings, among gradeschool-aged children in the school environment. SOCOM may serve as a reliable and useful tool in assessing COVID-19 mitigation procedures in schools. To safely plan for in-person learning, K-12 schools must understand mitigation strategy adherence among their students and staff. Because physical-distancing and face-covering behaviors are two recognized methods to prevent the spread of COVID-19 [36], real-time response to poor adherence is an important component of safely re-opening and keeping K-12 schools open for in-person learning. Following data collection, a verbal summary of the results was provided to each school and formal presentations of the findings and additional COVD-19 findings from the Safe School Restart Study are in progress. SOCOM can be applied by schools to monitor compliance, adjust mitigation strategy messaging, and contribute to informed school policies. The current study was limited to observing only traditional classrooms, communal dining, active recess, and PE classes. Nonetheless, we believe the tool can be used in other school settings, such as music classes, laboratories, teachers' lounges, and hallways as well as when students arrive at and depart the school grounds. Additional research, however, is needed to assess the viability of using SOCOM in these areas and times. Interrater reliability profiles for SOCOM were comparable to those found with SOPLAY and SOPARC [28]. After observations, the research team discussed the effectiveness of the SOCOM protocol, observation form, and experience. In general, heuristic assessments of SOCOM was positive with certain limitations in using the electronic form, including the need for access to the internet on the devices used. Although there were limitations with concurrent electronic data entry, observers had access to the paper data collection forms and were able to later enter data into the electronic form. SOCOM worked well, even within the constraints of school environments during the pandemic. However, our study did not train school personnel as observers. Although we have not yet trained in-school personnel to collect data, we believe the high reliability, ease of training and use, and the ability to modify the instrument, SOCOM could be used by school administrators and staff to help assess school reopening. Interventions (e.g., policies) could subsequently be implemented to further protect students, teachers, families, and the local community. Due to the complex, novel nature of capturing COVID-19 mitigation strategy adherence, training and observation preparation were important. Training materials for SOCOM were obtained from existing valid and reliable methods. Although previous training materials focused mainly on capturing PA levels, we found that training to observe physical-distancing and face-covering behaviors was readily adaptable. Field-based observational training using the protocol in real-time were imperative. Our training included an initial visit to each school to familiarize observers with features of the school's physical environment and review details of the school's COVID-19 schedule, rules, and restrictions. Visits permitted the development of appropriate weekly and daily observation schedules for each school and was welcomed by school leadership and staff. Visits were necessary for positioning observers in areas where they could have a clear view and to minimize interactions with students and staff. However, a limitation of SOCOM is the potential for observation bias as individuals may be inclined to be on their best behavior while under observation. To decrease observation bias in the future, observers should be school personnel. Training teachers, staff, and administration to use SOCOM in schools can also provide schools with real-time feedback that may impact practices and policies for in-person learning. Conclusions Despite fears of possible surges of COVID-19 cases due to SARS-CoV-2 variants, millions of children and staff will be in schools under various conditions. Moreover, COVID-19 the vaccination of adults in the USA is proceeding, the vaccination of enough people to approach herd immunity has yet to be achieved. At the time of this writing, a vaccine has not been approved to be used among children <16 years old and no child under the age of 12 years has been enrolled in any safety or efficacy vaccine trials, thus the potential for widespread COVID-19 vaccinations in school-aged children to diminish the need for mitigation procedures in schools is many months away. Quantifying the success of SARS-CoV-2 transmission mitigation in school settings is likely to be useful for the foreseeable future. Standardized methods of measuring the fidelity of mitigation procedures will likely aid in identifying the most effective ways to minimize SARS-CoV-2 viral transmission. SOCOM is a relatively inexpensive tool that can be implemented by schools in various settings, including the school day, after school programs, and school sports and competitions, to determine mitigation strategy adherence to help students return to school safely and slow the spread of COVID-19.	2021-03-15T19:08:05.012Z	2021-03-15T00:00:00.000	{ "year": 2021, "sha1": "5327a8c93e8027ac7728c2bef3db19e0ed8fae84", "oa_license": "CCBY", "oa_url": "https://www.cambridge.org/core/services/aop-cambridge-core/content/view/E2056BEC608C9DE01383E8FFB0DFBE78/S205986612100786Xa.pdf/div-class-title-development-of-the-systematic-observation-of-covid-19-mitigation-socom-assessing-face-covering-and-distancing-in-schools-div.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "7ba46b7ddd5c30dd691927aa85fd1e49073bb497", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [ "Medicine", "Sociology" ] }
231886877	pes2o/s2orc	v3-fos-license	Integrative analysis of physiological responses to high fat feeding with diffusion tensor images and neurochemical profiles of the mouse brain Background Obesity proceeds with important physiological and microstructural alterations in the brain, but the precise relationships between the diet and feeding status, its physiological responses, and the observed neuroimaging repercussions, remain elusive. Here, we implemented a mouse model of high fat diet (HFD) feeding to explore specific associations between diet, feeding status, phenotypic and endocrine repercussions, and the resulting microstructural and metabolic alterations in the brain, as detected by diffusion tensor imaging (DTI) and neurochemical metabolic profiling. Methods Brain DTI images were acquired from adult male C57BL6/J mice after 6 weeks of HFD, or standard diet (SD) administrations, both under the fed, and overnight fasted conditions. Metabolomic profiles of the cortex (Ctx), hippocampus (Hipc), and hypothalamus (Hyp) were determined by 1H high-resolution magic angle spinning (HRMAS) spectroscopy, in cerebral biopsies dissected after microwave fixation. Mean diffusivity (MD), fractional anisotropy (FA) maps, and HRMAS profiles were complemented with determinations of phenotypic alterations and plasma levels of appetite-related hormones, measured by indirect calorimetry and multiplex assays, respectively. We used Z-score and alternating least squares scaling (ALSCAL) analysis to investigate specific associations between diet and feeding status, physiological, and imaging parameters. Results HFD induced significant increases in body weight and the plasma levels of glucose and fatty acids in the fed and fasted conditions, as well as higher cerebral MD (Ctx, Hipc, Hyp), FA (Hipc), and mobile saturated fatty acids resonances (Ctx, Hipc, Hyp). Z-score and ASLCAL analysis identified the precise associations between physiological and imaging variables. Conclusions The present study reveals that diet and feeding conditions elicit prominent effects on specific imaging and spectroscopic parameters of the mouse brain that can be associated to the alterations in phenotypic and endocrine variables. Together, present results disclose a neuro-inflammatory response to HFD, characterized primarily by vasogenic edema and compensatory responses in osmolyte concentrations. Introduction Obesity and overweight are thought to develop from unhealthy life style habits, combining the exacerbated consumption of diets rich in fats and sugars with sedentary behaviors [1]. These circumstances lead, ultimately, to impairments in the regulation of the cerebral mechanisms controlling global energy balance and addictive behavior [2], favoring a phletora of life-threatening comorbidities which have reached pandemic proportions worldwide [3,4]. Initial evidences suggested that obesity developed from endocrine alterations in perypheral signals, essentially insulin and leptin, controlling the balance between hypothalamic orexigenic and anorexigenic responses, mainly through the melanocortin pathway [5,6]. Briefly, long-term increases in the plasmatic levels of leptin or insulin, resulted in insulin or leptin resistance, paving the way to diabetes or fat accumulation [7]. More recently, dietary saturated fatty acids (SFA) have been shown to permeate through the blood-brain barrier (BBB) triggering an NFκB-orchestrated neuroinflamatory response in the hypothalamus, and other appetite regulating regions in the brain, that precedes weight gain [8][9][10]. Moreover, considerable evidence accumulated, revealing important morphological and metabolic alterations in the brain of obese individuals, as well as in animal models of obesity. These included in general, gray matter reductions [11] and cognitive impairment [12], as well as increases in tricaboxylic acid (TCA) cycle flux, anaplerosis, and GABA production [13,14]. Magnetic resonance imaging (MRI) methods have contributed considerable progress to the understanding of global energy metabolism in the brain, and its disturbances in vivo [15]. Briefly, diffusion weighted imaging (DWI) revealed alterations of brain microstructure in rodents and humans subjected to feeding/fasting paradigms [16][17][18][19]. In addition, 1 H high-resolution magic angle spinning (HRMAS) and in vivo 13 C magnetic resonance spectroscopy, unveiled considerable changes in the cerebral neurochemical profile in mice subjected to high fat diet (HFD), or increases in hypothalamic TCA cycle activity and GABAergic neurotransmission, in leptin-deficient Ob/Ob mice [14]. Diffusion tensor imaging (DTI) approaches have added, recently, considerable potential to evaluate white matter microstructural changes in obese humans, but their application to animal models of obesity, more specifically to mouse models, remained to be explored [20]. Animal models have played a fundamental role in the understanding of the physio-pathological mechanisms underlying obesity and the development of therapeutic interventions [21]. In particular, a large variety of genetic models, including leptin-deficient Ob/Ob mice, or obese Zucker rats, have been crucial to reveal some of the molecular and cellular signatures involved in obesity. However, obesity and overweight result most frequently, from pleiotropic polygenic interactions and environmental determinants such as the diet type, or the level of physical activity [1,22]. Diet-induced obesity models appear, from this perspective, more adequate to evaluate dynamically the effects of excessive caloric intake during obesity development [1,23]. Among these, the HFD model has been primarily used in rodents [24], presenting higher body weight gain than controls [25], increased glucose and insulin levels [26], and insulin and leptin resistance [27]. Moreover, HFD models allow to investigate feeding/fasting paradigms superimposed to the dietary manipulations, an acute nutritional intervention currently considered to promote activation of a critical neuro-inflammatory profile, that precedes weight gain [28]. However, despite the wealth of information accumulated in HFD rodent models, no studies to our knowledge, have addressed from an integral perspective, the plethora of specific associations occurring among diet and feeding conditions, its phenotypic or endocrine consequences, and the resulting alterations in cerebral imaging and metabolic parameters. On these grounds, we aimed here to contribute an integrative analysis of the relationships between physiopathological variables and the corresponding imaging and spectroscopic parameters in the brain of C57BL6/J mice subjected to HFD. Animals were euthanized with a high-power microwave fixation system focused in the brain (TMW-4012C 5 kW, Muromachi Kikai Co. Ltd., Japan). Fixed brains were then isolated from the skull, the hypothalamus (Hyp), hippocampus (Hipc), and temporal cortex (Ctx) dissected, and the samples kept at −80°C for subsequent HRMAS acquisitions. Design and reporting adhered to the ARRIVE guidelines [29]. Physiological characterization Phenotyping Animals were individually evaluated in a phenotyping system that monitored automatically food and water consumption, as well as spontaneous respiratory and motor activities [30]. Endocrine profile Leptin, insulin, ghrelin, PYY, glucagon, and GLP-1 concentrations were determined in plasma samples obtained from HFD or SD mice, under the fed or fasted conditions, using the MILLIPLEX MAP Mouse Metabolic Hormone Magnetic Bead Panel (MMHMAG-44K Multiplex-assay), using the Luminex ® platform (see Supplementary information). Diffusion tensor imaging Anesthetized animals underwent MRI acquisitions on a Bruker 7T BioSpec system (Bruker Biospin, Ettlingen, DE), after determining blood glucose levels with a standard glucometer (Accu-Chek ® Aviva, Roche) in samples extracted from the tail vein. MRI studies acquired T 2 morphological images to identify the section containing hypothalamus, and a set of diffusion weighted images were acquired using a DTI sequence [31], with diffusion gradients applied in six directions. Diffusion images were analyzed with a homemade software package by fitting, voxel by voxel, the diffusion signal decay to a mono-exponential model, yielding mean diffusivity (MD) and fractional anisotropy (FA) maps [32]. Regions of interest including Ctx, Hipc, and Hyp were manually selected from the parametric maps by using ImageJ software (National Institutes of Health, Bethesda, MD, USA, ImageJ) overlaid over a mouse brain atlas [16,19]. H high-resolution magic angle spinning spectroscopy Small biopsies of Hyp, Hipc, and Ctx were used to obtain 1 H HRMAS spectra using a Bruker AVANCE 11.7T equipped with a HRMAS accessory. Studies were processed and quantified with LCModel (linear combination of model spectra) [33], which fits the HRMAS spectrum as a linear combination of individual model spectra contained in a database of cerebral metabolites (see additional experimental details in Supplementary information). Statistics In all cases, data that did not achieve the quality requirements according to SNR in MR images and restriction coefficients in fitting processing, as well as outliers, were excluded from the statistical analyses. Univariate analysis Physiologic and phenotyping statistics were calculated using GraphPad Prism 6 (GraphPad Software, San Diego, CA). Body weight and blood glucose differences were evaluated, where indicated, with multiple unpaired t-tests or two-tailed paired t-tests. Generalized linear model Statistical analyses of MRI data, HRMAS metabolites, and blood biochemical parameters were performed using the IBM SPSS package (IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY, IBM Corp). Evaluations of MRI-derived parameters were performed using the generalized linear model and generalized estimating equations (GEE), which allows for the analysis of repeated measures [34]. Data from HRMAS and blood analyses were evaluated with a linear mixed model. In all cases, p values < 0.05 were considered statistically significant. Z-score evaluation Z-scores were calculated from the mean values of each variable (MRI parameters, relative concentration of brain metabolites from HRMAS, plasmatic hormone levels, and phenotypic data) from the four experimental conditions (SD-fed, SD-fasted, HFD-fed, HFD-fasted), respectively, regarding their respective sample mean between the four conditions. Four independent bar graphs, corresponding to four experimental conditions, contain the Z-scores from all individual variables, allowing to overview of the patterns of change among them. Alternating least squares scaling (ALSCAL) ALSCAL is a nonmetric multidimensional scaling method that finds the structure of a set of distance measures between objects or cases, by assigning observations to specific locations in a new conceptual space (usually two-dimensional), such that the distances between points in the new space match the given similarities/dissimilarities as closely as possible. In this study, it was firstly calculated the Euclidean distance between the Z-scores of the original variables (MD, FA, GABA, Glu, Gln, Glc, Myo, NAA, Cr, GPC + Cho, Lip09, Lip13, relation Lip13/09, glucagon, insulin, leptin, PYY, weight, glucose, drink, food, RER, DistK, and calories), as measured in the four experimental conditions (SD-fed, SD-fasted, HFD-fed, HFD-fasted). We then transformed the multidimensional Euclidean distance space into a new two-dimensional space, using a Euclidean model, yielding a conceptual representation of the relationships among all variables and the corresponding diet/ feeding conditions (additional information in Supplementary information). In this representation, two variables with similar behavior are depicted in the same area, while variables with distinct trends are separated by longer distances. Physiological and imaging characterization of the animal model Animals fed with HFD depicted larger increases in body weight than mice receiving SD (Fig. 1A). Notably, these differences became significant after the first week of diet diversification, reaching 10-14% higher values after the 6week period. Body weight values measured before MRI performance (Supplementary information, Table 1) were also significantly higher in HFD mice as compared to SD animals, both in the fed and fasting conditions (Fig. 1B). In both cohorts, overnight fasting caused a statistically significant decrease in body weight, more pronounced in the SD group (11%), than in HFD animals (6%). Blood glucose levels measured prior to the MRI acquisitions were significantly higher in the fed state of HFD animals (p < 0.05), and decreased upon fasting (p < 0.05) (Fig. 1C) (Supplementary information, Table 1). Mice evaluations in the phenotyping system did not reveal significant differences between either the diets, or the feeding conditions (Fig. 1D, F). However, the respiratory exchange ratio (RER) (VCO 2 /VO 2 ) of SD mice exhibited periodical oscillations around 1, with RER values being higher at night (active period) and lower during the day Fig. 1 Physiological characterization of the C57BL6/J mouse model receiving high fat, or standard diets. A Time course of changes in body weight along HFD (red), or SD (green) administrations; B box plots of body weights before imaging sessions under fed or fasted conditions; C box plots of blood glucose levels before the imaging sessions under fed or fasted conditions. In each box plot, the central bar indicates the median, and the bottom and top edges refer to the 25th and 75th percentiles, respectively. The upper and lower limits of the box extend to the most extreme data points not considered outliers, which are plotted individually using the "•" symbol. Time course (90 h) of phenotyping variables from mice subjected to HFD or SD diets in the Phenomaster ® system (D caloric intake; E water consumption; F locomotor activity (measured as distance walked), G RER values). * /# p < 0.05, ## p < 0.01, *** /### p < 0.001. (resting period) (Fig. 1G). Notably, food removal eliminated the above-mentioned RER fluctuations, decreasing RER values until almost 0.7. The circadian oscillations were not observed in the animals fed with HFD for 6 weeks, with RER values approaching 0.7, during the complete experimental period. Hormonal profiles determined in blood samples ( Fig. 2A-F) revealed that leptin levels were significantly higher in the HFD than SD groups both in the fed and fasted conditions ( Fig. 2A). The fed state showed significantly increased insulin concentrations in both diet groups (Fig. 2B). Ghrelin concentration showed a tendency to increase upon fasting in the SD group, but the opposite was observed in the HFD group, with SD-fasted values significantly lower than HFD-fasted ones (Fig. 2C). PYY levels decreased significantly upon fasting in both diet cohorts (Fig. 2D). On the contrary, glucagon increased upon fasting in both diet groups, with the increases in SD animals reaching significance (Fig. 2E). Finally, GLP-1 levels augmented significantly after overnight fasting in SD, but not in HFD animals (Fig. 2F). Figure 3 summarizes the effects of HFD or SD, and the corresponding fed or fasted conditions, on parametric maps of MD of the mouse brain. Figure 3A illustrates the anatomical localization of the slice selected and different cerebral structures investigated (Supplementary information). Although parametric maps show a shift to light blue colors in HFD animals, consistent with increased MD (Fig. 3B), comparisons of average MD values through the whole brain in the slice did not reach statistical significance (Supplementary information, Table 2). However, statistically significant differences were detected when different regions were considered. Particularly, the GEE statistical analysis revealed significant effects of diet (p = 0.002), region (p < 0.001), and the product diet × region × condition (p < 0.001) on the MD coefficients. Pairwise comparisons showed statistically remarkable differences between MD values of the two diet groups in the fed state, in the Ctx and Hipc regions. The representation of MD values in box plots (Fig. 3D) confirms the higher MD values in HFD animals, as compared to SD mice. Besides, MD of HFD-fed animals was significantly higher in Hipc than in Ctx and Hyp. However, pairwise comparisons between SD-fasted and HFD-fasted did not reveal significant differences (Fig. 3E), except a significant increase in HFD-fasted Hipc, as compared to HFD-fasted Ctx. Figure 3 also shows results from the assessment of FA, with representative cerebral maps from mice subjected to SD or HFD, under both feeding conditions (Fig. 3C). Comparisons of average FA values through the whole brain did not show significant differences ( Supplementary Fig. 2 Endocrine profiles in the plasma of C57BL6/J mice receiving high fat or standard diets, under fed or fasted conditions. Appetite-related hormones were determined (pg/ml) with multiplex assays in plasma samples, obtained and processed as indicated in Table 2). However, the statistical assessment of the FA coefficients revealed a significant effect of the product diet × region × condition (p = 0.005), while diet, region, and feeding condition individually did not induce significant FA changes. The corresponding box plots (Fig. 3F, G) revealed in general a tendency to increase FA in HFD animals, which became significant in the Hipc of fasted animals only. Figure 4 summarizes the neurochemical profiles from brain biopsies of hypothalamus, hippocampus, and cortex, under the different diets and feeding conditions investigated, as obtained by 1 H HRMAS spectroscopy (Supplementary Fig. 2). Statistical analysis revealed significant changes within diets, feeding status and anatomical regions, involving mainly GABA (Ctx and Hyp), Gln (Hyp), GPC + Cho (Hyp), NAA (Hipc and Hyp), Tau (Hyp), Glc (Ctx, Hipc, and Hyp), Glu (Ctx and Hyp), and Lip09 (Ctx and Hipc) concentrations (Fig. 4). More specifically, HFD feeding increased the relative concentrations of mobile lipid methyl (at 0.9 ppm, Lip09) groups in all cases, with more pronounced effects in the Ctx and Hyp under fasting conditions (Fig. 4A). The ratio between lipid resonances (Lip13/Lip09) remained constant with diet and feeding condition (not shown), revealing no significant change in the average length of the fatty acyl chains. Moreover, no resonances from the vinyl protons were detected in the 5-6 ppm region ( Supplementary Fig. 2), revealing that the Lip09 and Lip13 resonances correspond to SFA. Glucose (Glc) levels were significantly increased in HFD feeding mice as compared to standard diet, and decreased in all regions and both cohorts (Fig. 4B). Relative GABA concentration decreased significantly during fasting in the Ctx and Hyp of the HFD cohort, and in the Hyp of SD animals. In this region, also significant inter diet group differences were detected in both feeding conditions (Fig. 4C). Glutamine (Gln) (Fig. 4D) and glutamate (not shown) concentration decreased significantly during fasting in the HFD mice Hyp. Similarly, GPC + Cho experienced a significant decrease only in the hypothalamus of HFD animals (not shown). Taurine (Tau) concentration, which was similar among diet groups in the fed state, decreased significantly with fasting in the HFD hypothalamus, remaining remarkably lower than the corresponding fasted SD counterparts (Fig. 4E). Interestingly, NAA hypothalamic concentration was significantly different in the sated state, with higher concentrations being detected on HFD animals and NAA values increasing with fasting in the Hipc of HFD mice (Fig. 4F). Statistical analysis The Z-scores method normalized the investigated variables in terms of their standard deviations, identifying those that present similar, or opposite, responses to HFD or SD, or to feeding/fasting conditions (Fig. 5). Z-scores for MD, FA, and Lip09 were negative for SD mice but positive for HFD animals, with close to (−1), or to (+1) values, respectively, in an independent manner from the feeding condition. Thus, these imaging and spectroscopic variables provide adequate parameters to distinguish the cerebral effects of both diets, independently of the feeding status. On the contrary, Myo showed positive Z-scores for SD mice but negative for HFD animals, both under feeding or fasting conditions, offering additional criteria to identify the different diets administered, from the observed neurochemical profiles ex vivo. Interestingly, other variables changed the sign of corresponding Z-scores depending only on the feeding/fasting conditions, independently of the diet consumed. Such is the case for the plasma levels of glucose and insulin, which depicted high-positive Z-scores on the fed state, and negative values after 16 h fasting. This provides a measure of confidence on the performance of this methodology, confirming the dominant role of these variables in the discrimination between the feeding conditions. Additionally, Z-scores for glutamate, PYY, food, and calories were negative in the fasted measurements, but positive in the fed, or NAA and glucagon with positive values at fasting but negative at feeding. Gln or ghrelin (to a lesser extent) depicted a crossed-response trend of Z-scores, being negative for the SD-fed and HFD-fasted states, and positive in the HFD-fed and SD-fasted conditions. This reveals that the Z-score analysis is able to detect additional biomarkers defining the feeding or fasting conditions. ALSCAL analysis led to a new two-dimensional space representation integrating all investigated variables and their specific associations to the four possible combinations of diet and feeding conditions (Fig. 6). In this space, each variable is represented with respect to the others by the transformation of the Euclidean distance between each pair of variables. This makes possible to group those variables that are at close range, or represent better, the four diet/feeding conditions. In particular, glucagon, Lip13, and NAA defined a cluster that is more closely associated to the HFD-fasted situation, while glucose, insulin, and weight clustered closer to HFD-fed. Similarly, GABA, PYY, drink, RER, and GPC + Cho were more closely associated to SD-fed, while ghrelin and Gln lied to SD-fasted. Similar ALSCAL analyses were performed in Hyp, Hipc, and Ctx regions showing analogous general trends, but also some specific characteristics ( Supplementary Fig. 3). In particular, Glc, insulin, and glucose or weight and leptin small distance was maintained in all three structures. RER, drink, and PYY also presented adjacent positions in cortex, hippocampus, and hypothalamus. In the cortical region, food, Glu, and calories and the lipid ratio (Relation), Myo, Lip13, and Gln depicted close positions. In the hippocampus, MD, Lip09, and DistK; leptin, and weight; glutamate and calories; RER and ghrelin groupings exhibited small distances between their variables. Finally, hypothalamus presented close positions in the groups formed by Lip09, Lip13, and lipid relation; GLP and NAA; Tau and Gln; GPC + Cho and Glu; Myo and GABA. Discussion Physiological and neuroimaging characterization of the HFD mouse model HFD feeding increased significantly caloric intake and body weight, together with hyperglycemia, hyperinsulinemia, and hyperleptinemia under the fed condition, which decreased appreciably upon fasting. Together, these results revealed that the model used, reproduced the expected behavioral and endocrine adaptations [35][36][37][38][39][40][41][42], as well as the development of an insulin and leptin resistant phenotype, also observed in obese individuals [42]. Similarly, the feeding/ fasting paradigm resulted in phenotypic and endocrine responses similar to those described in earlier studies [43], providing a solid frame supporting further analyses. DTI provides information on the alterations of the mouse brain microstructure as revealed by the MD and FA parameters. Cerebral MD measurements represent the net balance of Brownian water movements through the extracellular and intracellular compartments of the brain [44]. In this sense, net increases in MD reveal a dominant contribution of fast diffusion movements through the extracellular space, normally associated to alterations of BBB integrity and vasogenic edema. On the contrary, net decreases in MD reflect predominance of slower intracellular diffusions, typically associated with neurocellular swelling episodes, or cytotoxic edema. Our results revealed that HFD induced net increases of MD in the Ctx and Hipc of fed C57BL6/J mice, a difference that was not maintained in the fasting state. Since fasting has been shown to decrease apparent diffusion coefficient (ADC) values in these regions [16,19], the reversal of increased MD in the cortex of HFD mice under fasting, reveals the effective cancellation of increased MD values derived from HFD, with the decreases in MD under the fasted condition. This compensatory response appears to be heterogeneous through the brain [19], providing only partial cancelation in the Hipc, where mild net increases in MD persist in the HFD-fasted condition. Notably, the absence of significant MD changes in the Hyp does not imply the lack of HFD effects in this structure but rather, an effective compensation of the opposite ADC changes induced by both diets/feeding conditions. Together, these results are consistent with previous works reporting increased diffusion coefficients and vasogenic edema during obesity, even under normal feeding regimes [20,45,46], while reveal for the first time to our knowledge, partial or total reversal of such increases after a fasting period. FA measurements provide information on the anisotropic orientation of neuronal tracts through the white and gray matters [46,47]. Present results reveal that HFD mildly increases the average anisotropic orientation of axons observed in the Ctx, Hipc, and Hyp of SD animals. In this sense, FA increases detected in all structures suggest that the corresponding neurocellular responses to HFD, result in small, albeit potentially important, changes in neuronal tract orientation in all structures investigated, reaching statistical significance only in the hippocampus of fasted animals. Our results are consistent with previous works reporting increased cerebral diffusion during obesity in humans [45] but reveal, for the first time, the response of the mouse brain to HFD under the fed or fasted conditions, as disclosed by MD and FA measurements. The absolute quantification of HRMAS spectra provide the most reliable information of changes in metabolite concentrations. When this is not achievable, it is crucial to deal with the potential effects of the experimental conditions on the normalization process and the metabolite used to that. In our case, we opted for a relative quantification once previous studies stated the nonexistence of alterations in the total creatine content either due to HFD feeding [18] or fasting state [48]. The neurochemical profiles detected by HRMAS closely reflected the effects of different diets and feeding conditions in the mouse brain. Notably, HFD resulted in increased SFA in all regions investigated, more pronounced under fasting. SFA are known to trigger a neuro-inflammatory response mediated by binding to Tolllike receptors and the activation of the NfκB transcription cascade in the hypothalamus [49], a crucial event underlying the complex edema responses of MD and FA discussed above [50]. Remarkably, HFD induced also similar responses to diet/feeding conditions in the osmolytes Tau [51], Gln [52], and NAA of the Hyp. These reactive changes in osmolyte concentration disclose a complex and heterogeneous neuro-inflammatory response to increased SFA in the Hyp and Hipc, with significantly smaller effects in the Ctx. Finally, HFD showed reductions in the GABA concentrations [53], more pronounced in the fasted state, disclosing an important role for GABA as a novel biomarker for the different diet/feeding conditions. Integrative analysis of neuroimaging and physiological variables Z-scores provide a convenient statistical method to investigate complex responses to a given perturbation in large collections of multiple interacting variables. Briefly, it reduces the complexity of the universe of variables by identifying those that respond similarly to the investigated diet/feeding condition. Z-scores data revealed that MD, FA, and lipid resonances responded to the perturbations together, as a group, suggesting that the changes in these imaging and spectroscopic variables follow a coordinated mechanism. Concerning feeding conditions, Z-scores for plasma insulin increased, as expected, under feeding and decreased after 16 h fasting, providing a measure of confidence on the ability of the Z-score method to identify correctly the variable changes, and confirming the dominant role of insulin in the discrimination between the feeding/ fasting conditions [37]. Similarly, Z-scores for glutamate, PYY, glucose, food consumed, and caloric intake decreased after fasting, disclosing that these variables may share, with insulin, a common response mechanism to the diet administered. On the contrary, NAA showed increased Z-scores in fasting state. Finally, ASLCAL analysis supported the Zscores results; identifying groups of variables more closely representing the SD-fed and HFD-fasted states as the most separated profiles. In particular, glucagon, NAA, and Lip13 grouped closer the HFD-Fasted condition, while GABA, PYY, GPC + Cho, drink, and RER grouped close to the SD-Fed, suggesting that these groups variables are those that define more adequately these conditions. Similarly, glucose, weight, and insulin, or ghrelin, and Gln, provided adequate biomarkers for HFD-fed, or SD-fasted situations, respectively. Concluding remarks In summary, we report that the cerebral imaging parameters MD and FA, and the neurochemical profile, including mainly SFA and osmolyte variations, provide adequate biomarkers to define the effects of HFD in the brain of adult C57BL6/J mice. In particular, these imaging and spectroscopic alterations unveil a characteristic neuroinflammatory response of the mouse brain to HFD feeding that can be modulated by fasting. Similarly, the variations in phenotypic or endocrine variables, as food and water consumption, or insulin, leptin, and glucagon, are found to provide suitable biomarkers for the feeding/fasting transition. Taken together, the present findings reveal that diet and feeding conditions elicit important effects in physiological parameters, imaging and metabolic profiles of the mouse brain, providing the specific integrative associations. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.	2021-02-12T15:27:58.444Z	2021-02-11T00:00:00.000	{ "year": 2021, "sha1": "dd1ecf6a9a9e3675510d81fe6621acb6d0a74c0d", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41366-021-00775-9.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "dd1ecf6a9a9e3675510d81fe6621acb6d0a74c0d", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
258339357	pes2o/s2orc	v3-fos-license	Dermoscopic Findings in Clinically Diagnosed Cases of Plantar Warts, Corns, and Calluses: A Cross-Sectional Study Introduction: With the naked eye, it can frequently be challenging to tell a plantar wart from a corn or callus. A non-invasive diagnostic method called dermoscopy allows for the inspection of morphological features that are not apparent to the unaided eye. This study aimed to examine the dermoscopic findings in pared and unpared cases of palmoplantar warts, corns, and calluses. Methods: Seventy patients who had palmoplantar warts, corns, and calluses were included in this study. A predesigned structured format was used to document the dermoscopic findings. Result: The majority of patients (51.4%) had warts followed by callus (28.6%) and corn (20%). On dermoscopic examination, all unpared and pared cases of warts had homogenous black/red dots. Translucent central core was present in 92.85% unpared and 100% pared lesions of corns. Homogenous opacity was present in 75% unpared and 100% pared cases of callus. There was no association between unpared and pared lesions (p>0.05). Conclusion: The accuracy of identifying various clinical types of cutaneous warts, calluses, and corns can be improved by dermoscopy without paring. Introduction In dermatology practice, papules and plaques that affect the palmar as well as plantar aspect of the hand and foot are frequently seen, which can make it difficult to carry out daily tasks. The most prevalent lesions are calluses, corns, and palmoplantar warts [1]. Warts can occasionally be difficult to distinguish from other skin conditions, such as calluses and corns. Dermoscopy is intended to help distinguish between similar skin lesions, such as palmoplantar warts, corns, and calluses rather than to replace other diagnostic techniques [2]. The distinction between calluses and corns, each of which displays uniform opacity or a translucent center, is also made easier by this technique [3]. In the past few years, dermoscopy has dramatically increased in popularity, and numerous lesions have been investigated. To illuminate a lesion's finer details, the device uses polarized light and magnification. Additionally, polarized light when used in a non-contact fashion can seep through the epidermis with minimal reflection, enabling the visualization of deeper structures. A link between macroscopic clinical dermatology and microscopic dermatopathology is created by dermoscopy which is a non-invasive, straightforward, and reasonably priced diagnostic procedure that allows imaging of morphological aspects that are not visible to the naked eye [4]. Due to paucity of studies on this topic in the literature, we focused on studying the dermoscopic findings in pared and unpared cases of palmoplantar warts, corns, and calluses. Materials And Methods After getting written informed consent, 70 patients visiting OPD of tertiary healthcare setting with a clinical diagnosis of palmoplantar warts, corns, or calluses were enrolled in the study. Prior to starting the research, it received approval from the institutional ethics committee. Children less than 12 years of age, pregnant and lactating females, and hypertensive and diabetic patients were excluded from the study. Sample size of 70 was calculated by the following formula: z 2 pq/d 2 , where z=1.96 with 95% confidence interval. Dermoscopic examinations of the patients were performed both before and after paring. Dermoscopic images were captured using Dino-Lite digital dermatoscope (New Taipei City, Taiwan: AnMo Electronics Corporation) with 10× magnification. All findings were entered in a Microsoft Excel sheet. The significance of variation in the frequency distribution of the data was determined using the chi-square test. Statistical significance was defined as a p-value of 0.05 or lower. Data were expressed as percentage and mean±SD and analyzed using unpaired or paired t-tests as applicable. SPSS version 21.0 for Windows (Armonk, NY: IBM Corp) was used to perform the statistical analysis. Results A total of 70 patients were recruited in our study. Mean age of patients with callus, corn, and wart was 42±11.85 years, 32.29±11.15 years, and 32.42±13.51 years, respectively. Overall, majority of patients were male (52.9%) pertaining to age group interval of 19-30 years (42.9%). Callus and corn cases were males in majority (32.4% and 21.6%, respectively), while warts cases had females in majority (57.6%). A majority of patients had warts (51.4%) and the rest were suffering from calluses (28.6%) and corns (20%). All patients of callus as well as corn had absent homogenous black/red dots in unpared and pared lesions, while they were present in 100% unpared and pared cases of warts (Figures 1a-1c). In all patients with calluses and corns, papilliform surfaces were absent in both unpared and pared lesions, while in 55.56% of unpared and 66.67% of pared cases of warts, papilliform surfaces were present. All patients of callus had intact skin lines in unpared and pared lesions. Skin lines were interrupted in 14.29% unpared and pared cases of corn and all lesions of warts. Majority of patients had absent red linear vessels in 100% unpared and pared cases of callus as well as corn, while presence in 16.67% unpared and 30.56% pared cases of warts. All patients with callus and wart had absent translucent central core in unpared and pared lesions, whereas it was present in 92.85% unpared and 100% pared lesions of corn (Figures 2a-2c). The majority of patients had homogenous opacity in 75% unpared and 100% pared cases of callus whereas it was absent in all unpared and pared lesions of corn as well as wart ( Discussion In dermatology practice, palmoplantar warts, corns, and calluses are frequently seen, which can make it difficult to carry out daily tasks. The three most typical plantar lesions are corns, calluses, and plantar warts [4]. Although they can typically be diagnosed clinically, there may be some doubt in some circumstances [5]. With the naked eye, it can occasionally be challenging to distinguish a plantar wart from a callus or corn. Differentiating them is likewise a difficult task [6]. In the past 10 years, dermoscopy has been more frequently used to assist in the identification of a variety of dermatological problems. It is being used more often to help with the diagnosis of different infectious dermatoses [7]. The structures that comprise the epidermis and dermal papilla can be seen during dermoscopy which makes the skin's top layer, the stratum corneum translucent. Dermoscopy can be utilized as an auxiliary tool for the confirmation of the diagnosis in doubtful instances, particularly when the patient refuses or is unable to undergo a histopathological investigation [8]. It can be used without running the danger of spreading infection, which is crucial when treating infections like warts [9]. There are few literature-based dermoscopic descriptions of the aforementioned conditions [10]. Here, we attempted to appraise the dermoscopic findings in pared and unpared cases of palmoplantar warts, corns, and calluses. In the present study, cases from age group interval of 19-30 years had maximum cases (42.9%). The mean age of patients diagnosed with callus, corn, and wart was 42±11.85 years, 32.29±11.15 years, and 32.42±13.51 years, respectively. Gender-wise evaluation appraised slight male sex predilection with majority of males (52.9%) and rest females (47.1%). Age and sex can both be thought of as proxies for unmeasured underlying causative factors, such as variations in men's and women's footwear and rising age-related prevalence of typical co-morbidities. In the study conducted by Al Rudaisat and Cheng, patients' average ages were 36±15.3 years. The mean age of instances of common warts was 51.3 years, and the mean ages of cases of palmar and plantar warts were 34.1±11.3 years and 29.5±5.7 years, respectively [11]. Aqil et al. also appraised the average age of 32.8 years (10-65) for the cases of warts [12]. These results were in consensus with our study data and strengthened our results. Dermoscopic characteristics of common warts were characterized by Lallas et al. as tightly clustered papillae with a central red dot or loop encircled by whitish haloes [13]. Dermoscopy of palmoplantar warts frequently shows numerous obvious hemorrhages inside a well-defined, yellowish papilliform surface where skin lines are broken, according to published reports. This pattern allows them to be distinguished from callus, which lacks blood spots and instead displays a center reddish to blue structure less pigmentation [14]. Furthermore, four distinct dermoscopic patterns-unspecific, fingerlike, mosaic, and knoblike patterns which can occur in a single wart were discovered in a study involving a significant number of individuals [15]. Corn had a yellow area, a whitish annular band, and a translucent inner center, according to dermoscopy by Ankad et al. It was typical to find dermatoglyphics preserved. The yellow or white halo and red dots were noticeably lacking. Under dermoscopy, the stratum corneum thickening manifests as a yellow region. Localized fibrosis is the cause of the central transparent core that serves as the nucleus of the corn. The white ring and prominent collagen thickening are interrelated which similar to the findings of previous reports [1,4]. The callus was also clarified by Ankad et al. under dermoscopy, which revealed a noticeable opaque yellowish region with retention of dermatoglyphics. There were observed focal white regions [1]. These results are consistent with Sonthalia et al's earlier description [16]. In contrast to plantar warts, there were no vascular components. Hyperkeratosis, orthokeratosis, and acanthosis caused the yellow spots. Focal white spots that resembled white scales were caused by focal parakeratosis. Plantar warts had vascular components, although corn and callus did not. It results from dermal papillae that extend upward and have dilated and thrombosed capillaries [1]. Even though plantar papule paring may show thrombosed vessels, the non-invasive evaluation of vessels for the distinction among different lesions and the confirmation of clinical diagnoses using a dermoscope is very useful. The therapeutic prognosis of a plantar wart is significantly influenced by these vascular patterns. The resolution of the lesion is shown by the removal of dotted vessels [9]. Conclusions The accuracy of identifying various clinical forms of cutaneous warts, as well as assistance in separating them from other similar skin lesions like calluses and corns may be enhanced by dermoscopy without paring. It can be a quick clinical diagnostic tool in distinguishing it from close differentials. Therefore, dermoscopy, as a non-invasive diagnostic aid, can annihilate the anxiety related to invasive procedures like skin biopsy. Although it is a prefatory and introductory study, we recommend more quantitative research to substantiate and corroborate the benefits of dermoscopy. Additional Information Disclosures Human subjects: Consent was obtained or waived by all participants in this study. NKP Salve Institute of Medical Sciences and Research Centre and Lata Mangeshkar Hospital, Nagpur, Maharashtra issued approval #20/2021. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.	2023-04-27T15:15:59.852Z	2023-04-01T00:00:00.000	{ "year": 2023, "sha1": "a9f0e154be3e4a0cc7043782e24538910eb20156", "oa_license": "CCBY", "oa_url": "https://assets.cureus.com/uploads/original_article/pdf/151471/20230425-17572-17jzjil.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8ac4edc7fb7804c80e5b26b69448f072653dcfcf", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
53683886	pes2o/s2orc	v3-fos-license	Uniform Equicontinuity for a family of Zero Order operators approaching the fractional Laplacian In this paper we consider a smooth bounded domain $\Omega \subset \R^N$ and a parametric family of radially symmetric kernels $K_\epsilon: \R^N \to \R_+$ such that, for each $\epsilon \in (0,1)$, its $L^1-$norm is finite but it blows up as $\epsilon \to 0$. Our aim is to establish an $\epsilon$ independent modulus of continuity in ${\Omega}$, for the solution $u_\epsilon$ of the homogeneous Dirichlet problem \begin{equation} \left \{ \begin{array}{rcll} - \I_\epsilon [u] \&=\&f \&\mbox{in} \ \Omega. \\ u \&=\&0 \&\mbox{in} \ \Omega^c, \end{array} \right . \end{equation} where $f \in C(\bar{\Omega})$ and the operator $\I_\epsilon$ has the form \begin{equation} \I_\epsilon[u](x) = \frac12\int \limits_{\R^N} [u(x + z) + u(x - z) - 2u(x)]K_\epsilon(z)dz \end{equation} and it approaches the fractional Laplacian as $\epsilon\to 0$. The modulus of continuity is obtained combining the comparison principle with the translation invariance of $\I_\epsilon$, constructing suitable barriers that allow to manage the discontinuities that the solution $u_\epsilon$ may have on $\partial \Omega$. Extensions of this result to fully non-linear elliptic and parabolic operators are also discussed. Introduction. Let Ω ⊂ R N be a bounded open domain with C 2 boundary, f ∈ C( Ω) and ǫ ∈ (0, 1).In this paper we are concerned on study of the Dirichlet problem where I ǫ is a nonlocal operator approaching the fractional Laplacian as ǫ approaches 0. We focus our attention on I ǫ with the form (1.3) where, for σ ∈ (0, 1) fixed, K ǫ is defined as Notice that for each ǫ ∈ (0, 1), K ǫ is integrable in R N with L 1 norm equal to Cǫ −2σ , where C > 0 is a constant depending only on N and σ.We point Date: May 16, 2014.out that operators with kernel in L 1 , like I ǫ , are known in the literature as zero order nonlocal operators. Operator I ǫ is a particular case of a broad class of nonlocal elliptic operators.In fact, given a positive measure µ satisfying the Lévy condition R N min{1, \|z\| 2 }µ(dz) < ∞, and, for each x ∈ R N and u : R N → R bounded and sufficiently smooth at x, the operator I µ [u](x) defined as (1.4) has been a subject of study in a huge variety of contexts such as potential theory ( [28]), probability ( [13,31]) and analysis ([32, 33, 3, 14, 15]).An interesting point of view of our problem comes from probability, since (1.4) represents the infinitesimal generator of a jump Lévy process, see Sato [31]. In our setting, the finiteness of the measure is associated with the so-called Compound Poisson Process.Dirichlet problems with the form of (1.1)-(1.2) arise in the context of exit time problems with trajectories driven by the jump Lévy process defined by K ǫ (z)dz, and the solution u ǫ represents the expected value of the associated cost functional, see [29]. We may start our discussion with a natural notion of solution to our problem (1.1)-(1.2):we say that a bounded function u : R N → R, continuous in Ω, is a solution of (1.1)-(1.2) if it satisfies (1.1) pointwise in Ω and u = 0 on Ω c .As we see in Section §2, this problem has a unique solution, more interestingly, through an example we will see that such a solution may not be continuous in R N , since a discontinuity may appear on the boundary of Ω. See Remark 2.4.This situation is in great contrast with the limit case ǫ = 0, where the kernel becomes K(z)dz = \|z\| −(N +2σ) dz and the associated nonlocal operator is the fractional Laplacian of order 2σ, denoted by −(−∆) σ , see [23].In this case, the corresponding Dirichlet problem becomes where C N,σ > 0 is a normalizing constant.In the context of the viscosity theory for nonlocal equations (see [3,32,33]), Barles, Chasseigne and Imbert [4] addressed a large variety of nonlocal elliptic problems including (1.5).In that paper, the authors proved the existence and uniqueness of a viscosity solution v ∈ C( Ω) of (1.5) satisfying v = 0 on ∂Ω that is, consequently, continuous when we regard it as a function on R N .This result is accomplished by the use of a nonlocal version of the notion of viscosity solution with generalized boundary conditions, see [21,2,8] for an introduction of this notion in the context of second-order equations. Additionally, fractional problems like (1.5) enjoy a regularizing effect as in the classical second-order case.Roughly speaking, for a right-hand side which is merely bounded, the solution v of (1.5) is locally Hölder continuous in Ω, see [34].In fact, we should mention here that interior Hölder regularity for more general fractional problems (for which (1.5) is a particular case) has been addressed by many authors, see for instance [4,5,9,14,15,16,34] and the classical book of Landkof [28], for a non-exhaustive list of references.The interior Hölder regularity is accomplished by well established elliptic techniques as the Harnack's inequality ( [14,10]) and the Ishii-Lions method ( [4,27]).In both cases, the nonintegrability of the kernel plays a key role.Hölder regularity for problems like (1.5) can be extended up to the boundary, as it is proved by Ros-Oton and Serra in [30], where a boundary Harnack's inequality is the key ingredient (see also [12]).Naturally, as a byproduct of these regularity results, compactness properties are available for certain families of solutions of fractional equations.For instance, the family {v η } of functions solving For zero order problems, regularizing effects as arising in fractional problems are no longer available (see [18]).In fact, the finiteness of the kernel of zero order operators turns into degenerate ellipticity for which Ishii-Lions method cannot be applied.Thus, "regularity results" for zero order problems like (1.1)-(1.2) are circumscribed to the heritage of the modulus of continuity of the right-hand side f to the solution u ǫ as it can be seen in [17].However, the modulus of continuity found in [17] depends strongly on the size of the L 1 norm of K ǫ , which explodes as ǫ → 0. A similar lack of stability as ǫ → 0 can be observed in the Harnack-type inequality results for nonlocal problems found by Coville in [20].Hence, none of the mentioned tools are adequate for getting compactness for the family of solutions {u ǫ } of problem (1.1)-(1.2),which is a paradoxical situation since, in the limit case, the solutions actually get higher regularity and stronger compactness control on its behavior. In view of the discussion given above, a natural mathematical question is if there exists a uniform modulus of continuity in Ω, for the family of solutions {u ǫ } to (1.1)-(1.2),and consequently compactness properties for it.In this direction, the main result of this paper is the following Theorem 1.1.Let Ω ⊂ R N a bounded domain with C 2 boundary and f ∈ C( Ω).For ǫ ∈ (0, 1), let u ǫ be a solution to problem (1.1)-(1.2).Then, there is a modulus of continuity m depending only on f , such that The proof of this theorem is obtained combining the translation invariance of I ǫ and comparison principle, constructing suitable barriers to manage the discontinuities that u ǫ may have on ∂Ω and to understand how they evolve as ǫ approaches zero, see Proposition 3.2. As a consequence of Theorem 1.1 we have the following corollary, that actually was our original motivation to study the problem. Corollary 1.2.Let u ǫ be the solution to equation (1.1), with f and Ω as in Theorem 4.1, and let u be the solution of the equation (1.5), then u ǫ → u in L ∞ ( Ω) as ǫ → 0. We mention here that the application of the half-relaxed limits method introduced by Barles and Perthame in [7] (see also [6,11,3]) allows to obtain in a very direct way locally uniform convergence in Ω in the above corollary.At this point we emphasize on the main contribution of this paper, which is the analysis of the boundary behavior of the family {u ǫ } of solutions to (1.1)-(1.2) coming from Theorem 1.1 and the subsequent global uniform convergence to the solution of (1.5). There are many possible extensions of Theorem 1.1, for example, it can be readily extended to problems with the form with {f ǫ } ⊂ C( Ω) having a common modulus of continuity independent of ǫ ∈ (0, 1).It can also be extended to fully nonlinear operators and to parabolic equations, as we discuss in Section §6.We could also consider different families of approximating zero order operators, but we do not pursue this direction.There are many other interesting lines of research that arises from this work.From the discussion given before Theorem 1.1, questions arises with respect to Harnack type inequalities and its relation with regularity and compactness properties of solutions, when ǫ → 0. Regarding operators I µ , where µ might be singular with respect to the Lebesgue measure, an interesting question that arises is if the main results of this article can be extended to this case.The paper is organized as follows: In Section §2 we establish the notion of pointwise solution and the comparison principle.Important estimates for the discontinuity of the solution at the boundary are given in Section §3, and the boundary equicontinuity result is presented in Section §4.The interior modulus of continuity is easily derived from the boundary equicontinuity, and therefore the proof of Theorem 1.1 is given in Section §5.Further related results are discussed in Section §6. 1.1.Notation.For x ∈ R N and r > 0, we denote B r (x) the ball centered at x with radius r and simply B r if x = 0.For a set U ⊂ R N , we denote by d U (x) the signed distance to the boundary, this is d U (x) = dist(x, ∂U ), with d U (x) ≤ 0 if x ∈ U c .Since many arguments in this paper concerns the set Ω, we write d Ω = d.We also define Concerning the regularity of the boundary of Ω, we assume it is at least C 2 , so the distance function d is a C 2 function in a neighborhood of ∂Ω. More precisely, there exists δ In our estimates we will denote by c i with i = 1, 2, ... positive constants appearing in our proofs, depending only on N, σ and Ω.When necessary we will make explicit the dependence on the parameters.The index will be reinitiated in each proof. Notion of Solution and Comparison Principle. In the introduction we defined a notion of solution to problem (1.1)-(1.2),which is very natural for zero order operators and allows us to understand the main features of the mathematical problem that we have at hand.However, this notion is not suitable for a neat statement of the comparison principle and it is not adequate to understand the limit as ǫ → 0. For this reason, from now on, we adopt another notion of solution which is more adequate, that is the notion of viscosity solution with generalized boundary condition defined by Barles, Chasseigne and Imbert in [4]. We remark that results provided in this section are adequate for problems slightly more general than our problem (1.1)-(1.2).We will consider J ∈ L 1 (R N ) a nonnegative function, and we define the nonlocal operator associated to J as (2.1) for u ∈ L ∞ (R N ) and x ∈ R N , and a Dirichlet problem of the form with f ∈ C( Ω).Since we are interested in a Dirichlet problem for which the exterior data plays a role, we assume J and Ω satisfy the condition (2.4) inf In this situation, a bounded function u : R N → R, continuous in Ω is a viscosity solution with generalized boundary condition to problem (2. 2)-(2.3) if and only if it satisfies The sufficient condition is direct from the definition and the necessary condition follows from the lemma: where the above inequality is understood pointwise.Let x 0 ∈ ∂Ω and assume there exists a sequence {x k } ⊂ Ω such that Here and in what follows the considered measure is the Lebesgue measure. Proof.Consider {x k } ⊂ Ω as in (2.8).Then, we can write Hence, taking limsup in both sides of the last inequality, by (2.8) and the continuity of f , we arrive to where the exchange of the integral and the limit is justified by Fatou's Lemma.Then, using (2.9), we conclude the result. We continue with our analysis with an existence result for (2.2)-(2.3). Proof.According with our discussion above, we need to find a solution to (2.5)-(2.6).Consider the map T a : C( Ω) → C( Ω) defined as We observe that u ∈ C( Ω) is a fixed points of T a if and only if u is a solution to problem (2.5)-(2.6).Therefore, the aim is to prove that for certain a > 0 small enough, the map T a is a contraction in C( Ω).By (2.4), there exists Then, for all x ∈ Ω we have that is, T a is a contraction in C( Ω).From here existence and uniqueness follow. Remark 2.3.We observe that u : R N → R, a viscosity solution with generalized boundary condition to problem (2.2)-(2.3),may be redefined on the boundary ∂Ω as u = 0, to obtain a solution to (2.2)-(2.3) in the sense defined in the introduction. Remark 2.4.Let u be a solution of (2.2)-(2.3) in the sense defined in the introduction, with f ≥ ̺ 0 > 0. Our purpose is to show that u has a discontinuity on the boundary of Ω.Let us assume, for contradiction, that u : R N → R is a continuous function. Then u ≥ 0 in Ω, otherwise there exists x 0 ∈ Ω such that u(x 0 ) = minΩ{u} < 0 and evaluating the equation at x 0 we arrive to which is a contradiction to (2.4).Then, from the equation, we have for each x ∈ Ω the inequality Since u and f are continuous and u = 0 on ∂Ω and using that u ≥ 0 in Ω, we obtain that, for each x ∈ ∂Ω which is a contradiction.Thus, u > 0 on ∂Ω which implies that u is discontinuous on ∂Ω. In what follows we prove that a solution to (2.2)-(2.3), in the sense defined in the introduction, can be extended continuously to Ω. Proof: By contradiction, assume the existence of a point in Ω where u is different from v. Defining w = u − v, we will assume that (2.10) since the case inf Ω {w} < 0 follows the same lines.Moreover, we assume that the supremum defining M is not attained, since this is the most difficult scenario.Let η > 0 and let where Ω η was defined at the end of the introduction.We clearly have w(x η ) → M as η → 0 and since we assume M is not attained, then x η → ∂Ω as η → 0. Now, using the equations for u and v at x η ∈ Ω, we can write and by (2.4) and the fact that w(x η ) → M as η → 0, we have (2.11) where o η (1) → 0 as η → 0. But writing by the boundedness of w and the integrability of J, the second integral term in the right-hand side of the last equality is o η (1), meanwhile, using the definition of x η we have the first integral is nonpositive.Thus, we conclude and replacing this into (2.11),we arrive to By making η → 0, we see that this contradicts (2.10), since ν 0 > 0. As a consequence of the last proposition, we have the following 3) in the sense defined in the introduction.Moreover, v is uniformly continuous in Ω and its unique continuous extension to Ω coincides with the unique viscosity solution to (2.2)-(2.3). The main tool in this paper is the comparison principle, and here the so-called strong comparison principle is the appropriate version to deal with discontinuities at the boundary. R be bounded, upper and lower semicontinuous functions on Ω, respectively.Assume u and v satisfy (2.12) Proof: Assume by contradiction that there exists x 0 ∈ Ω such that Evaluating inequalities in (2.12) at x 0 and substracting them, denoting w = u − v, we arrive to and therefore, using that x 0 is a maximum point for w in Ω and that w ≤ 0 in Ω c , we can write w(x 0 ) and using (2.4) we arrive to a contradiction with the fact that w(x 0 ) > 0. As a first consequence of this comparison principle, we obtain an a priori L ∞ ( Ω) estimate for the solutions u ǫ of (1.1)-(1.2),independent of ǫ. Estimates of the Boundary Discontinuity. The aim of this section is to estimate the discontinuity jump on ∂Ω of the solution u ǫ of (1.1)-(1.2).For this purpose, a flattening procedure on the boundary is required. Recall that δ 0 > 0 is such that the distance function to ∂Ω is smooth in Ω δ 0 , and for x ∈ Ω δ 0 we denote x the unique point on ∂Ω such that d Ω (x) = \|x − x\|.We can fix δ 0 small in order to have the existence of three constants R 0 , r 0 , r ′ 0 > 0 depending only on the regularity of the boundary, satisfying the following properties: (ii) If we define the function Φ x as where ν ξ ′ is the unit inward normal to ∂(Ω − x) at (ξ ′ , ϕ x (ξ ′ )) and denoting Notice that Φ x (0 ′ , 0) is the origin and therefore R x is an R N -neighborhood of the origin. (iii) The constant r 0 > 0 is such that B r 0 ⊂ R x for all x ∈ Ω δ 0 . (iv We may assume 0 < r ′ 0 ≤ r 0 ≤ δ 0 .In addition, by the smoothness of the boundary there exists a constant C Ω > 1 such that The following Lemma is the key technical result of this paper Lemma 3.1.Let Ω ⊂ R N be a bounded smooth domain and ǫ ∈ (0, 1).For β ∈ (0, 1), consider the function where d = d Ω is the distance function to ∂Ω.Then, there exists δ ∈ (0, δ 0 ), β 0 ∈ (0, min{1, 2σ}) and a constant c * > 0, depending only on Ω, N and σ, such that, for all β ≤ β 0 we have Proof: We start considering δ < r ′ 0 and x ∈ Ωδ.We split the integral where In what follows we estimate each I i (x), i = 0, 1, 2, 3. Since ψ is bounded in R N independent of ǫ, β when ǫ, β ∈ (0, 1), we have where c 1 > 0 depends only on Ω and N .For I 1 (x), by the symmetry of K ǫ we have Then we consider the function θ(z) = ψ(x + z) + ψ(x − z) − 2ψ(x), which is smooth in Bd(x)/2 and therefore, we can write by Taylor expansion where z, z ∈ B d(x)/2 .With this, since we assume β < 1, by the smoothness of the distance function d inherited by the smoothness of ∂Ω we have where c 2 = C Ω β > 0 depends on the domain, but not on ǫ or d(x).From this, we get and since K ǫ (z) ≤ K 0 (z), we conclude that, for a constant c 3 > 0, we have Now we address the estimates of I 2 (x) and I 3 (x).For I 2 (x), recalling the change of variables Φ x , we have With this, using the change of variables Φ x and applying (3.2), we have But there exists a constant c 4 > 0, depending only on N and σ, such that and with this, defining ρ(ǫ, s) = (ǫ 1+2σ + \|s\| 1+2σ ) 1/(1+2σ) , we can write Finally, making the change t = −s/(ǫ + d(x)), we conclude (3.7) where τ = ǫ/(ǫ + d(x)) ∈ (0, 1).At this point, taking δ small in order to have ǫ + d(x) < 2/r ′ 0 , we find that the interval has at least lenght 1.Hence, we conclude the existence of c 7 > 0, depending only on Ω, N and σ, such that It remains to estimate I 3 (x).Defining D + (x) = {z : d(x + z) ≥ d(x)}, we clearly have and since we have Thus, making a change of variables we have Since Φ x is a diffeomorphism, there exists a constant c 8 > 0 such that and a constant λ ∈ (0, 1) small, depending only on the smoothness of ∂Ω, such that B λd(x) ⊂ Φ −1 x (B d(x)/2 ).Using this and (3.2) we arrive to At this point, we remark that for each M > 2, we have where c 9 > 0 depends only on N, σ and Ω.On the other hand, for each M > 2 there exists β = β(M ) > 0 small such that where c 7 > 0 is the constant arising in (3.8).From the last two estimates, we conclude that for each M > 2, there exists β small such that (3.9) where c 10 > 0 depends only on N, σ and Ω. Putting together (3.5), (3.6), (3.8) and (3.9), and fixing M = max{2, r 0 }, we have where c 11 > 0 depends only on N, σ and Ω.Hence, fixing β > 0 smaller if it is necessary, we can write Finally, taking ǫ+d(x) small in terms of c 7 , c 11 , r 0 , β and σ (and therefore, depending only on N, σ and Ω), we conclude (3.4), where c * = c 7 /8. Proof: Let β 0 as in Lemma 3.1, ψ = ψ β 0 as in (3.3) and consider the function Observing that ζ = ψ in Ω d 0 ∪ Ω c and ψ ≥ ζ in R N , we easily conclude that , for all x ∈ Ωd 0 , and using Lemma 3.1 we get Let C > 0 be the constant in Proposition 2.8 and define the function z+ = (Cd By construction of z+ , we have and z+ ≥ u ǫ in Ωc d 0 , and therefore, applying the comparison principle, we conclude u ǫ ≤ z+ in Ωd 0 .Similarly, we can conclude the function z− = −z + satisfies z− ≤ u ǫ in Ωd 0 , from which we get the result. Boundary Equicontinuity. In this section we establish the boundary equicontinuity of the family of solutions {u ǫ } ǫ∈(0,1) of problem (1.1)-(1.2).The main result of this section is the following The idea of the proof is based on the fact w(x) = u(x + y) − u(x), where y is fixed, satisfies an equation (near the boundary) for which the comparison principle holds.Using this, we get the result constructing a barrier to this problem, independent of ǫ and associated to m in Theorem 4. 1.In what follows we discuss the precise elements on the proof.We consider y ∈ R N with 0 < \|y\| < δ/2, with δ as in Lemma 3.1.Define the sets Notice that w ≡ 0 in R N \ ( Ō ∪ U ) and, by Proposition 3.2, there exists C 0 , β 0 > 0 such that \|w(x)\| ≤ C 0 (ǫ + \|y\|) β 0 for all x ∈ U .Since we have that w satisfies denoting by m f the modulus of continuity of f , we conclude that w ∈ C( Ō) satisfies the inequality (4.2) and the exterior inequality Let ζ and η the functions defined as and consider the function where A > 0 and m is a modulus of continuity satisfying m(\|y\|) ≥ m f (\|y\|).We have the following Proposition 4.2.There exists A > 0 large, depending on Ω, N and σ, such that for all ǫ ∈ (0, δ), with δ given in Lemma 3.1. Proof: Without loss of generality we may assume the existence of a number 0 < α < min{1, β 0 } and a constant c 1 such that m(t) ≥ c 1 t α , for all t ≥ 0. (4.5)By linearity of I ǫ , we have Thus, we may estimate each term in the right-hand side separately. Then, applying Lemma 3.1, for all ǫ small we have for some c * > 0 not depending on d(x), \|y\| or ǫ.In fact, for all ǫ ∈ (0, 1) the term (ǫ + d(x) − \|y\|) −ǫ is bounded below by a strictly positive constant, independent of ǫ, driving us to (4.6) On the other hand, when x ∈ Ω \ Ωδ, for all ǫ ∈ (0, 1) we have and therefore, there exists c 2 > 0, not depending on ǫ, d(x) or \|y\|, such that Since \|y\| ≤ δ/2, making c * smaller if necessary, the last inequality and (4. We start considering the case x ∈ Ω \ Ωδ, where we have dist(x, U ) ≥ δ/2 and then, there exists a constant c 3 > 0 depending only on δ (which in turn depends only on the smoothness of the domain), such that Using this, we have By the boundedness of Ω, there exists c 4 > 0 depending only on N such that Vol(U − x) ≤ c 4 \|y\|.Using this and (4.5), we conclude that (4.9) where c 5 > 0 depends only on N, σ and Ω.Now we deal with the case x ∈ O ∩ Ωδ (notice that in this case we are assuming d Ω (x) > \|y\|).Using (4.8) and recalling the change of variables Φ x introduced in (3.1), we can write where R x was defined at the beginning of Section §3.Using a similar analysis as the one leading to (4.9), there exists a universal constant c 6 > 0 such that and therefore, applying the change of variables Φ x and the estimate (3.2), we arrive to and from this, using a similar argument as the one leading to (3.7) to treat the last integral term, and applying (4.5), we conclude that (4.10) Now, the core of this estimate is the computation of the last integral.Denoting we claim the existence of a constant c 9 > 0 not depending on ǫ, d(x) or \|y\| such that (4.11) We get this estimate considering various cases.When \|y\| ≤ ǫ and d(x)−\|y\| ≤ 2ǫ we write and using that m(\|y\|) ≥ \|y\| α for some α ∈ (0, β 0 ), we have and from this, we conclude (4.12) for some constant c 10 > 0. for some constant c 1 > 0. Since a similar lower bound can be stated, by the arbitrariness of y we conclude the result with m 0 = c 1 Am. Proof of Theorem 1.1. Consider δ as in Lemma 3.1, let y ∈ R N such that \|y\| ≤ δ/8 and consider the sets Notice that Σ 4 ⊂ Σ 2 ⊂ Σ 1 .In addition, notice that if z ∈ Σ 3 , then z + y and z cannot be simultaneously in Ω.We also have where A > 0 is a constant to be fixed later.Notice that for each x ∈ Σ3 , we have (5.1) At this point, we remark that there exists a constant c 1 > 0, independent of ǫ, y or x, such that On the other hand, since dist(x, 3 ) ≥ δ/2 we have K ǫ (z)1 Σ 3 −x ≤ c 2 for some constant c 2 > 0, and by the boundedness of Ω, Vol(Σ 3 − x) ≤ c 3 \|y\| for some c 3 > 0. Using these facts on (5.1) and applying (4.5), we arrive to Thus, taking A large in terms of c 1 , c 4 , we conclude that − By the very definition of w, we have Then we have that −I ǫ [Z] ≥ −I ǫ [w] in Σ4 and by definition of W and the bounds of w in Σc 4 stated above, we conclude that w ≤ W in Σc 4 .Using the comparison principle, we conclude w ≤ W in Σ4 .A similar argument states the inequality −W ≤ w and the result follows. 6.1.Fully Nonlinear Equations.The result obtained in Theorem 4.1 can be readily extended to a certain class of fully nonlinear equations.For example, consider two sets of indices A, B and a two parameter family of radial continuous functions a αβ : R N → R satisfying the uniform ellipticity condition and with this, for a suitable function u and x ∈ R N , define the linear operators and the corresponding Isaacs Operator Under these definitions, we may consider the corresponding nonlinear equation Existence and uniqueness of a pointwise solution u ǫ to (6.2), which is continuous in Ω can be obtained in a very similar way as in the linear case, and Proposition 2.1 can be adapted to this nonlinear setting.This allows us to use the comparison principle stated in Proposition 2.7 as well. The lack of linearity can be handled with the positive homogeneity of these operators and the so called extremal operators since, for two functions u 1 , u 2 and x ∈ R N , these operators satisfy the fundamental inequality A priori estimates for the solution as it is stated in Proposition 3.2 can be found using the same barriers given in the proof of that proposition, as the following useful estimates hold: For each Using these inequalities and (6.1), we can use the same barriers appearing in the proof of Theorem 1.1 (Theorem 4.1 included) and get similar result.Moreover, the same modulus of continuity for the linear case can be obtained in this nonlinear framework, up to a factor depending on λ 1 and λ 2 . Similar problem is adressed by the authors in [24] for the Cauchy problem in all R N .Inspired by techniques used by Ishii in [26], a modulus of continuity in time can be derived once a modulus of continuity in space is found.So, the key fact is the modulus in x and this can be obtained in the parabolic setting noting that Theorem 4.1 readily applies considering equations with the form λu − I ǫ [u] = f in Ω for λ > 0 and that each time Z(x) is a suitable barrier for this problem, then the function (x, t) → e t Z(x) plays the role of a barrier for the evolution problem (6.3).6.3.Convergence Issues.The proof of Corollary 1.2 is standard in the viscosity sense, once the uniform convergence is stated.However, following the ideas of Cortázar, Elgueta and Rossi in [19], and also in [24], under stronger assumptions over the regularity of u in Corollary 1.2, we can find a rate of convergence.Since we know that \|w\| ≤ Cǫ β 0 on ∂Ω, by Proposition 3.2, we can get the result proceeding exactly as in the proof of Proposition 2.8.6.4.An example of a scheme without boundary equicontinuity.In this subsection we consider the reverse scheme, that is approximating zero order equations by fractional ones and we prove the absence of uniform modulus of continuity in Ω.For this, we recall some facts of Section §2.Let f ∈ C( Ω) with f ≥ ̺ 0 > 0, J : R N → R + integrable and I J as in (2.1).Consider the associated problem (2.2)-(1.2),that is in Ω c (6.4) As we saw in Remark 2.4, the unique solution u ∈ C( Ω) for this problem is such that u > 0 in ∂Ω. Consider J, f as above, with J such that J ≥ m in B r , for some r, m > 0. For ǫ ∈ (0, 1) and α > 1 consider the family of kernels in Ω u = 0, in Ω c (6.5) it is known that the unique viscosity solution u ǫ of (6.5) agrees the prescribed value of the equation on the boundary, and then u ǫ = 0 on ∂Ω for all ǫ ∈ (0, 1), see for example [4].We have {u ǫ } is uniformly bounded in L ∞ ( Ω) and therefore, the application of half-relaxed limits together with viscosity stability results in [3], imply u ǫ → u locally uniform in Ω as ǫ → 0, where u is the unique solution to (6.4).Since u is strictly positive in ∂Ω, the convergence of u ǫ to u cannot be uniform in Ω, and therefore the family {u ǫ } is not equicontinuous in this case. In this case, the family (u ǫ ) is not equicontinuous too, see [1]. 2 D for all h : D → R bounded nonnegative function, and D hK αβ,ǫ (z)dz ≤ λ hK ǫ (z)dz for all h : D → R bounded nonpositive function. 6. 2 . Parabolic Equations.Let T > 0, f : Ω × [0, T ] → R be a continuous function.A result similar to Theorem 4.1 can be readily obtained for the parabolic nonlocal equation 	2014-05-15T06:13:26.000Z	2014-05-13T00:00:00.000	{ "year": 2014, "sha1": "4d9069a8c20e81499d7cbeef9e76fe40edf17e5c", "oa_license": null, "oa_url": "https://arxiv.org/pdf/1405.3261", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "4d9069a8c20e81499d7cbeef9e76fe40edf17e5c", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics", "Physics" ] }
55163863	pes2o/s2orc	v3-fos-license	Experimental analysis of system parameters for minimum cutting fluid consumption when machining Ti-6Al-4V using a novel supply system This paper presents the development of a new controlled cutting fluid impinging supply system (Cut-list) to deliver an accurate quantity of cutting fluid into machining zones through precisely oriented coherent round nozzles. The performance of the new system was evaluated against a conventional system during the step shoulder milling of Ti-6Al-4V using a water-miscible vegetable oil-based cutting fluid, which was phase 1 in this comprehensive study. The use of Cut-list resulted in a significant reduction up to 42% in cutting fluid consumption as well as reductions in cutting force, tool flank wear, average surface roughness (Ra) and burr height (Gariani et al. in Appl Sci 7(6):560, 2017). This paper details phase 2 of the study which was aimed to investigate the effects of working conditions, nozzle positions/angles and impinging distances on key process measures including cutting forces, workpiece temperature, tool wear, burr formation and average surface roughness of the machined surface. Feed rate showed a significant effect on mean values of cutting force, burr formation and surface roughness, whereas average workpiece temperature and flank wear values are very sensitive to cutting speed. Nozzle position at a 15° angle in the feed direction and 45°/60° against feed direction assisted in minimising workpiece temperature. An impinging distance of 55/75 mm is also necessary to control burr formation, workpiece temperature and average surface roughness. It can be concluded that Cut-list gave promising results compared to conventional flood cooling systems in terms of the evaluated machining outputs. Therefore, the new system can be considered as a feasible, efficient and ecologically beneficial solution, giving less fluid consumption in machining processes. Introduction Historically, manufacturing by machining has prospered from the use of cutting fluids. Cutting fluids are supplied to the machining zone in order to improve machining performance. Cooling and lubrication are the key functions of cutting fluids. Additionally, they transport chips away from the machining zone, minimise the built-up edge (BUE) and protect machined components and machine tool parts from corrosion [1,2]. It is reported that the costs related to cutting fluids are approximately 16.9% of the total manufacturing costs, compared to tooling costs which represent about 7.5% [3]. This cost can be increased to up to 30% for machining refractory materials such as titanium and nickel-based alloys [4]. It is also estimated that the annual consumption of cutting fluids is about 100 million gallons in the USA [5]. A more effective cutting fluid supply method will significantly contribute to reducing fluid consumption and improving cutting performance. Currently, conventional flood cooling is the most common method used on machine shop floors, in which a large quantity of cutting fluid (e.g. up to 225 l/min for multiple teeth tool cutters) is continuously supplied into the machining zone [6]. However, high fluid consumption and low penetration ability, particularly at high cutting speeds, are the main disadvantages of this technique [7,8]. High fluid consumption results in increasing disposal and maintenance costs, particularly when mineral/ petroleum oil-based fluids are used. These types of fluids are claimed to cause human and environmental hazards due to their high content of toxic elements such as chemical agents, hydrocarbons and extreme pressure (EP) additives [9,10]. Between 13 and 32% of cutting fluids used in the EU and USA are disposed of without treatment [11]. Thus, additional processing for fluid waste (e.g. oil/water separation, ultrafiltration, conditioning and incineration) prior to disposal is always required [5,10]. In order to address these issues, minimum quantity lubrication (MQL) and minimum quantity cooling lubrication (MQCL) have been introduced to minimise fluid consumption in the machining operations. Fluid is atomised by missing with compressed air and is supplied as an aerosol. The cutting fluid in MQL and MQCL reaches the tool-workpiece interface through capillarity forms a film at the interface. This helps in lubrication and reducing contact pressure. Good penetrability of MQL and MQCL was also assisted to minimise cutting temperature in machining zone (particularly MQCL owing to its variant properties such as cooling compared to MQL), resulting in a reduction of tool wear and built-up edge formation. Improved surface integrity and desirable chip shape were also obtained when MQL and MQCL are used compared to dry and conventional flood cooling conditions [12][13][14][15]. Although MQL and MQCL are often used with biodegradable lubricants, resulting fumes by these systems particularly at higher cutting speeds is the main limitation [16]. High-pressure cooling (HPC), cryogenic cooling, oil/water mist and compressed air/gas/vapour supply systems have also been employed as alternative methods instead of conventional flood cooling supply systems to reduce fluid consumption and mitigate the hazards of using mineral oil-based fluids [17,18]. However, the high cost of the consumables involved (e.g. liquid nitrogen and gases), pumping and micro-particle filtration equipment are substantial limitations [19][20][21][22][23]. Additionally, the inaccurate estimation of cutting fluid flow rates during machining operations is also a clear disadvantage. In the same vein, vegetable oil (VO)-based cutting fluids have been introduced as potential alternatives to conventional fluid counterparts due to their high biodegradability (i.e. 100% within 28 days) [24] and superior physical and chemical properties [25,26]. The homogeneity and super-density of vegetable oil molecules can also help to form a thick, durable and strong film layer that affords VOs the ability to withstand high contact pressure [27,28]. Vegetable oil-based stocks have a high kinematic viscosity (e.g. 40.05 cSt at 40°C for sunflower oil) and high heat conductivity of up to 0.17 W/m.K compared to 20.06 cSt at 40°C and 0.12 W/m K for mineral oil-based fluids [29,30]. Thus, it is estimated that the global demand for biodegradable fluids is expected to increase by around 58% or 0.3 million tonnes in 2018 compared to 2011 [17]. Nozzle placement plays a crucial role, particularly in supply methods using less fluid consumption (e.g. MQL and oil/water mist). The impact of nozzle position on the turning of the AISI 1045 at an angle of 20°and 50°has been investigated [31] using MQL with palm oils and water/mist. Tests were performed at a cutting speed of 125 m/min, feed rate of 0.2 mm/rev and 0.2 mm depth of cut and 1000 mm cutting length. The results showed that nozzle placement at 20°produced lower tool flank wear of 63 and 71 μm for MQL and water/mist cooling, respectively, compared with 71 and 85 μm when a 50°angle was employed. In another study [32], nozzles positioned at 45°with respect to feed direction resulted in the best lubrication for the upmilling and downmilling of Inconel 182 using MQL (Fig. 1). The influence of nozzle position has also been evaluated when grinding 100Cr6 hardened steel using an MQL application [33]. It was found that MQL oil mist effectively penetrated into the boundary layer flow around the grinding wheel when the nozzle was positioned toward the wheel at an angle of 10°/20°to the workpiece surface. Additionally, titanium alloys are very attractive materials, particularly for aerospace and automotive applications, due to their superior inherent physical and mechanical properties (e.g.~50% density of steel, high strength, high operational temperatures and exceptional corrosion resistance). Ti-6Al-4V is the most widely used titanium alloy which is currently used in a wide range of engineering sectors including aerospace, automotive, medical, military aviation and sports equipment. It features relatively higher mechanical properties (i.e. high strength, low weight ratio and excellent corrosion resistance). However, Ti-6Al-4V has low machinability index compared to other metallic alloys such as steels owing to its low thermal conductivity (7.2 W/m.K for annealed Ti-6Al-4V) and high dynamic shear strength. This generates a large quantity of heat close to the machining zone, which leads to the use of relatively lower cutting speeds (i.e. 30-90 m/min) to remain economical [34,35] as well as has a knock on effect on cutting tool life. Additionally, the low E modulus of titanium alloys (~110 GPa for annealed Ti-6Al-4V) promotes tool rubbing, chatter and product geometry issues and contributes to higher tool wear rates [36,37]. On the other hand, increasing machining speed when cutting Ti-6Al-4V alloy with less manufacturing cost (e.g. using minimum amount of cutting fluid) is deemed necessary for process improvement. These reasons encouraged the authors to use Ti-6Al-4V as the workpiece material to examine the novel cutting fluid supply system. In the same vein, vibration is more prone to occur in milling titanium due to self-excited vibrations (i.e. chatter) between the workpiece and cutting tool (owing to its low E modulus) [38]. Localised shearing, which is correlated to the generation of cyclic force, is responsible for self-exited vibration. This vibration induces waviness of the surface in the first cut. In the following cut, the tool cuts into the wavy surface and generates a further variation of chip geometry/thickness and force that excites the structure, providing greater vibration between the workpiece and cutter, resulting in wave regeneration phenomenon [35,39]. Thus, rigid clamping is essential for avoiding effect of low E modulus of titanium alloys particularly for chatter-free surfaces [40]. Additionally, cutting tool path (i.e. contouring and ramping) and the inclined surfaces (e.g. concave and convex) can also affect cutting force, tool and workpiece deflection when milling titanium. It was revealed that the cutting force and tool deflection in contouring tool path style were found to be lower than that of ramping tool path mode. This was explained by the outcome that the contouring tool path style offers movements parallel to the inclined surface from the axis and ideal to move the chips away. In addition, the cutting forces obtained in the machining of convex inclined surfaces are found smaller in comparison to that obtained in the machining of concave inclined surfaces. This is because the chip was comfortably removed from the machining zone in the convex inclined surface. Furthermore, the cutting tool works on the inner surface and comes in contact with the workpiece with a longer cutting edge, resulting in increases in the cutting force and tool deflection values [41]. In a similar vein, it was indicated that cutting forces are strongly affected by the surface inclination angle (α) in milling operations. Influence of surface inclination angle on cutting forces was lower in the case of α ≥ 15, whilst the highest impact of inclination angle on cutting forces was found with the α = 0° [42]. Additionally, increasing the machining speed of titanium components is necessary for process improvement. Currently, a parallel machining strategy has been presented in order to extend metal cutting capacity and to minimise chatter. Also, parallel machining involves less bending force applied to the work part, affording additional improvements in product accuracy [43]. In the absence of coolant, titanium alloys are more disposed to react with atmospheric gases and cutting tool material at elevated temperatures, resulting in the degradation of machined surface quality as well as rapid tool wear [36]. Traditionally, using copious amounts of cutting fluid helps when machining titanium and is a dominant 'machining culture' on machine shop floors [44,45]. Thus, more attention is deemed necessary to reduce fluid consumption during machining titanium alloys, with a particular emphasis on the use of environmentally friendly cutting fluids. In this work, a novel controlled cutting fluid impinging supply system (Cut-list) is developed and applied to the shoulder milling of Ti-6Al-4V to reduce cutting fluid consumption. Key process measures include cutting force, workpiece temperature, tool flank wear, burr formation and surface roughness. The effects of working conditions (cutting speed and feed rate), nozzle position and impinging distances are also assessed employing the new supply system. The originality of the developed supply system depends on synchronisation between the calculated generated heat in the machining zone with an accurate cutting fluid quantity required to minimise its consumption and simultaneously enhance the cutting performance of titanium alloys. Furthermore, the developed system is beneficial to the machine tool industry, making it very viable in terms of the minimum fluid consumption for machining applications without substantial additional costs. Cut-list ( Fig. 2) was designed to supply cutting fluid in feed and against feed directions simultaneously. The angled overhead nozzle ring was placed on a vertical spindle head holding two round coherent nozzles for delivering fluid at three different impinging angles of 15°, 45°and 60°in the feed direction and similar angles against the feed direction as shown in Fig. 3. Cut-list was also developed to align nozzles in the tool-workpiece contact area at any given elevation angle relative to tool axis using an angled mounting wedge together with an adjustable nozzle holder. Additionally, the design of the adjustable nozzle holder allows the nozzle discharge tips to be placed away from the tool-workpiece contact zone at different impinging distances of 35, 55 and 75 mm. A closed loop Gusher vertical type coolant pump (Gusher Pumps Ltd., Wolverhampton, UK) was used to carry the cutting fluid. The pump was placed directly over the cutting fluid tank to minimise pressure drop in the system feed pipes. The output flow rate was controlled using an oval gear digital type flow meter/ regulator (Badger Meter Europa, Neuffen, Germany) located at 300 mm away from the coolant pump to ensure steady state flow conditions. The fluid pressure was monitored employing an OMEGA DPG digital-type and dual-scale pressure gauges (OMEGA Engineering Ltd., Manchester, UK), which were mounted directly after an in-line type filter (Magnom Corporation Ltd., Warwick, UK) and before the cutting fluid enters the nozzles. The new system nozzles were designed based on Webster nozzle geometry [46,47] to generate a high-quality coherent jet stream that affords low misting and minimum entrained air within the jet. These features help Cut-list to penetrate cutting fluid into the machining zone effectively. The internal dimensions of the manufactured coherent nozzle are aperture diameter (d = 1.75 mm), thickness (t = 2.5 mm) and an internal diameter of the feed pipe (D = 12 mm) (see Fig. 3). Step shoulder milling was selected as a cutting strategy in evaluating the new supply system, as shown in Fig. 4. During metal cutting, a greater proportion of the energy consumed (i.e. 90-98%) is converted into heat, whereas the remaining energy is retained as elastic energy in the chip [48][49][50][51][52][53]. In the present work, 90% of the total cutting power is considered to be converted into heat (i.e. the total heat generated in the primary, secondary and tertiary deformation zones). Equations 1 and 2 were used to calculate the metal removal rate (MRR) and cutting power, respectively. The accurate flow rate of the cutting fluid required to cool the machining zone is then computed according to Eq. 3 [54]. where MRR is the metal removal rate (mm 3 /min); a p and a e are the axial and radial depth of cut, respectively (mm); f is the feed rate (mm/rev); Z is the number of teeth; N is the spindle speed (rpm); K 1 is a compensation factor for minimising chip thinning (K 1 = 1.96) [55]; P c is the cutting power (W); U is the specific cutting energy (U =4W s/mm 3 for titanium) [56]; Q is the accurate flow rate (L/min); η nozzle is the coherent nozzle efficiency (0.95) [57]; △θ is the cutting fluid maximum tolerable temperature increase (Δθ = 3°C for wet machining) [58]; and ρ and C are the cutting fluid mass density and fluid heat capacity at 10% concentration (ρ = 0.988 g/m 3 and C =0.948 cal/g°C), respectively. Table 1 shows the cutting conditions used for accurate flow rate calculations. Additionally, the conventional flood estimations were based on 13 L/min per (kW) for cutting titanium as recommended by the Kennametal tool manufacturer [44]. The results showed that the fluid can be supplied at a flow rate of 8 L/min per (kW) using Cut-list with a reduction in cutting fluid consumption by up to 42% compared to the conventional system (see Fig. 5). Cutting fluid velocity and the minimal exit diameter of the round coherent nozzle were determined using Bernoulli's [46] and fluid continuity theories [57], respectively. Table 2 shows the experimental results for all cutting tests. To fulfil the flow coherency criterion, the contraction ratio (D/d) should be at least ≥ 2:1 and the actual nozzle exit diameter (d) must be greater than or equal to the theoretical minimal coherent nozzle exit diameter [57]. To obtain the highest jet stream quality, the actual nozzle exit diameter (d) was fixed at 1.75 mm for all trials, whereas the contraction ratio was set at~7:1 based on the calculated actual nozzle aperture diameter (i.e. 1.75 mm) and nozzle internal feed pipe diameter (i.e. 12 mm) which satisfy the above criterion. Experimental plan The experimental plan was divided into two main parts. Part I evaluated the effect of the settings of the new system on machining output, whereas part II focused mainly on a comparison between the proposed and conventional supply systems. Because the new system has the capability for more settings (i.e. nozzle angles and impinging distance) compared to the existing conventional flood supply system, Cut-list was initially evaluated at three impinging angles in the feed direction, three impinging angles against feed direction and three impinging distances, giving 27 tests at each setting of cutting speed and feed rate (i.e. two levels each). This gives a total of 108 experiments conducted using the new system. Table 3 shows the evaluated factors with their corresponding levels. All trials were carried out using a CNC Cincinnati 750-Sabri vertical milling machine (Cincinnati Machine UK Ltd., Birmingham, UK) with a maximum spindle power of 11 kW. Step shoulder downmilling was performed on annealed Ti-6Al-4VASTM B 265 grade 5 rectangular blocks, each having a width of 11 mm, height of 25 mm and length of 103 mm. Each trial involved cutting a length of 103 mm, and a new cutting insert was used to avoid the accumulation of wear from different tests. The axial (a p ) and radial (a e ) depths of cut and nozzle elevation angle (α) of 5 mm, 1.3 mm and 40°, respectively, were maintained for all tests. Sandvik H13A coarse grain uncoated tungsten carbide inserts with a positive rake angle and a nose radius of 0.8 mm were used. These inserts were mounted on a ∅18.5 (i.e. D c ) × 110-mm-long square shoulder milling tool holder (Sandvik Coromant, Halesowen, UK), implying a major cutting edge angle κ = 90°with an overhang distance of 60 mm (i.e. to eliminate chatter). Figure 6 shows the experimental setup using the new Cut-list system. The single tooth cutter (i.e. Z = 1) was used to facilitate tool wear measurements. A commercial (Vasco1000) watersoluble vegetable oil-based cutting fluid (Jemtech Ltd., East Sussex, UK) containing 45% pure vegetable oil was used. The fluid was blended at 10% concentration and regularly checked using a portable refractometer (Cromwell Tools, Luton, UK). Table 4 details the chemical composition and thermo-physical properties of the cutting fluid used throughout the experimentation. An Alicona Infinite Focus G4 optical microscope (Alicona UK Ltd., Kent, UK) was utilised to capture the average tool flank wear (VB). Average tool flank wear (VB) was measured following each trial in accordance with the ISO 8688-2 (1989) standard. The average surface roughness (R a ) of machined samples was measured using a Taylor Hobson Surtroni 3+ (Taylor Hobson, Leicester, UK). Three R a readings (at the beginning, middle and end of the cut) were recorded, and an average was then computed (see Fig. 7 Flow rate (L/min) Fig. 5 Flow rate versus cutting conditions and generated heat for the two systems Four Ttype thin sensing probes with Ø 1.0 mm diameter × 10 mm probe length × 2 m extension cable length were inserted into Ø 1.0-mm drilled holes in each sample at 0.5 mm from the machined surface and 20 mm apart. A distance of 20 mm was also allocated before passing over the first thermocouple for ensuring stable steady state thermal conditions (see Fig. 7). Prior to each test, each workpiece material was left for a few seconds to reach ambient temperature (~19°C). It is worth mentioning that the lowest values of process outputs (i.e. tool wear, R a and burr height) are crucial for the quality improvement of the machined parts and reducing manufacturing costs. For this reason, 'the-lower-the-better' criterion was adopted in this study. For workpiece temperature, surface roughness, burr height and tool wear, three measurements were taken for each test and average was calculated. Process analysis and effect of the new system (part I) Cutting force Figure 8 shows the effect of process variables on the cutting force. The best settings to minimise cutting forces when shoulder milling Ti-6Al-4V were found to be 95-m/min cutting speed, 0.1-mm/rev feed rate and nozzle position at 15°in the feed direction, 45°against feed direction and 75-mm impinging distance. Feed rate was found to have a considerable influence on the cutting force over the evaluated range; i.e. cutting forces increased with the increase of feed rate. This is due to the high correlation between cutting force and cutting area (where uncut chip thickness is part of it) and thus to feed rate. It was also noticed that cutting force increased with higher cutting speed. This agrees with the assumption that more energy is required to remove a higher volume of material and hence higher cutting force [59,60], although this is in disagreement with the finding that higher cutting speeds may cause material softening, and hence lower cutting forces when machining steel alloys as described by Veiga et al. [36]. Additionally, variations in nozzle positions/ angles and impinging distance had only a limited impact on cutting force. Workpiece temperature Figure 9 presents the variation of average workpiece temperatures functions of all process variables evaluated in this research. Average workpiece temperature values ranged between 23.5 and 27.5°C. This is probably because only a small proportion (~20%) of the heat generated when cutting titanium is conducted into the workpiece and chip whilst 80% of the heat are expected to be transferred to the cutting tool [61]. In addition, the impinging fluid jet on targeted heat-affected zones helped to dissipate more than 30% of the heat generated during the cutting process [35]. Additionally, the decrease in workpiece temperature with higher cutting speeds can be attributed to the increase in cutting fluid flow rate associated with increasing cutting energy (in accordance with increasing working conditions). Increasing the flow rate resulted in improving the cooling capacity of the cutting fluids which assisted to transfer more heat from the workpiece to the cutting fluid, hence a reduction in workpiece temperature. Unexpectedly, the optimal (low) workpiece temperature was recorded at the higher cutting condition and nozzle position at an angle of 15°in the feed Int J Adv Manuf Technol direction and 45°/60°against feed direction, and an impinging distance of 55 mm. This is due to the increase in the fluid flow rate associated with increasing cutting energy (see Fig. 5), which improved the fluid's ability to transfer more heat from the workpiece to the cutting fluid. Feed rate, impinging distance and nozzle positions, particularly in the feed direction, showed a considerable response to average workpiece temperature. Tool flank wear Figure 10 demonstrates the effects of control factors on mean values of tool flank wear (VB). A cutting speed of 95 m/min, feed rate of 0.15 mm/rev and nozzle location at 15°in the feed direction and 45°against feed direction and an impinging distance of 75 mm can be selected as the optimal cutting conditions for controlling tool wear. However, the use of the controlled cutting fluid through the developed Cut-list system meant that tool wear increased rapidly with increased cutting speed owing to the rise in heat generated. The heat generated at the tool edge would have softened the insert edge, causing tool flank wear to sequentially increase [62]. Tool flank wear values seem to be independent of feed rate, nozzle position and impinging distance. It can also be concluded that the traditional understanding that cutting speed is the dominant variable affecting tool wear is still applicable to the Cut-list cutting fluid supply system. Figure 11 shows an SEM analysis of the used uncoated WC cutting tool edges at various cutting conditions. Wear occurred mainly on the flank face of the cutting tool where uniform abrasive wear was observed at low cutting speed, whereas adhesion has already taken place at higher cutting speed. The adhered substances were examined using EDX, and the presence of titanium element in the adhered materials on the rake face of the tool substrate was found, as shown in Fig. 12. In addition, a few elements such as tungsten (W) and cobalt (Co) from the tool substrate were found in the adhered workpiece material at higher cutting speed, which proves that diffusion took place. The increase in heat generated with increased cutting energy may offer a good atmosphere for the diffusion of tool material atoms across the tool-workpiece interface, and thus, by impairing bonding strength in the tool materials, diffusion wear has occurred [63]. Figure 13 presents the variation in average burr height measured at the edge of the machined surface as a function of various process variables assessed in this work. The smallest burr height can be achieved at 200-m/min cutting speed, 0.15-mm/rev feed rate, nozzle location at 45°in the feed direction and 45°against feed rate direction and an impinging distance of 55 mm. It was observed that burr height tends to decrease rapidly with increased cutting speed and feed rate, and it is relatively sensitive to impinging distance rather than nozzle positions/angles. This is deemed to be due to the impinging distance affecting the fluid velocity [64], which led to maintaining the cutting edge in a sharper condition with less metal seizing. In addition, cutting speed and nozzle angle do not show a significant impact on burr height. Figure 14 shows burrs formed on the milled top surfaces of Ti-6Al-4V at various cutting conditions. Surface integrity Average surface roughness (R a ) results at various process and system levels are shown in Fig. 15. Minimum surface roughness was achieved at a cutting speed of 200 m/min, feed rate of 0.1 mm/rev, nozzle positions of 45°a nd 60°in feed and against feed direction, respectively, and 75-mm impinging distance. Surface roughness values were found to decrease with rises in cutting speed and decreased feed rates. This can also be attributed to the increase in cutting fluid flow rate associated with increased cutting speed (see Fig. 5). A comparable finding was also reported recently [64] when Xiao et al. investigated the end-milling of Ti-6Al-4V under MQL at four different fluid supply rates (2, 6, 10 and 14 ml/h). However, the effect of feed rate on mean values of R a seems much higher than that of cutting speed. This is likely to be due to the theoretical surface roughness being directly proportional to the square of the feed per revolution. In the same vein, decreased feed rate possibly gave the cutting fluid enough time to carry away the heat from the machining zone, and led to a low material removal rate and the accumulation of chip in the tool-workpiece zones, which resulted in an improved surface finish. It was noted that the major effective parameter to reduce R a is the feed rate, whilst cutting speed and impinging distance showed less noticeable effects on R a values. Additionally, SEM images for titanium samples before and after the machining trials confirmed that no major changes in the micro-structure as well as no signs for surface or subsurface damage are seen in Fig. 16. Burr formation Nozzle position effect Workpiece temperature was found to be influenced by nozzle position/angle, in particular in the feed direction, as can be seen in Fig. 9. Seemingly, locating nozzle at 15°in the feed direction helped to create efficient fluid trapping between the tool and workpiece surfaces which, in turn, led to improving the accessibility of cutting fluid into the machining zone to perform both cooling and lubrication functions adequately. A similar phenomenon was observed in another study [65], where it was found that shifting the nozzle from a 45°angle to an acute angle of 12.5°in the feed direction when end-milling H13 steel under MQL assisted in increasing the amount of cutting fluid reaching the machining zone. Additionally, the proximity of the nozzle positioned at an angle of 15°from the tool-workpiece contact point contributed in minimising the fluid particle dispersion caused by tool rotation, which allowed them to adhere to the tool and workpiece surfaces effectively and to persist in working as a lubricant in the machining zone. Conversely, at the nozzle positions of 45°or 60°, more cutting fluid particles are driven away from the tool surface whilst the cutting tool rotates in a cyclic loop process [66]. In addition, nozzle placement at an angle of 45°or 60°against feed direction tends to offer enough space to assist in chip evacuation and this helped to minimise the interference between the impinging jet and the removed chip, which led to better lubricating and cooling ability and consequently improved machined surfaces. Impinging distance effect According to Figs. 9, 13 and 15, impinging distance had a noticeable impact on workpiece temperature, burr formation and average surface roughness. According to Bernoulli's equation (Eq. 4) [66], the hydraulic head (h) (where in this case, the impinging distance is equal to the hydraulic head) has an effect on jet velocity, and this consequently affected fluid penetration efficiency. The cutting fluid jet velocity (V j ) increases as the impinging distance decreases. However, too short impinging distance has a negative effect on the cutting fluid droplets owing to their high levels of rebounding from the workpiece and cutter surfaces [66]. Therefore, impinging distance should be controlled at an optimal level, as the impact of shorter impinging distance would be to conspicuously affect the workpiece temperature, burr formation and average surface roughness. Since the optimal impinging distances obtained for controlling the aforementioned responses were 55/75 mm, they tend to have a combined action by improving the fluid penetration ability with less fluid dispersion and spring-back effect, which in turn assisted the cutting fluid droplets to adhere firmly to the workpiece and cutter surfaces. 4.2 Performance comparison with a conventional flood system (part II) Data collected using the new system was compared with a conventional (uncontrolled) flood supply system. The best results were considered (i.e. amongst every 27 tests conducted at the same cutting speed and feed rate). In general, the new system (Cut-list) showed better performance compared to the conventional system although the 42% reduction in cutting fluid consumption achieved by the Cut-list. Cutting force ranged between 1600-1903 N and 1340-1879 N for the conventional and new system, respectively. This provided a reduction of 16% in cutting force, particularly at a low feed rate (0.1 mm/rev) owing to the good penetrability of the new system, which led to forming the boundary of the oil film between the workpiece and cutting tool surfaces, resulting in reducing contact pressure. Additionally, a significant reduction in tool wear up to 46.77% was achieved at the higher cutting condition (i.e. 200 m/min and 0.15 mm/rev) utilising the Cut-list. Impinging the cutting fluid in both directions (with and against feed) helped to penetrate the fluid effectively into machining zone and cool the tool insert at the end of the cyclic process. In terms of burr formation, burr height ranged between 0.11-0.41 mm and 0.1-0.17 mm for the conventional and new system, respectively. This afforded a significant reduction up to 31.70% in burr height, particularly at higher cutting speed (i.e. 200 m/min) owing to the high velocity of the jet (i.e. 10 m/s). In addition, no substantial differences were found between systems in workpiece temperature results (maximum of 1.5°C), whilst average surface roughness (R a ) values were relatively smaller with the use of the new system. Conclusions This paper presents a comprehensive experimental evaluation of a new cutting fluid supply system (Cut-list) when machining Ti-6Al-4V using vegetable oil-based cutting fluid. In general, the new system (Cut-list) was found to perform favourably as compared to the conventional system. It significantly reduced cutting fluid consumption up to 42%, cutting force by 16.41%, tool flank wear by 46.77% and burr formation by 31.70%. However, no notable discrepancy in workpiece temperature values was found between the two systems [67]. Feed rate was the dominant factor affecting mean cutting force, burr formation and surface roughness, whilst cutting speed has the greatest impact on workpiece temperature and tool flank wear. The cooling performance of the new system is relatively sensitive to nozzle positions and impinging distance with respect to workpiece temperature, burr formation and surface roughness (R a ). Lastly, it can be concluded that coherent round nozzle can be used effectively to replace conventional sloped nozzle in milling operations. Funding This work is sponsored by the Libyan government and technically supported by the Mechanical and Construction Engineering Department, Northumbria University at Newcastle, UK. Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.	2018-12-05T06:00:30.642Z	2018-03-01T00:00:00.000	{ "year": 2018, "sha1": "8bc35d5b7897b9de61063e5fc0ee1e4704dafe9d", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s00170-017-1216-y.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "32619beeb6752dae1e8805881a349e2ced928ca7", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Engineering" ] }
253237147	pes2o/s2orc	v3-fos-license	The Near Infrared Imager and Slitless Spectrograph for JWST -- V. Kernel Phase Imaging and Data Analysis Kernel phase imaging (KPI) enables the direct detection of substellar companions and circumstellar dust close to and below the classical (Rayleigh) diffraction limit. We present a kernel phase analysis of JWST NIRISS full pupil images taken during the instrument commissioning and compare the performance to closely related NIRISS aperture masking interferometry (AMI) observations. For this purpose, we develop and make publicly available the custom"Kpi3Pipeline"enabling the extraction of kernel phase observables from JWST images. The extracted observables are saved into a new and versatile kernel phase FITS file (KPFITS) data exchange format. Furthermore, we present our new and publicly available"fouriever"toolkit which can be used to search for companions and derive detection limits from KPI, AMI, and long-baseline interferometry observations while accounting for correlated uncertainties in the model fitting process. Among the four KPI targets that were observed during NIRISS instrument commissioning, we discover a low-contrast (~1:5) close-in (~1 $\lambda/D$) companion candidate around CPD-66~562 and a new high-contrast (~1:170) detection separated by ~1.5 $\lambda/D$ from 2MASS~J062802.01-663738.0. The 5-$\sigma$ companion detection limits around the other two targets reach ~6.5 mag at ~200 mas and ~7 mag at ~400 mas. Comparing these limits to those obtained from the NIRISS AMI commissioning observations, we find that KPI and AMI perform similar in the same amount of observing time. Due to its 5.6 times higher throughput if compared to AMI, KPI is beneficial for observing faint targets and superior to AMI at separations>325 mas. At very small separations (<100 mas) and between ~250-325 mas, AMI slightly outperforms KPI which suffers from increased photon noise from the core and the first Airy ring of the point-spread function. INTRODUCTION The recently commissioned James Webb Space Telescope (JWST ) is a joint NASA/ESA/CSA flagship science mission to explore the beginnings of the Universe, the assembly of galaxies, the birthplaces of stars, and planetary systems and the origins of life (Gardner et al. 2006). For the direct observation and characterization of giant exoplanets and circumstellar dust, JWST is equipped with multiple coronagraphic imaging modes in the near-and mid-infrared as well as a non-redundant mask (NRM) operating between ∼ 2.8-4.8 µm that transforms the 6.5 m primary mirror into an interferometric array of seven subapertures (Artigau et al. 2014). It is the first time in history that such an aperture mask is available on a space-based telescope. By exploiting the interferometric capabilities of this mask, the NIRISS instrument (Doyon et al. 2012) is expected to enable high-contrast imaging up to ∼ 10 mag contrast at small angular separations of ∼ 70-400 mas (Greenbaum et al. 2015), well inside the inner working angle (IWA) of the NIR-Cam coronagraphs (half width half maximum of ∼ 6 λ/D, where λ is the observing wavelength and D is the telescope primary mirror diameter, Krist et al. 2009Krist et al. , 2010. The unique parameter space accessible with the NIRISS NRM enables advances in exoplanet and planet formation as well as galaxy evolution science by studying close-in stellar and substellar companions, warm exozodiacal dust around nearby stars, transitional disks, and feedback in active galactic nuclei for instance (Sivaramakrishnan et al. 2022). While the NIRISS NRM transforms JWST into an interferometer and thereby enables highcontrast imaging down to the Michelson diffraction limit (∼ 0.5 λ/D), the NRM also blocks ∼ 85% of the incoming light and thus significantly reduces the sensitivity of the observations (Artigau et al. 2014). An alternative technique that is not affected by such a big throughput loss is called "kernel phase imaging" (KPI) and has been introduced by Martinache (2010). KPI uses Fourier quantities of full pupil images to achieve the same high resolution (∼ 0.5 λ/D) as aperture masking interferometry (AMI). The basic idea of KPI is to discretize the full pupil into an interferometric array of "virtual" subapertures during post-processing. However, due to the high redundancy of the full pupil, KPI requires high-Strehl images (which are naturally given from space in the absence of a disturbing atmosphere) so that the image Fourier phase can be linearized and the contributions from individual baselines can be disentangled. In this linear regime, the relationship between the image Fourier phase φ and the pupil plane phase ϕ reads where A is the matrix mapping baselines in the pupil plane to spatial frequencies in the image Fourier plane, R is the diagonal matrix encoding the redundancy of each baseline, and φ obj is the image Fourier phase intrinsic to the observed object. One can then multiply Equation 1 with R from the left, obtain the kernel K of the baseline mapping matrix A via singular value decomposition, and derive the kernel phase which is independent of pupil plane phase errors to first order (higher order terms appear because the linearization applied here is only an approximation, Ireland 2013). Therefore, kernel phase has similar properties as closure phase in AMI and Ireland (2016) has shown that kernel phase is a generalization of closure phase to the case of redundant apertures. Hence, KPI with JWST is expected to achieve similar performance as AMI except for an increased sensitivity to faint targets due to its increased throughput. With NIRISS, KPI can be used with the same four filters as AMI, which are F277W, F380M, F430M, and F480M, albeit the usefulness of F277W is limited given that the NIRISS detector is undersampled at this wavelength. The AMI observing template, which is also used for KPI observations, provides repeatable target acquisition on a predefined detector position to subpixel accuracy (typically < 0.1 pixels Rigby et al. 2022). Using the same detector position for the science and the PSF reference target will help mitigating systematic errors (e.g., from flat-fielding). The methods presented here are based on a monochromatic description of the image and given the ∼ 6% bandwidth of the medium-band filters mentioned above, we expect some level of spectral decoherence for both AMI and KPI techniques. While a detailed study of this effect is beyond the scope of this paper, we note that kernel phase tech-niques have been successfully applied to Hubble Space Telescope (HST ) wide-band (F110W) images by Pope et al. (2013). To minimize calibration errors, we recommend to use a PSF reference target with a similar spectral type as the science target. In the model fitting process with fouriever (Section 2.3), it is possible to apply bandwidth smearing to account for the finite filter bandpass of the observations. Finally, since KPI can be applied to any full pupil image, it can be used with all instruments onboard JWST opening up the entire wavelength range of ∼ 0.7-25.5 µm for high-resolution imaging. KPI's increased uv-coverage with respect to AMI also enables high-contrast image reconstruction of complex scenes down to the Michelson criterion. For a more detailed description of kernel phase we refer the reader to Martinache (2010) and Martinache et al. (2020). The first application of KPI can be found in Martinache (2010) who detected a previously known companion to the star GJ 164 at a contrast of ∼ 9 : 1 and a separation of ∼ 0.6 λ/D in archival HST NICMOS data, clearly demonstrating the super-resolution power of KPI. Later, Pope et al. (2013) discovered five new brown dwarf companions with separations ranging from ∼ 0.4-0.6 λ/D, also in archival HST NICMOS data. The first time that KPI was applied behind an adaptive optics system from the ground was in Pope et al. (2016) who observed the known binary α Oph with both KPI and AMI and found excellent agreement between the binary parameters derived from both techniques. Kammerer et al. (2019) and Wallace et al. (2020) went one step further and searched for close-in substellar companions in an archival VLT NACO dataset of nearby field stars and a Keck NIRC2 dataset of young stars in the Taurus Molecular Cloud where they achieved contrast limits of up to ∼ 7 mag at ∼ 1 λ/D in the L-band. Kammerer et al. (2019) and Laugier et al. (2019) came up with different methods to extend the dynamic range of KPI observations beyond the saturation limit in the central few pixels of the core of the point-spread function (PSF). The method from Kammerer et al. (2019) is also implemented in the JWST kernel phase pipeline presented here and explained in more detail in Section 2.1.1. Similar to angular differential imaging (Marois et al. 2006) in classical high-contrast imaging, Laugier et al. (2020) also derived self-calibrating angular differential kernel phase observables, but these are more suitable for datasets with larger field rotation than JWST can provide. To measure the photometry of the individual components of the T Tau triple star system at ∼ 10 µm, Kammerer et al. (2021) applied KPI to VLT VISIR-NEAR observations in the mid-infrared, demonstrating the feasibility of KPI with instruments such as JWST MIRI. Recently, pioneering work from Pope et al. (2021) has shown that automatic differentiation methods can be used to extract kernel phase observables from any differentiable optical system, such as a Lyot coronagraph for instance, which has previously been impossible given that focal plane masks destroy the linearity of the Fourier phase observables. Albeit beyond the scope of this paper, automatic differentiation methods are a viable alternative beyond the classical KPI methods presented here. The paper is structured as follows. Section 2 describes the methods that we use to reduce and analyze the JWST NIRISS KPI commissioning data. In particular, Section 2.1 introduces our customly developed and publicly available stage 3 kernel phase data reduction pipeline that can be used to extract kernel phase observables from JWST images in a similar fashion as the other STScI jwst 1 data reduction pipelines. Section 2.2 motivates the need for a dedicated kernel phase data exchange format and describes the KPFITS file format that we are proposing here. Section 2.3 introduces our publicly available model fitting toolkit fouriever that can be used to search for companions or determine detection limits from OIFITS (a widely used file format for exchanging AMI and long-baseline interferometry data, Pauls et al. 2005;Duvert et al. 2017) and KPFITS files. The NIRISS KPI commissioning observations are described in Section 3 and our results from their analysis are presented and discussed in the context of the NIRISS AMI commissioning observations in Section 4. Finally, Section 5 summarizes our work and draws conclusions for the application of AMI and KPI with JWST. METHODS For reducing and analyzing the JWST NIRISS KPI commissioning data, we develop a variety of custom and user-friendly software that we make publicly available on GitHub. This enables other JWST observers to use our tools for proposal planning and for getting science-ready data products and plots out of their own programs. Kernel phase stage 3 pipeline The STScI jwst data reduction pipeline is organized into three stages. Stage 1 performs detector level calibrations such as bias correction and jump detection, stage 2 performs photometric calibrations, and stage 3 performs highlevel calibrations that are specific to each of the observing modes supported by JWST (e.g., AMI, coronagraphic imaging, integral field spectroscopy). To enable the community a straightforward access to kernel phase observables from JWST images, we develop a custom stage 3 pipeline that we name the Kpi3Pipeline 2 . This pipeline can be used for extracting kernel phase observables from full pupil NIRISS and NIR-Cam images, support for MIRI will be added 2 https://github.com/kammerje/jwst-kpi in a future update. Before the JWST images can be fed into the Kpi3Pipeline, they need to be processed with the jwst stage 1 and 2 pipelines. For those, similar as with AMI data, we recommend to skip the inter-pixel capacitance, the photometry, and the resample step since Fourier plane imaging techniques in general are highly sensitive to variations in the pixel-by-pixel response of the detector (e.g., Ireland 2013). Skipping these steps avoids systematic errors being introduced by the pipeline corrections. Instead, the approach with NIRISS KPI and AMI observations is to center the science and the reference targets on the exact same detector pixel and thereby minimize flat-fielding errors. The available detector positions were carefully chosen to provide both a good uvcoverage of the pupil support (which is challenging with NIRISS given its coarse sampling) and a clean detector region with only few bad pixels (Sivaramakrishnan et al. 2022). Dithering is hence not recommended for NIRISS KPI (and AMI) observations. While the backend of the Kpi3Pipeline is based on XARA 3 (Martinache 2010(Martinache , 2013Martinache et al. 2020), the frontend is designed similarly to the STScI jwst pipelines to provide a uniform experience for the user. Similar to the VLT NACO kernel phase data reduction pipeline developed by Kammerer et al. (2019), the Kpi3Pipeline consists of five steps: 1. bad pixel fixing step, 2. recentering step, 3. windowing step, 4. kernel phase extraction step, 5. empirical uncertainties step. An intermediate data product can be saved after each step so that the individual steps can be run separately and each of the five steps can generate a diagnostic plot for validation purposes. Each step and its diagnostic plot is described in more detail below. A list of the tunable parameters of each step can be found in Appendix B. In principle, all steps except for the fourth one can be skipped although it is highly recommended to run at least steps 1, 2, and 4 to ensure that the kernel phase extraction step is provided with properly preprocessed data. An additional input that is required for running the Kpi3Pipeline is a discrete model of the pupil. We note that due to different pupil plane masks, the NIRISS, NIRCam, and MIRI instruments all see slightly different pupils. We provide default pupil models for the NIRISS CLEARP and the NIRCam CLEAR pupils but the user can also provide a custom pupil model if desired. For the default pupil models, we distribute subapertures on a hexagonal grid so that 19 subapertures fall inside one JWST primary mirror segment. To obtain an isotropic grid, we also distribute subapertures on top of the gaps between the individual primary mirror segments. We are using a "gray" pupil model so that the subapertures on top of the gaps have a slightly reduced transmission. Then, we apply the transmission of the instrument-specific pupil mask and discard all subapertures with a total transmission of < 0.7. This results in the NIRISS CLEARP pupil model shown in Figure 1. This model consists of 349 individual subapertures spanning 948 distinct baselines. After discarding all baselines with a redundancy of less than 10, 800 distinct baselines yielding 452 individual kernel phases remain. Discarding baselines with low redundancy helps to avoid the longest (and most noisy) baselines at the edge of the primary mirror. We find that a redundancy threshold of 10 is sufficient to avoid Fourier phases > 0.5 rad and entering the nonlinear regime. Bad pixel fixing step Bad pixels can have a ruinous impact on Fourier plane imaging since an individual bad pixel contributes noise to all spatial frequencies of the image. However, Ireland (2013) has shown that bad pixels do not affect kernel phase observables if they are properly corrected. In the Kpi3Pipeline, bad pixels can be fixed using two different methods: "medfilt" or "fourier". In both cases, the locations of the bad pixels are obtained from the jwst stage 1 pipeline. As a default, all pixels flagged as "DO NOT USE" in the data quality array are considered as bad pixels but the user may specify other data quality flags 4 that shall be considered as bad pixels. With the "medfilt" method, bad pixels are simply replaced with the median filtered image using a kernel size of five pixels. With the "fourier" method, bad pixels are fixed by minimizing their Fourier power outside the region of support permitted by the pupil geometry. This method was introduced by Ireland (2013) and starts by computing the matrix B Z which maps the bad pixel values x onto the Fourier domain Z (the complement of the pupil support), so that their Fourier power \|f Z \| is given by where b are the corrections to be made to the bad pixels and Z is remaining noise. These corrections are obtained by computing the Moore-Penrose pseudo inverse of B Z and solving for where the star denotes the complex conjugate. This algorithm has been successfully applied to fix bad and reconstruct saturated pixels in kernel phase observations by Kammerer et al. (2019) and is described in more detail in Sivaramakrishnan et al. (2022). While the "fourier" The left panel shows the bad pixel map from the jwst stage 1 pipeline considering the specified data quality flags as bad pixels (here DO NOT USE), the middle panel shows the frame in a logarithmic color stretch before fixing the bad pixels, and the right panel shows it after fixing the bad pixels using the "medfilt" method. The bottom five rows are reference pixels for correcting bias drifts which are not used for science. method is particularly suited for reconstructing saturated PSFs, we do not have any saturated PSFs in the NIRISS KPI commissioning data and therefore use the "medfilt" method for simplicity. The diagnostic plot of the bad pixel fixing step is shown in Figure 2. Recentering step As mentioned in Section 1, the image Fourier phase needs to be in the linear regime in order for the kernel phase technique to be applicable. Hence, recentering the images is important since a PSF being offset from the image center by only half of a pixel would already result in a linear ramp in the image Fourier phase from −π/2 to +π/2 across the support of the telescope pupil (i.e., the modulation transfer function of the pupil). While the kernel phase observables are independent of first order pupil plane phase errors (and thus a linear ramp in the image Fourier phase, Martinache 2010), there are virtually always systematic phase errors that can easily cause the image Fourier phase wrapping around the ±π discontinuity if added on top of a linear phase ramp and thereby destroying the properties of the kernel phase observables. To avoid this, the Kpi3Pipeline uses the Fourier phase norm minimization (FPNM) method implemented in XARA that recenters the images using a leastsquares gradient descent minimization of the norm of the image Fourier phase within the support of the telescope pupil. This method was shown to be more robust, especially in the presence of low-contrast companions at small angular separation (Kammerer et al. 2019), albeit slightly slower than the other two methods (BCEN = centroid of the brightest speckle & COGI = center of gravity of the image) that are also implemented in XARA. We note that before recentering the images, we trim them to their maximum possible square size (constrained by the location of the detector edges) around the expected PSF center (the reference pixel position where JWST aims to place the PSF during target acquisition 5 ). The diagnostic plot of the recentering step is shown in Figure 3. Windowing step Windowing with a smooth function is an effective method to mitigate artifacts when numerically Fourier transforming images with sharp edges. Since XARA uses a Fourier transform to extract kernel phase observables from the telescope images, we window (i.e., multiply) them with a super-Gaussian windowing function w of shape before Fourier transforming them, where x and y are the pixel coordinates with respect to the image center and r is the radius of the super-Gaussian windowing function in units of pixels. The radius r is chosen adaptively as the smaller of 40 pixels and a fourth of the trimmed square image size to make sure that the window is not larger than the image itself. If the images are large enough, a radius of 40 pixels gives access to separations of at least 5 λ/D (sometimes more depending on the observing wavelength). At these separations, classical highcontrast imaging techniques such as coronagraphy are typically superior to KPI. The NIRISS full pupil images considered for the KPI analysis here do only have a native size of 80 by 80 pixels and we are using a constant window radius of r = 15 pixels for them resulting in a field-of-view of ∼ 2 arcsec, which is more than sufficient for the region where NIRISS KPI is superior to NIRCam coronagraphy (Kammerer et al. 2022). The diagnostic plot of the windowing step is shown in Figure 4. Kernel phase extraction step Once the bad pixels are fixed and the images are recentered and windowed, kernel phase observables can be extracted using XARA. First, the image Fourier phase φ at the discrete spatial frequencies of the pupil model is obtained using a linear discrete Fourier transform. Then, the kernel phase θ is computed as whereK = K · R and K is the kernel of the pupil model A as described in Martinache (2010). We also decided to normalize the rows of theK matrix to one so that the amplitude of the kernel phase signal is no longer depending on the geometry of the pupil model. This enables a more direct comparison between kernel phase observables obtained from different telescopes/instruments. To analytically estimate the kernel phase uncertainties, we first need to linearize the relationship between the kernel phase θ and the image I, so that where F denotes the linear discrete Fourier transform at the spatial frequencies of the pupil model and the fraction denotes element-wise division (Kammerer et al. 2019). Then, the kernel phase covariance Σ can be computed from the image covariance Σ image as where Σ image is assumed to be uncorrelated with the square of the "ERR" extension of the stage 2-reduced pipeline product on the diagonal. If the recentering step is being run in conjunction with the kernel phase extraction step, a more accurate method is used to extract the kernel phase observables where the integer pixel recentering is performed by rolling the image and the subpixel recentering is performed by adding a linear ramp to the extracted image Fourier phase. This method circumvents interpolation errors when performing subpixel shifts of the images. We note that for extracting the kernel phase uncertainties, we still use the images that have been recentered with subpixel precision. The diagnostic plot of the kernel phase extraction step is shown in Figure 5. Empirical uncertainties step A single JWST data product (here called an exposure) typically consists of a large number ( 10) of individual integrations. While all previous pipeline steps can be performed on hundreds of individual images within timescales of seconds to minutes, it can be very time-and memory-consuming to perform model fitting on such a large number of individual data points, especially if accounting for correlated uncertainties. Hence, it is advisable to average individual integrations within a single exposure into one final data point. This is achieved in the Kpi3Pipeline by computing the covarianceweighted mean of the kernel phase observables according to where θ i and Σ i are the kernel phase and its covariance of the individual integrations and is the mean covariance matrix (Kammerer et al. 2019). Another advantage of having several individual integrations is that the uncertainties can be estimated empirically as the standard deviation of the kernel phase observables over these individual integrations. This can be especially helpful if the kernel phase observables are dominated by systematic errors (e.g., Kammerer et al. 2019;Wallace et al. 2020) which are not reflected in the analytical error estimation from the previous pipeline step. An empirical estimation for the kernel phase covariance can then be obtained by computing the correlation matrix C mean of Σ mean (which only includes image pixel uncertainties) and multiplying it with the empirically estimated kernel phase standard deviation σ emp , that is Here, m and n denote the indices of the matrices. This procedure ensures that the kernel phase uncertainties remain consistent with the square root of the diagonal of the kernel phase covariance matrix. The diagnostic plot of the empirical uncertainties step is shown in Figure 6. A common and well-defined data exchange format has proven highly valuable for sharing astronomical data and reproducing observational results. In the optical and nearinfrared (long-baseline) interferometry community, the OIFITS file format (Pauls et al. 2005;Duvert et al. 2017) has established itself as a golden standard that is used by most major observatories and instruments (e.g., CHARA/MIRC, Monnier et al. 2004 niques (see, e.g., Ireland 2016), there are a few subtle differences that strongly motivate the need for a distinct file format for KPI data. Firstly, as shown in Section 1, the kernel phase θ is obtained from a special linear combinatioñ K of the image Fourier phase φ. Thus, the kernel matrixK is of fundamental importance in KPI and is repeatedly used in the model fitting process to project image Fourier phase onto kernel phase. Fast and easy access to this kernel matrixK is therefore vital. While it is in principle possible (albeit time-consuming) to recompute this matrix from the geometry of the pupil array that can be saved in the OI ARRAY extension of an OIFITS file, we strongly prefer to save this matrix directly in one of the FITS file extensions, not least because handling hundreds of individual subapertures becomes impractical using the OI ARRAY extension. Secondly, recent work from Martinache et al. (2020) has shown that the use of "gray" pupil models featuring subapertures with a continuous transmission 0 < T ≤ 1 is highly desirable in KPI as they help to reduce the systematic kernel phase signal of unresolved and point-like PSF reference targets. However, gray pupil models are not supported by the OIFITS file format due to the third dimension required to store transmission. Finally, the Karhunen-Loève calibration method applied in Kammerer et al. (2019), Wallace et al. (2020), and Kammerer et al. (2021) can be used implicitly if the kernel phase θ and the kernel matrixK are saved in matrix format. This is because the Karhunen-Loève calibration ) is a linear projection represented by a matrix P so that replacing the kernel phase and the kernel matrix with P · θ and P ·K, respectively, will ensure that any model fitting code will automatically run correctly with a calibrated dataset. Based on the aforementioned issues with the OIFITS file format, the kernel phase community has expressed the need for a dedicated data exchange format in the framework of the Masking/Kernel Phase Hackathon 7 held virtually in mid 2021 to ensure compatibility among a variety of data reduction, calibration, and model fitting tools. The kernel phase FITS (KPFITS) file format proposed here is based on the XARA file format developed by Martinache (2010) and Martinache (2013). However, based on recent developments from Kammerer et al. (2019) and Martinache et al. (2020) several modifications were made to the original XARA file format in consultation with kernel phase experts from the global community. For completeness, we also added an optional extension for saving kernel amplitude observables as introduced in Pope (2016). The structure of the KPFITS file format is described in Table 1 and shall be used as a standard for saving and exchanging kernel phase data in the future. The exact order of the FITS file extensions is in principle irrelevant as extensions shall be referred to in the code with their extension names, the numbering in Table 1 can hence be regarded a suggestion. We note that in principle, AMI data can also be exchanged using the KPFITS file format in a more efficient way. To ensure that the information necessary to consistently reprocess the kernel phase data is present, the KPFITS file format also requires a PRIMARY FITS header with the header keywords specified in Table 2. While several of these keywords are optional, others are strictly required to rerun the Kpi3Pipeline and extract kernel phase observables or to aid the model fitting procedures described in Section 2.3. Model fitting with fouriever There are several publicly available model fitting toolkits that understand OIFITS files and can be used to analyze long-baseline interferometry and AMI data (e.g., LITpro, Tallon-Bosc et al. 2008, CANDID, Gallenne et al. 2015, PMOIRED, Mérand 2022. However, none of these toolkits accounts for correlated uncertainties in the fits. Recently, Lachaume et al. (2019) and Kammerer et al. (2020) developed methods to extract and model correlations in long-baseline interferometry data and Kammerer et al. (2020) showed that accounting for them in the fits can improve VLTI/GRAVITY companion detection limits by a factor of up to ∼ 2. More importantly, they also found that widely used detection criteria based on χ 2 -statistics are only valid when accounting for correlations and yield an excessive number of false positive detections otherwise. In light of these findings, we develop the fouriever toolkit which provides a single solution for analyzing KPI, AMI, and long-baseline interferometry data while accounting for correlated uncertainties in the fits. The toolkit also enables modeling correlations in long-baseline interferometry and AMI data as described in Kammerer et al. (2020) and calibrating science data using a Karhunen-Loève projection based on calibrator data as introduced in Kammerer et al. (2019). The fouriever toolkit is written in Python and can be obtained from GitHub 8 . Many functionalities were inspired by CANDID but have been modified to improve the performance given that fits accounting for correlations involve significantly more complex matrix multiplications. In the following, we briefly outline the search for companions and the estimation of detection limits with fouriever and highlight similarities and improvements with respect to CANDID. We also note that fouriever comes with tutorials and test data for JWST NIRISS AMI, VLT NACO and Keck NIRC2 KPI, and VLTI PIONIER and VLTI GRAVITY long-baseline interferometry applications. Companion search The first step in the analysis of high-contrast imaging data usually consists of the search for one or multiple companions. For this purpose, fouriever can compute a χ 2 -detection map based on binary model fits similar to the fitMap in CANDID but accounting for correlated uncertainties in the fits. This χ 2 -detection map is obtained from a set of least squares gradient de- Table 1. Structure of the KPFITS file format. "Ind" denotes the index of the FITS file extension, "Opt" specifies whether an extension is optional or not, N f denotes the number of frames, N λ denotes the number of spectral channels, N pix denotes the number of pixels per image axis, N sap denotes the number of subapertures, N uv denotes the number of uv-points and N ker denotes the number of kernels. We note that the telescope images are optional and the PRIMARY extension can in principle be an empty array. Ind Opt Name Type Dimensions Description where R = D − M are the residuals between data and model and Σ is the data covariance matrix. For simplicity, other toolkits assume that there are no correlations and that the matrix Σ is diagonal. The model observables are obtained from the complex visibility of a binary source where V 1 and V 2 are the complex visibilities of the primary and the secondary, f is the relative flux of the secondary with respect to the primary, ∆ RA and ∆ DEC are the right ascension and declination offset between the primary and the secondary, u and v are the Fourier plane coordinates for which the complex visibility shall be obtained, and λ is the observing wavelength (e.g., Berger 2003). For the case of an unresolved companion considered here, we set where J 1 denotes the first-order Bessel function of the first kind and b = √ u 2 + v 2 denotes the length of the baseline for which the complex visibility shall be obtained, so that the primary is described by a uniform disk with angular diameter ϑ and the secondary is described by an unresolved point-source (e.g., Berger 2003). The model observables M in the form of squared visibility amplitudes (v2), closure phases (cp), or kernel phases (kp) are then obtained via v2 = \|V \| 2 , where ∠ denotes the phase of a complex number and C andK denote the closure and kernel matrices, respectively, whose rows contain the linear combinations of Fourier phases forming closure and kernel phases. The detection significance is obtained using χ 2 -statistics and computed via where CDF ν denotes the cumulative distribution function of a χ 2 -distribution with ν degrees of freedom, χ 2 r,ud is the reduced χ 2 of the uniform disk only model (i.e., without a companion), and χ 2 r,bin is the reduced χ 2 of the binary model. As in CANDID, fouriever also supports numerical bandwidth smearing which helps to prevent underestimating the companion flux. Moreover, an on-sky search region can be specified where fouriever is looking for companions. This feature is particularly useful if an already known companion resides outside the diffraction field-of-view (FoV) of 0.5λ min /B min and creates aliasing artifacts that could be mistaken for a true companion, where λ min is the shortest observing wavelength and B min is the smallest baseline of the pupil or interferometric array. Searching for additional companions is possible by repeating the computation of the χ 2 -detection map after analytically subtracting the best fit companion model from the data. The uncertainties derived from least squares gradient descent minimizations are usually unreliable if the uncertainties in the underlying data have been wrongly estimated. While CANDID employs the bootstrapping (with replacement) method (Efron & Tibshirani 1986) to extract the uncertainties and correlations of the model parameters, fouriever employs the MCMC method (using emcee, Foreman-Mackey et al. 2013) with a temperature T χ 2 by default equaling the reduced χ 2 of the best fit binary model obtained from the χ 2 -detection map to account for potential over-or underestimation of the uncertainties in the underlying data (e.g., Andrae 2010). The log-likelihood function that is being sampled by the MCMC method is thus given by Both the bootstrapping and the MCMC method yield comparable results and an advantage of the latter is that it can also be used to fit more complex models such as multiple companions simultaneously or geometric disk and ring models to the data (see, e.g., Kammerer et al. 2021;Blakely et al. 2022). Figure 13 shows a companion search in VLTI/PIONIER data of AX Cir from Gallenne et al. (2015) with fouriever and CANDID. For a more direct comparison, we did assume uncorrelated uncertainties and numerical bandwidth smearing evaluated at three uniformly spaced wavelength nodes across the observing bandpass in both cases. The recovered model parameters (host star uniform disk diameter ϑ/diam*, relative companion flux f , right ascension offset of the companion ∆ RA /x, and declination offset of the companion ∆ DEC /y) from the MCMC fit with fouriever and the bootstrapping with CANDID agree well within their uncertainties. Both approaches also reveal a correlation in the model parameters between the host star uniform disk diameter and the relative companion flux. We observe a similar computation time for fouriever and CANDID, although we note that fouriever natively uses a higher resolution grid than CANDID when computing the χ 2detection map and also achieves a similar performance with correlated uncertainties. Detection limits Estimating companion detection limits (also known as contrast curves) is one of the most fundamental pathways to assess the sensitivity of high-contrast imaging observations and to quantitatively interpret non-detections. For this purpose, fouriever offers the same two methods to estimate these limits as CANDID, namely the "Absil" and the "Injection" method. "Absil" method: this method has been introduced by Absil et al. (2011) and refined by Gallenne et al. (2015) and computes the detection limit as the relative companion flux f at which the binary model deviates by a certain probability (e.g., 99.73% for 3-σ) from the uniform disk only model according to that is assuming that the binary model is the true model (see Gallenne et al. 2015 for a more detailed discussion of this choice). This condition is evaluated on a grid around the science target and azimuthally averaged to obtain a contrast curve. "Injection" method: this method has been introduced by Gallenne et al. (2015) and has been reported to be more robust than the "Absil" method if the data is biased or affected by correlations. It consists of analytically injecting a fake companion into the data and then computing the significance of the best fit uniform disk only model over the best fit binary model fitted to the synthetic data with the injected companion. In this case, P bin (Equation 22) sets the detection limit in the form of relative companion flux f . This method is computationally more expensive than the "Absil" method but it resembles more closely the procedure that is undertaken if a real companion is detected. The fouriever toolkit can compute these detection limits accounting for correlated uncertainties in the fits and uses a more aggressive smoothing when azimuthally averaging the detection maps because the sparse uv-coverage especially in long-baseline interferometry observations often results in detection maps showing strong changes in sensitivity at high spatial frequencies (aliasing). This can be seen in the top panels of Figure 14 which also compares the detection limits of the VLTI/PIONIER data of AX Cir from Gallenne et al. (2015) obtained with fouriever and CANDID. For better comparability, we apply the same azimuthal averaging that is being used in fouriever when computing the CANDID detection limits shown in Figure 14. To illustrate how accounting for correlated uncertainties in the fits improves the detection limits, we also model the correlations among the closure phase observables of the AX Cir data considering that two telescope triplets of closing triangles at the VLTI always share one of their three baselines and are therefore not mathematically independent (e.g., Monnier 2007). This results in the correlation models from Section 2.2 of Kammerer et al. (2020) with x = y = 0. Then, we repeat the computation of the detection limits with fouriever using these correlation models in the fits. For the AX Cir data with only four closing triangles and three spectral channels, the correlations are small and the detection limits improve by only ∼ 5% (Figure 14). JWST NIRISS OBSERVATIONS At the high Strehl and the unparalleled thermal stability that can be achieved from space, JWST is an ideal observatory for kernel phase imaging (Sivaramakrishnan et al. 2022). Based on simulations, Sallum & Skemer (2019) predict contrast limits of up to ∼ 8 mag in 90 min of observations (including overheads) at separations of 200 mas with NIRCam which is comparable to the performance achieved by NIRISS AMI for bright targets and more than 1 mag better for faint targets. We note that for NIRISS KPI, Here, we analyze NIRISS CLEARP (full pupil) images that have been taken as part of the instrument commissioning on 23 May 2022 and 5 June 2022 (PID 1093, PI Deepashri Thatte). Besides the NRM, NIRISS is also equipped with a full pupil mask (Figure 1) that can be used for full pupil kernel phase or reference star direct imaging. Four targets were observed, each with two exposures consisting of 232-241 individual integrations resulting in an effective exposure time of ∼ 227-254 s (see Table 3). Each exposure was designed to collect 1e8 photons on the detector according to the JWST Exposure Time Calculator 9 . Since the target acquisition did not work as expected during the first set of observations on 23 May 2022, three of the four targets were reobserved on 5 June 2022. The fourth target was not reobserved because a low-contrast close-in companion candidate was detected around it in the first set of observations so that the target is not useful for estimating companion detection limits for the NIRISS KPI mode. All data were processed with the jwst stage 1 and 2 pipelines and then fed into our custom kernel phase stage 3 pipeline as described in Section 2.1. For the analysis presented here, we assume a central 9 https://jwst.etc.stsci.edu/ wavelength of 4.813019 µm 10 (Rodrigo & Solano 2020) for the F480M bandpass and a detector pixel scale of 65.6 max/pix. Although we only analyze NIRISS data here, we note that the Kpi3Pipeline also supports NIRCam data and a pupil model for the NIRCam full pupil is provided. For the instrument commissioning, two different dither positions on the detector were explored: POS 1 and POS 2 (Figure 7). For each observed target, the first exposure uses POS 1 and the second exposure uses POS 2. However, due to malfunctioning target acquisition, the PSF was accidentally placed on different detector positions during the first of the two runs, so that in total four different dither positions were explored during instrument commissioning. The performance of these four different dither positions is analyzed in Section 4.2. We note that due to the high temporal stability of the observatory and the precise target acquisition procedure, dithering is not recommended for NIRISS KPI (and AMI) observations. Instead, it is recommended to select one dither position and use it for all science and reference star observations throughout the entire observing run. As mentioned in Section 3, we report the discovery of a low-contrast close-in companion candidate around CPD-66 562 in Section 4.1, a target that was chosen for the NIRISS instrument commissioning program because it was believed to be an isolated point-source. Moreover, we investigate the stability of the kernel phase observables throughout the observations and report faint source detection limits for the other three targets in Section 4.2. A low-contrast close-in companion candidate around CPD-66 562 When inspecting the jwst pipeline-processed commissioning data by eye it is already possible to identify a low-contrast close-in companion candidate around CPD-66 562. This ob- servation is supported by the raw kernel phase observables extracted with the Kpi3Pipeline which show a much larger scatter (±0.152 rad) for CPD-66 562 than for the other three targets (±0.007 rad, Figure 8). We note that the theoretically expected kernel phase signal of a centro-symmetric point-source is zero so that larger scatter in the kernel phase observables is always indicative of a detection (e.g., Martinache 2010). A companion search with fouriever reveals the precise parameters of the detected companion candidate. Before, though, we calibrate the raw kernel phase observables of CPD-66 562 using the other three targets as point-source references. Here, we consider the data from both dither positions of the first run on 23 May 2022. Using the Karhunen-Loève calibration class (klcal) in fouriever, the Karhunen-Loève basis of the kernel phase observables of the three reference targets is computed and clipped to K klip = 3 principal components Kammerer et al. 2019). Then, the kernel phase observables of CPD-66 562 are projected into an N ker −3-dimensional subspace that is orthogonal to (and thus independent of) these first three principal components of the reference target kernel phase observables. Then, the location of the companion candidate is derived by computing a χ 2detection map from the calibrated kernel phase observables of CPD-66 562 as described in Section 2.3.1. Finally, the companion parameters including their uncertainties are estimated using an MCMC fit initialized around the best fit position from the χ 2 -detection map (Figure 9). The χ 2 -detection map shows a highly significant detection reaching the numerically set limit of N σ = 8.0. The companion candidate is fairly bright with a relative flux of ∼ 20% of the primary and separated by ∼ 151 mas which corresponds to only ∼ 1 λ/D at λ = 4.8 µm. The small relative uncertainties in the flux of < 1% and in the position of < 1 permille obtained from the MCMC fit demonstrate the high precision at which the kernel phase technique can resolve companions at the diffraction limit. KPI detection limits To evaluate the performance of JWST NIRISS full pupil KPI, we compute 5-σ companion detection limits using the Absil method in fouriever. For this purpose, we only consider the three point-source reference targets that were reobserved on 5 Jun 2022 with successful target acquisition achieving a precision of < 0.1 pixels (Rigby et al. 2022). This also means that we exclude the low-contrast binary CPD-66 562 reported in Section 4.1. First, we investigate the raw kernel phase signal of the three point-source reference targets from the second run averaged over both dither positions ( Figure 10). The average observed scatter is ±0.008 rad and thus comparable, albeit slightly larger, to the first run. This small difference can stem from unflagged (and thus uncorrected) bad pixels in the data from the sec-ond run (e.g., from cosmic rays) or the temporally varying wavefront quality of JWST (due to e.g., wing tilt events, Rigby et al. 2022). Since kernel phase observables are often dominated by systematic errors from higher order phase aberrations in the system (e.g., Kammerer et al. 2019;Martinache et al. 2020), it is common practice to use reference targets for calibrating out these systematic errors. The stability of the system can then be assessed by considering the difference between kernel phase observables from different reference targets. For this purpose, the right panel of Figure 10 shows the kernel phase residuals between each pair of targets observed in the second run (again averaged over both dither positions) and reveals that the signal measured on 2MASS J062802.01-663738.0 seems to be an outlier. Since two slightly different dither positions were used for the two exposures on each target, we repeat the same analysis for each dither position separately. Figure 15 shows 2MASS J062802.01-663738.0 as an outlier in both dither positions. A companion search with fouriever using the other two targets as calibrators reveals that this outlier is roughly consistent with a source at ∼ 240 mas separation and ∼ 0.6% flux ratio (Figure 17). At a contrast of ∼ 5.6 mag, this companion candidate is above the 5-σ detection threshold measured for the other two targets. To exclude the possibility that this detection is caused by wavefront drift between the observations of the different targets, we also analyze the data from the first run for an additional companion candidate around 2MASS J062802.01-663738.0. Figure 16 compares the kernel phase residuals between each pair of targets from the first run and also reveals 2MASS J062802.01-663738.0 as an outlier in both dither positions. A companion search using only the data from the first run also yields a detection towards the South-East = 107.208 +0.038 0.036 Figure 9. Kernel phase detection of a low-contrast close-in companion candidate around CPD-66 562 in the JWST NIRISS full pupil KPI commissioning data. The top two panels show the χ 2 -detection map and the data vs. model kernel phase signal of the best fit binary model obtained with fouriever. In the top left panel, the host star is located in the center and highlighted by a black star and the best fit companion position is highlighted by a black circle. The bottom panel shows the corner plot from an MCMC fit initialized around the best fit position from the χ 2 -detection map with more credible parameter uncertainties than the ones from the least-squares gradient descent minimization shown in the top panels. of 2MASS J062802.01-663738.0, albeit at significantly different separation and flux ratio. As a final check, we use classical PSF subtraction methods to search for the companion candidate in the image plane using only the data from the second run. We build a PSF library with the images of the other two targets (TYC 8906-1660-1 and CPD-67 607) and use the publicly available pyKLIP package (Wang et al. 2015) to subtract the first 20 Karhunen-Loève modes of the PSF library from the science target (2MASS J062802.01-663738.0). The bottom panel of Figure 17 shows the PSF-subtracted image and clearly reveals a point-source at a po- (2) and (3) show a larger systematic kernel phase signal than target (1), but their signals do also agree much better resulting in smaller kernel phase residuals between them and ultimately a much better calibration. sition that is consistent with the best fit from the kernel phase reduction. Hence, the detection around 2MASS J062802.01-663738.0 appears to be real and could be either a companion or a background source. The agreement between the kernel phase and the classical PSF subtraction techniques is a great confirmation of our methodology and a more detailed comparison between Fourier plane and classical imaging techniques is planned for a future publication. Finally, comparing the four dither positions tried during instrument commissioning suggests that the first dither position from the second run should be avoided since it yields a ∼ 3 times larger scatter in the raw kernel phase and a ∼ 2 times larger scatter in the kernel phase residuals. We note that the RMS of the kernel phase uncertainty estimated from the standard deviation over the data cubes is on the order of ∼ 0.0034 rad for all targets and thus slightly larger than the calibration residuals between the best two targets, meaning that with 2e8 collected photons the observations are not yet limited by systematic calibration errors. Next, we compute companion detection limits for each of the three point-source reference targets from the second run. For each of the three targets, we use the other two targets as references and apply the Karhunen-Loève calibration in fouriever Kammerer et al. 2019) with K klip = 2 to calibrate out systematic errors. Then, we use the Absil method in fouriever to compute companion detection limits from the calibrated kernel phase observables of each target. For 2MASS J062802.01-663738.0, we analytically remove the best fit companion before computing the detection limits. Figure 11 shows the detection limits obtained from this procedure. The KPI detection limits show a prominent bump just inside of 300 mas separation which is caused by increased photon noise from the first Airy ring of the PSF. This is highlighted by the gray shaded background showing the azimuthal average of a NIRISS CLEARP F480M PSF computed with WebbPSF 11 (Perrin et al. 2012) in a logarithmic color stretch. For the best target, the detection limits reach ∼ 6.5 mag at ∼ 200 mas and ∼ 7 mag at ∼ 400 mas. These limits agree well with the expectation from the kernel phase calibration residuals of σ KP ∼ 0.002 rad between TYC 8906-1660-1 and CPD-67 607 as shown in Figure 10 and translating into a contrast limit of ∼ 6.75 mag. Compared to the theoretical KPI detection limits predicted by Ceau et al. (2019) using their binary test T B (which is similar to our detection criterion), our on-sky limits are ∼ 1 mag worse. This is consistent with the ∼ 1 mag difference between the theoretical AMI detection limits from Ireland (2013) and the limits measured onsky (see Figure 12). We expect that some fraction of this difference is caused by charge migration which has not been accounted for in the simulations and theoretical predictions. However, a more detailed analysis of the systematic errors is still ongoing. For comparison, Figure 12 shows the detection limits achieved with the NIRISS AMI F480M commissioning observations of AB Dor together with our KPI detection limits. Both the KPI and the AMI observations were designed to collect 2e8 photons on the detector and use the same F480M filter. The AMI data (observations 12 and 13 of PID 1093) were reduced with the jwst stage 1 and 2 pipelines and closure phases and visibility amplitudes were extracted from the cleaned interferograms using a custom version of ImPlaneIA 12 (Greenbaum et al. 2015). Next, the observables of AB Dor were calibrated against those of the two observed reference targets (HD 37093,observations 15 and 16,and HD 36805,observations 18 and 19) using median subtraction. Then, the best fit parameters of the known close-in companion AB Dor C (separation ∼ 326 mas) were obtained with fouriever as discussed in Section 2.3.1 using both closure phases and vis- The gray shaded background shows the azimuthal average of a NIRISS CLEARP F480M PSF computed with WebbPSF in a logarithmic color stretch where darker means brighter to highlight regions of increased photon noise. For comparison, the reference star differential (RDI) imaging contrast limits from the PSF subtraction with 2MASS J062802.01-663738.0 are also shown. We note that this reduction suffers from imperfect image registration and the limits at small separations could likely still be improved. ibility amplitudes in the fit. Finally, the best fit companion was analytically subtracted from the data before computing the detection limits using the Absil method in fouriever. The NIRISS AMI commissioning data reduction and analysis is described in more detail in Sivaramakrishnan et al. (2022). Figure 12 shows that AMI (light blue curve) reaches deeper contrasts than KPI, especially at small angular separations 400 mas. This is the case since the NRM only collects photons on non-redundant baselines whereas the vast majority of the photons collected in KPI with the full pupil are affected by redundancy noise, so that the AMI observations achieve better de-tection limits than the KPI observations with the same number of collected photons. For a fair comparison, it needs to be considered that the NRM needs 5.6 times more time to collect the same number of photons than the full pupil since the NRM has an optical throughput of only 15% while the CLEARP full pupil mask has an optical throughput of 84%. Assuming that the contrast scales with the square root of the number of collected photons (e.g., Ireland 2013) and multiplying the AMI detection limits by 0.84/0.15 results in more comparable limits between AMI (dark blue curve) and KPI. While the scaled AMI contrast curve still achieves better limits at ∼ 250-325 mas where KPI is suffering from increased photon noise from the first Airy ring of the PSF, KPI now achieves better limits beyond ∼ 325 mas where AMI is limited by the reduced throughput and uv-sampling. Finally, we note that the unscaled AMI detection limits (light blue curve) are still ∼ 1 mag above the fundamental noise floor according to Ireland (2013) and future efforts will aim at further improving the performance of NIRISS AMI and KPI (Sivaramakrishnan et al. 2022). SUMMARY & CONCLUSIONS Fourier plane imaging techniques such as KPI and AMI are vital to explore close-in circumstellar environments with JWST in order to directly detect substellar companions or study circumstellar dust (Artigau et al. 2014). In this paper, we present the Kpi3Pipeline data reduction pipeline for kernel phase imaging with JWST and the fouriever model fitting toolkit to search for companions and compute detection limits from KPI, AMI, and long-baseline interferometry data while accounting for correlated uncertainties in the fits. Then, we use these tools to reduce and analyze the JWST NIRISS full pupil KPI data taken during the instrument commissioning. We also motivate and describe the new KPFITS file format for efficiently exchanging KPI and AMI data. Among the four KPI targets observed during the instrument commissioning, we discover a low-contrast close-in companion candidate around CPD-66 562. Due to the low contrast (flux ratio of only ∼ 20%), this companion candidate lies in the stellar and not the substellar regime. Moreover, with 2e8 photons collected on the detector at 4.8 µm, NIRISS KPI achieves 5-σ companion detection limits of ∼ 6.5 mag at ∼ 200 mas and ∼ 7 mag at ∼ 400 mas. This is ∼ 1 mag worse than theoretical predictions, consistent with the ∼ 1 mag difference between theoretically predicted AMI detection limits and those measured on-sky. A detailed analysis of the kernel phase observables extracted from each of the four different dither positions that were tried during the instrument commissioning suggests that the first dither position of the second run yields an increased scatter in the raw and calibrated observables. Furthermore, we find a high-contrast (∼ 0.6% flux ratio) companion at ∼ 240 mas separation around 2MASS J062802.01-663738.0 using both kernel phase as well as classical PSF subtraction techniques. The host star is flagged as a giant in the TESS-HERMES survey (Sharma et al. 2018) so that the detected companion candidate (if not a background source) would also be in the stellar regime. A more detailed analysis of the detection and a comparison between Fourier plane and classical imaging techniques is planned for a future publication. A comparison with NIRISS AMI commissioning observations of AB Dor shows that when correcting for the different throughput between the NRM used for AMI and the CLEARP full pupil mask used for KPI, both techniques perform similar. We find that AMI achieves slightly deeper limits at the smallest separations of 100 mas and between ∼ 250-325 mas where KPI suffers from an increased photon noise from the core and the first Airy ring of the PSF, respectively. In the other regions, KPI performs slightly better due to its better uv-coverage and more compact PSF. However, we note that a major difference between NIRISS AMI and KPI is the range of targets they can observe. While AMI is well suited for bright and nearby targets (bright source limit of m = 3 mag in F480M) for which KPI might saturate quickly, KPI is beneficial for observing faint and more distant targets due to its 5.6 times higher throughput. In comparison with NIRCam KPI, NIRISS KPI offers a more precise target acquisition allowing to center the PSF on the exact same detector pixel every time which is not possible with NIR-Cam KPI. However, NIRCam KPI observations enable simultaneous collection of data in the short wavelength and the long wavelength channels, roughly doubling the observing efficiency. Compared to ground-based extreme adaptive optics-fed instruments, JWST performs better for stars fainter than ∼ 10 mag in the L-and M-band. NIRISS AMI and KPI are expected to outperform current ground-based NRM and PSF subtraction methods at small separations within ∼ 100/150 mas in the L/M-band due to JWST 's high thermal stability. In the future, however, METIS at the E-ELT is expected to reach contrasts of ∼ 2 × 10 −5 at similar separations (Brandl et al. 2021). While fouriever is a stand-alone toolkit for performing calibrations, modeling correlations, and searching for companions in KPI, AMI, and long-baseline interferometry data, we specifically develop this toolkit to enable a uniform and state-of-the-art analysis of both JWST AMI and KPI observations. In combination with the Kpi3Pipeline kernel phase stage 3 pipeline also introduced in this work, fouriever provides a powerful and easy-touse solution for a kernel phase data reduction and analysis of JWST full pupil images to the community. While the current version of the Kpi3Pipeline is compatible with NIRISS and NIRCam images, support for MIRI will be added in a future version. Finally, we collect a number of recommendations mentioned throughout the paper for the convenience of future observers interested in using the KPI technique with JWST : • KPI requires observations of a PSF reference target. To minimize calibration errors, this target should be of similar spectral type as the science target. • Dithering is not recommended with NIRISS. The target acquisition accuracy and pointing stability of NIRISS are sufficient to repeatedly put a target on the same detector position. This is not nec-essarily true for other instruments and observing modes onboard JWST which might be used for KPI in the future. • While offsets can be used to place targets anywhere on the NIRISS detector, it is recommended to chose one of the two predefined positions since these are located on a clean region close to the center of the detector. During commissioning, we found that the first dither position should be avoided since it yields a ∼ 3 times larger scatter in the raw kernel phase and a ∼ 2 times larger scatter in the kernel phase residuals. • The Kpi3Pipeline introduced here extracts kernel phase observables from stage 2-reduced JWST images. Before running the Kpi3Pipeline, users should reduce their data with the jwst stage 1 and 2 calibration pipelines while skipping the inter-pixel capacitance, the photometry, and the resample step. • Users should run at least steps 1 (bad pixel cleaning), 2 (recentering), and 4 (kernel phase extraction) of the Kpi3Pipeline to ensure that they obtain scientifically useful data products (or perform their own custom bad pixel cleaning and recentering before extracting kernel phases). ACKNOWLEDGMENTS These observations were made possible through the efforts of the many hundreds of people in the international commissioning team for JWST. This work is based on observations made with the NASA/ESA/CSA James Webb Space Telescope. The data were obtained from the Mikulski Archive for Space Telescopes at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-03127 for JWST. These observations are associated with program #1093. Support for programs #1194, #1411, and #1412 was provided by NASA through a grant from the Space Telescope Science Institute, which is operated by the Association of fouriever CANDID Δ !" = 6.2 ± 0.1 mas Δ #$% = −28.5 ± 0.1 mas Figure 13. Comparison of a companion search in VLTI/PIONIER data of AX Cir with fouriever (left panels) and CANDID (right panels). While the χ 2 -detection map from fouriever (top left panel) shows the reduced χ 2 of the binary model, the one from CANDID (top right panel) shows the ratio of the reduced χ 2 of the binary model and the best fit uniform disk only model. The reduced χ 2 of the best uniform disk only model is 0.975 in both cases. The best fit companion position is highlighted with a black circle and a red crosshair, respectively. The uncertainties in the top left panel are obtained from a least squares gradient descent minimization and are typically unreliable while the uncertainties in the bottom panels quote the 16th and 84th percentiles of the posterior distribution from the MCMC and the bootstrapping, respectively, and are more credible. Considering these uncertainties, fouriever and CANDID agree with each other to within one sigma. Detection limits (3-) Figure 14. Comparison of the 3-σ companion detection limits obtained for VLTI/PIONIER data of AX Cir with fouriever and CANDID after analytically removing the best fit companion shown in Figure 13. The top left and right panels show the 2D detection limit maps obtained with the "Absil" and the "Injection" method using fouriever and the bottom panel shows the contrast curves (azimuthal averages) obtained from these maps with solid lines. The same two methods using CANDID yield the detection limits shown with solid transparent lines. The differences between fouriever and CANDID (especially at small angular separation) are caused by the detection limits being evaluated on slightly different grids (fouriever centers the host star in the center of a single pixel while CANDID centers it in between four pixels). The dashed lines show how the detection limits improve by ∼ 5% when accounting for basic correlations in the fits with fouriever (cf. Section 2.3.2). Point-source companion fit Figure 17. Companion search around 2MASS J062802.01-663738.0, the (supposedly) point-source reference target that was identified to be an outlier in both NIRISS KPI observing runs. The top left panel shows the χ 2 -detection map and the top right panel shows the data vs. model kernel phase signal as in Figure 9, obtained from a binary model fit to the data from the second run. The bottom panel shows a PSFsubtracted image of 2MASS J062802.01-663738.0 obtained with pyKLIP (20 KL modes). A companion candidate consistent with the one from the kernel phase reduction is clearly detected (white arrow). The image pixel scale is equivalent to the NIRISS detector pixel scale (∼ 65 mas).	2022-11-01T01:16:13.518Z	2022-10-31T00:00:00.000	{ "year": 2022, "sha1": "12c5b9ac3f58d386d639b889910c6cd5d6c73f5a", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "12c5b9ac3f58d386d639b889910c6cd5d6c73f5a", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
247713625	pes2o/s2orc	v3-fos-license	TeachMe: a web-based teaching system for annotating abdominal lymph nodes The detection and characterization of lymph nodes through interpreting abdominal medical images are significant for diagnosing and treating colorectal cancer recurrence. However, interpreting abdominal medical images manually is labor-intensive and time-consuming. The related radiology education has many limitations as well. In this context, we seek to build an extensive collection of abdominal medical images with ground truth labels for lymph nodes recognition research and help junior doctors to train their interpretation skills. Therefore, we develop TeachMe, which is a web-based teaching system for annotating abdominal lymph nodes. The system has a three-level annotation-review workflow to construct an expert database of abdominal lymph nodes and a feedback mechanism helping junior doctors to learn the tricks of interpreting abdominal medical images. TeachMe’s functionalities make itself stand out against other platforms. To validate these functionalities, we invite a medical team from Gastrointestinal Surgery Center, West China Hospital, to participate in the data collection workflow and experience the feedback mechanism. With the help of TeachMe, an expert dataset of abdominal lymph nodes has been created and an automated detection model for abdominal lymph nodes with incredible performances has been proposed. Moreover, through three rounds of practicing via TeachMe, our junior doctors’ interpretation skills have been improved. Colorectal cancer (CRC) is one of the most common causes of cancer death in China and many other countries around the world [1][2][3] . To prevent CRC recurrence and achieve the best prognostic, the detection of abdominal lymph nodes (LNs) is of great importance [4][5][6] . Generally, contrast-enhanced computed tomography (CE-CT) scan of the whole abdomen is a crucial means to identify whether abdominal LNs have metastasis and diagnose CRC. However, there are many limitations when interpreting CE-CTs manually. Firstly, misdiagnosis and missed diagnosis often happen, which dramatically delays the golden time limit for treatment. Besides, it will take a considerable labor cost if all interpretations need to be done by professional senior doctors. Moreover, qualified doctors are limited in some rural areas, for which reason many patients have difficulties seeing a doctor in the early stage of the disease. To address the problem presented above, medical schools must strengthen personnel training to improve junior doctors' professional quality. As for medical education, radiology education is a vital part of the undergraduate medical curricula. It is all about the essential skills to diagnose CRC and other diseases. Worse still, radiology education for junior doctors is also limited at present. Since the skills of interpreting medical images rely significantly on experience, junior doctors need much training to accumulate knowledge to achieve an expert level. But they are often time-poor as they need to balance clinical practice with medical theory learning and other responsibilities. Many of them can not get enough practice at school consequently. Still, their learning outcomes sometimes are not assessed by their tutors in time. Therefore, to promote radiology education in medical school, it is necessary to improve the traditional teaching way in class and create a new teaching method. Furthermore, to solve the limitations in diagnosing CRC fundamentally, creating a support to assist doctors in interpreting medical images is in demand as well. With the rapid development of artificial intelligence and Internet technology, a computer-aided system may provide an outlet for these issues. For one thing, previous studies [7][8][9] have proved that deep neural networks have had tremendous success in solving medical problems over the past few years. It suggests that trianing an accuracy neural network model using a large dataset with precise www.nature.com/scientificreports/ labels is an effective way to reduce doctors' working intensity and improve the efficiency. For another, previous studies 10 have demonstrated the efficacy of radiology-themed online e-learning platforms for senior medical students. Those e-learning platforms provide a more flexible learning style for junior doctors. Motivated by the potential, we intend to develop a computer-aided system to collect an expert dataset of abdominal lymph nodes supporting the research of automated lymph nodes detection models and assist junior doctors to practice their interpretation skills. With this purpose, we do the following analysis and investigation. To start with, what matters first for constructing a dataset of abdominal lymph nodes is a convenient annotating tool for doctors to cooperate with the collection process. Nevertheless, the existing annotating tools are not proper to satisfy the need. Generally, the annotating tools used in hospitals are the customized system. Most of them are desktop applications, which must be part of the hospital's network to gain direct access to the picture archiving and communication systems (PACS). The networking requirement reduces efficiency and limits flexibility. Though there are some other informal annotation tools, such as LabelMe 11,12 , ITK-SNAP 13 and 3D-Slicer 14 , they are still not appropriate to annotate abdominal lymph nodes. LabelMe 11,12 is a web-based annotating tool providing polygon labeling and other functions. But it aims to annotate natural images and is unsuitable for medical images. ITK-SNAP 13 and 3D-Slicer 14 can browse and annotate DICOM (Digital Imaging and Communications in Medicine) images. They also provide much functionality, such as medical image visualization, manual segmentation, and semi-automatic segmentation. However, they do not provide a unified standard of annotations and cannot centrally store the annotation information, which are not convenient for several annotators to collaborate with each other. On the other hand, we have investigated the existing e-learning platforms of radiology education and not found an interactive platform specializing in training interpretation skills for abdominal CE-CTs either. As far as a great teaching platform's concerned, one of the most critical elements is the platform's content provided to the students. Since our purpose is to construct a platform for junior doctors to practice specialized skills in detecting abdominal lymph nodes through CE-CTs, the content must be very reliable and advanced. Furthermore, to achieve better teaching effect, the platform should be interactive. However, the following six existing e-learning platforms are not perfect. Firstly, MyPACS.net 15 and COMPARE Radiology 16 are web-based authoring tools that teachers can create teaching content using medical images. But both of them only focus on passive theoretical information delivery, not providing functionalities for students to practice skills. Though ELERA 17 and Radiology ExamWeb 18 can provide the functionalities to create tests or give the learners guided instruction and feedback, they still focus on the basic concepts, not the expert diagnosis skills. Similarly, RadStax 19 and RadEd 20 can create labels on regions of interest of the medical image and teach how to detect them. But the former is only concerned with introducing information related to these labels, and the latter's teaching content is relatively simple. All in all, these existing e-learning platforms are online classrooms essentially but not clinical practice tutorials. They are not the appropriate platforms for training the skills of abdominal lymph nodes detection, which are complicated and tricky. Based on the points discussed above, this paper introduces TeachMe. TeachMe is not just an annotation tool for annotating abdominal lymph nodes but also a teaching platform providing instruction on practicing abdominal medical images interpretation skills. Junior doctors can conduct specified training of interpretation skills in this system. They can advance their interpretation skills by repeatedly practicing via this system with error informations provided. Thus, the goals of TeachMe are summarized as follows: (1) To provide annotating functionality for making accurate annotations on abdominal CE-CTs; (2) To archive the precise locations and labels of the annotations; (3) To interact with junior doctors and help them improve their interpretation skills for detecting abdominal lymph nodes. To achieve these goals, TeachMe is developed as a web-based teaching system for annotating abdominal lymph nodes with three main modules: a construction process of the expert database for abdominal lymph nodes, an expert database for abdominal lymph nodes, and a feedback mechanism providing the corrections for junior doctor users. To construct the expert databse, TeachMe adopts a three-level annotation-review workflow to collect the annotated data with the gold standard and ensure the expert database's accuracy. The annotations of abdominal lymph nodes stored in the database and the stored information can be exported as a traning dataset easily. TeachMe can teach junior doctors via the feedback mechanism using the stored expert content, which is the most significant difference between TeachMe and other platforms and makes itself stand out as a novel teaching system. To validate TeachMe's functionalities, several gastroenterology doctors in our medical team have used the system, from whom a dataset of abdominal CE-CTs with accurate lymph nodes annotations called PLNDataset has been created. With the dataset, an effective neural network model for detecting abdominal lymph nodes has been proposed in 21 . Moreover, with TeachMe's feedback mechanism, the junior doctors in our medical team has improved their interpretation skills through three rounds of practicing. In this work, the proposed TeachMe provides functionalities to collect an expert dataset of abdominal lymph nodes with the gold standard and tutor junior doctors according to the constructed expert knowledge. The development of TeachMe is the critical first step to promote the research of interpreting CE-CTs automatically and solve the limitations from clinical work. More importantly, it provides a novel way to advance radiology education for junior doctors. System description This section presents TeachMe's architecture with its main modules and its functionalities. Firstly, the architecture of TeachMe is shown in Fig. 1. The main components of the architecture are the client, the back-end, and the database. The client provides functionalities that are realized in the user interfaces through web browsers. The back-end processes users' requests from the client. And the database stores all annotations with gold www.nature.com/scientificreports/ standards and other related information. To get the web page implemented with HTML (the HyperText Markup Language), the client sends requests to the back-end. The web server based on Node.js handles and responds to these requests, providing related data to the client. Moreover, some asynchronous requests realized with AJAX (Asynchronous JavaScript and XML) are sent to the server to retrieve data in JSON (JavaScript Object Notation) format and update the HTML web pages dynamically, without reloading the entire page. As for the requests handling process, the server queries the requested data from the database and provides the processed data to the client. Besides, some functionalities provided by TeachMe are implemented with Python. So the server will call the related built-in programs to realize these functionalities once the client sends the corresponding request to the back-end. Overall, the key technologies to realize TeachMe's architecture are summarized as follows: TeachMe follows a Browser/Server architecture (described in Fig. 1), which is an effective improvement of the Client/Server architecture with the development of Internet technology. The web server is based on Node.js, which is an open-source, cross-platform, back-end JavaScript runtime environment. And the database is based on MongoDB, which is a non-relational database with great performance and expansibility. To implement the interfaces and the application's logic, we used HTML5 and JavaScript, respectively. Moreover, we use Python to write corresponding scripts realizing some functionalities of the back-end, such as merging annotations and the feedback mechanism. As for the development environment, we deploy TeachMe using tmux on the server with a Linux operating system. Since TeachMe follows the Browser/Server architecture, TeachMe and its functionality will be delivered to client machines exclusively through a web browser such as Chrome and Mozilla Firefox. Secondly, Fig. 2 describes the platform's main modules and their functionalities. There are three main modules integrated into TeachMe. The first is the annotating process to construct an expert database, the second is the expert database itself, and the third is a feedback mechanism for correction. All of them are indispensable for teaching purposes. Especially, the feedback mechanism is established on the basis of the constructed expert database because the system computes the feedback information according to the content in the expert database. Thus, constructing the expert database is the first step. Once one case gets annotated and its annotations get reviewed, the expert knowledge of the case will be stored and used for correcting junior doctors' annotations as the "answers". Thirdly, as shown in Fig. 3, the platform supports three user types: primary annotator users, superior annotator users, and junior doctor users. TeachMe provides different functionality for them. Both primary annotator users and superior annotator users participate in the construction of the expert database for abdominal lymph nodes. The primary annotators are responsible for submitting the preliminary annotation results. The superior annotators are responsible for reviewing and confirming the final results. In contrast, junior doctor users are the service object of the system. The system serves the junior doctor users based on the knowledge in the expert database. They are just like the examinees who take part in an exam of detecting abdominal lymph nodes. So they are welcome to give their answers as many as they can. The system will make corrections by comparing the results with the annotations stored in the expert database. To introduce more details, the following subsections present TeachMe's three main modules, respectively. Three-level annotation-review workflow. The three-level annotation-review workflow is realized by the human experts and a well-trained automoted detection model together. They work together to construct the expert database of abdominal lymph nodes. Human experts refer to primary annotators and superior annotators. First and foremost, paired primary annotators annotate a given CT study independently. When they finish the annotations, TeachMe merges the same annotations and keeps the different annotations of the paired www.nature.com/scientificreports/ primary annotators. Next, the system marks the annotators' names for each annotation as a signature. Then TeachMe stores the annotation information, creating the prototype of the expert database. Based on this prototype, TeachMe compares the predictions of the automated detection model with the first-level annotation results to obtain the candidates of lymph nodes that all first-level annotators have missed. In the end, a given superior annotator will check the annotations that have been calibrated by the model and make corrections. They need to confirm if there are incorrect annotations or omitted lymph nodes. Thus, the accuracy of the expert database System allocates cases for annotators. Two primary annotator users annotate one case severally. System merges the annotations. System makes the predictions of AI as a supplement to the results. One superior annotator user review the results. System stores the final audited results as expert knowledge. One junior doctor select one case to practice. The junior doctor user annotates the case. System compares the results with the annotations stored in expert database. System sends the error information to the junior doctor user. Expert database of abdominal lymph node. Apart from the information of locations and classifications of lymph nodes, the expert database includes other essential details. For one thing, the annotated start slice and end slice are important parts of the expert knowledge. The start slice refers to the frame when the aorta starts to separate to the right common iliac artery and left common iliac artery. And the end slice refers to the section when the symphysis pubis part starts to bifurcate. For another thing, TeachMe labels a specific index based on a particular sorting rule for all annotations. Every time annotators create a new annotation, TeachMe will calculate its index according to its location and classification. Here is the sorting rule: if there is more than one annotated region of the same type almost at the same horizontal level on one slice, the order of these annotations will ascend from left to right breadthways; If there is more than one annotated region of the same type, almost at the same vertical level on one slice, the order of these annotations will be descending bottom to top lengthways. Besides, the signatures of annotations are also important information of these annotations. Correction for junior doctors' annotation results. When a junior doctor user uses TeachMe to practice the skills of detecting abdominal lymph nodes, he/she should choose a study, view the study frame by frame and make annotations as much as possible by oneself. When he/she submits the final results, TeachMe will compare the results with the gold standard knowledge stored in the expert database. Every time a junior doctor user finish an exercise, TeachMe will analyze and record the neglected region and the wrong labeled region. To analyze the annotation results, TeachMe excludes the missed annotations and the wrong labeled annotations, so the rest annotation regions are the correct regions included in the gold standard annotations. Then TeachMe assesses if the rest annotations' classification labels are accurate. After this process, TeachMe replies to the junior doctor with a report about how to correct his/her annotations. Thus, junior doctors can improve their ability to detect the abdominal lymph nodes through the annotation practice in TeachMe again and again. What is more, TeachMe can also reply with the correct start slice and the end slice if the junior doctor has labeled them wrong. User interfaces of TeachMe There are two parts of the user interface (UI) in TeachMe: the UI of management of annotation tasks and the UI of annotation canvas. Moreover, there are three main roles in TeachMe: primary annotators users, superior annotators users, and junior doctor users. Figure 4 shows the annotation task management page. The reviewing task management page approximately the same, listing the table of annotation/review studies the annotator needs to annotate. Annotators can click the corresponding study and enter the annotating/reviewing page. The junior doctor users' annotation task management page slightly differs as shown in Fig. 5. It has the dialog box indicating which exercise TeachMe has corrected. The junior doctor user can click the corresponding one to enter into the viewing page and check. The UI of the annotation canvas roughly the same. Figure 6 illustrates the annotating/reviewing page. The annotation canvas is on the left, displaying the medical images of the selected study. Users create all annotations on the canvas. Users can scroll the upright progress bar to slide the CT images and get a quick look through. When users find out an abdominal lymph node, they just need to drag the mouse drawing a box and the region will be annotated. If the annotation is not proper, they can Figure 10 shows the annotating page of the working interface. The dialog box on Fig. 10 shows the detailed correction information for the junior doctor user. Each line in the dialog box displays a message of one annotation, and when the junior doctor user clicks one of them, TeachMe will highlight the corresponding incorrect annotation. The correction information includes which annotations the junior doctor omitted, mistaken, and not quite right. For the record, the omitted annotations refer to those lymph nodes not annotated by the junior doctor user but exist in the gold standard. The incorrect annotations refer to the annotated region that the junior doctor user drew that does not contain gold standard lymph nodes. And the annotations not quite right refer to the annotations' labels are wrong compared with the gold standard. Results and discussions User impressions. To collect the dataset of abdominal lymph nodes and construct the expert database, we finish three rounds of data collection with the participation of gastroenterology doctors from West China Hospital. We invite six PhDs as primary annotators and two gastroenterology experts as superior annotators. They participated in the annotation-review workflow and finished the first round of data collection, creating a dataset consisting 103 CE-CT studies with 2171 abdominal lymph nodes annotated and reviewed. Then, we use the collected dataset to construct a small-scale expert database and train a neural network model. In the subsequent two rounds of data collection, the neural network model started participating in the annotationreview workflow supporting the doctors. In the end, a large-scale of dataset has been created successfully, which is introduced in subsequent subsection. Besides, to test the feedback mechanism of TeachMe, we invite three undergraduates in our medical team to annotate different assignments independently. When they finish each of the studies, TeachMe will contrast their annotations with the "answer" stored in the expert database and provide them immediate feedback, showing them the error information. Long-term exercising and error correction allow them to discover their areas of weakness and come to understand what they should concentrate on when restarting new exercising. As shown in Fig. 11, two users' accuracy of detecting the abdominal lymph nodes has improved significantly after three rounds of exercises. Though user C got his worst performance in the second round, the difference in his accuracy between the first two rounds is not significant and in a normal range. The possible reason for this situation is that the junior doctor's state fluctuated at that time affected by some disturbances. Moreover, after using TeachMe for a period of time, all doctors expressed their impressions on the platform. All of them considered TeachMe is practical and easy to use. For the annotators, they think TeachMe is valuable to collect medical datasets and helpful for their medical research. For the junior doctors, they agree that TeachMe can tell them some tiny lymph nodes that they should have detected but omitted and it does help them to improve their skills. Comparison with other systems. The following features distinguish TeachMe from other annotating tools: www.nature.com/scientificreports/ (1) Three-level annotation-review workflow to ensure the accuracy of the expert database; Primary annotators annotate each study first. A neural network model successively supplies candidates omitted. Superior annotators confirm the annotations at last. The three-level annotation-review workflow plays a role as a peer-review mechanism to guarantee the content's reliability. (2) A Built-in unified sorting rule for annotation; Each annotation has a given serial number based on a specific law. It is convenient for doctors to track the lymph nodes for the follow-up study. Moreover, it provides a straightforward way of referring to a specific lymph node during a consultation. (3) A feedback mechanism to make assessment and interact with junior doctors; TeachMe can make assessments and reply to the junior doctor users with the corrections of annotations. It is convenient for junior doctors to discover their weakness and improve their interpretation skills. www.nature.com/scientificreports/ In Table 1, we compare TeachMe with other platforms we mentioned in the Introduction. Equipped with the three-level annotation-review workflow to peer-review the teaching content, a unified annotating rule to guarantee teaching content's consistency and a feedback mechanism to make assessments, TeachMe is more professional as a teaching platform for practice advanced clinical skills. Thus, more considerate functionalities make TeachMe stand out. Dataset and model. After three rounds of data collection, PLNDataset is constructed. It contains 236 CRC patients' CE-CT with 6880 abdominal lymph nodes' position and classification information in total. As mentioned above, the 6880 abdominal pelvic lymph nodes' annotations have been made and checked strictly by professional gastroenterology doctors via the three-level annotation-review workflow. After annotators draw the bounding box of the lymph node using the annotation tool in TeachMe, they will select the label to which the lymph nodes' region belongs. After careful consideration, our gastroenterology experts decided to divide the abdominal pelvic lymph nodes into seven regions, which we call the seven partitions of abdominal lymph nodes. Figure 12 illustrates the partition distribution of annotated lymph nodes in the three rounds of data collection. These information plus the pelvic region's start and end frame are the final expert knowledge of abdominal lymph nodes collected by TeachMe. With these knowledge, more valuable research can be conducted. We propose an automated lymph node detection method in 21 PLNDataset. The proposed method uses the spatial prior knowledge to detect the pelvic region on the CE-CT images at the first stage. Then, a neural network model with two main pathways is applied to detect and locate lymph nodes all over the pelvic area. The model is trained using the annotated data without reviewed in the first place to obtain a corase detector. Later on, we use the annotated data reviewed by superior annotators to train the model again to improve the model's performance. Table 2 shows the model's performances with metrics of sensitivity and mFROC. For the record, sensitivity and mFROC are effect evaluating indicator frequently used in image recognition tasks. To be specific, the metric sensitivity is defined as the proportion of all the true lymph nodes detected to all the real lymph nodes annotated. The free-response receiver operating characteristic curve (FROC) describes the variation of the sensitivity as the average number of false-positives (FPs) per CT study changes. Furthermore, the metric mFROC (mean FROC) is defined as the average sensitivity at eight default false-positive rates, which are 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 FPs per CT study. The higher of the two metrics that the neural network model can achieve, the more accurate the model can obtain. As shown in Table 2, the model without calibration achieved 79% in sensitivity and 35.1% in mFORC. The benchmarks are increased to 83% and 39.1% with calibration. The results indicates the model's performance got promoted using calibrated data. It implies the expert database of authentic annotated medical www.nature.com/scientificreports/ images is valuable, for the reviewing process can contribute to the model's performance. We are now making great efforts to decrease the model's FP predictions and perform some partition detection research. With the help of TeachMe, it is expected that more incredible method of detecting abdominal lymph nodes will be proposed. What is more, as the technologies of model compression and acceleration such as knowledge distillation 22 progress, to deploy the detection model on mobile devices can be expected soon. Ethical declarations. This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare that all methods were carried out in accordance with relevant guidelines and regulations. All medical images in the study are retrieved from West China Hospital's picture archiving and communication system (PACS). All experimental protocols were approved by West China Hospital. The study is a retrospective study, which does not involve clinical intervention. All the private and sensitive information of the medical images are eliminated. The Ethics Committee on Biomedical Research, West China Hospital of Sichuan University, has approved that the qualifications of researchers and the research approach meet ethical requirements. Moreover, they have confirmed the ethical approval that exempts this study from the signature of written informed consent. Future work TeachMe is a web-based teaching system for annotating abdominal lymph nodes, easy to access without installation. It provides a three-level annotation-review workflow to collect an expert dataset for automated abdominal lymph nodes research. Also, it is a novel teaching platform for junior doctors to train their interpretation skills via its feedback mechanism. However, there are some objectives that TeachMe should fulfill for improvements: (1) At present, TeachMe doesn't support DICOM format medical images directly. Medical images demonstrated in TeachMe are with fixed settings like window width, window level. Users can not adjust the brightness and contrast to get the most transparent view for deliberating the tricky regions. Thus, supporting the DICOM format is a demand to improve the experience of annotating. (2) Since we design TeachMe for the detection of abdominal lymph nodes at the outset. The rectangle annotation tool can be completely satisfied for this demand. But when it comes to abdominal lymph nodes segmentation, it is more appropriate to annotate the abdominal lymph nodes with annotation tools like bidirectional cross-shaped annotation tool or free sketch annotation tool. Thus, TeachMe should provide more types of annotation tools for different annotation tasks, which will make the system more flexible. (3) As TeachMe is custom-designed for gastroenterology surgery doctors, the labels set in TeachMe are the classifications of abdominal lymph nodes that the superior annotators formulated and confirmed by all the experts in our medical team. At present, TeachMe is only suitable for abdominal lymph node annotation tasks. If the system enables the superior annotators to formulate customized labels, TeachMe can be ideal for more scenarios.	2022-03-27T06:22:37.848Z	2022-03-25T00:00:00.000	{ "year": 2022, "sha1": "68818ea40f5799e45a360688e79d7d514324b662", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "ea438aa89da99ba086662d9584c724ea134f09d3", "s2fieldsofstudy": [ "Medicine", "Computer Science" ], "extfieldsofstudy": [ "Medicine" ] }
103647192	pes2o/s2orc	v3-fos-license	A fluorescence and UV/vis absorption dual-signaling probe with aggregation-induced emission characteristics for specific detection of cysteine Biological thiols with similar structures, such as glutathione (GSH), N-acetyl-l-cysteine (NAC), homocysteine (Hcy) and cysteine (Cys), play important roles in human physiology and are associated with different diseases. Thus, the discrimination of these thiols is a great necessity for various biochemical investigations and the diagnosis of related diseases. Herein, we present a new dual-signaling probe consisting of a typical aggregation induced emission fluorogen of a tetraphenylethylene group and 2,4-dinitrobenzenesulfonyl moiety. The probe can be used to selectively and quantitatively detect Cys over a variety of bio-species, including GSH, NAC and Hcy, from both UV/vis absorption and fluorescence channels. The mechanism study showed that the fluorescence and UV/vis absorption were turned on as the probe undergoes displacement of the 2,4-dinitrobenzenesulfonyl group with Cys, where the UV/vis and fluorescence signals originate from the dinitrophenyl-containing compounds and aggregates of TPE-OH, respectively. In addition, the discrimination of Cys was achieved by more rapid intramolecular displacement of sulfur with the amino group of Cys than NAC, Hcy and GSH. Moreover, the probe shows ignorable cytotoxicity against HepG2 cells, which demonstrates the great potential of the probe in selectively detecting Cys in vivo. Introduction Biological thiols such as glutathione (GSH), N-acetyl-L-cysteine (NAC), homocysteine (Hcy) and cysteine (Cys) play essential roles in human physiology. [1][2][3][4][5][6] Although these thiols have similar structure, their roles in biological processes are diverse and associated with different diseases. For instance, GSH is a pivotal indicator of the maintenance of xenobiotic metabolism, intracellular signal transduction and gene regulation, which is highly involved in the aging and the pathogenesis of many diseases. 1,2 Since NAC was found to be a precursor of GSH in cells and a thiol antioxidant, it shows the potential to prevent cancer and other mutation-related diseases. 6 Hcy is a risk factor for Alzheimer's and cardiovascular diseases. 3,4 Abnormal level of Cys would lead to slow growth, skin lesions, liver damage and edema etc. 5 Due to the critical role of these thiols in human health, the selective detection of these thiols is of great importance and a necessity for various biochemical investigations and the diagnosis of related diseases. However, it is challenging to selectively and quantitatively detect these thiols because of the structural similarity of Cys, Hcy, NAC and GSH. [7][8][9][10] In the past few years, some uorescent and colorimetric probes have been developed to selectively detection Cys in terms of high sensitivity and simplicity of operation. 10,11 However, most of these probes send out only one signal based on the same chemo-sensor molecule, 12-14 which may be inuenced by possible variations in the sample environment, especially in biological surroundings. In order to improve the accuracy of the results, the utilization of dual signaling probes is one of effective alternatives, which allows mutual correction on the same target analyte, yet through different transduction channels. [15][16][17][18][19][20] However, dual signaling probes for Cys are still very limited so far. 10,11,21 In 2001, Ben Zhong Tang reported an abnormal phenomenon of aggregation induced emission (AIE) of silole, which shows nonuorescence in solutions but strong emission once aggregated. [22][23][24] By taking advantage of attractive AIE phenomenon, a large number of intelligent AIE uorogens susceptible to external stimuli, such as bio-species, mechanical force, temperature change, pH variation, toxic vapor, and photonic irradiation have been explored and developed. 25 Tetraphenylethylene (TPE) is a typical AIE uorogen, 26-30 which has been widely used for the construction of various uorescent sensors due to its signicant increase in uorescence upon aggregation, low cytotoxicity and ease in synthesis and modication. [31][32][33][34][35][36][37] Just recently, Wang and co-workers reported a new TPE-based AIE probe bearing a 2,4-dinitrobenzenesulfonyl pendant, which is able to detect Cys selectively over Hcy and GSH in PBS. 38 In this work, they found that uorescence would signicantly increase upon the substitution reaction between the probe with Cys, which would result in the leave of uorescent quencher of 2,4dinitrobenzenesulfonyl group from TPE moiety. Although previous works showed that some 2,4-dinitrobenzenesulfonylbased probes were not able to discriminate Cys from GSH or Hcy, 39,40 this probe demonstrated a strong selectivity to Cys over Hcy and GSH. All these results showed that the whole structure of probes, besides 2,4-dinitrobenzenesulfonyl itself, also affect the reactivity of substitution reaction toward thiol compounds, especially the reaction kinetics. [38][39][40] Herein, a TPE-based dual signaling probe was synthesized by introducing a 2,4-dinitrobenzenesulfonyl group to a TPE moiety (Scheme 1). 41 It is assumed that the 2,4-dinitrobenzenesulfonyl group would not only make the TPE-based probe more soluble in aqueous media, but also quench the possible uorescence of TPE through photo-induced electron transfer (PET). Thus, the replacement of 2,4-dinitrobenzenesulfonyl group by Cys would not only signicantly increase the uorescent of TPE by the combination of AIE and PET effect, but also enhance the UV/vis absorption due to the producing of dinitrobenzene-based compound. In addition, it is hypothesized that the discrimination of Cys could be achieved by more rapid intramolecular displacement of sulfur with amino group of Cys than NAC, Hcy and GSH. Therefore, the probe would be able to selectively detect Cys over NAC, Hcy and GSH through the combination of uorescence and UV/vis absorption from two separated transduction species. To test this hypothesis, the performance of TPE-NP for selectively detecting Cys over other various biospecies, including NAC, Hcy and GSH were examined by Fluorescent and UV/vis absorption spectroscopy and the mechanism behind was also investigated by the combination of HPLC-MS and 1 H NMR. It should be pointed out that although the strategy is similar to that of previous report, 38 where only uorescent channel was employed to detect Cys, a dual signaling approach was develop in the current work, which will allows to mutual correction on the same target analyte to get more reliable results. In addition, the reaction mechanism for selectively detecting Cys was systematically investigated in the current work, which is of great benet to design highly selective and sensitive dual signaling probes for detecting biological thiols. Instrumentation All 1 H and 13 C NMR analyses were performed on a JEOL resonance ECZ 400S (400 MHz) in DMSO-d 6 , tetramethylsilane was used as internal standard. Electron impact ionization mass spectrometry (EI-MS) was performed by an Agilent 5937N system. FT-IR spectra were recorded on a Nicolet AVATAR-360 spectrophotometer with a 4 cm À1 resolution. UV/vis absorption spectra were recorded on a Hitachi U-2910 spectrophotometer. Fluorescence spectra were measured by a Hitachi F-2700 uorescence spectrophotometer with a band width of 2.5/5.0 nm. UV/vis and uorescence measurements were performed under room temperature. Synthesis of 4-(1,2,2-triphenylvinyl)phenol (TPE-OH) Benzophenone (7.26 g, 39.85 mmol), 4-hydroxybenzophenone (7.90 g, 39.85 mmol), zinc powder (11.46 g, 175.34 mmol) and THF (200 mL) were added to a three-neck ask equipped with a magnetic stirrer under N 2 . The mixture was cooled to 0 C followed by adding TiCl 4 (16.63 g, 87.67 mmol) slowly via a constant pressure drop funnel while the temperature was kept under 0 C. The suspending mixture was warmed to room temperature, stirred for 0.5 h and then heated at reux for another 15 h. The mixture was cooled to room temperature and charged with HCl (320 mL, 1 M). The mixture was extracted by CH 2 Cl 2 and the organic layer was dried over MySO 4 and concentrated. The crude product was puried by silica column chromatography (eluent: n-hexane/ethyl acetate, v/v ¼ 4/1) to give 4-(1,2,2-triphenylvinyl)phenol (TPE-OH) (9.02 g, 65%) as a white powder. 1 Cytotoxicity assay in vitro against HepG2 cells The cytotoxicity of the samples were assessed against HepG2 cells with the CCK-8 assay. In brief, the HepG2 cells were respectively incubated with the TPE-NP with different concentrations for 24 h. Aer that, the cells were resuspended in fresh medium and treated with DMEM solution. Furthermore, the control group was only treated with physiological saline. The evaluation of cytotoxicity was determined by cell viability which was calculated on the basis of the absorbance of control group and the absorbance of treated group. Selectively and quantitatively detection of Cys by TPE-NP Aqueous solution of TPE-NP (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) is colorless because of the absence of absorption features in the visible region ( Fig. 2A). The solution exhibits no uorescence, probably due to relatively high solubility of TPE-NP in the media and photo-induced electron transfer between tetraphenylethene group and dinitrophenyl moiety ( Fig. 2C). 15,42,43 Upon the addition of Cys (200 mM), the colorless solution became bright yellow. This observation showed that TPE-NP could be a "naked-eye" probe for Cys. Meanwhile, the absorption band at around 350 nm increased steadily over time and reached plateau aer about 2 h (Fig. 2B). Upon the addition of Cys, a new emission peak located at 450 nm emerged and a dramatic uorescence enhancement was observed in Fig. 2C. The time-dependent uorescence response was also detected, which showed that the uorescence intensity at 450 nm almost does not change before 50 min but then increased sharply to about more than ten times of the weak background aer 80 min (Fig. 2D). Although the steady increase of absorption at 350 nm indicated that the displacement reaction occurred continually upon the addition Cys, the product of TPE-containing moiety did not aggregate until its concentration is higher than its critical aggregation concentration. This might be the reason why the uorescent intensity just increased obviously aer around 50 min upon the treatment with Cys. We then incubated the solution with various concentrations of Cys (20-400 mM) for 2 h. One can notice that both UV/vis absorption and uorescent intensity of TPE-NP solution increase with the increasing in the concentration of Cys ( Fig. 3A and B). We plotted the absorbance intensity at 350 nm and uorescence intensity at 450 nm of TPE-NP solution as This journal is © The Royal Society of Chemistry 2018 a function of the concentration of Cys. Both absorbance and uorescence intensities are linearly proportional to the concentration of Cys (insets of Fig. 3A and B), resulting the detection limit of Cys in PBS low to 0.21 mM (S/N ¼ 3) from uorescent channel. In previous reports on the detection of Cys by uorescent sensors, the detection limit is in the range from 0.121 mM to 1 mM. [44][45][46] Especially, the detection limit of Cys for a TPE-based probe reported by Wang and co-workers is about 0.18 mM, close to the value of TPE-NP. 38 All these results show that Cys can be quantitatively detected on the basis of the change in UV/vis absorption and uorescence intensities. Especially, GSH, Hcy and NAC, three thiol-containing compounds as Cys, did not cause obvious change in uorescent emission, even the amount of Hcy and GSH are ten times of that of Cys ( Fig. 2D and 4C). All these results show that TPE-NP has a high selectivity toward Cys over Hcy, NAC, GSH and other related bio-species, even if the only difference of Hcy and NAC from Cys is a methylene or an acetyl group at their residues. Mechanism of detecting Cys by TPE-NP Since dinitrophenyl group is a well-known uorescent quencher, the uorescence of TPE-NP is probably quenched by dinitrophenyl group due to PET effect. 15,42,43 On the basis of previous reports, 15,42,43 we speculated that the displacement of dinitrophenyl group of TPE-NP upon the treatment with Cys gave TPE-OH and dinitrophenyl-containing compounds (Scheme 1). Thus, uorescence emission of TPE-OH at 450 nm was turned on and the produce of dinitrophenyl-based compound resulted in the yellow color of solution (Scheme 1). In order to testify the yellow color originated from dinitrophenyl-containing compound, we synthesized a new sensor of 4-phenyl 2,4-dinitrobenzenesulfonate (phenol-NP) by bio-species, including amino acids (aspartic acid, glutamic acid, phenylalanine, tyrosine, threonine, arginine, histidine, asparagine, leucine, alanine, proline, valine, glycine, lysine, glutamine, methionine, isoleucine, serine, tryptophan, GSH and Hcy), metal ions (Na + , K + , Ca 2+ and Mg 2+ ), H 2 O 2 , vitamin C and NAC for 2 h, respectively. the reaction between phenol, instead of TPE-OH, and 2,4-dinitrobenzenesulfonyl chloride (Fig. 5A). Aer the aqueous solution of phenol-NP (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) was treated with Cys (200 mM), the colorless solution turned yellow (Fig. 5B) and the absorption with a maximum wavelength at about 350 nm obviously increased (Fig. 5A). Whereas, aqueous solutions (PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) of TPE-OH and phenol is colorless (Fig. 5B). Thus, these results indicate that the uorescence and UV/vis absorption (yellow) are originated from the aggregates of TPE-OH and dinitrophenylcontaining compounds, respectively. This means that the dual signals of UV/vis absorption and uorescence for detecting Cys quantitatively and simultaneously based on TPE-NP probe are originated from two separated signaling molecules. Thus, the cross-referencing of the obtained results can be realized by this approach, which undoubtedly makes the results more reliable. To verify the mechanism of the reaction of TPE-NP with Cys, the products of the reaction were analyzed by HPLC and 1 H NMR ( Fig. 6A and B). We can see that a peak at 20.1 min appears in HPLC curve (Fig. 6A) aer TPE-NP was incubated with Cys for 2 h, where the retention time is the same as that of TPE-OH (20.1 min). Moreover, the molecular weight of this compound is 348.1 g mol À1 , consistent with that of TPE-OH (348.1 g mol À1 ). One also can notice that two characteristic proton resonance signals of TPE-OH at 6.47 and 6.70 ppm and three peaks originated from the protons of dinitrophenyl group at 8.95, 8.86 and 7.23 ppm appear aer the incubation (Fig. 6B). These observations show that TPE-OH is one of the products of the reaction and the up-shi of signals from dinitrophenyl moiety suggests the leave of electron withdrawing group. Since all of Cys, Hcy, NAC and GSH contain a thiol group, one inevitably raises the question why Cys has much higher reactivity toward TPE-NP than Hcy, NAC and GSH. On the basis of previous reports on the detection of thiols using nitrophenylcontaining sensors 15,42,43 and the above observations, a possible mechanism is proposed as shown in Fig. 7A. At pH ¼ 7.4, the deprotonated thiols of Cys, Hcy, NAC and GSH would serve as active nucleophile to attack the dinitrophenyl of TPE-NP for generating a sulfur-substituted intermediate (Fig. 7A). The primary amine of Cys, Hcy and GSH would further attack the dinitrophenyl to form thermodynamic ve-, six-or tenmembered cyclic transition state by intramolecular cyclization, which would assist the leave of TPE-containing moiety to give TPE-OH and SO 2 . Since ve-membered ring is much more favored conformation than six-and ten-membered ring, the reaction rate for Cys would be much faster than those of Hcy and GSH. To verify the importance of amino group in accelerating the displacement reaction, TPE-NP solution was incubated with butanethiol and 3-mercaptoisobutyric acid for 2 h. In comparison to Cys, both butanethiol and 3-mercaptoisobutyric acid almost did not lead to obvious change in uorescence and UV/vis spectra ( Fig. 7B and C). The low reactivity of TPE-NP toward NAC further shows the importance of amino group in accelerating the displacement reaction. All these results demonstrate that the proposed reaction process is reasonable. In vitro cytotoxicity of TPE-NP Finally, we performed proof-of-concept in vitro experiments on the examination of the in vitro cytotoxicity of TPE-NP against the HepG2 cells, which was evaluated by means of a standard CCK-8 cell assay. It could be seen that the cell viability of TPE-NP is higher than 95% for HepG2 cells in a range of concentrations from 5 to 50 mM, respectively. These results show that TPE-NP have ignorable cytotoxic effect on the HepG2 cells in a range of concentration (5-50 mM, 2.89-28.9 mg mL À1 ), which demonstrates the potential of TPN-NP in selectively detecting Cys in vivo (Fig. 8). Conclusions In summary, a simple colorimetric and uorescent dual signaling probe has been developed, and the probe can selectively and quantitatively detect Cys from other common biospecies including Hcy, NAC and GSH. The uorescence and UV/vis absorption are originated from the aggregates of TPE-OH and dinitrophenyl-containing compound, respectively. This allows the mutual correction through different transduction channels and signaling reporters, which will make the result more reliable. It is proposed that the discrimination of Cys from Hcy, NAC and GSH is achieved through the faster displacement of sulfonate attaching to dinitrophenyl with thiolate for Cys, followed by subsequent substitution of the thiolate by the amino group of Cys. Given the low cytotoxicity of TPE-NP, the interesting reaction mechanism provides a strategy for designing highly selective and sensitive dual signaling probes for detecting Cys in vitro and in vivo. Conflicts of interest There are no conicts to declare.	2019-04-09T13:11:14.267Z	2018-07-02T00:00:00.000	{ "year": 2018, "sha1": "01e3b88387a2ed6903c34e04bfa8d51d6cf04a3f", "oa_license": "CCBY", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2018/ra/c8ra03756f", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "825dcd8566356a60c6db3902f1b32a4c95a1091a", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
19012419	pes2o/s2orc	v3-fos-license	Transcriptional Consequence and Impaired Gametogenesis with High-Grade Aneuploidy in Arabidopsis thaliana Aneuploidy features a numerical chromosome variant that the number of chromosomes in the nucleus of a cell is not an exact multiple of the haploid number, which may have an impact on morphology and gene expression. Here we report a tertiary trisomy uncovered by characterizing a T-DNA insertion mutant (aur2-1/+) in the Arabidopsis (Arabidopsis thaliana) AURORA2 locus. Whole-genome analysis with DNA tiling arrays revealed a chromosomal translocation linked to the aur2-1 allele, which collectively accounted for a tertiary trisomy 2. Morphologic, cytogenetic and genetic analyses of aur2-1 progeny showed impaired male and female gametogenesis to various degrees and a tight association of the aur2-1 allele with the tertiary trisomy that was preferentially inherited. Transcriptome analysis showed overlapping and distinct gene expression profiles between primary and tertiary trisomy 2 plants, particularly genes involved in response to stress and various types of external and internal stimuli. Additionally, transcriptome and gene ontology analyses revealed an overrepresentation of nuclear-encoded organelle-related genes functionally involved in plastids, mitochondria and peroxisomes that were differentially expressed in at least three if not all Arabidopsis trisomics. These observations support a previous hypothesis that aneuploid cells have higher energy requirement to overcome the detrimental effects of an unbalanced genome. Moreover, our findings extend the knowledge of the complex nature of the T-DNA insertion event influencing plant genomic integrity by creating high-grade trisomy. Finally, gene expression profiling results provide useful information for future research to compare primary and tertiary trisomics for the effects of aneuploidy on plant cell physiology. Introduction Aneuploid cells contain a chromosome variant with chromosome number other than a multiple of the basic (monoploid) chromosome number (x), which may alter large-scale gene expression leading to cellular malfunction and diseases due to genomic imbalance [1,2]. Aneuploidy results from failure of the chromosomes or chromatids to separate properly to opposite poles during meiosis or mitosis. Trisomy (2n52x+1) is a type of aneuploidy that involves an extra copy of a particular chromosome rather than the normal number of 2 [2]. In humans, trisomy causes severe developmental defects in fetuses, and only newborns with a few types of trisomy can survive [3]. The most commonly known disease caused by trisomy in humans is Down syndrome, which affects individuals with an extra chromosome 21 in whole or in part [4]. Recent work has suggested that the unbalanced genome in aneuploidy may result in loss of genomic integrity and epigenetic changes in many organisms [5][6][7]. Genes on the aneuploid chromosomes frequently show a dosage compensation effect that probably helps mitigate the harmful consequences of genomic imbalance. Despite the detrimental effects in cells, aneuploidy sometimes provides a means for adapting to selective pressure [8]. Aneuploidy is highly associated with poor prognosis, high malignancy and increased drug resistance in tumorgenesis [9][10][11]. Studies of humans, mice and yeast suggest that an abnormality in chromosome number or structure alters cellular physiology by resulting in changes in genome stability, imbalanced protein homeostasis and numerous dysfunctional growth characteristics (see reviews in [10,12,13]). Whole-chromosome aneuploidy, which can be used to examine the physiological effects in aneuploid cells, is relatively easy to generate and maintain than high-grade aneuploidy [5,14]. However, the molecular mechanisms and transcriptional signatures underlying the organismic physiological alterations of aneuploidy are not fully understood [14]. Considering the diverse phenotypes induced by different degrees of aneuploidy, whether the knowledge from the studies of simple aneuploidy can be informative for understanding the role of complicated aneuploidy in cancer and genetic diseases is unclear [15]. Thus, further studies of the cellular physiology of aneuploidy will increase our understanding of the causal effects and consequences of genomic imbalance in eukaryotic cells. Plants are more tolerant than animals to genomic imbalance caused by aneuploidy [1] and can be manipulated to generate different karyotypes for viable individuals [1,12]. Thus, research of plants has provided an excellent opportunity to study the transcriptional consequence of different types of aneuploidy. Trisomy in the 5 chromosomes in Arabidopsis (Arabidopsis thaliana) has been described, and different types of trisomy can be distinguished morphologically as compared with diploid (2n52x) plants [1,16]. Different types of trisomy have been documented in plants primarily by genetic, cytogenetic and morphological analyses. Trisomic plants produce offspring with various karyotypes; most of the progeny are diploid and some are trisomic. Tetrasomics (2n52x+2) are extremely rare, and some types of tetrasomy have never been observed [16]. Tertiary trisomy (2n52x+T) is present in individuals carrying an extra copy of a chromosome consisting of portions of 2 non-homologous chromosomes in the genome [2]. Transgenic plants carrying T-DNA insertions have been widely used in plant research for forward and reverse genetics to generate mutants for functional studies [17]. A T-DNA inserted in genomic sequence should disrupt or reduce the function of a gene if insertion is near or within the coding sequence. However, several reports have documented T-DNA insertions resulting in or associated with chromosomal abnormalities such as sequence deletion, duplication, the addition of unknown filler sequences, or chromosome rearrangement in plants [18,19]. We have limited information about the physiological consequences of numerical abnormalities in chromosomes resulting from T-DNA insertions. A previous study demonstrated that a transgenic tobacco line with a T-DNA insertion generated trisomic and tetrasomic offspring [6,7]. However, the cause of the aneuploid progeny was unclear. In this study, we uncovered an example of high-grade aneuploidy (tertiary trisomy 2) when characterizing a T-DNA insertion allele in the Arabidopsis AURORA2 locus at chromosome 2. The aur2-1 mutation was associated with defective development in male and female gametophytes and showed unusual genetic transmission. Additionally, we found more genes on the non-triplicated chromosomes that were mis-expressed in the high-grade trisomy than the primary trisomy 2; the former carried a chromosomal translocation that would have a significant impact on cell physiology. Finally, transcriptome and gene ontology (GO) analysis revealed overrepresentation of genes involved in the stress response, plastids and mitochondria in two primary and one tertiary trisomics, which supported increased energy demand and cellular metabolism as a common response to different types of stress resulting from genomic imbalance in aneuploidy [20]. Pollen viability and in vitro germination assays Staining for pollen viability was as described previously [22] with minor modifications. Briefly, fluorescein diacetate (FDA) (Sigma) was dissolved in 0.5% (w/v) acetone and diluted (1:49) in 10% sucrose as a working solution. Mature pollen grains were incubated with FDA staining solution for 15 min in the dark before observation under a fluorescence microscope (Axio Scope A1, Zeiss). The number of viable and non-viable pollen grains was counted by use of ImageQuant TL (GE Healthcare). The in vitro germination assay was performed as described [23]. The morphologic features of germinating pollen and pollen tube elongation were examined and scored under a microscope. The length of the elongated pollen tube was measured by use of ImageJ (http://rsbweb.nih.gov/ij/). Morphologic analysis of pollen grains, ovules, embryogenesis and seed formation For analysis of tetrads in the qrt2-1/qrt2-1 and aur2-1/+ qrt2-1/qrt2-1 plants, mature pollen grains from open flowers were transferred to filtered water and observed on slides under a microscope. Mature pollens treated with 49,6diamidino-2-phenylindole (DAPI) (Sigma) were placed on a slide and examined under a florescence microscope as described [24]. To observe the phenotype of mature ovules, we emasculated flowers at stage 12 [25], then fixed carpels for 2 d to ensure that the maximum number of ovules reached maturity. To observe the developing embryos, flowers were manually pollinated and siliques were fixed 6 or 10 d after pollination (DAP). Carpels or siliques were fixed and cleared as described [26]. Ovules or developing seeds were mounted in the clearing solution and examined under a microscope with differential interference contrast (DIC) optics. To evaluate fertilization and seed formation, self-or manually pollinated flowers were further grown for 10 d. Siliques were collected and dissected to score seed sets and unfertilized ovules. Chromosome preparation for cytological analyses The chromosome spreads for DAPI staining in pollen mother cells (PMCs) and shoot apical meristem (SAM) cells were performed as described [27,28]. PMC and SAM cells were fixed with fresh Carnoy fixative [ethanol-acetic acid (3:1, v/v)] at 4˚C overnight and stored at 20˚C. The tissues were washed once with filtered water and twice with citrate buffer (10 mM sodium citrate, pH 4.8) and incubated in digestion solution [0.02% (w/v) cellulase ONOZUKA R-10 and 0.2% (w/v) Macerozyme R-10 (both from Yakult, Japan) in citrate buffer] at 37˚C for 1 h. Then tissues were washed with filtered water, decomposed in a drop of 60% acetic acid, fixed on slides, and stained with DAPI solution for fluorescence microscopy. Fluorescence in situ hybridization (FISH) of mitotic cells in SAM was performed as described [29]. All images were acquired by use of AxioVision software (Zeiss) and analyzed by use of Adobe Photoshop CS2. Cryo-scanning electron microscopy (Cryo-SEM) Fully open flowers were dissected and loaded on stubs, then samples were frozen with liquid nitrogen slush and transferred to a preparation chamber at 160C. After 5 min, the temperature was raised to 85C and sublimed for 15 min. After being coated with platinum (Pt) at 130C, samples were transferred to the cold stage in the SEM chamber and observed at 160C by use of Cryo-SEM (FEI Quanta 200 SEM/Quorum Cryo System PP2000TR FEI) at 20 KV. Comparative genome hybridization with Arabidopsis tilling array (array CGH) Genomic DNA (gDNA) was extracted from rosette leaves by use of the DNeasy Plant Mini Kit (Qiagen). Affymetrix Arabidopsis Tilling 1.0R arrays were used for hybridization with labeled gDNA prepared from Col-0 and trisomic plants (AAA and AAa, see above). DNA labeling, hybridization, and detection followed the manufacturer's instructions (Affymetrix). Probe sequences from the BPMAP specification of the array (At35b-MR_v04-2_TIGRv5) were mapped to the TAIR10 genome by use of BLAT [30]. Intensity normalization involved the AffyTiling package of BioConductor. Probe locations and normalized signals were joined and sorted by use of an in-house perl script. Raw data for the tilling array are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi. nlm.nih.gov/geo/) (accession nos. GSM980297, GSM980298 and GSM980299). Whole-genome transcriptome analysis For expression microarray analysis, samples of total RNA were prepared from 26d-old plants of the wild type (Col-0), trisomy 2 (AUR2, round leaves, AAA genome), and tertiary trisomy 2 (aur2-1; round leaves, AAa genome), respectively. The karyotype and genotype were first scored by leaf morphology (round leaves), then underwent genotyping before sample collection. RNA samples from the rosette leaves of multiple plants (21-35 plants) were extracted by use of the RNeasy Plant Mini Kit (QIAGEN). Florescence labeling, hybridization to probes and detection followed the manufacturer's instructions (Affymetrix). The sequence annotation for the Affymetrix ATH1 oligos was downloaded from TAIR and processed by use of Excel (Microsoft); probe sets not mapping to two or more gene loci were selected [5]. Sequence-specific probe effects were removed by use of the GC-RMA package from R software [31]. The 14,976 probe sets for genes on chromosomes 1, 4, and 5 and those on chromosome 3 from the 39 end of At3g20040 (AUR2) to the end were selected for model fitting by use of vsn package [32] and the hybridization intensities were then normalized by using the fitted model [5]. Genes on the non-triplicated chromosome with sufficiently strong expression were isolated as described ( Figure S4 in S1 File) [5,14]. Changes in gene expression and gene ontology (GO) analysis involved use of GeneSpring (Agilent) and ArgoGO [33]. The raw data for Affymetrix ATH1 arrays are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm. nih.gov/geo/) (accession no. GSE54827). Identification of a trisomic Arabidopsis plant with a T-DNA insertion In this study, we discovered two types of aneuploidy in a heterozygous T-DNA insertional mutant (aur2-1/+) (Figures S1A and S1B in S1 File) and its descendants. The proportion of undeveloped seeds in self-fertilized plants was 18.6% and 1.2% for aur2-1/+ and the wild type (WT5Col-0), respectively, under the same growth conditions (Table S1 and Figure S1C in S1 File), which suggests defective fertilization or embryo development or both in aur2-1/+. The round shape of rosette leaves in the progeny of aur2-1/+ is consistent with the description of an aneuploid mutant with trisomy 2 in Arabidopsis [1,16,34] ( Fig. 1A-iv). Thus, we examined the metaphase chromosome spread of mitotic cells to confirm this possibility. WT cells had 10 chromosomes (2n52x), and aur2-1/+ cells had 11 (2n52x+1) (Fig. 1B). We next analyzed the meiotic chromosome spreads in pollen mother cells (Fig. 1C). The number of DAPIstained chromosomes differed between WT and aur2-1/+ as early as diakinesis stage. Chromosome spreads showed 1 univalent plus 5 bivalents or 1 pentavalent plus 3 bivalents for aur2-1/+ but 5 typical bivalents in the WT (Fig. 1C, viii-a and viii-b vs. ii). The WT showed the 5 bivalents aligned along an equatorial plate, whereas an extra univalent out of the metaphase plate appeared in aur2-1/+ at metaphase I (Fig. 1C, iii vs. ix). Furthermore, in the WT, 5 pairs of sister chromatids were segregated to each daughter cell, whereas aur2-1/+ showed an extra DAPI dot in each nucleus during sporogenesis from anaphase-II and telophase-II to pollen tetrads (Fig. 1C, v-vi vs. xi-xii). Additionally, we analyzed the karyotype of aur2-1/+ by using 5S and 45S rDNA probes labeled with different dyes in dicolor FISH (Fig. 1D, right panel) [35]: aur2-1/+ showed 11 DAPIstained chromosomes from shoot apical meristem cells and 3 sets of green florescence dye (45S rDNA) at chromosome 2 ( Fig. 1D, left panel). In sum, both the rosette leaf phenotype and cytological studies demonstrated that aur2-1/+ is trisomy 2 and the additional chromosome appears to have a functional centromere for chromosome segregation during meiosis and mitosis. Genotype and karyotype of aur2-1/+ progeny On genotyping nearly 1,000 plants of the self-fertilized heterozygous aur2-1/+ mutant, we did not find any homozygous progeny, which suggests that the aur2-1 allele may have an unusual inheritance in the trisomic genome. To understand the genetic behavior and genomic constitution of aur2-1/+, we performed genotype and karyotype analyses of aur2-1/+ progeny. By reciprocal crosses between the aur2-1/+ and WT, we found a 25.2% transmission rate of aur2-1 from aur2-1/+ ovules, which was comparable to that of aur2-1 in F1 progeny of self-fertilized aur2-1/+ plants (27.1%); however, we found no aur2-1 allele in F1 progeny when WT ovules were pollinated with aur2-1/+ pollen (Table 1). Therefore, pollen failed to transmit the aur2-1 allele to offspring and the inheritance of aur2-1 allele is mainly through maternal transmission. Because the round leaf phenotype is a morphological characteristic of Arabidopsis trisomy 2 plants, we used it as a reference to visually assess the karyotype of plants [16,34]. We found 3 major types of aur2-1/+ progeny from both self-fertilization and reciprocal crosses: plants carrying a WT AUR2 with WT leaves (AUR2, W) or round leaves (AUR2, R) and aur2-1/+ plants with round leaves (aur2-1/+, R) ( Table 2; Fig. 1A); however, we found no aur2-1/+ plants with WT leaves. In addition, we found a few plants (7/242) from self-fertilized aur2-1/+ with extremely small and tiny (T) round leaves that died before bolting ( Table 2, Fig. 1A-v). Tetrasomics grow slowly and show an enhanced phenotype as compared with corresponding trisomics in Arabidopsis and other species [16,36]; thus, the plants with tiny rosette leaves may be tetrasomics. Chromosomal translocation in the aur2-1/+ trisomy Two types of trisomy 2 progeny with round leaves were derived from selffertilized aur2-1/+ plants: WT (AAA) and aur2-1/+ (AAa) (Fig. 1A, iii and iv). For simplicity, A and a hereafter represent chromosome 2 (Chr.2) carrying an AUR2 and an aur2-1 allele, respectively. To survey possible additional mutation(s) and validate an extra copy of Chr.2 in aur2-1/+, we used DNA tiling arrays for CGH (array CGH) to comprehensively inspect the whole genomes of WT trisomy (AAA), aur2-1/+ trisomy (AAa), and diploid Col-0 (AA) ( Fig. 2A). In comparing the signal intensity of hybridized probes in Col-0 and the trisomic genomes (AAA and AAa), array CGH confirmed the chromosomal imbalance in the trisomic mutants. The ratio of probe intensities in AAA to Col-0 (red dots) indicates a duplication of Chr.2 in the AAA genome ( Fig. 2A, left column). However, in the aur2-1/+ genome, only part of Chr.2 (11 Mb), including the whole short arm (2S) and part of the long arm (2L), showed increased signal intensity ( Fig. 2A, blue dots). The signals of the remaining long arm of Chr.2 (8 Mb) were comparable to those for the WT genome ( Fig. 2A). These results suggest that the extra Chr.2 in aur2-1/+ was broken in the middle of long arm and the region extending from the breakpoint was deleted. We found increased signals in the short arm of The findings are consistent with the cytogenetic data supporting that aur2-1/+ is tertiary trisomy 2. The extra chromosome could presumably be paired with the segments of the regular chromosome 2 and 3 to form a pentavalent at the diakinesis stage (Fig. 1C, viii-b). We further determined the potential breakpoint in Chr.2L and Chr.3S ( Fig. 2A, shaded regions) by plotting the ratio of hybridization signals against an 80-kb genomic region in the two trisomic genomes, AAa and AAA ( Figure S2A in S1 File). The translocation breakpoint was mapped and plotted against the annotated genes. The AUR2 locus (At2g25880) was located proximal to the Chr.2L breakpoint ( Figure S2B in S1 File, upper panel) and the 3S breakpoint was at the 59 end of At3g20040 ( Figure S2B in S1 File, lower panel). The T-DNA insertion site of the AUR2 locus was in close proximity to the breakpoint, which explains the tight association of aur2-1 allele with the chromosomal translocation ( Figure S2B in S1 File). The interchanged chromosome structure in the aur2-1/+ genome may have originated from a T-DNA insertion event. Interestingly, we found increased intensity of the tiling probes representing genes in the chloroplast (plastid) and the mitochondrial genomes in both the Table 2. Genotype and rosette leaf phenotype in the progeny of self-fertilized and backcrossed aur2-1/+. tertiary (aur2-1/+) and primary (AAA) trisomy as compared with the diploid WT (Fig. 2C). Array CGH of AAA and AAa genomes revealed a comparable increase in chloroplast genomic sequences, which suggests the presence of homologous sequences in the trisomic genomes. Alternatively, the number of chloroplasts may be increased in the aneuploid cells. A 620-kb sequence proximal to the centromere region of Arabidopsis Chr.2 contains highly homologous sequences to the 350-kb mitochondrial genome [37,38]. Thus, the increased mitochondrial sequences in the array CGH analysis of the two trisomics may result from the imbalanced copy number of Chr.2. However, a portion of the mitochondrion genome is not significantly enriched in the AAa genome, probably because of a translocation of the sub-chromosomal region of Chr.3 in the genome. Phenotypic consequences of chromosome translocation in aur2-1/+ We showed a higher ratio of undeveloped seeds or unfertilized ovules in tertiary trisomy 2 (AAa, aur2-1/+) than WT plants (18.6% vs. 0.8%) (Table S1 and Figure S1C in S1 File). To determine which source of gametophytes, parental or maternal, was involved in the phenotype, we performed reciprocal crosses between aur2-1/+ and WT. Manually crossing aur2-1/+ with WT pollen (aur2-1/+ 6 Col-0) or ovules (Col-06aur2-1/+) resulted in 14.7% and 8.3% undeveloped seeds, respectively, in F1 progeny (Table S1 in S1 File), which suggests that mutations in aur2-1/+ cause fertility defects in both parental and maternal gametophytes. To investigate whether the fertility phenotype was attributed to the trisomic genome or any additional mutations associated with aur2-1/+, we examined seed development in siliques of WT (Col-0), diploid sibs of WT (AA), trisomy 2 (AAA), and tertiary trisomy 2 (AAa) plants. We found undeveloped seeds and deformed siliques of irregular size along the stems in both AAa and AAA but not AA or WT plants, which suggests that the trisomic genome is mainly responsible for the phenotype (Figs. 3A and 3B). Abnormal transmission of the aur2-1 allele in self-fertilized aur2-1/+ may result from defective gametophytes, thus leading to sterility. To investigate this possibility, we examined the gametogenesis of ovules and pollen. We first inspected unfertilized ovules of AAa and WT by DIC light microscopy. The secondary endosperm nucleus (SEN) and egg nucleus (EN) in the embryo sac are clear, but in most cases, only 1 of the 2 synergid cell nuclei (SN) can be found in the same focal plane [39]. We found several types of abnormal ovules in both the WT and AAa plants (Fig. 3C). Degraded ovules still have the normal shape of Characterization of an Arabidopsis Tertiary Trisomy ovules, but the boundaries of cells were unidentifiable (Fig. 3C, Degraded). Some of the ovules do not have an SEN and SN but have one EN (Fig. 3C, One nucleus), and some do not have any visible nucleus (Fig. 3C, No nucleus). A small number of ovules showed fusiform morphologic features (Fig. 3C, Fusiform). AAa plants appeared to have more ''degraded'' and ''one-nucleus'' ovules than the WT (Fig. 3C). Most of the WT ovules (96%) showed normal features as compared with Aaa (78.2%), so aur2-1 is associated with defective ovule development. This observation agreed with findings that only 25.15% of the progeny carried the aur2-1 allele transmitted from maternal gametophytes ( Table 1). We next examined the morphology and viability of pollen grains by Cryo-SEM and staining with FDA, respectively. AAa produced more aborted and shrunken pollen grains than did AAA and WT (Fig. 3D). Consistently, the proportion of viable pollen grains was comparable in WT and AA (approximately 90%) and slightly decreased in AAA (83.2%) but significantly reduced to 58.8% in AAa plants (Fig. 3E). Thus, although the trisomic genome (AAA) causes slightly reduced fertility, chromosome translocation associated with the aur2-1 allele apparently results in a severe defect in pollen development. In addition to pollen development, the failed transmission of aur2-1 allele by pollen might result from defects in pollen tube germination or elongation. The in vitro germination assay revealed more germinated pollen and elongated pollen tubes in the WT than in AAa (83.5% vs. 30.8%) ( Figure S3A in S1 File). Therefore, both viability and germination of pollen grains were lower in AAa than the WT. We introduced quartet2 (qrt2) to generate the aur2-1/+ qrt2-1/qrt2-1 double mutant for tetrad analysis. QRT2 is essential for separating pollen grains from the 4 tetrads of meiosis during pollen development, so use of qrt2 allows for analysis of allele segregation in aur2-1/+ pollen [40,41]. We found 3 types of pollen tetrads in aur2-1/+ segregating at 4:0 (30/53556.6%), 3:1 (10/53518.9%), and 2:2 (13/ 53524.5%) viable to non-viable pollen (Fig. 3F, upper panel). Furthermore, DAPI staining of nuclear DNA revealed WT-like pollen grains in tetrads with 1 vegetative and 2 sperm nuclei, which were absent in degraded pollen grains (Fig. 3F, lower panel). Taken together, our results from genetic and morphological analyses of ovules and pollen indicate that both types of gametophytes in aur2-1/+ are defective to different degrees, which is likely responsible for the impaired transmission of aur2-1 allele. Additionally, we sought to determine whether embryo development was affected by the tertiary trisomy 2. The ovules from WT or AAa were manually fertilized with WT pollen and developing embryos were examined between 2 and 6 DAP. Most of the embryos (256/269595.2%) were successfully developed from ovules of AAa fertilized with WT pollen as compared with embryos (242/ 243599.6%) from fertilized WT ovules; however, the embryo development was slightly delayed for fertilized AAa ovules. For developing embryos at 6 DAP, 69.1% (186/269) and 25.7% (69/269) of fertilized ovules in AAa developed to the heart and torpedo stages, respectively, as compared with 37.9% (92/243) and 61.7% (150/243) of WT embryos, respectively ( Figure S3B in S1 File). Therefore, the undeveloped seeds shown in Figure S1C in S1 File are not due to aborted embryo development but most likely to defective gametophytes for effective fertilization. Gene expression profiles in primary and tertiary trisomy 2 Phenotypic alterations resulting from genomic imbalance in aneuploidy involve changes in gene expression [1,[42][43][44]. To evaluate the global effect of the wholechromosome and high-grade aneuploidy on gene expression, we used microarray assay to analyze the transcriptome profiles of WT, trisomy 2 (AAA), and tertiary trisomy 2 (AAa). Consistent with observations from array CGH ( Fig. 2A), we found upregulated expression of genes in the triplicated chromosomes of the AAA and AAa corresponding to the trisomic chromosomal regions (Fig. 4A). We next investigated the expression patterns of genes on non-triplicated chromosomes [Chr.1, 3(3'), 4, 5] by normalization with the whole genome that excluded the trisomic regions as previously described [14]. To identify differentially expressed genes, we compared the gene expression profiles in the two trisomy 2 examples, AAA and AAa, with that in the WT (Fig. 4B, p,0.05). Interestingly, the number of misregulated genes in AAa (1358) was 1.7 times more than that in AAA (796), which indicates that the chromosomal translocation in the tertiary trisomy (AAa) affects more genes than the simple trisomy (AAA) (Fig. 4B). We further categorized 325 genes that were misregulated in both AAA and AAa into four groups based on the expression patterns and assigned GO terms to define functional properties (Fig. 4C). Approximately 78% of genes showed expression regulated by both AAa and AAA (Groups II and IV), whereas 22% were inversely regulated (Groups I and III). Because AAa is a tertiary trisomy 2 harboring a translocated chromosome 3 (Fig. 2B), we further examined the enriched GO terms present only in AAA or AAa (Table S2 in S1 File). In total, 471 and 1033 genes showed misregulated expression specifically in AAA and AAa, respectively (Fig. 4B). We found several expression. (B) Venn diagram shows the specific and overlapping genes with significant misregulated expression compared to wild-type diploid (Col-0) between trisomy 2 (AAa) and tertiary trisomy 2 (AAA) (p,0.05). (C) Clustering and heat map of the 325 misregulated genes common to the two trisomics (AAA and AAa). Four groups of genes based on expression patterns are shown on the right. doi:10.1371/journal.pone.0114617.g004 Table 3. Groups of enriched gene ontology (GO) terms for differentially expressed genes common to both trisomy 2 and tertiary trisomy 2. Characterization of an Arabidopsis Tertiary Trisomy GO cellular component terms related to organelles including plastid, mitochondrion, vacuole, endoplasmic reticulum, plasma membrane and cell wall were specifically enriched in AAa (Table S2 in S1 File). Furthermore, genes involved in several metabolic processes such as glycosinolate biosynthesis, sulfur metabolism, carbohydrate catabolism and metabolism were revealed among those misregulated specifically in AAa. Finally, GO terms related to biological process including cell cycle, cell death, proteolysis and chlorophyll metabolism were enriched only in AAa. The difference in specific gene sets misregulated in AAA and AAa likely results from the non-reciprocal translocation of Chr.3 to Chr.2 to generate the tertiary trisomy 2. Trans effect of trisomy 2 and 5 on gene expression profiles We next asked whether different trisomics would show a similar trans effect on changes of transcriptional signatures in Arabidopsis. By using the annotation enrichment tool (agriGO) we analyzed genes that were significantly misregulated (FDR,0.05) in the two trisomy 2 examples (AAA and AAa) and the trisomy 5, a primary trisomy with transcriptome data available [5]. Notably, we found GO terms related to response to stress and various types of endogenous and external stimuli and involved in catalytic activity were significantly enriched in both upregulated and downregulated groups of genes for all three trisomics ( Fig. 5; Tables S3 and S4 in S1 File). Although several GO terms were commonly detected, misregulated genes in certain biological processes were specific to different trisomics. Genes involved in the flavonoid metabolic process and external encapsulating structure were upregulated and enriched only in trisomy 2 (AAA and AAa) but not trisomy 5 (Tables S3 and S4 in S1 File). Furthermore, genes related to transcription factor activity and DNA binding were upregulated and enriched only in primary trisomy 2 (AAA) and trisomy 5 but not tertiary trisomy 2 (AAa), while those involved in proteolysis, proteasome accessory complex, aging, cell death, cell cycle process, glucosinolate biosynthesis, carbohydrate metabolism and catabolism were specifically enriched in AAa ( Fig. 5; Tables S3 and S4 in S1 File). Moreover, we found significant enrichment of several catalytic activity terms only in AAa, including that of oxidoreductases, transferases, and kinases (Table S4 in S1 File). In contrast, genes involved in nucleolus, ribosomal biogenesis, translation, ncRNA metabolism, several developmental processes, organelle and photosynthesis were overrepresented only in trisomy 5 ( Fig. 5; Tables S3 and S4 in S1 File). Our observations agree with previous studies showing downregulation of genes related to cell growth, metabolic processes and proliferation in aneuploid cells, and upregulation of those associated with stress responses in eukaryotic organisms, including yeast, mice and humans [14,[45][46][47]. , AAa (black) and trisomy 5 (gray) were derived by use of agriGO [33]. The p values for GO terms are shown inlogarithmic scale. Data for trisomy 5 are from [5]. Complete lists are presented in Tables S3 and S4 in S1 File. doi:10.1371/journal.pone.0114617.g005 Nuclear-encoded genes related to organelles are misregulated in the trisomics Large-scale chromosomal changes in aneuploidy are proposed to affect the biogenesis and depletion of organelles that cause energy consumption, proteotoxic and aneuploidy-associated stresses [8,15,48]. The status of genomic unbalance urges cells to consume energy to overcome the detrimental effects of genetic abnormality in aneuploidy [20]. The plastids and mitochondria are the energy sources in plant cells [49][50][51][52] and the peroxisomes are essential organelles mediating many important metabolic pathways [53]. To understand the effect of trisomy on cellular energy and metabolism in Arabidopsis, we examined the expression profiles of nuclear-encoded genes related to plastids, mitochondria and peroxisomes [5]. In the non-triplicated WT chromosomes, 10.1%, 4.6%, and 2.1% of genes account for functions related to plastids, mitochondria and peroxisomes, respectively (Table 4, Wild type). However, in the non-triplicated chromosomes of trisomy 2, tertiary trisomy 2, and trisomy 5, the number of plastid-related genes increased to 12.4%, 14.6%, and 18.7%, respectively (Table 4). Additionally, mis-expressed genes related to mitochondria and peroxisomes were overrepresented in the genomes of tertiary trisomy 2 and trisomy 5, but not in trisomy 2 (Table 4). Consistent with the analysis of enriched GO terms, the number of differentially expressed genes related to organelles important for cellular energy and metabolism were significantly increased in at least two of the three trisomics examined, which suggests that aneuploid cells altered the expression of genes involved in these organelles to adapt to energy stress resulting from the imbalanced genome. Aneuploidy derived from T-DNA mutagenesis Tertiary trisomy has been found in animals and plants and is considered to originate from the product of the malsegregation of interchange heterozygotes [2,54]. Thus, the aur2-1/+ mutant may originate from a chromosome rearrangement that was likley induced by a T-DNA insertion event, followed by chromosome non-disjunction to become tertiary trisomy. However, with aur2-1/ +, the malfunction of the T-DNA inserted gene may have increased the frequency of this phenomenon. AUR kinases play an important role in controlling the function of centrosomes, chromosome segregation and bipolar spindle assemble in yeast and mammalian cells [55][56][57]. Disruption in AtAUR2 by the T-DNA insertion (aur2-1) may cause missegregation of chromosomes during gametophyte development, thus leading to trisomy. However, the reduced fertility in aur2-1/+ does not seem to directly associate with the aur2-1 T-DNA insertion allele. We characterized another mutation in AtAUR2 (aur2-2, GK403B02) and found that aur2-2 was fertile in both male and female gametophytes, which is consistent with a previous report [21]. Therefore, the defect in transmission of aur2-1 allele from male or female gametes is associated with aur2-1/+ trisomy but not aur2-1 per se. The results from array CGH showed increased signal intensity in chloroplast and mitochondrial genomes for both aur2-1/+ (AAa) and primary trisomy 2 (AAA) (Fig. 2B). The homologous sequences of mitochondrion on the extra copy of Chr.2 may have enhanced the signal intensity of mitochondrial sequences in the trisomics on array CGH [37,38]. Alternatively, the enhanced probe signals may indicate increased contents of both organelles in aneuploid cells. In fact, several types of aneuploidy in Cyphomandra betacea showed a higher copy number of chloroplasts than the diploid WT [58]. Recent reports indicated that aneuploid cells show increased need for energy that may help overcome the detrimental effects of overproduced proteins from the extra copies of genes [14,20]. Both chloroplasts and mitochondria are the energy sources of plant cells; their maintenance and operation highly depend on proteins translated from nuclearencoded genes [49][50][51][52]. Changes in large-scale gene expression in the aneuploidy may affect the replication and operation of these two organelles. This finding was supported by the gene content analysis in the Arabidopsis genome [38]. More than 10% of the annotated genes on Chr.2 are functionally associated with chloroplasts and mitochondria [38]. Disturbing the balance of these nuclearencoded genes may also lead to changes in number of these two organelles. AAa genome is preferentially inherited in the offspring of tertiary trisomy 2 Trisomy produces various progeny with different karyotypes and does not exclusively generate trisomic offspring [16]. The tertiary trisomy 2 (AAa) was intentionally and stably maintained in generations by selecting the aur2-1 allele in this study. Because the aur2-1 allele was present only in heterozygosity, the aur2-1/+ trisomy could be simplex (Aaa) or duplex (AAa). In the process of meiosis (M Fig. 6), the haploid tetrads carrying a chromatids are expected to be degraded in subsequent microgametogenesis [59,60]. The duplex genome (AAa) will show a ratio of 2:2 or 4:0 for viable to non-viable pollen following the rule of independent assortment (Fig. 6A), and 3:1 or 4:0 if a crossing-over takes place (Fig. 6B). In contrast, the simplex genome (Aaa) is expected to generate 2:2 and 1:3 patterns in pollen tetrads. Thus, our observation supports that aur2-1/+ is a duplex (AAa) (Fig. 3F). An inheritance model by calculating the theoretical segregation ratio of progeny from self-fertilized aur2-1/+ (AAa) is shown in a Punnett square (Table S5 in S1 File). Among all possible combinations of progeny, the tetrasomics (AAAA, AAAa, and AAaa) would be rare because of the poor transmission of n+1 gametes [2,16]. Reciprocal crosses showed no transmission of Aa or a from the male gametes (Table 1) and no a gametes from the female side (Table 2). Therefore, we found no trisomic (Aaa) or diploid (Aa and aa) progeny in the self-fertilized aur2-1/+ (Table S5 in S1 File, shaded boxes). Thus, 3 major types of progeny would be generated -AAA, AAa and AA (Table S5 in S1 File, white boxes). Theoretically, the number of trisomic progeny carrying genotypes AAA and AAa should be equivalent (AAA:AAa5(2/36+2/36): 4/3651:1). Because the male n+1 gametes (AA) were rarely transmitted [16], the expected ratio between the AAA and AAa offspring would be skewed and close to 1:2 (AAA:AAa52/36:4/3651:2). Surprisingly, we found 11:72 and 4:26 ratios of AAA:AAa in the progeny of selffertilized aur2-1/+ and aur2-1/+ ovules crossed with WT pollen, respectively (Table 2); therefore, the AAa genome is preferentially inherited in the offspring of aur2-1/+. The preferential inheritance of the T-DNA insertion (aur2-1) may be due to its translocated chromosomal structure (Fig. 2B), which contains portions of Chr.2 and Chr.3, and will not be a perfect pairing partner to either one. The WT chromosomes may pair in higher frequency and ensure that each copy moves to the opposite poles in first division. If this is the case, the T-DNA carrying chromosome (a) is rarely alone and expected to accompanied by a normal chromosome (A) in one side [2]. Our findings suggest that the unique genomic structure of the tertiary trisomy 2 likely provided a selective advantage among progeny. Poor transmission of the T-DNA-inserted chromosome (a) in both male and female gametes resulted in limited genotypes in the offspring of self-fertilized aur2-1/+ (Table S5 in S1 File). Preferential inheritance from female gametes led to better survival of the T-DNAcarrying progeny (AAa) than the other trisomic offspring (AAA). Chromosome translocation is frequently associated with T-DNA insertion mutagenesis, about 19% [18]. Similar cases may show preferential inheritance in other chromosomal rearrangements resulting from T-DNA insertion. Chromosome non-disjunction may arise occasionally and thus led to tertiary trisomy. Further studies to investigate the occurrence of aneuploidy in the T-DNA insertion mutant library will be required to test this possibility. Transcriptional consequences of aneuploidy in Arabidopsis By using expression microarray analysis, we found a trans effect of triplicated genes on the diploid chromosomes to reveal genes involved in stress response, organelle-related functions, and transcription factors were misregulated and overrepresented in the trisomy (Fig. 5; Tables S2-S4 in S1 File). Our observations are consistent with previous reports of aneuploidy in plants and other organisms [5,14,42,61]. Our analyses of the global gene expression profiles support a hypothesis that, regardless the type of trisomy, genes involved in stress response, defense response, energy and metabolism processes are significantly affected by aneuploid genomes. Previous studies showed increased transcription of environmental stress response (ESR)-like genes in all types of aneuploid cells in yeast [14,62], plants [5,61], mice and humans [14,63]. The trans effect on gene expression may be attributed to regulatory proteins encoded by some of the triplicated genes to modulate concurrently on overlapping targets. Alternatively, the differential gene expression in aneuploid cells is a consequence, rather than the cause, of transcription failure. Several specific GO terms were identified for misregulated genes in different trisomics. For example, the genes related to transcription were overrepresented in primary trisomics (trisomy 2 and trisomy 5) but not tertiary trisomy 2. In contrast, genes related to proteolysis, aging and cell death were significantly enriched in tertiary trisomy 2 but not the other primary trisomics. Furthermore, tertiary trisomy 2 seemed to show specific involvement in regulation of cell cycle regulation and carbohydrate metabolism. Mouse models of Down syndrome, Ts65Dn and Ts1Cje, show a connection between the high-grade aneuploidy and cellular alterations in protein degradation and aging [64]. The Ts65Dn mouse is a tertiary trisomy containing the distal region of mouse Chr.16 (92 genes homologous to nearly two-thirds of the human Chr.21) and Chr.17, whereas the Ts1Cje mouse carries a smaller region of mouse Chr.16 (67 genes homologous to the human Chr.21) and telomeric region of Chr.12. Both mouse models showed a combinational effect on phenotypes involving impaired protein homeostasis phenotype, such as cognitive abnormalities, age-related atrophy, degeneration of cholinergic neurons, age-related endosomal pathologies and protein misfolding [64]. Aneuploid cells may require more time to properly align chromosomes at metaphase. As a consequence of having extra chromosomes, tumor cells would be more prone to chromosome missegregation and aberrant in cell cycle progression because of checkpoint abrogation (reviewed in [48,65]). Thus, the high-grade aneuploidy may affect additional genes located on other chromosomes, which combined with a gene dosage effect, influences gene expression patterns. Whether the genomic instability evoked by aneuploidy contributes to the affected plant phenotypes or such transcriptional changes are the result of differential growth fitness remains to be tested. Taken together, the tertiary trisomy aur2-1/+ (AAa) has multiple traits: round leaves, aborted seeds, underdeveloped siliques with irregular size, defective ovule development, poor pollen viability, germination and pollen tube elongation, and abnormal pollen morphology. These phenotypes appeared to be caused by several factors. First, the most prominent round leaf phenotype was attributed to the effect of chromosomal imbalance by an extra Chr.2 [16]. In AAa, the extra chromosome contained 7 Mb chromosome 3S and the whole short arm 2S recombined with half of the long arm 2L (Fig. 2B and Figure S2 in S1 File). Large chromosomal deletion combined with translocation should affect more genes in AAa than AAA [66]. This possibility is supported by a previous report that the longer chromosomal component in the trisomic chromosome provided more profound morphological effect than the short one in tomato [67]. Second, the poor pollen viability and defective male gametophyte development may be caused by the translocated structure of the extra chromosome in aur2-1. Tertiary trisomy resulting in low pollen fertility was found in pearl millet (7%) [68] and lentil (9%) [69]. In mammals, most trisomy causes adverse effects on embryo development and is fatal to fetuses, leading to spontaneous abortion [8]. Finally, gene expression profiling provided useful information to compare and contrast the effects of different degrees of aneuploidy on plant cell physiology. Supporting Information S1 File. Figure S1, Seed development in self-fertilized and manually crossed aur2-1/+. Figure S3, Pollen germination and embryo development in tertiary trisomy 2. (A) Pollen grains from aur2-1/+ show impaired pollen germination and pollen tube growth. The in vitro pollen germination of pollen grains from the wild type (Col-0) and aur2-1/+ were scored by microscopy and measured by use of ImageJ after 8-h incubation in germination agar medium. Data are mean¡SD from 3 independent experiments (n5400500). (B) Representative differential interference contrast (DIC) images of developing embryos in wild type (Col-0) and tertiary trisomy 2 (aur2-1/+; AAa) whose ovules were both manually pollinated by WT pollen. Quantitative data of the embryos at 6 days after pollination (DAP) are shown. Scale bar, 100 mm. and orange lines, moving average. Upper and lower lines of the purple or orange lines, moving average ¡ SD. Sufficiently strongly expressed transcripts (the dots to the right of the dash lines) were isolated to perform the transcriptional signature analysis in this study. The methodology of data analysis is from Huettel et al. (2008) [5]. Table S1, The ratio of undeveloped seeds in the progeny of selffertilized and manual crosses of aur2-1/+ and wild-type (Col-0) plants. Table S2, Enriched gene ontology (GO) terms of genes that are misregulated specifically in the trisomy 2 or tertiary trisomy 2. Table S3, Enriched GO terms of significantly upregulated genes in the trisomy 2, tertiary trisomy 2, and trisomy 5. Table S4, Enriched GO terms of significantly downregulated genes in the trisomy 2, tertiary trisomy 2, and trisomy 5. doi:10.1371/journal.pone.0114617.s001 (PDF)	2018-04-03T05:38:48.727Z	2014-12-16T00:00:00.000	{ "year": 2014, "sha1": "a2f0fa4f0f6019674847684ec7f6788c25b5b641", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0114617&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a2f0fa4f0f6019674847684ec7f6788c25b5b641", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
51709533	pes2o/s2orc	v3-fos-license	An Explorer of Chemical Biology of Plant Natural Products in Southwest China, Xiaojiang Hao Abstract Xiaojiang Hao, who obtained Master Degree from Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS) in 1985, and Doctor in Pharmacy degree in Pharmacy from Institute for Chemical Research, Kyoto University, in 1990, was born in Chongqing in July, 1951. In 1991, he returned to KIB, CAS, as an Associate professor and served as the chair of the Department of Phytochemistry. In 1994, he was promoted to a full professor at the current institute. He served as the Deputy Director of KIB and the Director of Open Laboratory of Phytochemistry from 1995 to 1997, and the Director of KIB from 1997 to 2005. Professor Hao has published more than 450 peer-reviewed SCI papers, which have been cited over 6000 times. He has obtained one PCT patent and 23 patents in China. Due to his tremendous efforts, one candidate drug, phenchlobenpyrrone, has entered the Phase II clinical trail for the treatment of Alzheimer’s disease. Moreover, he won the First Prize of Natural Sciences in Yunnan Province for three times, and Ho Leung Ho Lee Fund Science and Technology Innovation Award in 2017. Graphical Abstract Introduction China is one of the countries in the world with the most abundant plant resources, and Yunnan Province is known as the ''Kingdom of Plants'' as well as the ''Treasure house of Drugs''. A wide variety of medicinal plants are the cornerstones of the formation and development of Chinese medicine. China has long lived in harmony with nature. The rich natural resources and diverse plants have brought endless supply for medicinal herbs to the Chinese people. With the development of modern medical sciences, phytochemistry is also playing an increasingly important role. For 40 years, Professor Hao has been an enthusiastic explorer in the field of Natural Product Sciences of Plants and written a profound chapter for the plant resources in Southwest China. Professor Hao has dedicated his life to medicinal plant chemistry and natural product research to meet the health care needs of the country. After graduating from Department of Chemistry at Guizhou University in 1976, he began to engage in phytochemical research and quickly realized the great potential and prospects of medicinal plant chemistry. He determined to continue his studies in this area from the very beginning of his career. He was admitted to the graduate program at KIB in 1982, and after receiving his MS degree in science, he stayed at KIB as an Assistant Professor. In 1986, he was selected to study at Institute of Chemical Research, Kyoto University in Japan. Four years later, he received PhD in Pharmacy with outstanding achievements and immediately returned to the motherland, where he applied his knowledge and expertise to the needs of the motherland. After returning to China, Professor Hao took the responsibility for the construction of disciplines in phytochemistry. He In spite of the heavy daily administrative work, Professor Hao always insists on the scientific research and graduate student training. His research team has always been at the front-line of his research field. His hardwork and efforts have produced a series of important research achievements and cultivated a new generation of natural product researchers. He has published over 450 papers in SCIoriginal academic journals which has been cited more than 6000 times. Additionally, he is granted with 1 PCT international patent which was licensed to a company and 23 national invention patents. Mining Novel Phytochemical Structures from Diverse Plants in Southwest China Since 1991, the Hao's team has studied the chemical components of more than 200 species of plants and identified more than 4600 chemical constituents including 1400 new structures and 90 new scaffolds, part of which were cited by ''Hot off the Press'' in Natural Product Reports ( Fig. 1) . The studies include the elucidation of complex chemical structures, exotic and novel backbones, and novel biogenetic mechanisms for Daphniphyllum alkaloids. The Hao Lab has become one of the most important discovers of new Daphniphyllum alkaloids . ''The research on novel structure alkaloids of Daphniphyllum'' he presided over won the First Class Award of Natural Science of Yunnan Province in 2009. These results were actually inspired by the hypothesis of the biosynthetic pathway of Daphniphyllum alkaloids that is the ''premature'' involvement of amines in the formation of molecular skeletons, which lead to the structural diversity of Daphniphyllum alkaloids in Daphniphyllum species. The structural diversity of plant natural products derived from the diversity of biosynthetic pathways is also mirrored by other new natural products, such as alkaloids [63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80], terpenoids , polyphenols [114][115][116][117][118][119][120][121][122][123], and steroids [124][125][126][127][128] rooted from the plentiful plant resources in Southwest China. Exploring Phytochemical Geography of the Spiraea japonica Complex In his long history of scientific research practice, Professor Hao never hesitated to learn new knowledge and explore beyond his comfortable zones, which has led him to new research systems and strategies and helped him continuously achieve innovative results. For example, he discovered that only the compound of Spiraea japonica L.f. contained diterpenes and diterpene alkaloids among more than 100 species of the genus Spiraea. This unusual phenomenon prompted him to select the Spiraea japonica compound to carry out exploratory research by combining plant resources and distribution, diterpenoids and alkaloids as well as biology to reveal several scientific issues of plant populations at different levels. He discovered and carried out the biomimetic and biosynthetic pathways from diterpenoids to diterpene alkaloids. These results revealed a high degree of harmonization and correlations between plant population evolution, plant faunal evolution, and chemical composition types, which exemplified a novel research model of phytochemistry (Fig. 2) (Fig. 3) [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67], and the research has led to the First Class Award of Natural Science of Yunnan Province in 2013. than a decade, he has actively promoted co-operations with biologists, developed the research direction of chemical biology with the use of small molecule natural products as chemical probes, and obtained preliminary results in the research of the mechanism of action related to tumors, neuroprotection, and viruses [187][188][189][190][191][192][193][194][195][196][197][198][199][200][201]. New areas of chemical biology research have been established with biologists including Prof. Li Lin [187,190,192,196,200], Prof. Chen Quan [194,195,197,199,201] and Prof. Yang Chonglin [188,191]. The collaborations have provided new strategies, new potential targets, and lead compounds for the treatment of related diseases (Fig. 4). Although the research interests are in many areas, the main thrust of Professor Hao's efforts has always been the study and development of innovative drugs and pesticides. Hao designed and synthesized a new anti-AD drug called Fenkeluotong with independent intellectual property rights. Phase II clinical trail is underway for this candidate drug. He directed the research on the mechanism of action, largescale synthetic technology, safety evaluation and preparation study of the new chemical activator AHO of plant systemic acquired resistance, and the field experiment of five consecutive years was significant for the prevention and treatment of disease effects. These honors have become a source of motivation for continual exploration and innovation in Professor Hao's research team. Today, Professor Hao is still presiding over and undertaking many important scientific research projects such as the State Key Program of National Natural Science Foundation of China, research and development projects of new drugs and new pesticides. In spite of the heavy work, he devoted his energies to training students. He has trained 61 doctors, of whom 30 have been promoted to professors and become leaders in scientific research and academic institutions. More importantly, 36 of the former students serve in universities and research institutes among the Yunnan and Guizhou areas have made tremendous contributions to the development of the remote Southwest China. Despite of the life-long outstanding achievements, Professor Hao continues to maintain a modest and low-key style. He often said that the achievements do not belong to himself, but the result of the team's joint efforts. He can do more for the scientific research for the motherland and benefit the people is his highest wish. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.	2018-08-06T13:17:57.005Z	2018-07-21T00:00:00.000	{ "year": 2018, "sha1": "dddc82c0203a4af632138bf79fb29dc6788f48b5", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s13659-018-0184-8.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fc3df78f56f946e996dc1807eff987e8a8047bef", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Political Science", "Medicine" ] }
218945778	pes2o/s2orc	v3-fos-license	Rapidly Developing Methicillin-Resistant Staphylococcus Aureus Pericarditis and Pericardial Tamponade Methicillin-resistant staphylococcus aureus (MRSA) pericarditis is a rare life-threatening infection. A 46-year-old female with hypertension, acquired immunodeficiency syndrome (AIDS) and recurrent neck abscesses, presented with a neck abscess and sepsis. Bloody purulent drainage from the abscess was found and antibiotics were started. Drainage was positive for MRSA. Four days after, course was complicated by acute pericarditis and pericardial tamponade; pericardial fluid was drained and was positive for MRSA. Vancomycin was continued, and aspirin and colchicine were started. Two days later, there was a recurrent pericardial fluid collection with loculation. Surgery was thought to be dangerous in the setting of CD4 count of 12. She was managed conservatively thereafter, with vancomycin, aspirin and colchicine, and was successfully discharged from the hospital. Introduction The pericardium is a fibroelastic sac composed of visceral and parietal layers separated by a potential space, the pericardial cavity, which can normally have up to 50 mL of fluid. Acute pericarditis is an inflammation of the pericardium. It has been reported in 0.1%-0.2% of hospitalized patients, and 5% of patients presenting to the emergency department with a nonischemic chest pain [1,2]. Pericardial disease can occur secondary to inflammatory, neoplastic, vascular, iatrogenic, and idiopathic causes [3][4][5]. Cardiac tamponade is one of the complications of acute pericarditis. In a case series, it occurred in 14% of patients with idiopathic pericarditis and 61% of patients with neoplastic, tuberculous, or purulent pericarditis [6]. Methicillin-resistant staphylococcus aureus (MRSA) pericarditis is a rare life-threatening infection, with only few cases reported in literature [7,8]. Mortality rates for purulent pericarditis were reported to be as high as 40% in treated population [9]. In immunosuppressed patients or following thoracic surgery, staphylococcus aureus (30%) and fungi (20%) are more common [10]. We report a case of a rapidly developing MRSA pericarditis and tamponade in a patient with acquired immune deficiency syndrome (AIDS). Case Presentation A 46-year-old female with AIDS (CD4 count of 12), presented with fever and pleuritic positional chest pain. She was found to have a neck abscess and to be in sepsis (temperature 38.6 C, heart rate 114 bpm and white blood cell (WBC) count of 12.2 x 10 9 /L). Neck exam was remarkable for an anterior fluctuant neck mass. Cardiovascular exam on the day of admission showed regular rate and rhythm, normal heart sounds, and no jugular venous distention (JVD). CT scan of the neck was done, which showed the abscess on the right side ( Figure 1). Her neck abscess was drained in the emergency department, and samples were sent for evaluation. Blood cultures were drawn, and the patient was started empirically on vancomycin, and piperacillin/tazobactam for treatment of sepsis. Blood and abscess fluid cultures grew MRSA. Thus, vancomycin was continued. On the day of admission, electrocardiogram (EKG) (Figure 2, Panel A) showed sinus tachycardia, 1 mm ST segment elevation in inferior leads. Chest X-ray ( Figure 2, Panel B) showed pulmonary venous congestion, but no cardiomegaly, no focal consolidations. Chest CT without intravenous contrast ( Figure 2, Panel C) showed mild pericardial effusion, but was unremarkable otherwise. Four days later, she developed an acute hypoxic respiratory failure and had increased oxygen requirements reaching 8 L of oxygen-mask, and she was transferred to the medical intensive care unit (MICU). Exam showed tachycardia, tachypnea and increased JVD to 12-15 cm. EKG (four days after initial EKG, Figure 2, Panel D) showed sinus tachycardia, 1.5 mm ST segment elevation in inferior and lateral leads. Chest X-ray (four days after initial chest X-ray, Figure 2, Panel E) showed left lower lobe consolidation, with greater left than right pleural effusion. Transthoracic echocardiogram (TEE) was done and showed a moderate-tolarge pericardial effusion. There were echocardiographic indications for tamponade. The inferior vena cava diameter was 2.1 cm with minimal respiratory variation. There was a circumferential pericardial effusion with a thickness of about 2 cm (Figure 3). Pericardiocentesis with a pericardial drain was then performed, fluid was yellow and cloudy, with WBC count of 302/mm 3 , 81% neutrophils, lactate dehydrogenase was 1339 unit/L. Pericardial fluid culture was positive for MRSA. Aspirin and colchicine were started. TEE: Transthoracic echocardiogram Less than 48 hours after the drain was placed, it stopped draining and the patient had an increased JVD. At the time pulmonary embolism (PE) was to be ruled out. Thus, CT chest with intravenous contrast obtained (six days after initial CT, Figure 2, Panel F) showed an expanding pericardial effusion, and mild pleural effusion, but was negative for PE. Attempts to exchange the drain failed. Cardiothoracic surgery felt she was not a surgical candidate, given her CD4 count. Condition was managed conservatively, thereafter, with six weeks of vancomycin, aspirin and colchicine. Discussion MRSA pericarditis is a rare, potentially fatal disease. Route of infection is usually either by hematogenous or contiguous spread from retropharyngeal space, cardiac valves and below the diaphragm [11]. We believe route of infection in our case was by hematogenous spread, secondary to disseminated MRSA bacteremia due to untreated recurrent neck abscesses. Bacterial pericarditis typically presents with fever and chills, chest pain (often with dyspnea), and tachycardia. The presentation is always acute, with fevers occurring at regular intervals and frank rigors. Tachycardia is often due to the febrile response, but it may be an effort to compensate for decreased cardiac output from reduced ventricular filling due to cardiac tamponade. Tamponade can develop rapidly, as an effusion of 500 mL can accumulate over several days [12]. Chest pain is usually substernal pleuritic, often radiating to the scapular ridges due to the phrenic nerves passing through the anterior pericardium and innervating the trapezius ridges, which is consistent with our patient's presentation [13]. Tuberculous pericarditis was also suspected given the patient's severe immunosuppression, but acid-fast bacilli culture, and adenosine deaminase were negative which ruled out tuberculosis. Management of MRSA pericarditis was done through pericardial drain, with some cases treated with pericardial window (Class I recommendation for pericardial window, and Class IIa for subxiphoid pericardiotomy by 2015 ESC guidelines for the diagnosis and management of pericardial diseases: The task force for the diagnosis and management of pericardial diseases of the European Society of Cardiology) [14]. Our case was managed by pericardial drainage alone, and although she had recurrence of pericardial effusion, pericardiotomy was felt to be very risky given her AIDS with CD4 count <50. Thus, the team decided to proceed with medical management and pericardial drain insertion instead of the more aggressive option of pericardial window, and the patient's condition improved with antibiotics, anti-inflammatory medications and pericardial drainage within days, in spite of recurrence of effusion and loculation, and the patient was discharged. Conclusions MRSA pericarditis is a rare, possibly fatal, disease. It is usually a result of the spread of a remote infection (either hematogenously or contiguously). Prompt identification and diagnosis are key. Management is usually by drainage and/or pericardial window. Long-term intravenous antibiotics are usually required as well. Additional Information Disclosures Human subjects: Consent was obtained by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.	2020-05-21T00:10:23.607Z	2020-05-01T00:00:00.000	{ "year": 2020, "sha1": "bb79e27928d22a324bf01309a3f0080491578aec", "oa_license": "CCBY", "oa_url": "https://www.cureus.com/articles/31116-rapidly-developing-methicillin-resistant-staphylococcus-aureus-pericarditis-and-pericardial-tamponade.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "9dbf2e6b372892fe90af91277ef36e6e30998810", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
244710307	pes2o/s2orc	v3-fos-license	Aggressive containment, suppression, and mitigation of covid-19: lessons learnt from eight countries Shishi Wu and colleagues examine three distinct response strategies for covid-19 in eight countries and argue that aggressive containment is the optimal approach to limiting loss of lives and livelihoods and achievable in the absence of vaccines and effective therapies Defining response strategies Aggressive containment is usually defined as zero community transmission for more than 28 days, with the understanding that outbreaks might arise from border control failure. 4 6 This strategy rapidly reduces disease burden by lowering covid-19 cases, deaths, and the risk of overwhelming the health system. After community transmission is eliminated, domestic control measures can be lifted, and daily activities can resume largely without restriction. Suppression, on the other hand, aims to minimise the number of covid-19 cases without expecting to end community transmission. Modelling studies indicate that suppression cannot prevent an epidemic but can delay it until population immunity is reached through immunisation or natural infection. 1 5 7 Mitigation aims to minimise the effects of the pandemic on vulnerable populations and to avoid overwhelming health systems by permitting controlled transmission in low risk groups. 8 Different response strategies are put into use through combinations of public health interventions, which can be categorised into three groups: case based (such as case detection, contact tracing, and isolation), population based (such as mask wearing and physical distancing), and border control measures (such as travel bans and mandatory quarantine requirements). 9 Community engagement, whereby individuals and groups are involved in delivering services and supporting the uptake of control measures, was commonly included in the response strategy. We looked at the public health interventions and community engagement activities implemented in eight countries with different response strategies (box 1). We also examined the preceding determinants to developing and sustaining these interventions. Box 1: How countries were categorised by their response strategies We purposively selected eight countries and categorised their responses to the initial stages of the pandemic in 2020 into three distinct strategic approaches. For countries that did not explicitly announce their response strategies, the categorisation was derived from position statements on the goals of national responses by government officials or the intention of public health interventions implemented in the first wave of the pandemic. The strategies were defined as: • Aggressive containment-countries aimed to eliminate community transmission and achieved elimination status for 28 consecutive days by implementing public health interventions • Suppression-countries aimed to suppress and minimise community infections by implementing public health interventions • Mitigation-countries aimed to avoid overwhelming health systems by flattening the epidemic curve or achieving herd immunity in the population. The public health interventions focused on protecting vulnerable and high risk groups while allowing transmission among low risk groups. Public health interventions were categorised as: • Population based interventions-such as lockdowns, face masks, social distancing, and personal hygiene • Case based interventions-include case detection, contact tracing of confirmed cases, isolation, and surveillance of cases • Border control measures-such as travel restrictions for travellers from high risk countries or mandatory quarantine requirements. Aggressive containment Aggressive containment was the predominant strategy in Asian and Pacific countries, such as China, New Zealand, and Singapore (table 1). With clearly defined targets, these countries achieved zero community cases in the first year of the pandemic. Case based interventions Strict measures to identify, trace, and isolate covid-19 cases were commonly implemented in these countries. 14 Surveillance needs to be sensitive and geographically comprehensive to provide a timely and accurate understanding of disease burden. 15 In addition to passive testing of symptomatic people, these countries also took an active surveillance approach. In China, multiple rounds of population testing were conducted for all residents in cities where outbreaks occurred. Surveillance testing of high risk groups was also implemented in China, New Zealand, and Singapore, aiming to identify community cases at an early stage. These countries made great efforts to increase capacities for testing, contact tracing, and isolation. China and Singapore ramped up test kit production to meet accelerating demand. All three countries scaled up laboratory capacity, including building new laboratories, decentralising testing services, and training laboratory personnel. Contact tracing was implemented stringently: all three countries required contact tracing for all cases using a forward tracing approach that looks for case contacts from symptom onset. Close contacts were isolated either in government designated facilities or at home. In Singapore, spot checks were carried out to ensure people adhered to quarantine orders. Innovative technologies were widely used to facilitate contact tracing. 16 An app that allows people to scan a QR code at businesses they visit was used in all three countries to document their location and visit time. 17 Population based interventions Countries that aimed for containment implemented intensive population level interventions to curb community transmission early in the pandemic. China, New Zealand, and Singapore implemented lockdown 18 either in high burden regions or nationally. Initial lockdowns were more stringent and longer than in countries pursuing other strategies-venues and events involving gatherings were banned, and mobility restrictions were imposed, such as limited access to public spaces except for essential business. China implemented mandatory mask wearing soon after the outbreak in Wuhan in January 2020. Singapore and New Zealand adopted it in April and June 2020, respectively. To mitigate the effects of population based interventions, all three countries launched socioeconomic support for businesses, households, and the economy, such as subsidised loans for small businesses (New Zealand), poverty subsidies (China), or job support programmes (Singapore). These countries viewed the route to economic recovery as only possible by containing the pandemic. Border control measures Countries implemented strict travel restrictions to keep their borders closed to most visitors. Testing and mandatory 14 day quarantine were required for travellers entering China, New Zealand, and Singapore. The governments provided surveillance testing for workers at ports of entry; phone based technology was used to monitor the health status of inbound travellers in quarantine. Community engagement All three countries rolled out comprehensive media campaigns across multiple platforms urging the public to adhere to prevention measures. They also proactively engaged communities to plan, deliver, or promote adherence. China used community workers to educate residents about covid-19 prevention measures, provide directions to nearby testing and quarantine facilities, and distribute necessities to residents. Singapore used volunteers to educate older people who lived by themselves about prevention measures. Factors influencing strategic choices and actions A strong commitment to tackling covid-19 from the heads of government prompted immediate action at the beginning of the pandemic. Learning from past experiences with the SARS outbreak in 2003, China and Singapore had strengthened their preparedness for public health emergencies. The Singaporean government was committed to improving pandemic preparedness and constructed a fit-for-purpose infectious diseases centre to be used in times of emergencies. Policy makers were receptive to scientific input from expert groups and committees, enabling these countries to formulate evidence driven national covid-19 responses-for example, an expert committee of infectious disease experts, public health specialists, and clinical infectious disease doctors was formed during the first month of the outbreak in Wuhan, which played a central role in making decisions on response strategies. Suppression Countries that adopted the suppression strategy include Argentina, Uganda, and the United States (table 2). As of 16 November 2021, Argentina (2549.9) and the US (2300.3) were among the countries with the most cumulative deaths per million people; Uganda (68.8) had much lower cumulative deaths per million people than other countries using the suppression strategy. 13 Table 2 \| Performance of countries that adopted the suppression strategy in the first year of the pandemic Results Quarterly GDP change in 2020 versus the same period in 2019 (%) 11 Case based interventions Guidance on testing eligibility was based on available resources and the epidemiological situation in each country. Argentina and the US prioritised testing for high risk groups in the first half of 2020 and decentralised testing services to private laboratories. Uganda surged testing capacity by modifying existing laboratories to make them appropriate for testing covid-19 and purchased test kits on the international market to ensure adequate stocks. The testing capacity in these countries was stretched by limited testing sites and laboratories and shortage of testing kits and technicians. Contact tracing was implemented with different approaches and stringency in each country. Argentina did not implement consistent contact tracing until May 2020 with the Detectar programme. 20 In the US, contact tracing was the responsibility of states and counties, which used different implementation models, funding, workforce, and policies. 21 As cases increased, financial shortage and overwhelming workload were the common challenges for implementing contact tracing stringently in the US and Uganda. In the US, reluctance to provide contacts was an additional challenge, leading to less than one contact per case identified on average in some regions. 21 To support its surveillance system, the US launched syndromic surveillance, which monitors cases that meet the clinical definition of covid-19 without confirmation by testing. Population based interventions These countries implemented intensive population interventions to contain community transmission in the early phase of the pandemic (see supplementary file for specific interventions). In Argentina, a national lockdown started in March 2020, with exemptions for people providing essential services. Many states in the US issued "stay at home" orders in March or April 2020, which remained in place until mid-May. Schools and non-essential businesses were closed. A national lockdown was imposed in Uganda at the end of March 2020. But lockdown in these countries was relaxed before community transmission was eliminated. As evidence on face masks emerged, Argentina, Uganda, and many US states introduced mandatory mask wearing in April and May 2020. Countries that aimed for suppression saw a trade-off between public health measures and economic growth, emphasising economic recovery during relaxation periods. Despite this, multiple epidemic waves and subsequent tightening of restrictions resulted in all countries providing fiscal stimulus for businesses and employment protection. Border control measures Travel restrictions, such as suspension of flights from high risk countries and temporary border closure, were imposed in all three countries in the early months of the pandemic but were gradually lifted towards the end of 2020. Although proof of negative test results was commonly required for inbound travellers, quarantine requirements varied in stringency across countries. All travellers to Uganda were required to isolate at government facilities for 14 days, whereas home quarantine was allowed in the US and Argentina. Community engagement Countries aiming for suppression developed risk communication strategies that leveraged media platforms to reach the masses while local non-governmental organisations reached out to communities on the ground. Community engagement activities were also common in these countries. In Uganda, for example, community volunteers warned the public about the consequences of violating control measures when fewer deaths and cases led to a rise in complacency among the public. Factors influencing strategic choices and actions Partisan politics in countries such as the US led to mixed messages and lack of commitment from the government to mount a timely and effective response to the pandemic, resulting in high loss of lives. 22 US political leaders discredited scientific advisers, contributing to delay and lack of coordination in its covid-19 response. 23 Uganda was relatively better prepared for public health emergencies, having previously dealt with Ebola outbreaks. Argentina and the US were less prepared, partially owing to limited experiences of responding to large scale epidemics or underfunded public health systems. Mitigation Sweden maintained a mitigation strategy from the start of the pandemic; the United Kingdom initially aimed for mitigation but shifted towards suppression as the pandemic escalated (table 3) Case based interventions Countries that aimed for mitigation delayed efforts to increase capacity for testing and contact tracing capacity. Sweden introduced testing for travellers returning from high risk countries in February 2020. After the eligibility for testing was expanded to priority groups, insufficient access to testing was reported in many regions. In the UK, people with symptoms suspected of covid-19 were not eligible for testing in the early months of the pandemic. After the government decided to increase testing capacity in May 2020, eligibility expanded. Contact tracing policies were inconsistent over the course of the pandemic, with delayed efforts to increase capacity. In Sweden, contact tracing was implemented at the start of the pandemic but was abandoned after a few weeks due to stretched human resources. In the UK, full contact tracing was abandoned in line with the move from contain to delay phase in March 2020. After receiving additional funding from the government, a new contract tracing system was launched in May 2020. However, it did not function effectively as expected, as tracers failed to get in touch with one in eight confirmed cases. 21 Both countries took measures to strengthen surveillance of covid-19 cases, such as conducting large-scale surveillance studies or deploying sentinel surveillance. Population level interventions Countries that adopted the mitigation strategy had less stringent population level interventions (see supplementary file for specific interventions) but gradually tightened them as the pandemic evolved. Instead of imposing lockdowns, Sweden issued recommendations for maintaining physical distance in schools, workplaces, and public places. Social and mass gatherings were banned in both countries. As daily cases increased, the UK moved towards a suppression strategy, marked by the announcement of lockdown in March 2020. The lockdown relaxed in June 2020 as the epidemic curve flattened. Mask wearing was not recommended for the public in these countries in the first six months, given the lack of evidence on its effectiveness. There was no national recommendation for mask wearing for the public in Sweden. In the UK, mask wearing was not mandatory until June 2020. Both countries offered socioeconomic support, such as the furlough scheme that paid wages of employees who lost their jobs owing to the pandemic (UK) and small businesses loans (Sweden). Border control measures Sweden did not require testing for symptom-free travellers before entering, and arriving travellers were not obliged to quarantine. The UK initially had no specific testing requirements for travellers. The government advised against non-essential travelling to high risk countries but did not impose travel bans or border closure. In June 2020, however, the UK tightened measures at the borders, including 14 day self-isolation for arriving travellers. Community engagement Risk communication was largely conducted on media platforms with limited community engagement. Press conferences by the Swedish Public Health Agency were held regularly and broadcast on television and the internet; digital platforms and hotlines were established to help people with migrant backgrounds access covid-19 information. Similarly, in the UK, updates on the epidemic situation or control measures were disseminated through regular press briefings. Factors influencing strategic choices and actions There was broad consensus on adopting a more relaxed response to covid-19 among politicians in Sweden, in line with its constitution, but policy makers in the UK took a "wait and see approach" through public statements, reassuring the public despite increasing domestic cases. When public health interventions were enacted, the population had already heard mixed messages about the need for a response. Benefits, challenges, and trade-offs The three strategies have their own benefits, challenges, and trade-offs (table 4). Although factors such as culture, demographics, and geography have contributed to how the pandemic and responses unfolded, countries that opted for aggressive containment had lower deaths per million than those that took other approaches. Requires resources and infrastructure to build increased capacities in a short time. Lowest deaths per million. Quicker economic recovery. Aggressive containment Stringent mobility restrictions may increase unemployment and reduce access to social and health services. Easing control measures before eliminating community transmission can lead to resurgence and excessive deaths. Recurrent public health interventions can cause fatigue, undermining the effectiveness of the interventions. Reduces covid-19 burden in the short term, thus avoiding overwhelming the health system. Suppression High death rates comparable to countries following the suppression strategy. But contrary to common belief, they also suffered from economic contraction and a slow recovery in the first year. Older people are not completely protected from infection, so the health system can be overwhelmed, resulting in excessive deaths. Preserves freedom of movement. Mitigation A concern of aggressive containment is the potential economic cost attributable to the maintenance of stringent measures. We found, however, that countries using all strategies needed to introduce similar socioeconomic support measures. This supports emerging research that containment might actually protect the economy by reducing uncertainty and allowing for a faster recovery, resulting in less spending on socioeconomic measures and better economic returns owing to lower morbidities and mortalities. 24 -26 Most countries employing aggressive containment achieved economic growth in the third and fourth quarters of 2020 (table 1), whereas countries following other strategies showed a sustained decline in gross domestic product (GDP) (table 2, table 3). Overall, countries that implemented aggressive containment saw less fluctuation in weekly GDP and quicker recovery in the first year of the pandemic, which supports the strategy's potential to protect lives and livelihoods better than suppression or mitigation approaches. 27 Successful containment is underpinned by a country's ability to rapidly mobilise resources and expand health system capacity, so lower income countries might face challenges. Even if countries manage to implement aggressive containment measures, some trade-offs remain. Despite favourable health and economic outcomes, aggressive containment has been criticised for restricting civil liberties and raises concerns about privacy infringement when aggressive contact tracing measures are taken. 28 But civil liberties can be restored quickly in countries that achieved containment, whereas research shows that liberties were severely impacted in countries following other strategies where mobility restrictions were often implemented more strictly. 29 Aggressive containment also disrupts employment and reduces access to social and health services, bearing social and economic costs that need to be balanced with the cost of lives lost. 30 Suppression can reduce covid-19 burden in the short term, but easing control measures before eliminating community transmission can lead to a resurgence of cases, resulting in epidemic waves and excessive deaths. Recurrent public health interventions can cause fatigue in the population, leading to higher non-compliance in subsequent pandemic waves. 31 Countries employing the suppression strategy, such as Argentina and the US, had some of the highest mortalities in the world and also experienced steep economic downturns and slow recovery in 2020 (table 2). Having much lower deaths per million, Uganda seems to be more resilient to the health effects of the pandemic, possibly as lower income countries have younger populations that are less affected by the disease than higher income countries. 7 Owing to a large informal labour sector, however, lower income countries are more vulnerable to the socioeconomic repercussions of stringent public health interventions. The governments often lack fiscal space and an accountable administrative system to tackle these unintended consequences. 32 33 Thus, the benefits of adopting the suppression strategy to reduce mortality might be less substantial when compared to the corresponding economic loss. 34 Countries that opted for mitigation preserved the freedom of movement of their citizens, but this seems risky, particularly in higher income countries with older populations, where health systems might get stretched or even overwhelmed, resulting in excessive deaths. 2 7 Both Sweden and the UK had death rates comparable to countries adopting the suppression strategy (table 3). These countries also experienced a slow economic recovery after a steep economic decline in the second quarter of 2020, as seen in Sweden's and the UK's economic contraction in the third and fourth quarter of 2020 (table 3). Steps to achieving successful containment Our analysis shows that containing community transmission is the optimal strategy to save lives and protect the economy and is achievable in the absence of vaccines and effective drugs. We identified the interventions, actions, and determinants that contribute to successful containment (fig 1). Determining when public health interventions are implemented and lifted is central to success. Countries must take immediate action in response to a disease outbreak. Premature lifting of public health interventions leads to epidemic waves, causing even more deaths and damage to the economy. 35 36 Countries that aimed for containment clearly defined the goal of eliminating community transmission and made exit decisions based on explicit indicators. Countries need to implement all three categories of public health interventions comprehensively, with support measures for increasing capacity and mitigating the socioeconomic repercussions. Countries successful in containment implemented intensive population based interventions, such as multiple national or subnational lockdowns to curb community transmission. They delivered a range of socioeconomic support to provide safety nets for affected and vulnerable populations and quickly introduced measures to identify, trace, and isolate cases in communities and at borders. To ensure these measures could be delivered comprehensively and stringently, these countries made efforts to increase public health capacity and strengthen border control as the pandemic escalated. Support packages also improved the effectiveness of case based interventions like isolation. In the US, for example, provision of food deliveries and additional financial support to those in isolation was found to enhance compliance. 37 Countries that opted for mitigation or suppression focused on population based interventions, but they often sidelined or were hesitant towards case based and border control measures-most of these countries delayed efforts to increase public health capacity, suspended contact tracing, and maintained relaxed border control. Successful containment is underpinned by the public's trust in policy makers and the government. Effective community engagement, achieved through strong community leadership and coordination, was fundamental to Ebola responses, leading to increased trust in health authorities. 38 -40 During the covid-19 pandemic, New Zealanders trusted and supported the interventions implemented by their government, resulting in high compliance initially, which declined over time owing to fatigue and complacency. 41 To maintain adherence, two way community engagement through a whole-of-society approach ensures that all stakeholders understand the rationale for tightening or sustaining measures. Notably, trust and community engagement are integral when technology is involved in contact tracing. The US and UK faced challenges in their populations' trust in the government's data management and privacy regulations, which affected the uptake of contact tracing applications and thus reduced their effectiveness. 42 43 By engaging the community and implementing privacy safeguards, contact tracing applications offer faster tracing and ringfencing, and is therefore postulated to contribute to successful containment. 44 Three factors influence strategic choices and the success of containment. First, strong political will and commitment to tackling covid-19 prompted immediate actions, multi-sectoral collaboration, and rapid resource mobilisation through a whole-of-government, whole-of-society governance approach in successful countries. 45 Second, a country's response capacity depends on its public health preparedness. 46 The effectiveness of responses depends on the country having a well maintained public health system to perform its core functions and manage logistics in emergencies. 47 Countries have been asked to strengthen their preparedness for public health emergencies by implementing the Global Health Security Agenda and International Health Regulations. 48 But most countries lack robust preparedness plans and core public health capacities, as their public health systems are vastly underfunded and neglected. 49 50 Successful countries drew lessons from past outbreaks to inform decisions on control measures and adapt their public health systems quickly. 51 The lack thereof, coupled with the absence of aggressive containment measures, has been documented to overwhelm health systems, increasing the mortality risks for patients with covid-19. 50 Finally, successful containment is more likely to be achieved with scientific input. Up to date research evidence is the foundation for making optimal decisions on control measures to be implemented or relaxed, but the supply of scientific evidence does not guarantee its uptake. Hence it is critical to establish mechanisms to ensure that evidence is incorporated in policy making. In countries adopting the aggressive containment strategy, independent expert committees, formed after the declaration of the pandemic, commonly spearheaded the formulation of response strategies and took on key advisory roles in high level policy decision making. Going forward As the world proceeds into covid-19 endemicity, aggressive containment might not be the best way forward. Some, but not all, countries that permitted controlled spread of the virus (thus generating higher natural immunity levels) and had robust vaccination campaigns have been seen to relax public health interventions and are reopening sooner than countries that used aggressive containment and have more naive populations that rely predominantly on the rapid expansion of vaccination coverage. 52 53 Countries that are reopening faster have paid the price in terms of a higher death toll in the earlier stages. The containment strategy was arguably intended to provide time to develop covid-19 treatments and vaccines, to strengthen health systems, and for informed decision making but not to be a long term response. 36 54 As we cross the pandemic's two year mark, a more sustainable approach that amalgamates acceptable levels of community transmission and high vaccination rates may be the best way for countries that have used aggressive containment to reach a new equilibrium. 55 56 Implication of our analysis Containing community transmission is the optimal strategy for new emerging infectious hazards and achievable in the absence of vaccines. Our findings further bolster recent research in favour of more stringent responses and extends those findings by including a diverse geographic, sociocultural, and economic sample of countries to analyse response strategies. 27 30 36 The lessons drawn from eight countries are useful to guide the development of a comprehensive response to covid-19 and future pandemics. Although the development of vaccines has raised the hope of returning to normal life, relying solely on vaccines to control the pandemic is uncertain given the inequitable global vaccination rollout. Implementing the aggressive containment strategy-which uses public health interventions built on trust and community engagement coupled with strong political will, health system preparedness, and receptiveness to scientific inputs-will reduce the impact on lives and livelihoods, particularly at the earlier stages of the pandemic. As more countries, including those successful in containment, transition to covid-19 endemicity, continuing investments and efforts are needed to reduce inequalities, enhance health system capacities, and strengthen public health preparedness in the event of potential emergent strains and waning vaccine immunities. Key messages • Aggressive containment of community transmission is the optimal strategy in emerging pandemics to save lives and protect the economy and achievable in the absence of vaccines and treatments • Successful containment requires countries to take immediate action in response to emerging outbreaks and clearly define the targets for relaxing interventions • A comprehensive package of public health interventions needs to be implemented stringently with support measures for mitigating the adverse effects and increasing capacity • Success is underpinned by trust and community engagement and facilitated by strong political commitment, well prepared public health systems, and scientific input into policy making • Aggressive containment might not be sustainable in the long term. A more sustainable approach which amalgamates acceptable levels of community transmission and high vaccination rates may be the best way forward. Provenance and peer review: Commissioned; externally peer reviewed. Contributors and sources: SW and HL-Q conceived and planned the manuscript. SW, RN, and HL-Q drafted the manuscript with inputs from all authors. All authors contributed to revising the manuscript and approved the final version. This analysis was part of the work commissioned by the Independent Panel for Pandemic Preparedness and Response, which reviewed the national responses of 28 countries. Data used for the analysis were collected through three complementary methods: literature review of peer reviewed papers, policy documents, public reports, and articles that examined national and subnational policy responses; semi-structured interviews with country experts and submissions written by the national governments of selected countries about their own account of the measures implemented to contain covid-19; and validation of country specific data by experts through written consultation and round	2021-11-29T14:06:41.965Z	2021-11-28T00:00:00.000	{ "year": 2021, "sha1": "18ae280b5930f8c65a6354865580759ee63daae4", "oa_license": "CCBYNC", "oa_url": "https://www.bmj.com/content/bmj/375/bmj-2021-067508.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "18ae280b5930f8c65a6354865580759ee63daae4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
228081014	pes2o/s2orc	v3-fos-license	Encapsulation Preserves Antioxidant and Antidiabetic Activities of Cactus Acid Fruit Bioactive Compounds under Simulated Digestion Conditions Cactus acid fruit (Xoconostle) has been studied due its content of bioactive compounds. Traditional Mexican medicine attributes hypoglycemic, hypocholesterolemic, anti-inflammatory, antiulcerogenic and immunostimulant properties among others. The bioactive compounds contained in xoconostle have shown their ability to inhibit digestive enzymes such as α-amylase and α-glucosidase. Unfortunately, polyphenols and antioxidants in general are molecules susceptible to degradation due to storage conditions, (temperature, oxygen and light) or the gastrointestinal tract, which limits its activity and compromises its potential beneficial effect on health. The objectives of this work were to evaluate the stability, antioxidant and antidiabetic activity of encapsulated extract of xoconostle within double emulsions (water-in-oil-in-water) during storage conditions and simulated digestion. Total phenols, flavonoids, betalains, antioxidant activity, α-amylase and α-glucosidase inhibition were measured before and after the preparation of double emulsions and during the simulation of digestion. The ED40% (treatment with 40% of xoconostle extract) treatment showed the highest percentage of inhibition of α-glucosidase in all phases of digestion. The inhibitory activity of α-amylase and α-glucosidase related to antidiabetic activity was higher in microencapsulated extracts than the non-encapsulated extracts. These results confirm the viability of encapsulation systems based on double emulsions to encapsulate and protect natural antidiabetic compounds. Introduction In Mexico, there are about 3600 to 4000 species of medicinal plants, of which Mexicans empirically use approximately 500 plant species, principally the families Asteraceae (47 species), Fabaceae (27), Solanaceae, Euphorbiaceae (10), Lamiaceae (9) and Cactaceae (16). These plants are potential sources of hypoglycemic drugs and are widely used in several traditional systems of medicine to prevent diabetes [1]. Chemical compounds related to the antidiabetic activity of these plants include polysaccharides, alkaloids, glycopeptides, terpenes, peptides, amines, steroids, phenolic compounds Droplet Morphology and Encapsulation Efficiency The double emulsions obtained were microspheres due to the complex distribution of the drops that make up the internal aqueous phase of all the treatments. After 48 days of storage, no flocculation or coalescence of the globules was observed. The smallest sizes were observed in ED20% and in the control ( Figure 1) there were no statistical differences (p > 0.05) between these treatments. The drop size of the treatments was proportional to the percentage of encapsulated xoconostle. The highest Molecules 2020, 25 Stability of Bioactive Compounds in Multiple Emulsions At time zero, the content of total phenols in all the encapsulates showed significant differences (p < 0.05) between all the treatments. After 48 days of storage, the lowest percentage of loss of total phenol content was the ED40% treatment, presenting 22.7%. The flavonoid content was not significantly different between the ED60% and ED40% treatments. Which could be due to the encapsulation efficiency obtained for these two treatments. The ED40% treatment had a high encapsulation efficiency of 95.91 ± 2.26%, while ED60% had lower values, 66.32 ± 1.85%. Both the content of betacyanins and betaxanthins decreased significantly (p < 0.05) during storage and showed statistical differences between treatments (p < 0.05) ( Table 1). Stability of Bioactive Compounds in Multiple Emulsions At time zero, the content of total phenols in all the encapsulates showed significant differences (p < 0.05) between all the treatments. After 48 days of storage, the lowest percentage of loss of total phenol content was the ED40% treatment, presenting 22.7%. The flavonoid content was not significantly different between the ED60% and ED40% treatments. Which could be due to the encapsulation efficiency obtained for these two treatments. The ED40% treatment had a high encapsulation efficiency of 95.91 ± 2.26%, while ED60% had lower values, 66.32 ± 1.85%. Both the content of betacyanins and betaxanthins decreased significantly (p < 0.05) during storage and showed statistical differences between treatments (p < 0.05) ( Table 1). Antioxidant and Antidiabetic Activity of Xoconostle Extract All treatments showed a decrease in antioxidant activity level during storage ( Table 2). The ED40% treatment had the highest antioxidant activity. Regarding the inhibition of the radical 2,2 azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) (%), significant differences (p < 0.05) were observed between treatments and during storage. After 48 days of storage ED40% showed the highest antioxidant activity (45.35 ± 0.26%). At day zero, no statistical differences (p > 0.05) were observed in the percentage of inhibition of α-amylase between the treatments with encapsulated xoconostle extract. ED20% and ED40% remained without significant changes during the first 12 days (Table 2). Results are shown as mean ± standard deviation of tests performed in triplicate (n = 3). Averages with the same lowercase letters in the same column do not show statistical differences (p > 0.05) between the days of storage of the same treatment. Means with the same capital letters in the same row do not show statistical differences between treatments on the same day (p > 0.05). DPPH: inhibition of the 2,2-diphenyl-1-picrylhydracil radical expressed as a percentage (%). ABTS: inhibition of the 2,2 azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid radical expressed as a percentage (%). Control: double emulsion with 40% deionized water in the internal aqueous phase. ED20%: double emulsion with 20% xoconostle extract in the internal aqueous phase. ED40%: double emulsion with 40% xoconostle extract in the internal aqueous phase. ED60%: double emulsion with 60% xoconostle extract in the internal aqueous phase. Acarbose was used as a second control at the time of analysis of all samples. The ED40% treatment remained with the highest inhibitory power against α-amylase (30.46 ± 1.27%) which is congruent with the high EE%. The α-glucosidase inhibition percentages showed statistical differences (p < 0.05) between treatments and during storage time (Table 2). At day zero, this inhibition percentage was proportional to the amount of encapsulated extract; the highest antidiabetic power was found in ED60% treatment (78.84 ± 1.17%). At the end of the trial, after 48 days ED60% showed the greatest losses of biological activity (≈85%) and ED40% had the highest antidiabetic activity (30.19 ± 0.67%) after day 48. Release of Fatty Acids during Digestion In the first 20 min there were no apparent differences between the ED40%, ED60% and control treatments, while ED20% showed a slower but progressive release of fatty acids, probably due to the greater amount of oil phase present in this formulation ( Figure 2A). In the first 20 min there were no apparent differences between the ED40%, ED60% and control treatments, while ED20% showed a slower but progressive release of fatty acids, probably due to the greater amount of oil phase present in this formulation ( Figure 2A). Bars with the same lowercase letters do not show statistical differences (p > 0.05) between the digestion phases of the same treatment. Bars with the same capital letters do not show statistical differences between treatments in the same digestion phase (p > 0.05). DPPH: inhibition of the 2,2-diphenyl-1-picrylhydracil radical expressed as a percentage (%). ABTS: inhibition of the 2,2′azinobis-(3-ethylbenzothiazoline) -6-sulfonic acid radical expressed as a percentage (%). SD: no digestion. GF: gastric phase. FI: intestinal phase. Control: double emulsion with 40% deionized water in the internal aqueous phase. ED20%: double emulsion with 20% xoconostle extract in the internal aqueous phase. ED40%: double emulsion with 40% xoconostle extract in the internal aqueous phase. ED60%: double emulsion with 60% xoconostle extract in the internal aqueous phase. Bars with the same capital letters do not show statistical differences between treatments in the same digestion phase (p > 0.05). DPPH: inhibition of the 2,2-diphenyl-1-picrylhydracil radical expressed as a percentage (%). ABTS: inhibition of the 2,2 azinobis-(3-ethylbenzothiazoline) -6-sulfonic acid radical expressed as a percentage (%). SD: no digestion. GF: gastric phase. FI: intestinal phase. Control: double emulsion with 40% deionized water in the internal aqueous phase. ED20%: double emulsion with 20% xoconostle extract in the internal aqueous phase. ED40%: double emulsion with 40% xoconostle extract in the internal aqueous phase. ED60%: double emulsion with 60% xoconostle extract in the internal aqueous phase. Antioxidant Activity All treatments showed an increase in antioxidant capacity (DPPH and ABTS) as the digestive process advanced and there were significant differences between treatments (p < 0.05). The antioxidant activity measured as inhibition of the radical DPPH showed a maximum of 73.72 ± 0.99% (ED40%) after the intestinal phase (FI), without showing statistical differences (p > 0.05) with ED60% (72.15 ± 1.15%). This progressive increase in antioxidant activity during digestion is due to the hydrolysis of the canola oil ( Figure 2B) that forms the microspheres and the consequent release of biocomposites from the xoconostle extract present in the innermost part of the emulsions. The inhibition of the radical ABTS in general did not show statistical differences (p > 0.05) between the samples submitted to gastric digestion (GFR) and the undigested samples (SD), however, it increased with the intestinal phase due to the hydrolysis of the lipid component and release of xoconostle extract ( Figure 2C). In the samples without digestion (SD), the inhibition of α-amylase was proportional to the fraction of extract used as internal phase and decreased after digestion. Statistical differences (p < 0.05) were observed between the treatments after the intestinal phase (FI), however, the ED40% treatment showed the highest percentage of inhibition (29.10 ± 2.43%) ( Figure 2D). The ED40% treatment also showed the highest percentage of inhibition of α-glucosidase in all phases of digestion. The treatments with extracts not subjected to digestion (SD) had around 50% inhibition of α-glucosidase and after digestion their inhibitory activity decreased, the ED40% treatment again showing the highest percentage of inhibition after intestinal digestion ( Figure 2E). Composition of the Cactus Acid Pear (Xoconostle) Extract In respect to the results of total phenols and flavonoids contents of the extracts, Morales et al. [24] found 59.48 mg GAE/g total phenolic content and 58.40 mg QE/g total flavonoids content when they analyzed extracts from seeds of O. matudae fruits, and Osorio-Esquivel et al. [25] found similar results when analyzed Opuntia joconostle. Other authors, Hernandez-Fuentes et al. [26], reported similar results with the whole fruit, and they concluded that the difference in the total phenolic content could be due to the genotype effect, both of the species and the crops, as well as the growth conditions of the fruits. Bioactive compounds, such as phenolic compounds, act as antioxidants, which delay or prevent oxidation of the substrate, and they are strongly correlated to antioxidant activity. The consumption of xoconostle fruits may contribute to increasing the protective effect of antioxidants in the diet [27]. Figure 3 shows the droplet size results of the different treatments and Figure 1 shows the micrographs obtained throughout storage. There are similar reports when encapsulating phenolic compounds [28] and other materials [29]. The presence of spherical drops of oily phase (canola oil) dispersed in a continuous phase that in turn contain smaller drops of extract inside, in addition to the Brownian movement observed under the microscope, confirm the successful formation of double water-type emulsions, water-oil-water (W 1 /O/W 2 ) [30]. Regarding their morphology, main types of double emulsions have been reported ( Figure 1) according to the type and quantity of drops present in the internal aqueous phase: (1) microcapsules, which contain only one drop of internal aqueous phase, (2) multivesicles, which contain several drops defined in their innermost phase (W1) and (3) microspheres, in which, unlike the previous ones, their internal structure is very complex [31]. These results suggest the formation of double microsphere-type emulsions due to the complexity shown in the internal aqueous phase of all treatments. Similar observations have been reported when encapsulating phenolic compounds [28] and other materials [32]. There are many phenomena of destabilization of double emulsions, one of the most common being the gravitational separation of phases (creaming or sedimentation). To counteract this phenomenon, drop size decrease has been reported as an effective strategy [33]. Therefore, it is accepted that there is an inverse relationship between the initial drop size and the stability prognosis of double emulsions, in the same way, changes in drop size during storage are related with loss of stability [34] and, therefore, less protection of encapsulated bioactive compounds. Droplets (W 1 ) with high internal phase concentrations, tend to bind together and form larger droplets that decrease the stability of multiple emulsions [25]. Droplet Morphology and Encapsulation Efficiency the same way, changes in drop size during storage are related with loss of stability [34] and, therefore, less protection of encapsulated bioactive compounds. Droplets (W1) with high internal phase concentrations, tend to bind together and form larger droplets that decrease the stability of multiple emulsions [25]. Stability of Bioactive Compounds in Multiple Emulsions The encapsulation of bioactive compounds susceptible to damage caused by environmental factors is a widely used, efficient technique. However, it has been reported that the success of encapsulation depends largely on the wall material used [35] and the suitable selection and proportion of surfactants of the primary emulsion. Furthermore, it has been suggested that a high encapsulation efficiency (EE) is a direct consequence of a good stability of double emulsions [31]. In this work, the highest encapsulation efficiency was observed in ED40% and the lowest in ED60%. This value can be explained by the fact that, in emulsions with high internal phase concentrations, the W1 droplets tend to bind together and form larger droplets that decrease the stability of multiple emulsions [36]. Shaddel et al. [37] suggest that double emulsions with more than 50% internal phase are unfavorable for the protection of the compounds since, as the encapsulated volume increases, the protective matrix decreases. EE values similar to those calculated for ED20% were reported by Silva et al. [38] encapsulating gallic acid in double emulsions (80%). Treatment ED40% showed an EE similar to that reported by de Almeida et al. [39] by preparing double emulsions with anthocyanin as internal phase W 1 (90.6%). Other authors reported EE values (62%) similar to treatment ED60% (≈66%) when encapsulating other materials [30]. In all treatments, the content of total phenols, flavonoids and tannins showed a tendency to decrease during storage despite encapsulation. Guzmán-Díaz et al. [40] reported percentages of loss of green tea polyphenols encapsulated within double emulsions of 15.13% after 35 days of storage at 4 • C. These results are similar to the present study-the storage temperature of the double emulsions influences the stability of the encapsulated compounds during storage. Kim et al. [28] reported that the effectiveness of double emulsions to protect bioactive compounds during storage can be influenced by their concentration in the internal phase (W 1 ), by the type of encapsulated antioxidants and by the pH of the samples. There was also a significant decrease in the stability of the encapsulated kojic acid within double emulsions when the pH was closer to 7.0, a mean stability at pH 4.5 and the highest stability at acid pH (3.7). This behavior could partly explain the degradation of the encapsulated xoconostle compounds in our study. Betalains are pigments that have antioxidant activity and this gives them multiple biological activities. The most important betalains in cactus fruits are betacyanins. Both the betacyanin (BTC) and betaxanthin (BTX) content decreased significantly during storage. Cenobio-Galindo et al. [15] reported similar results of total betalain values (BTC + BTX when applying double emulsions incorporated in a fermented yogurt-type beverage (0.46-1.4 mg/100 mL). Antioxidant and Antidiabetic Activity of Xoconostle Extract The antidiabetic activity of natural extracts has been widely reported [12,16,41,42]. This property has been attributed mainly to the content of polyphenols and associated chemical families such as flavonoids and hydroxycinnamic acids. The antidiabetic activity of these phytochemicals affects glucose homeostasis at different points in metabolism due to their ability to inhibit enzymes such as α-amylase and α-glucosidase. Through different computational techniques, enzymatic inhibition mechanisms have been elucidated, which depend on the nature of the phytochemical. For example, it has been reported in the literature that the 3 ,4 -dihydroxyl groups of the B ring of certain flavonoids interfere with the amino acids that form the active site of these enzymes, preventing their biocatalysis [43]. Regarding the antidiabetic power of compounds such as phenolic acids, their activity has been reported. However, little is known about their mechanisms of action, although it is suggested that the acetylated groups present in these and other compounds play an important role in the mechanisms of inhibition [41]. Wang et al. [44] reported the inhibition of -amylase of in vitro assay due to the content of anthocyanins of the fruits of cultivated Lycium ruthenicum Murray. Pinto et al. [45] found 65% as the maximum inhibition of α-amylase, when it was tested in the Physalis peruviana L. extract. Other studies also reported that natural polyphenols have been reported to inhibit the activity of α-amylase and β-glucosidase [46][47][48][49]. Medina Pérez et al. [16] measured inhibitory activities of extracts obtained from different anatomic origin (pericarp, endocarp, mesocarp and whole fruit) of xoconostle fruits and found significant differences (p < 0.05) between extracts, the inhibition of α-amylase and β-glucosidase were higher in whole fruit extracts (25 mg/mL for α-amylase and 30 mg/mL for β-glucosidase inhibition). The inhibitory activity of α-amylase and α-glucosidase related to antidiabetic activity was higher than that reported by [50] when encapsulating phenolic compounds using the complex coacervation method (α-amylase and α-glucosidase), which reaffirms the viability of encapsulation systems based on double emulsions to encapsulate and protect natural antidiabetic compounds. Antioxidant Activity All treatments showed an increase in antioxidant capacity (DPPH and ABTS) after the intestinal phase. This increase in antioxidant activity after the intestinal phase may be due to different reasons. The digestion of the lipid fraction with pancreatin released the xoconostle extract, which contains different molecules with antioxidant activity. Later, the hydrolysis of the triacylglycerols that make up canola oil could cause the release of antioxidant compounds trapped in the oily matrix that contributed to the antioxidant power. In addition, it has been shown that the digestion of dairy proteins (such as whey protein isolate (WPI) used as a continuous phase in our treatments) with pepsin and pancreatin, releases peptides with amino acids such as Trp and Tyr which contain phenolic and indole groups with the ability to donate hydrogens, which in turn gives them high ABTS + radical elimination activity [51]. Pasukamonset et al. [50] found percentages inhibition of α-amylase similar to our results after digesting an extract of polyphenols from Clitoria ternatea trapped within alginate microcapsules (28.87 ± 0.09%). Links et al. [52] also reported a decrease in the percentages of inhibition of α-amylase and α-glucosidase after digestion of sorghum tannins encapsulated within microparticles of kafirin, attributing the degree of enzyme inhibition to the size and chemical structure of the polyphenols present in the extracts. The antidiabetic effect of the emulsion extract did not behave in a proportional way to the amount of encapsulated extract. Likewise, the antioxidant activity was not related to the inhibitory activity of the extract against any of the two digestive enzymes analyzed. Thus, the antidiabetic power seems to be mainly conditioned by the percentage of extract that actually remained within the oil drops (EE) and by factors such as globule size, that is, the smaller the drop size, the greater the gastric and intestinal resistance of emulsions against digestion. Fatty Acid Release During the simulation of the intestinal phase of digestion, the lipase present in pancreatin hydrolyzes the triacylglycerols present in the oily phase of the emulsions (canola oil), releasing the encapsulated content within the fat globules (xoconostle extract). As hydrolysis products, two free fatty acids and a monoacylglyceride are obtained [53]. Gasa-Falcon et al. [54] reported similar results to the present study: an accelerated lipid digestion in the first minutes of the trial and then a slower and progressive digestion period up to 120 min of digestion using double emulsions (using mandarin fiber as a biopolymer in the continuous phase). It should be noted that the ED40% treatment and the control (which were prepared with the same proportion of phases) did not show differences in hydrolysis throughout the experiment (p > 0.05). Because lipid digestion in double emulsions is considered an interfacial phenomenon that depends on the binding of the lipase-colipase complex on the surfaces of the emulsified fat droplets in the continuous phase, it can be concluded that the encapsulation of xoconostle extract did not cause a interfacial impediment that altered the behavior of lipid digestion of double emulsions [23]. Plant Material and Reagents Extracts from cactus pear (Opuntia oligacantha C.F. Först var. Ulapa) were obtained from physiological maturity fruits, acquired in the municipality of Tezontepec de Aldama Hidalgo, Mexico. Obtaining the Xoconostle Extract The methodology of Espinosa-Muñoz et al. [14] was used to extract the bioactive compounds of O. oligacantha. Lyophilized xocostle fruits were pulverized and passed through a 50 mm sieve and exposed to ultrasounds from the Vibra-cell VCX130 generator (Sonics, Newtown, CT, USA) for 20 min, at 130 W, 80% amplitude, and 20 kHz frequency. The final extract was filtered (Whatman No. 1) and centrifuged at 10,000 rpm for 15 min at 4 • C in a Z 36 HK centrifuge (Hermle, Baden-Wurtemberg, Germany). Samples were stored at −27 • C until analysis. Preparation of Double Emulsions The preparation of the double emulsions was carried out by the general two-stage method described by Cenobio-Galindo et al. [19], with modifications. In the first stage, a simple water-in-oil emulsion (W 1 /O) was prepared using xoconostle extract as the aqueous phase (W 1 ) and canola oil as the oil phase (O) and in the second stage, part of it was re-emulsified. A single emulsion was prepared from the second aqueous phase (W 2 ) to form the water-in-oil-in-water or W 1 /O/W 2 double emulsion systems. Simple W 1 /O emulsions were prepared in proportions of 20:80 (w/w), 40:60 (w/w) and 60:40 (w/w) (xoconostle extract: canola oil) and a control treatment with a 40:60 (w/w) ratio of distilled water and canola oil, respectively. Ten percent (w/w) of the oil phase of all treatments was replaced with 4 parts of lipophilic emulsifier (PGPR) and 1 part of hydrophilic emulsifier (DATEM), as described by Pimentel-González et al. [21]. Prior to the formation of the simple emulsions, the PGPR was dissolved in the canola oil and the DATEM in the xoconostle extract for 3 min at 4000 rpm in the dark by means of homogenization. The W 1 /O emulsions were formed using an Ultra-Turrax IKA T25 (IKA Works, Inc. Wilmington, NC, USA) homogenizer at 13,000 rpm for 15 min in an ice bath. To obtain the double emulsions W 1 /O/W 2 , 30% (w/w) of each single emulsion W 1 /O was taken and re-emulsified in 70% (w/w) of an aqueous dispersion (W 2 ) of 40% (w/w) whey protein isolate (WPI) previously prepared. The powder was slowly dispersed in distilled water by moderate magnetic stirring for 4 h and stored for 48 h at 4 • C. Before use, the powder was dissolved in 2.5% (w/w) of hydrophilic emulsifier PGPR in the dispersion. The double emulsions were obtained by low energy homogenization using an Ultra-Turrax IKA T25 (IKA Works, Inc. Wilmington, NC, USA) at 4000 rpm for 15 min in an ice bath. All treatments were stored at 4 • C and during days 0, 3, 6, 12, 24 and 48 of storage, aliquots were taken for the evaluation of the different parameters. Morphology and Droplet Size Drop morphology and size were analyzed according to the methodology reported by [21]. An aliquot of 100 µL of double emulsion was taken and dispersed in 900 µL of distilled water. A small drop was then taken from the mixture, placed on a slide and analyzed on an Olympus CX 31 optical microscope (Olympus Optical Co. Ltd., Tokyo, Japan) at 100× magnification. Photomicrographs were taken with a LUMENERA ® (Caltex Scientific, Irvine, CA, USA) camera coupled to the microscope and 30 random globular formations were measured for the determination of the droplet diameter using the Image-Pro Plus image processor (version 4.5, Media Cybernetics Inc., Silver Springs, MD, USA). Encapsulation Efficiency The encapsulation efficiency of the double emulsions (EE) was calculated according to the methodology reported by [23] and it was considered as the amount of total phenolic compounds of the xoconostle extract that was found in the simple emulsions (W 1 /O) after a process of separation of the external aqueous phase (W 2 ) on day 0 of storage. Ten milliliters of each treatment including the control were taken and centrifuged at 8000 rpm for 15 min at 4 • C. The supernatant external aqueous phase (W 2 ) was carefully removed, filtered with Whatman No. 40 paper and the total phenol content was determined by the Folin-Ciocalteu method. The total phenol content of the external aqueous phase (W 2 ) of each treatment was adjusted with the control using Equation (1): where FT is the phenol content in the external aqueous phase, FT Treatment is the phenolic content in W 2 of each treatment and FT Control is the total phenolic content W 2 in the control. Then, the value of Equation (1) was substituted into Equation (2) to determine the percentage of encapsulation efficiency (EE%): where FT Extract is the total phenolic content in the non-encapsulated extract. Bioactive Compounds For the determination of bioactive compounds, the methodology reported by [40] with modifications was used. Ten milliliters were taken from each double emulsion treatment and deposited in 50-mL centrifuge tubes. A 12-mL aliquot of an ethanol/methanol solution (50:50) was added to each tube and vortexed vigorously for 30 min in the dark and at room temperature. Finally, the mixture was centrifuged at 10,000 rpm for 30 min at 4 • C and the residue was discarded. To eliminate turbidity, the samples were centrifuged again for 15 min at 2 • C and the residue was discarded. The emulsion extract was then filtered with Whatman No. 4 paper, collected in clean tubes and stored at −75 • C until subsequent analysis for bioactive compounds and antioxidant and antidiabetic activity. Total Phenol Content The content of total phenols was quantified by the Folin-Ciocalteu method as described by [55], with minor modifications. An aliquot of 0.5 mL of the ethanolic/methanolic extract of emulsions was taken and mixed with 2.5 mL of the Folin-Ciocalteu reagent diluted with distilled water in a 1:10 (v/v) ratio. The samples were allowed to react for 7 min and 2 mL of 0.7 M Na 2 CO 3 were added. It was left without movement for 2 h in the dark and the content of total phenols was determined as mg EAG/100 mL double emulsion by interpolation of the absorbance to 765 nm on a gallic acid standard curve previously prepared using a JENWAY 6715 (Staffordshire, UK) spectrophotometer. Total Flavonoid Content The total flavonoid content was estimated using the method reported by [56]. A methanolic solution of AlCl 3 at 2% (w/v) was prepared and 2 mL of this and 2 mL of emulsion extract were added. The tubes were shaken for 15 s and left in the dark for a further 10 min. The flavonoid content was determined as mg equivalents of quercetin (EQ)/100 mL double emulsion by interpolation of the absorbance at 415 nm in a standard curve of quercetin prepared using a JENWAY 6715 (Staffordshire, OSA, UK) spectrophotometer. Total Betalains Determination To quantify total betalains, the protocol of [19] was followed, 1 mL of sample was taken and mixed with 20 mL of 20% PQ06121 (Fermont, Nuevo León, México). Absorbance readings were taken on a spectrophotometer JENWAY, 6715 (Staffordshire, OSA, UK) at a wavelength of 483 nm (betaxanthines) and 538 nm (betacyanins). The results were expressed in mg/g of film. Tannin Content The tannin content was determined using the methodology proposed by [57] with some variations. An aliquot of 200 µL of emulsion extract and 600 µL of 0.1 M FeCl3 were placed in test tubes and then allowed to react for 5 min in the dark. Then, 600 µL of 0.008 M K3Fe was added and left unmoved for 10 min and the samples were read at 720 nm in a JENWAY 6715 (Staffordshire, OSA, UK) spectrophotometer. Distilled water was used to prepare the blank. The absorbances were interpolated on a quercetin standard curve (EQ). DPPH Assay To determine antioxidant activity by inhibition of the DPPH radical, the DPPH solution was prepared by dissolving 7.8 mg of 1,1-diphenyl-2-picrylhydrazyl radical 80% methanol. The solution was agitated for 2 h in the dark [58]. Then, 2.5 mL aliquots of 6.1 × 10 −5 M methanolic DPPH solution were added to glass tubes and reacted with 0.5 mL of sample. The tubes were left to stand in the dark for 30 min. Subsequently, the samples were measured at 515 nm using a spectrophotometer (JENWAY, Model 6715, Dunmow, UK). The blank was an 80% methanol aqueous solution. The percent inhibition was calculated using Equation (3). ABTS + Assay Antioxidant activity was measured using the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) radical following [59] by reacting 10 mL of 7 mM ABTS solution with 10 mL of 2.45 mM (K 2 S2O 8 ) potassium persulfate. The mixture was stirred for 16 h in a container in complete darkness. The absorbance was adjusted with 20% ethanol to obtain a value of 0.7 ± 0.1. A total of 200 µL of sample was added to 2 mL of ABTS solution and allowed to react for 6 min; absorbance was measured at 734 nm in a spectrophotometer (JENWAY, Model 6705, Dunmow, UK). α-Amylase Inhibition In Vitro Assay The determination of α-amylase inhibition in aqueous extracts of whole xoconostle fruits was based on the method of [16] with slight modifications. A 100 µL aliquot of the extract obtained from emulsions was mixed with 100 µL of 0.02 mol/L sodium phosphate buffer (pH of 6.9) and 100 µL of buffer solution ofα-amylase (1 U/mL), and it was pre-incubated at 37 • C for 10 min. After a pre-incubation time, 100 µL of aqueous starch solution (0.1%) was added and incubated at 37 • C for 60 min. The reaction was stopped with 1 mL of dinitrosalicylic acid reagent. The test tubes were then incubated in a water bath set at 90 • C for 5 min and immediately cooled to room temperature in an ice bath. The reaction mixture was then diluted after by adding 3 mL distilled water, and absorbance was measured at 540 nm using a spectrophotometer (Jenway 6715, Staffordshire, UK). A dextrose calibration curve was developed to quantify the amount of reducing sugars obtained as a result of the hydrolysis of starch by the action of α-amylase. The results were expressed in the inhibition percentage. α-Glucosidase Inhibition In Vitro Assay The α-glucosidase inhibition assay was performed following [16] with slight modifications. Two-hundred microliters of extracts obtained from microemulsions were mixed with 100 µL of a stock solution of p-nitrophenyl-α-d-glucopyranoside (10 mg in 2 mL of phosphate buffer, pH 6.9) and then incubated at 37 • C for 10 min. Subsequently, 40 µL of a solution of α-glucosidase (5.7 U/mg, 2 mg in 1 mL of phosphate buffer, pH 6.9) was added and it was left to react for 20 min. To interrupt the enzymatic activity, 4 mL of 1 M Na 2 CO 3 and 5 mL of distilled water were added to later read the absorbance at 405 nm. The coloration is proportional to the enzymatic activity. The results were expressed in the inhibition percentage. Simulation Intestinal Conditions Simulated gastrointestinal digestion of the double emulsion was performed following the method described by [23] with some modifications. Two phases were established: (A) gastric phase; 20 mL of double emulsion was diluted (1:5) with distilled water and adjusted to pH 2 by addition of HCl 6 N and 20 mL of gastric liquid (16% of pepsin and 10% of NaCl in HCL 0.1 M). The mix was incubated at 37 • C for 2 h in a shaking water bath. When the trial finished, two aliquots were taken for the next step and the further analyses. (B) Second phase; the pH of samples from the first trial was adjusted to 7 with sodium bicarbonate (0.5 M), then, 1.25 mL of freshly prepared pancreatin-bile mixture (0.4 g of pancreatin and 2.5 g of bile salts in 100 mL of 0.1 M NaHCO 3 (pancreatic fluid)) was added. The mixture was incubated at 37 • C for 2 h in a shaking water bath. When the trial finished, an aliquot was taken for further analysis. At the end of the gastrointestinal digestion, the samples were heated in a boiling bath for 4 min to inactivate the enzymes and immediately centrifuged at 12,000 rpm for 10 min at 4 • C in a centrifuge Z 36 HK (HERMLE Labortechnik GmbH, Wehingen, Germany) for the analysis of total phenols, flavonoids, α-amylase inhibition, and α-glucosidase inhibition. Fatty Acid Release During intestinal digestion, the pH of the samples progressively decreased due to the release of free fatty acids by the activity of lipase present in pancreatin. To maintain the pH at 7.0 during the 120 min of intestinal digestion, a solution of NaOH (0.25 M) was added in the form of drops. The volume of NaOH spent at min 0, 10, 20, 30, 40, 60, 80, 100 and 120 was monitored and was used as an indicator of the digestibility of the lipid phase of the emulsions, as described by [54]. Antioxidant and Antidiabetic Activity during Digestion To evaluate the protection of the double emulsions on the biological activity of the compounds, the antidiabetic and antioxidant activity of the extract was evaluated before and after digestion. To this end, 7.5 mL of emulsions without digestion, emulsions after gastric digestion and emulsions after intestinal digestion were deposited into dialysis bags. Aliquots of 12 mL of ethanol/methanol (1:1) were added and the extracts were collected outside the bags. The extracts were brought up to 18 mL in all cases. These extracts were used for the determination of antioxidant and antidiabetic activity following the previously described methodology in each case. Statistical Analysis All experiments were replicated three times. The completely random design was used. All results were expressed as the mean ± standard deviation of triplicate measurements. When ANOVA had significant differences (p ≤ 0.05), the comparison means technique of Tukey was used. All analyzes were performed using IBM ® SPSS Statistics version 24 software (International Business Machines Corporation (IBM), Armonk, NY, USA). Conclusions Microencapsulation of xoconostle extract using double emulsion system could be an alternative for the preservation of xoconostle extract, since it maintains viable up to 60-80% of the bioactive compounds, in addition to its antioxidant and antidiabetic activity. Double emulsions with a higher amount of internal aqueous phase (xoconostle extract) are less efficient in protecting bioactive compounds and biological activity. The ED40% formulation was the most suitable for preservation of biocomposites and antidiabetic activity of the xoconostle extract. Simulated digestion in vitro increased the antioxidant capacity of the treatments but decreased their antidiabetic effect. In future studies, research could be carried out in in vivo systems, likewise, these results give guidance to new investigations in which the extracts encapsulated in double emulsions can be incorporated into food formulations of special diets for patients with diabetes.	2020-12-10T14:07:16.651Z	2020-12-01T00:00:00.000	{ "year": 2020, "sha1": "1f10bc4e0182e03d89140194f59ce0a0a7422a25", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1420-3049/25/23/5736/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8ca0896d485bc18f4872eb7e5157293ee4f4864a", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
237882365	pes2o/s2orc	v3-fos-license	EFFECT OF PAIRED FORMATIVE ASSESSMENT ON STUDENTS’ LEARNING Objective: To determine the effect of paired formative assessment on students’ learning. Study Design: Quasi experimental study. Place and Duration of Study: Islamic International Medical College Rawalpindi, from Jan 2018 to Jun 2018. Methodology: A total of 160 students who attended otolaryngology module were included in the study. Randomized allocation was done by computerized software programme and students were divided into 2 groups i.e. experimental (group A) and control group (group B). Later on in group A, pairing of students was done by lottery method. Group A and B were dealt with paired and individual formative assessment respectively. Effectiveness of both methods of learning was calculated on the basis of academic scores obtained in tests consisting of 20 MCQs from predefined and taught syllabus. A p-value were obtained by applying independent sample t-test and considered statistically significant at 0.05. Results: Out of 160 participants, 94 (58.7%) were females and 66 (41.3%) were males. In individual testing phase mean scores of group A was 13.36 ± 2.22 and mean scores of group B was 13.24 ± 2.5 (p 0.861). In paired formative assessment phase, mean scores of group A was 16.70 ± 1.94 (CI 95% 2.16-4.55) and mean of scores of group B was 13.40 ± 2.23 (CI 95% 2.16-4.55) p=0.001. Conclusion: The effectiveness of paired formative assessment. This method provides students a conducive environment to achieve learning objectives. INTRODUCTION Due to the rising pressure on the educational institutes to meet high accomplishment objectives, schools and universities have been executing rigid assessment platforms within their educational systems. Evaluation and assessment programs are a key to measure the growth towards meeting a learning target. Formative assessment is a planned development and should be assimilated during the course of learning cycle to regulate students' current instructional techniques 1 . It is an umbrella term used to promote students' learning by accomplishing key objective, understanding of learning objectives, exhibiting student's knowledge to the instructor, familiarizing them with the potential of summative assessment 2,3 . In order to alter the delimited and inhibited forms of summative assessment, educators are now being enthused to place more importance on interactions in classroom learning, inspiring students to reflect and achieving a process of co-assessment in their knowledge 4 . To improve the learning bases of students, teachers should use students-centred tactics in their teaching 5 . Formative assessment is a part of the develop-mental or on-going teaching learning process in which teachers take a positive approach and employ constructive communication techniques to provide guidance and continuous feedback to the students on their performance 6 . Paired formative assessment is a novel technique in which discussion and feedback process take place simultaneously amongst the students' pair, supervised by the teacher. Paired formative assessment is a potentially transformative instructional tool that aims to support learning. The growing interest of educational institutions to make students autonomous and lifelong learners, has motivated them to re-consider the association between learning and its assessment. We evaluated the effect paired formative assessment in this experimental study. METHODOLOGY Paired formative assessment was done at Islamic International Medical College Rawalpindi, from Jan to Jun 2018). The students of 3 rd year and 4 th year MBBS students participated in the study after approval of study by ethics review board (Riphah/ERC/18/0629). As the numbers of students in both 3 rd and 4 th year are 100, therefore the accessible population for the study was 200 students who attended Otolaryngology module. The sample of students was taken by simple random sampling technique. The minimum sample size was 134 which was calculated using the software for sample size calculation. Clinical.com (statistic>> sample size calculator). Randomized allocation was done by computerized software programme and students were divided into two groups i.e. experimental (group A) and control group (group B). Later on in group A, pairing of students was done by lottery method, after informed written consent. Students who did not attend tutorials of the pre-defined syllabus were excluded from the study. All students were informed to come prepared for formative assessment. In the individual phase of assessment both groups were given a test consisting of 20 MCQs from predefined and taught syllabus. Time allotted for test was 30 minutes. Students were given 30 minutes break. Same MCQs were rearranged for formative assessment phase. Students of group A were randomly paired using lottery method. Group A and B were given test of rearranged MCQs. Time allotted was 30 minutes. Scores of group A and B in individual and formative assessment phases were compared. Statistical analysis was done using SPSS-23. All the data was entered in a specially designed proforma and Statistical analysis was performed by using SPSS 23. Mean ± SD was calculated for quantitative variables. Frequency and percentage were calculated for qualitative variable. Before assessment, students were cautioned that pairing was not to promote cheating. Instead, it was intended to help everybody to learn. Teachers gave students enough time for discussion with partners as this made learning more meaningful. RESULTS The descriptive analysis presented in table-I below showed that out of 160 study sample, 94 (58.7%) were the females and 66 (41.3%) were the males. Paired formative assessments of experimental group showed high association between post-test marks and students learning. Students got maximum marks after pairing. The averages of the paired assessment and individual test marks also indicated that the participants obtained higher marks in the paired formative assess-ment than in the individual. Here we concluded that our hypothesis was true. Students performed better in paired formative assessment phase. DISCUSSION This study on paired formative assessment was unique and directed on medical college students in Islamic International medical college, where the conservative or traditional individual formative assessment was frequently used. Medical education is a dynamic process and its methods are constantly changing to achieve new learning objectives 7 . Student's centred methods are evolving to foster their learning and performance 8 . Assessment scores improve by involving students into discussion and formative process of feedback 9 . The growing need of progress of new assessment forms such as self, peer and co-assessment is anobligation to create future independent reflective practitioner 6 . Paired formative assessment is a new tactic. This study examined pair work from a new viewpoint such that its main impact is the students' learning. The union was put into its place to encourage students to learn from each other through communication. Thus, new knowledge can be combined and understood through collaborating social processes. The main goal of this research was to scrutinise the manner in which the 'peer interaction' is operationalized in a pair format task and to use this as the foundation for the expansion of Paired formative assessment 7 . The Appalachian education laboratory promotes active involvement of students in their own learning and paired formative assessment gives an excellent chance to achieve this goal 10 . This study arose from anapplied need to encourage students for formative assessment for making their interface in a paired task in a non-threatening environment. In peer learning, students build and negotiate their own connotation and understanding of content and perception 8,9 . Involving the same concept with pair work during assessment, students were involved in searching for, collecting, analysing, interrogating and applying information to complete an assignment or solve a problem 10 . This research project was established on the work of Panadero et al who employed similar pre-test and post-test to examine the effective strategies of co-operative learning to determine the learning outcomes 11,12 . These studies concluded that cooperative strategies used in the formative assessment improved students' who were revealed to paired strategies were able to achieve a higher score as compared to the ones included in controlled group which had completed task individually as an old-custom strategy 13,14 . Johnson and Johnson conducted 117 studies and observed the effects of competitive, individualistic and co-operative strategies on academic achievement ad established strong co-relation between co-operative paired learning than individualistic learning 15,16 . The main focus of our research was to explore the effectiveness of social interactions during assessment. Knowledge sharing/transfer is the main advantage of pair programming as reported in literature and our study results also confirm this finding that knowledge transfer/sharing is improved in pairing as compared to individual assessment 17 . This study was supported by the study which incorporated the idea of buddy quiz 18 . This approachrefers to a student assigned to another as a partner during quizzes, the two students or buddies are allowed to freely discuss and process information collaboratively about an item in a quiz. Exchanges led to an overall increase in participation, motivation and ultimately improve learning of the class 19 . In paired formative assessment the foremost key component is positive interdependence among paired students, so they understand that they linked together for a purpose that one cannot achieve success unless they both synchronize their efforts to accomplish the goal. Johnson et al noted positive interaction in pairs, providing each other the needed help, sharing concepts, challenging other's conclusion and reasoning the order to promote high order thinking to solve problem issues; and working constructively together to accomplish their mutual goal 20 . These results support the hypothesis that paired formative assessment has an overall positive effect of improving the academic performance of students. This study was a timely endeavour in present time of medical education reforms. Paired formative assessment proved to be a helpful arrangement for learners to be benefited by the quality of performance of other equal-status learners, because of the favourable and non-threatening environment. Implications for Future Research Firstly, future research need to look at other colleges in Pakistan, which have different settings and are located in different environments to determine the implication of paired formative assessment on the performance of students. Secondly, future studies should be emphasized on the long-term impact of paired formative assessment on students' performance. Thirdly, this study was conducted on students' academic performance only. The researchers expect that paired formative assessment impacts other variables. Additionally, future studies should examine how a paired formative assessment affects the productivity and impact on students' motivation. Lastly, future researches can also find the perception of students regarding paired formative assessment strategy. This can be done by looking at their participation, performance, enjoyment during assessment and challenges they face using paired strategies. Disclosure One of the author was working in Rawalpindi during the period of this study and is currently posted to Combined Military Hospital Lahore. CONCLUSION The practice of paired formative assessment seems to have positive impact on students learning. The results of our study showed that structured paired formative assessment in the class-room is a student centred instructions to maximize students' participation, sharing of knowledge, and enhanced learning and ultimately improve academic performance.	2021-09-01T15:03:29.444Z	2021-06-30T00:00:00.000	{ "year": 2021, "sha1": "1cfc616b7b46c20de0b74c5b96e9dde079611256", "oa_license": "CCBYNC", "oa_url": "https://www.pafmj.org/index.php/PAFMJ/article/download/3671/3421", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "b4943197f8bc6d0b96d06b22c30e5555de0ecde6", "s2fieldsofstudy": [ "Education", "Medicine" ], "extfieldsofstudy": [ "Psychology" ] }
206904765	pes2o/s2orc	v3-fos-license	Towards agrobots: Identification of the yaw dynamics and trajectory tracking of an autonomous tractor More efficient agricultural machinery is needed as agricultural areas become more limited and energy and labor costs increase. To increase their efficiency, trajectory tracking problem of an autonomous tractor, as an agricultural production machine, has been investigated in this study. As a widely used model-based approach, model predictive control is preferred in this paper to control the yaw dynamics of the tractor which can deal with the constraints on the states and the actuators in a system. The yaw dynamics is identified by using nonlinear least squares frequency domain system identification. The speed is controlled by a proportional-integral-derivative controller and a kinematic trajectory controller is used to calculate the desired speed and the desired yaw rate signals for the subsystems in order to minimize the tracking errors in both the longitudinal and transversal directions. The experimental results show the accuracy and the efficiency of the proposed control scheme in which the euclidean error is below $40$ cm for time-based straight line trajectories and $60$ cm for time-based curved line trajectories, respectively. Introduction One of the most important tasks in tractor operation is the accurate steering during field operations, e.g. accurate trajectory following during tillage, to avoid damaging the crop or planting when there is no crop yet. Besides, the rows must be parallel, and the distance differences between them must be equal with respect to each other during the planting. Moreover, the tractor has to cover the full field without overlap during other operations. However, the steering accuracy decreases when the operator gets tired or does more actions than driving the tractor like operating/controlling the implements. In order to automate the trajectory following problem and also increase the steering accuracy, several automatic guidance systems have been developed to avoid the problems mentioned above. There are various reasons why the control of tractors with a high efficiency is a challenging task. First, an autonomous tractor can be configured with different types of implements and also encounter various environmental conditions (such as humidity, temperature, etc.) during field operations. In such conditions, there is always a tradeoff between performance and robustness when a conventional controller, e.g. proportional-integral-derivative (PID) controller, is used. Since conventional controllers have time invariant coefficients and do not have the ability to adapt to changing conditions, they are not appropriate to be used in such agricultural production machines. Second, these machines show many nonlinear behaviors such as saturation, dead-time and time lags, which are difficult to handle with conventional control algorithms. Third, tractor navigation involves two subsystems, namely: the yaw dynamics and the longitudinal dynamics which make the control operation more challenging. There is also interaction apart from the hydraulic driveline as a change in the longitudinal speed will change the yaw dynamics and vice versa. In model-based control, the control performance highly depends on the accuracy of the model describing the system behavior. In the last decade, several models have been proposed where the yaw dynamics of a wheeled vehicle are described with a bicycle model. Simple kinematic models have been proposed in [25]. These models neglect the side-slip of the tires and the dynamics of the steering actuator. Therefore, they are not appropriate for slippery surfaces which are common in field conditions with loose or wet soil. As a solution to this problem, a bicycle dynamics model which takes the lateral forces into account is proposed in [26]. As the effect of side slip can be taken into account by this model, it covers a range of slippery and hard surfaces. However, in the previous approach, side slip angles cannot be calculated when the longitudinal speed is equal to zero. As a solution to this problem, the relaxation length approach is proposed in [12] to calculate the side-slip angles more accurately. Bevly [2] reported that the relaxation length for only the front tire is adequate in order to model the real-time system. Modeling of side-slip angle, which is the difference between the real and effective steering angle, and determining cornering stiffness values are very important steps in analyzing the yaw dynamics of autonomous vehicles. In [8], the cornering stiffness is estimated by a robust adaptive Luenberger observer and a sliding mode controller is designed based-on chained system theory. The proposed controller and observer were reported to be robust to time varying lateral disturbances and also inaccurate side-slip angles. As an alternative approach to controlling the agricultural production machines, model reference adaptive control approaches have been proposed in [7]. It is observed that the model reference adaptive control algorithm is able to adapt itself to various implementation configurations properly to control lateral position of a tractor for a straight path. In [10], the effect of the hitch point loading on the tractor dynamics is investigated by using a cascaded estimator approach. The experimental results show that the online estimation for the changes in the system provides the ability of adapting the controller gain to maintain the consistent yaw dynamic control of the tractor. Model predictive control (MPC) has been widely used in the chemical process industry since the 1980s. The main goal of MPC approach is to minimize a performance criterion with respect to constraints of a system's inputs and outputs. The future values of the system are calculated based on a model. The main advantages of MPC over conventional controllers for the control of agricultural machines are the ability to deal with constraints and with multi-input-multi-output controllers. Several successful applications on agricultural production machines have been reported in literature. An MPC design was implemented on the cruise control of a combine harvester [5] in which the speed model was developed based on relating the engine speed and the current to the hydraulic pump to the longitudinal speed. The engine speed and the pump settings were controlled simultaneously and this approach was tested experimentally on a New Holland combine harvester. The experimental results show that a satisfactory acceleration performance can be achieved even by keeping the engine speed low. In [20], an MPC strategy is described for the control of an autonomous tractor by using an extended kinematic model. This control scheme has been tested experimentally on a farm tractor whose realtime localization is achieved relying solely upon a real-time kinematic (RTK) global positioning system (GPS). However, the control accuracy is limited, because the model used is a kinematic model, and thus neglects the dynamic behaviour of the system. As an extension to MPC, a nonlinear MPC (NMPC) is proposed to obtain better lateral position accuracy of a tractor-trailer system in [1]. The lateral position error of the trailer was reported to be less than 10 cm in straight paths for a space-based trajectory in real-time experiments. Moreover, centralized, decentralized and distributed NMPC approaches have respectively been proposed in [17,15,18]. The drawback of these studies is the same as [20] which is that the model used does not include the dynamic behaviour of the system. Another NMPC algorithm is proposed for the yaw dynamics control of an autonomous vehicle in [4]. Although it was reported that the proposed controller would allow to use hard constraints for obstacle avoidance strategies, it does not include any real time experiments. Since more advanced control algorithms and mathematical models bring not only more accuracy, but also more computational burden to the real time systems, there always exists a trade-off between the complexity of the method and the computational efficiency of the overall system. The main contributions of this study beyond the state of the art are modeling the yaw dynamics of an autonomous tractor considering various definitions of side slip angles and controlling it with good computational efficiency. In order to achieve this, first, the yaw dynamics model of the autonomous tractor has been derived, the model structures have been validated, and model parameters have been estimated by using frequency response function (FRF) measurements. Finally, the nonlinear least square (NLS) frequency domain identification (FDI) approach is used to obtain the model parameters to determine which model is better for the tractor at hand. After the identification of the yaw dynamics, an MPC controller for the yaw dynamics is designed based on the identified model. Then, this yaw dynamics controller has been combined with a kinematic controller for the trajectory tracking in which the kinematic controller is used for compensating the errors both in the x-and y-axes. This paper is organized as follows: The experimental set-up is described in Section II. The kinematic model of the system and the mathematical model of the yaw dynamics are presented in Section III. In Section IV, the identification of the yaw dynamics is described. In Section V, the basics of the implemented MPC approach are given. The overall control structure and the real-time experimental results are presented in Section VI. Finally, some conclusions are drawn from this study in Section VII. Experimental Set-up Description The aim of this study is to track a time-based trajectory with a small agricultural tractor shown in Fig. 1. The GPS antenna is located straight up the center of the tractor rear axle to provide highly accurate position information for the autonomous tractor. The height of the antenna is 2 m above ground level. It is connected to a Septentrio AsteRx2eH RTK-DGPS receiver (Septentrio Satellite Navigation NV, Leuven, Belgium) with a specified position accuracy of 2 cm at a 20-Hz sampling frequency. The Flepos network supplies the RTK correction signals via internet by using a Digi Connect WAN 3G modem. The block diagram of the hardware is shown in Fig. 2. The GPS receiver and the internet modem are connected to a real time operating system (PXI platform, National Instruments Corporation, Austin, TX, USA) through an RS232 serial communication. The PXI system acquires the steering angle, the GPS data and controls the tractor by sending messages to actuators. A laptop connected to the PXI system by WiFi functions as the user interface of the autonomous tractor. The algorithms are implemented in LabV IEW T M version 2011 (National Instruments, Austin, TX, USA). They are executed in real time on the PXI and updated at a rate of 20-Hz. The designed MPC in Section 5 calculates the desired steering angle for the front wheels, and a low level controller, a PI controller in our case, is used to control the steering mechanism. In the inner closed loop, the steering mechanism is controlled by using an electro-hydraulic valve with a maximal flow of 12 liter/min. The electro-hydraulic valve characteristics are highly nonlinear and include a saturation and a dead-band region. The voltage limited between 0 − 12 volt and the steering angle limited between ±45 • became the input and the output for the steering system, respectively. The angle of the front wheels is measured using a potentiometer mounted on the front axle, yielding an angle measurement resolution of 1 • . The speed of the tractor is controlled by using an electro-mechanic valve. There are two PID type controllers in speed control. The PID controllers in the outer closed-loop and the inner closed-loop are generating the desired pedal position with respect to the speed of the tractor and voltage for the electro-mechanic valve with respect to the pedal position, respectively. Since the measured speed coming from the GPS is noisy, a discrete Kalman Filter (KF) was used to reduce noise. A position-velocity model described in [3] was used where vehicle velocity is assumed as a random-walk process. The KF assumes that the vehicle moves with a constant velocity between discrete-time steps. The state vector of the model used in the KF and the state transition matrix are written as follows: where Φ(T s ), v x,k and v y,k are the state transition matrix and velocities coming from the GPS, respectively. Kinematic Model The schematic diagram of an autonomous tractor is illustrated in Fig. 3. The linear velocitiesẋ andẏ at the rear axle of the tractor (point R) are written as follows:ẋ where v x , ψ, δ and L represent the longitudinal velocity, the yaw angle of the tractor, the steering angle of the front wheel, the distance between the front axle and the rear axle of the tractor, respectively. Instead of considering the point R in the dynamic equations and the trajectory tracking control, the center of gravity (CG) is preferred. Thus, the linear velocitiesẋ andẏ are projected onto the CG as follows: where v y is the lateral velocity of the tractor at the CG. Modeling of the Yaw Dynamics As the driving speed of the tractor is rather limited, it is reasonable to assume that the lateral forces on the right and left wheels are equal to each other and can be summed. Therefore, the tractor is modelled in 2D as a bicycle system. The velocities, the sideslip angles and the forces on the rigid body of an autonomous tractor are schematically illustrated in Fig. 4. The yaw dynamics models are derived based on the following assumptions: • The traction forces are neglected, • The aerodynamic forces are neglected, • The tire moments are small, such that these can be neglected, • The pitch and roll dynamics are neglected. The notations used in the following (see also Fig. 4) are summarized in Table 1. Vehicle Dynamics The lateral motion of the tractor is written as follows: Distance between the front axle and the CG l r Distance between the rear axle and the CG L Distance between the front axle and the rear axle Traction force on the front wheels F t,r Traction force on the rear wheels F l, f Lateral force on the front wheels F l,r Lateral force on the the rear wheels C α, f Cornering stiffness of the front wheels C α,r Cornering stiffness of the rear wheels α f Side slip angle of the front wheels α r Side slip angle of the rear wheels σ f Relaxation length of the front wheels σ r Relaxation length of the rear wheels where γ, m, F t, f , F l, f , F l,r represent the yaw rate and the mass of the tractor, the traction and lateral forces on the front wheel, and the lateral force on the rear wheel. The yaw motion of the tractor is written as follows: where l f , l r and I represent the distance between the front axle and the CG of the tractor, and the distance between the rear axle and the CG of the tractor, the moment of inertia of the tractor. Since it is a time consuming process to calculate the inertial moment of the tractor, an approximate value for the inertial moment can be calculated as follows [9]: Tire Model The lateral tire forces are calculated in a linear model in which they are assumed to be proportional to the slip angles [27,11] : where C α,i and α i i = { f , r}, represent the cornering stiffnesses of the tires and the side-slip angles, respectively. The tire side-slip angles must be calculated in order to determine the slip forces. The side slip angles of the front and rear tires have been considered in a linear form, and they are written as follows: As can be seen from the equations above, the side slip angles cannot be calculated when the longitudinal speed is zero. As a solution to this problem, the relaxation length is defined as the amount a tire rolls to reach the steady state side slip angle. Previous research in vehicles suggests that the relaxation length of a tire plays a very important role in the steering motion at high velocities [24,6]. This result motivates us to use this approach for the agricultural vehicles due to the fact that the calculation of the side-slip angles in (8) and (9) for very low velocities can go to infinity. Since a tire generates the steady state side slip angle simultaneously, a first order mathematical model is used to describe the slip angle dynamics through the relaxation length. A first order differential equation for the side slip angle can be written as follows:α A relaxation length of 1.5 times the tire radius has been proposed for agricultural vehicles as it allows to obtain similar changes for a similar increase in velocity [2]. For passenger vehicles which have higher velocity than agricultural vehicles, a factor larger than 2 is typically selected [21]. By combining equations (8), (9) and (10), the time derivatives of the side slip angles of the front and rear wheels can be written as follows:α where σ f and σ r represent the relaxation length of the front and rear tires of the tractor, respectively. Equations of Yaw Motion The tractor is driven on the field with a constant longitudinal velocity as generally required in automatic guidance of agricultural vehicles. For this reason, the time derivative of the longitudinal velocityv x can be set to zero and the longitudinal velocity v x can be assumed as a parameter. It is also assumed that the steering angle is sufficiently small to justify linearization of the equations. Thus, (4) and (5) can be written considering (7) as follows: By using different considerations, three different transfer functions can be written. Firstly, the traditional bicycle model for the yaw motion of the autonomous tractor can be obtained by combining (8), (9) and (13). The relation between the yaw rate of the autonomous tractor and the steering angle of the front wheels can be written in transfer function form as follows: Secondly, the bicycle model with relaxation length approach for only the front wheel can be obtained by combining (9), (11) and (13). The relation between the yaw rate of the autonomous tractor and the steering angle of the front wheels can be written in transfer function form as follows: Thirdly, the bicycle model with the relaxation length approach for the front and the rear wheels can be obtained by combining (11), (12) and (13). The relation between the yaw rate of the autonomous tractor and the steering angle of the front wheels can be written in transfer function form as follows: The relation between the physical parameters in the previous equations and the transfer function parameters in (15) and (16) is shown in Appendix A. These different models might be proper for different real-time systems or different cases of the same real-time system. In Section IV these transfer functions (14), (15) and (16) will be fit to the empirical transfer function estimate obtained from the frequency domain experiments to decide which is most appropriate for the real-time system considered in this study. Identification of the Yaw Dynamics It was observed during the identification experiments that the front wheels reached their limits due to drift when the excitation signal was applied to the steering mechanism in an open-loop fashion. As a solution to this drifting problem, the system was controlled with a P controller, and then the closed-loop system was identified. The schematic diagram of the identification processes is shown in Fig. 5. The steering mechanism of the tractor was identified in [16,14]. In this section, the nonlinearity of the yaw dynamics is checked by using an odd-odd multisine signal and a frequency content in which the linear contributions are dominant is determined for the identification process [29,28]. A multisine signal is applied to the steering angle controller, and the linear models with respect to Section 3.2.3 are obtained by using the NLS FDI method [14]. After the frequency domain analysis, a qualitative comparison of the linear models in time-domain is given, and the effect of the longitudinal speed is shown. After the design and the implementation of the steering angle controller, the frequency spectrum of the response of the yaw rate to a random odd-odd multisine excitation is shown in Fig. 6. It can be seen that the contribution of the nonlinearities to the total response is as large as the linear contribution after 2 Hz. Moreover, it is known that the noise in FRF measurements above 1.5 Hz will be due to the lack of input signal. As can be seen from Fig. 7, the range till 2 Hz is not enough to capture the peak, and the magnitude is still quite high. Since the nonlinear contributions are dominant after 2 Hz, a linear model can be derived until 2 Hz. Based-on the considerations above, a multisine signal with a frequency content between 0.02Hz and 2Hz has been applied to the system as an excitation signal. The model parameters are identified by using the NLS FDI approach based on FRF measurements. In Fig. 7, the measured FRF and the FRFs of identified models are shown. As can be seen from Fig. 7, since the traditional bicycle model in (14) consists of one zero and two poles such a system cannot have a frequency response like the one in Fig. 7, and thus it is not a candidate for the parameter estimation. The other two models in (15) and (16) can be proper candidates for the parameter estimation. After the identification process, the transfer functions of the identified yaw dynamics model are calculated respectively for the bicycle model with relaxation length approach for only the front wheel, the bicycle model with relaxation length approach for the front and the rear wheels, and a second order system as follows: G RLF (s) = 292s + 177 s 3 + 11.6s 2 + 249s + 150 (17) G RLFR (s) = 279s 2 + 335s + 27860 s 4 + 11.5s 3 + 347s 2 + 1311s + 23340 Even if the models derived in (15) and in (16) fit with the real-time FRF measurements, these models are higher order transfer functions. As a simplified model, a second order transfer function in (19) is fitted to FRF measurements. As can be seen from Fig. 7, the frequency domain response of the proposed transfer function in (19) is similar to the derived models in (17) and (18). It can be concluded that a second order transfer function can also be used to model the yaw dynamics of the tractor at hand. It should not go unnoticed that we do not throw the physical models away by proposing the second order empirical transfer function. The reason is that the empirical model might not be suitable for different soil conditions due to the higher values for the slip. For a better understanding, (11), (12) and (13) should be considered carefully in which there are four ordinary differential equations that describe the yaw dynamics behaviour of the tractor. In case of having small side-slip angles, the equations for the side-slips can be neglected. As a result, there will only be two ordinary differential equations for the yaw dynamics resulting in a second order transfer function. Another disadvantage of using the empirical model is that the side-slip angles cannot be estimated. A qualitative comparison of the three identified models in time-domain is given in Table 2. A multisine signal between 0 and 8 Hz is given to the system to check whether the models are appropriate for the real-time system at hand. As can be seen from Table 2, although the parameter estimation of the three models is done until 2Hz in frequency domain, the models still give reasonable results at higher frequencies. Moreover, it can be concluded that the addition of zeros and poles do not increase the accuracy of the identified models when the side-slips are negligible. Model Multisine The selected and estimated parameters for the bicycle model with the relaxation length approach for the front and the rear wheels are given in Table 3. It is assumed that the values of the mass m, the distance between the front axle and the CG l f , and the distance between the rear axle and the CG l r are known. The inertia moment I is calculated based-on (6). The values for the cornering stiffness and the relaxation length of the front and the rear wheels are approximately estimated based-on (18). On the other hand, since it is observed that the parameters are not realistic for the bicycle model with the relaxation length approach for the front wheel, they have not been able to be estimated. During the parameter estimation process, it has been observed that there are more than one solution for a specific parameter. When two separate solutions resulted in roughly the same values, the estimated parameters values have been said to be realistic. The effect of longitudinal velocity In order to be able to analyze the effect of the linear longitudinal velocity on the identified linear models, an additional experiment has been performed in which the linear velocity is varied from 1 m/s to 2 m/s. In Figure 8, it is illustrated how the poles and the zeroes of the three models mentioned above change with respect to the longitudinal velocity. As can be seen from Fig. 8, the bicycle model with RLFR and the bicycle model with RLF have pole-zero cancelation, and the poles go to the left on the s-plane, which is an expected case from the given transfer functions in (15) and (16), when the longitudinal velocity increases. Model Predictive Control MPC controllers predict the future system behavior based-on the system model, and calculate the optimal input sequence based on these predictions [22]. As it was stated in the previous section, among the identified models, the second-order empirical model gives similar performance accuracy with the other mentioned physical models to represent the autonomous tractor at hand. Thus, the second-order empirical model is used for the MPC design in this study. The objective function consists of a function of the states, the outputs and the inputs of the system. The control action is calculated by minimizing the cost function subject to the predicted behavior of the model and the system constraints. The states and the outputs are predicted over a given prediction horizon. The main equality constraint is the system model, and the inequality constraints are the state constraints, the output and the input constraints (actuators limits). In our case, there are no constraints on the system states, but the constraint on the input to the system, which is the steering angle, is defined in (21). A discrete-time linear invariant state-space model can be written as follows: where x(k) ∈ R n , y(k) ∈ R p and u(k) ∈ R m are the state, output and input variables, respectively. The pre-known matrices A, B, C and D are calculated considering the sampling time of the real-time system by using (19). The constraints are written for all k ≥ 0 as follows: −45 degrees ≤ u(k) ≤ 45 degrees −55 degrees/s ≤ ∆u(k) ≤ 55 degrees/s The cost function in its general form is written as follows: where N p = 8 and N c = 3 represent the prediction and control horizons, and U = [u T k , ..., u T k+N c −1 ] T is the vector of the input steps from sampling instant k to sampling instant k + N c − 1. It was reported that prediction and control horizons are related to the speed of vehicles for a stable performance [19]. Since a tractor is a slow vehicle, small prediction and control horizons are chosen to decrease computational burden in real-time applications. The first sample of U is applied to the plant: and the optimization problem is solved over a shifted horizon for the next sampling time. Q and R are positive-definite weighting matrices defined as follows: The following plant objective function is solved at each sampling time for the MPC: In our case, the designed MPC minimizes the error between the reference yaw rate and the measured yaw rate, and finds the desired steering angle δ desired to the real-time system. . Kinematic Controller The kinematic model in (3) is re-written in the algebraic model as follows: where the lateral velocity v y equals to γl r . An inverse kinematic model is needed to generate the desired longitudinal speed and the desired yaw rate for the tractor. The inverse kinematic model in this study is written as follows: Consideringẋ d =ẋ r + k s tanh (k c e x ) andẏ d =ẏ r + k s tanh (k c e y ), the kinematic control law proposed in [23,13] to be applied to the tractor for the trajectory tracking control is written as: where v x d and γ d are respectively the desired speed and the desired yaw rate, and e x = x r − x and e y = y r − y are the current position errors in the X− and Y − axes, respectively. The parameters k c and k s are the gain and the saturation constant of the kinematic controller, respectively. The advantage of the kinematic model used in this paper is that since it has a saturation, the generated yaw rate cannot have extremely large values. The coordinates (x, y) and (x r , y r ) are the current and the reference coordinates at the CG of the tractor, respectively. Considering a perfect velocity tracking (v x = v x d and γ = γ d ) which means that the dynamics effects are ignored, the stability analysis is done by using a Lyapunov function [23]. Dynamic Controllers The proposed control scheme used in this study is schematically illustrated in Fig. 9. A PID controller is used for the longitudinal velocity control. In the yaw dynamics control, an MPC controller is designed and its output is the desired steering angle for the front wheels. A low level PI controller is used to control the steering mechanism. State Estimation An Extended Kalman Filter (EKF) was used for the state estimation. Since only one GPS antenna was mounted on the tractor, the yaw angle of the tractor cannot be measured. It is to be noted that the yaw angle of the tractor plays a very important role in the accuracy of trajectory tracking control as the estimated yaw angle is used in the inverse kinematic model to generate the desired speed and the desired yaw rate for the system. Therefore, the inputs of the EKF are position and velocity values from GPS. The outputs of the EKF are the position of the tractor on x-and y-coordinate system and the yaw angle. In trajectory control, the estimated values are used. Since the GPS antenna was located at point R on the tractor, the kinematic model in (2) is used. The discrete-time kinematic model used by EKF is written with a sampling interval T s as follows: The general form of the estimated system model is written: where f is the estimation model for the system and h is the measurement function. The difference between the kinematic model and estimation model is the process noise w k and observation noise v k both in the state and the measurement equations. They are both assumed to be independent and zero mean multivariate Gaussian noises with covariance matrices Q k and R k , respectively: Experimental Results An 8-shaped trajectory with both straight and curved line geometries has been applied as the reference trajectory. The motivation of choosing an 8-shaped trajectory is that we can evaluate the performance of the controller both for Figs. 10 and 11, respectively. The experimental results show that the proposed control scheme is able to control the system. Since the given trajectory consists of linear and curvilinear lines, the optimal values of the Q and R weighting matrices of the MPC controller had to be tuned by making a trade-off between optimal performance on the straight and curvilinear lines. It was observed during the experiments that although the curvilinear trajectory tracking is better with aggressive controllers (having big gains) than the linear trajectory, these controllers give a large overshoot for the linear trajectory. Meanwhile, although the linear trajectory tracking is worse with an aggressive controller than the curvilinear trajectory, this controller gives large error values for the curvilinear trajectory. Thus, it can be concluded that if a reference signal consisting of only linear or curvilinear geometry is considered, more accurate results can be obtained. Figures 12 and 13 show the longitudinal velocity and the yaw rate responses of the autonomous tractor. As can be seen from Fig. 12, there is a steady state error for the control of the longitudinal velocity by the PID controller. On the other hand, there is no steady state error for the yaw rate control by the MPC controller. These figures show that the error in both the x-and y-axes come from the poor control performance of the PID controller in the longitudinal Reference yaw rate Actual yaw rate Figure 13: The reference and the actual yaw rate of the autonomous tractor dynamics, because it cannot cope with the strong high nonlinearities and interaction between the subsystems. Conclusion In this study, modeling, identification and control aspects of an autonomous tractor have been investigated. Three yaw dynamics models have been derived from the equations of motion of the system. The parameters of these transfer functions have been estimated through nonlinear least squares frequency domain identification. While the fourth order transfer function model derived from the equations of motion considering the cornering stiffness and relaxation length for both the front and rear wheels gave realistic parameters estimates, the transfer function could be reduced to a second order model as not all yaw dynamics could be excited by the steering mechanism. This reduced order transfer function was then incorporated in an MPC for the yaw dynamics control and combined with a PID controller for the longitudinal speed control and an inverse kinematic controller for the trajectory tracking. The performance of these controllers was evaluated during real-time tests. The yaw rate control by the MPC gave satisfactory results, while the PID control of the longitudinal velocity did not. Although the 8-shaped time based reference trajectory could be tracked reasonably well, the longitudinal speed control should be improved to obtain better trajectory tracking. In order to increase the control accuracy in the straight lines, a space-based trajectory in which the longitudinal speed is constant, but only the yaw rate of the tractor is controlled, can be preferred. The MPC presented in this study provides the ideal framework for this.	2018-01-23T22:42:50.244Z	2015-07-01T00:00:00.000	{ "year": 2021, "sha1": "ef5ae9395fff1f5f7053b65131ae6fb815cff665", "oa_license": null, "oa_url": "https://lirias.kuleuven.be/bitstream/123456789/524522/1/J5.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "e28ba49c9b01984cf4b3bf3a5aceeaa35976ebc8", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Engineering" ], "extfieldsofstudy": [ "Computer Science", "Engineering" ] }
221421740	pes2o/s2orc	v3-fos-license	Complementary Metal-Oxide Semiconductor and Memristive Hardware for Neuromorphic Computing The ever-increasing processing power demands of digital computers cannot continue to be ful ﬁ lled inde ﬁ nitely unless there is a paradigm shift in computing. Neuromorphic computing, which takes inspiration from the highly parallel, low-power, high-speed, and noise-tolerant computing capabilities of the brain, may provide such a shift. Many researchers from across academia and industry have been studying materials, devices, circuits, and systems, to implement some of the functions of networks of neurons and synapses to develop neuromorphic computing platforms. These platforms are being designed using various hardware technologies, including the well-established complementary metal-oxide semiconductor (CMOS), and emerging memristive technologies such as SiO x -based memristors. Herein, recent progress in CMOS, SiO x -based memristive, and mixed CMOS-memristive hardware for neuromorphic systems is highlighted. New and published results from various devices are provided that are developed to replicate selected functions of neurons, synapses, and simple spiking networks. It is shown that the CMOS and memristive devices are assembled in different neuromorphic learning platforms to perform simple cognitive tasks such as classi ﬁ cation of spike rate-based patterns or handwritten digits. Herein, it is envisioned that what is demonstrated is Introduction Conventional computing that involves storing data in, and retrieving it from, memory results in numerous interactions between the slow memory and the fast processors.3] This significantly affects the computation speed, in particular when a large amount of data is to be processed in a short time.Furthermore, the excessive data movement in conventional sequential-and parallelprocessing computers results in very high power consumption.Even though supercomputers with highly parallel processing capabilities are presently used to satisfy the high throughput of computationally complex tasks, they are extremely power hungry. [4]he prevalence of big data and a need for low-power and high-speed processing in everyday life and edge computing devices demands a shift to computers with elevated capabilities but with low power consumption.This may be achieved through unconventional computing methods, [5] among The ever-increasing processing power demands of digital computers cannot continue to be fulfilled indefinitely unless there is a paradigm shift in computing.Neuromorphic computing, which takes inspiration from the highly parallel, low-power, high-speed, and noise-tolerant computing capabilities of the brain, may provide such a shift.Many researchers from across academia and industry have been studying materials, devices, circuits, and systems, to implement some of the functions of networks of neurons and synapses to develop neuromorphic computing platforms.These platforms are being designed using various hardware technologies, including the well-established complementary metal-oxide semiconductor (CMOS), and emerging memristive technologies such as SiO x -based memristors.Herein, recent progress in CMOS, SiO x -based memristive, and mixed CMOS-memristive hardware for neuromorphic systems is highlighted.New and published results from various devices are provided that are developed to replicate selected functions of neurons, synapses, and simple spiking networks.It is shown that the CMOS and memristive devices are assembled in different neuromorphic learning platforms to perform simple cognitive tasks such as classification of spike rate-based patterns or handwritten digits.Herein, it is envisioned that what is demonstrated is useful to the unconventional computing research community by providing insights into advances in neuromorphic hardware technologies. which neuromorphic computing is a promising approach, where engineering inspirations are taken from the noise-tolerant, parallel, low-power, and high-speed signal processing abilities of biological brains. [6]euromorphic computing was coined by Mead, [7] when he envisioned that while exploiting the similarities between semiconductor physics and biological neural systems, one may develop brain-inspired computing platforms.20] In this article, as shown in Figure 1, we provide an overview of neuromorphic computing with CMOS, SiO x -based memristive, and mixed CMOS-memristive technologies.Our work covers neuro-mimicking designs, which are able to replicate some known aspects of biological neurons and synapses.We also use these neuro-mimicking components to show they can be used in simple cognitive tasks such as spike-based pattern classifications or image sensing.In addition, we use memristive devices to perform more complex classification tasks such as classifying Modified National Institute of Standards and Technology (MNIST) hand-written digits using networks developed mainly from memristive weight elements, where these elements show good performance despite device variations and nonidealities. Neuromorphic Components Design In this section we discuss some previously published as well as some new results on CMOS and memristive neuromorphic neurons and synapses.These designs are toward one of the main goals of neuromorphic engineering, i.e., replicating the underlying principles of neural systems, with the hope to understand them better and discover the use of technology for artificial neural components.Using these components, we will then, in the next section, design systems that perform neuromorphic learning and computation, which are essential purposes of neuromorphic systems. CMOS Synapse and Neuron Design Silicon technology that has reached maturity in the past 40 years has been widely used in neuromorphic computing, from the early works of Mead's team such as the Mahowald and Douglas silicon neuron [21] to cooperation between Mead's students and the wider neuromorphic community in constructing CMOS implementations of various neuron models. [22]In addition, synaptic plasticity, which is believed to play an essential role in learning and memory in the brain, has also been implemented in CMOS in various forms.27] When implementing CMOS-based neuronal and synaptic models, one could design a circuit that utilizes the above-threshold [23] or subthreshold [28] region of the operation of transistors to implement various mathematical expressions, which are mainly devised by computational neuroscientists. [29]One of the main advantages of analog CMOS designs for neuron and synapse circuits is that the designer can produce almost any behavioral characteristics of the neuron or synapse, which has been approximated and mapped through a mathematical model.In that case, the designer will route together transistors, and in some cases capacitors (which can be realized using transistors), to realize those rules. In the following sections we discuss the design and implementation of some previous neuron and synapse circuits implemented using CMOS technology and discuss their behaviors, which closely mimic the observations in biological experiments. CMOS Synapse Design Here, we describe the design of an analog synaptic circuit that closely mimics some of the known behavioral characteristics of synapses in the visual cortex and hippocampus. [30,31]The circuit that is shown in Figure 2a has been designed and fabricated in CMOS to implement the triplet spike timing-dependent plasticity (STDP) rule proposed in a previous study. [32]STDP is a recognized synaptic plasticity rule that alters the synaptic weight based on the timing differences between pre-and postsynaptic spikes.If a presynaptic spike arrives before a postsynaptic one, it can result in potentiation, whereas a reverse spiking order may cause depression.Triplet STDP, on the other hand, considers not only the timing differences between pre-and postsynaptic spikes, but it takes into account the timing difference between a pair of pre-or a pair of postsynaptic spikes in the presence of a post-or presynaptic spike, respectively. [32]ur newly proposed STDP circuit that resembles, but is simpler than a previous design presented in a previous study, [33] is capable of reproducing the outcome of a wide range of synaptic plasticity biological experiments, including spike pair, triplet, and quadruplet performed in the hippocampus, as shown in a previous study. [30]In addition, it can mimic frequency-dependent pairing experiments performed in the visual cortex, as shown in a previous study. [31]There are three different circuit parts shown in Figure 2a that each correspond to a different feature of a computational model which represents the triplet STDP rule proposed in a study by Pfister et al. [32] Here, the red middle part implements the pair-based dynamics of STDP.With the addition of two side parts, shown in blue, extra pre-pre or post-post interaction dynamics are added up to the base-line pair-based dynamics (shown in red).For more details on the circuit and its functionality, please refer to the study by Azghadi et al. [33] Figure 2b, c shows measurement results from the circuit in (a), which was fabricated using a 1-poly 6-metal 0.18 μm Austria Micro Systems (AMS) CMOS process.In this circuit, the synaptic weight is represented by the charge stored in the weight capacitor, C W . Here, the top purple trace in (b) shows the change in the synaptic weight due to a pre-post-pre triplet of spikes, when only the pair-based STDP (red part) of the circuit is active.In contrast, the bottom trace demonstrates the synaptic weight change in the results of the same spike combination and timings but when the complete triplet circuit, i.e., all red and blue parts, is active.Similarly, in the top purple trace in (c), the synaptic weight potentiation as a result of the pair-based pre-post interaction is shown to be smaller compared with the triplet-based weight change.The higher potentiation of the triplet-based STDP is due to the additional post-pre-post interaction, which is activated through the blue triplet parts of the circuit.This figure shows the first physical implementation of a very large-scale integration (VLSI) synaptic device that accounts for higher-order STDP rules. CMOS Neuron Design A CMOS-based circuit design of an adaptive exponential integrate and fire (IF) neuron circuit [22,34] is shown in Figure 3a.The circuit is composed of several parts including an input differential pair integrator low-pass filter (ML1-ML3), a second low-pass filter (MG1-MG6) which implements spike frequency adaption, a noninverting amplifier with current-mode positive feedback for address event representation (AER) (MA1-MA6), and a reset block (MR1-MR6) for resetting the neuron and implementing the required refractory period.Figure 3b shows measurement results from the neuron fabricated in a 0.35 um AMS CMOS process. [18]Here, the silicon neuron's membrane voltage stored on C men is shown in response to a constant current injection (I in ).This closely resembles the spiking behavior of cortical neurons measured in response to somatically injected currents. [35]As the figure shows, the neuron can be controlled to exhibit behaviors similar to biology. SiO x Memristive Synapse and Neuron Design Memristive and resistive random access memory (RRAM) devices have been extensively used to implement synapse and neuron circuits for neuromorphic devices, circuits, and systems. [36] In this article, we focus mainly on silicon oxide (SiO x ) memristive devices for neuromorphic computing. SYNAPSE SiO x is commonly used as a gate dielectric for metal-oxidesemiconductor field-effect transistors (MOSFETs) due to its stable physical and chemical characteristics, i.e., relative dielectric constant: 3.9, energy gap: 9 eV, dielectric strength 13.5 MV cm À1 , and thermal stability >1050 C. [37] In addition to its excellent insulating properties, CMOS compatibility, and controllability, SiO x -based resistive switching phenomena have recently been demonstrated in vacuum [38] and in ambient atmospheric conditions, [39] indicating that this traditionally passive material can be utilized as an active memory element (memristor), controlled by an external electrical field. [40,41]urthermore, the microstructure plays a crucial role in air-stable resistance switching in pure silicon oxide.The columnar structure in sputtered silicon oxide films provides preferential parts for filaments formation. [42]The control of the interface roughness between the bottom electrode and the switching layer affects both the switching voltages and endurance. [43]part from the intrinsic CMOS compatibility, switching properties of silicon oxide-based RRAMs compare favorably against other more commonly used RRAM materials.We direct readers to the extensive review. [39]These features make SiO x an attractive material for neuromorphic computing. Many different mechanisms could govern the resistance switching in silicon oxide.Generally, the filamentary resistance switching mechanisms are classified into those that are intrinsic to the oxide material-commonly known as valance change or intrinsic switching mechanisms-and those that involve metallic diffusion from electrochemically active electrodes (e.g., silver or copper) or metal doping to form metallic filaments-commonly known as electrochemical metalization, conductive bridge, or extrinsic switching mechanisms. [44] when all its parts, i.e., the triplet depression (left blue) and triplet potentiation (right blue), work in conjunction with the pair-based (red) part.Note that a pre-post-pre spike triplet b) can result in a higher synaptic depression in triplet STDP interactions (bottom trace), whereas it shows a lower depression (top trace) in the pairbased spike interaction, due to considering only the post-pre pair, and not the pre-post-pre triplet.Similarly, in the case of post-pre-post spiking in (c), higher potentiation is expected when considering the triplet combination (bottom trace), whereas lower potentiation is elicited when only a pre-post pair is considered (top trace). Here, we only focus on intrinsic switching in silicon oxide that can further be subdivided into air-stable switching and air-sensitive switching. [39][47] Air-sensitive switching occurs only in vacuum as the oxidation of conductive filaments occurs in an oxidizing ambient.The second type, air-stable switching, is possible either in oxygen-rich or nonoxidizing environments, as conductive filaments form far from surfaces and are more critically affected by the oxide microstructure. [42,43,48]The taxonomy of resistance switching in SiO x -based RRAMs is shown in Figure 4. SiO x Memristive Synapse Design SiO x memristive devices are used to implement both STDP [49,50] and synaptic weights in physical implementations of artificial neural networks. [15,51]STDP is often regarded as a key local learning rule in biological systems, modulating synaptic weights in accordance with the degree of temporal overlap between pre-and postsynaptic action potentials.It has been shown that memristive devices can implement a pulse timing-dependent change of resistance by suitably overlapping voltage pulses. [52]Most realizations use bipolar resistance switching, but unipolar SiO x -RRAMs can perform STDP plasticity by controlling set and reset processes. In this case, the STDP response is generated using identical square voltage pulses, where the post-synaptic pulse is modified by a capacitor and converted into a triangular pulse before being applied to an RRAM device.The purpose of pulse modification is to achieve the desired device response to leading and trailing pulse edge slopes.The concept is described in detail in a previous study. [50]Figure 5 shows the results of the implementation of STDP-like response in unipolar SiO x -RRAM devices.In this figure, (a) shows a nonidentical STDP-mimicking pulse set up.If a square pulse and a triangular pulse are below threshold there is no change in device resistance if a single pulse is applied.However, if the sum of these two pulses is above the threshold it is possible to adjust device resistance.In these examples the square pulse is a presynaptic spike and the triangle pulse is a postsynaptic spike.The presynaptic spike arrives earlier than the postsynaptic spike; the resulting sum is a slow leading edge above the threshold.This leads to a decrease in resistance (increase in conductance-set process).The postsynaptic spike arrives earlier than the presynaptic spike; the resulting sum is a slow trailing edge above the threshold.This causes an increase in resistance (decrease in conductance-reset process).Figure 5b shows the percentage of the expected successful operation, i.e., if a conductance decrease or increase is expected for a specific Δt, does that happen or not on the actual device? Figure 5c,d shows the resulting STDP synaptic weight (conductance) changes in response to Δt values in the range from À600 to þ800 μs. All the above memristive synapse results are for air-stable devices.However, one-diode-one-resistor (1D-1R) air-sensitive test structures can also be used for vacuum-type SiO x -based synapse device demonstration, as shown in Figure 6.In this figure, (a) demonstrates a secondary electron microscopy (SEM) image of the top-down view of the fabricated device, whereas (b) shows a crosssection image of the 1R device, including its layer information.The synapse device structure and characteristics of this 1D-1R design have been described in detail in a previous study. [53]o fabricate this device, the active SiO x memory layer is deposited to a thickness of 40 nm using plasma-enhanced chemical vapor deposition (PECVD).The reactive ion etch (RIE) step then clears out the SiO x layer inside the hole and creates a SiO x sidewall where the memory device is formed (Figure 6b).The active memory area of the 1 R device is 2 Â 2 μm 2 and the overall size including metal interconnects is 21.9 Â 21.9 μm 2 .The overall size of the 1D device is 41 Â 19 μm 2 . Figure 6c shows an endurance of sequential long-term potentiation (LTP) and long-term depression (LTD) behaviors using nonidentical pulses in 1D-1R architecture with different voltage increment steps for potentiation and depression: 0.1 V (top), 0.2 V (middle), and 0.3 V (bottom).For depression, the pulse height modulates from 11 to 17 V with 10 μs pulse width; for potentiation, the pulse height changes from 4.0 to 10 V with 10 μs pulse width.Such flexible artificial control built with synaptic devices could provide a suitable platform for a broad weight Figure 3. a) A neuron circuit implemented in CMOS (Adapted with permission. [34]Copyright 2014, IEEE.) is capable of reproducing b) the spiking behavior similar to that of biological neurons (Adapted with permission. [18]Copyright 2015, ACM).The inset shows the spiking behavior of neurons measured in response to somatically injected currents, whereas the spiking behavior of the neuron is also in result of increasing I in . range of computing applications.Some of the advantages that SiO x -based synaptic devices provide over other resistive switching materials include a higher dynamic range (10 4 ) and the potential to achieve as many as 10-60 multilevel states (dependant on the stability, e.g., retention and endurance) in both LTP and LTD by changing the increment/decrement of the voltage step, as shown here. Figure 6d,e shows that the SiO x -based 1D-1R synaptic device can mimic STDP.These figures show a total of ten different conductance levels of the device, when positive and negative spike timing differences, as well as spike widths, are used, to emulate the potentiation and depression window behavior of STDP, observed in experimental [30,49] and computational experiments. [32] SiO x Memristive Neuron Design Memristive and RRAM devices are more commonly used to implement synaptic weight plasticity in spiking neural networks (SNNs) or represent weights in artificial neural networks.However, unipolar SiO x -RRAMs have also been considered for modeling aspects of the electrical activity of the neuron.It has been demonstrated that specific operational procedures could lead to the generation of controlled voltage transients that resemble spike-like responses seen in biological neurons.Additionally, the integration and thresholding capabilities that are crucial for neuronal functionality have also been demonstrated.Further, redox-based resistance switching models can Lower LHS: Air-sensitive resistance switching: typically, electroforming and resistance switching occur only in devices with an exposed oxide surface and not in bulk devices.This is attributed to re-oxidation of surface-based silicon filaments by an oxidizing ambient.Lower RHS: Air-stable resistance switching.This type of switching occurs in ambient (oxidizing) conditions and is defined by the microstructure of the oxide material.Switching voltages are typically lower for air-sensitive switching.Reproduced with permission. [39]Copyright 2018, John Wiley and Sons. be related to the Hodgkin-Huxley (HH) [54] conductance model by analyzing the equivalent electrical circuits [55] and are found to be very similar. Figure 7 shows the thresholding, spiking, and integration capabilities of SiO x RRAM-based neurons.The main idea is to control the competing set and reset processes (the former being field driven, the latter current driven) by stressing devices with appropriate current inputs.A constant current bias is used to test thresholding and spiking functionality, whereas current pulses are used to implement integration. The unipolar switching in SiO x -RRAM devices is utilized to generate voltage transients (resembling voltage spikes) by applying a constant current bias.By applying currents larger than the reset current, the device is put in the metastable state, fluctuating between the low resistance state (LRS) and the high resistance state (HRS).As a result, the voltage spiking is measured at the output of the device.If the input current is lower than a threshold (reset) current, the resistance states are stable; therefore, no voltage spiking is observed.Figure 7a shows threshold spiking behaviour.Furthermore, the integration functionality is demonstrated by applying a train of excitatory current pulses while changing the timing between the pulses.A much smaller current pulse senses the voltage across the device.Figure 7b-f shows the obtained results.The frequency of input current pulses controls the rate of voltage spiking.The results resemble the leaky IF (LIF) model.A more in-depth analysis of the results and the experimental setup can be found in a previous study. [55]n addition to the neuron spiking behavior shown in Figure 7, a simple one-resistor-one-resistor (1R-1R) test structure can be used for air-stable type SiO x -based neuron device demonstration, as shown in Figure 8.The detailed neuron device structure and characteristics have been described in previous studies. [38,56]or this neuron design, two device structures have been fabricated: a) SiO x /HfO x stacking bilayer as shown in Figure 8a,b and b) vanadium electrode (V)/SiO x single layer shown in Figure 8d.For the SiO x /HfO x stacking bilayer structure, the SiO 2 layer of 30 nm thickness was sputtered and HfO 2 of 1 nm thickness (confirmed by the transmission electron microscopy (TEM) image, as shown in Figure 8a) was deposited using atomic layer dDeposition (ALD) at 250 C. The TaN layer of 170 nm that serves as a top electrode WAS deposited also through sputtering.Copyright 2018, The Authors, published by Frontiers Media. The SiO x /HfO x stacked structure exhibits excellent reliable threshold switching in air with an ultralow operation voltage (<0.5 V) (Figure 8b).The nonlinearity/selectivity of this device is about 180, which can stably operate at 0.3 V threshold voltage, whereas the holding voltage is 0.1 V, as shown in Figure 8c.The self-compliance current is also observed here, which is due to the internal filament limitation. [57]Figure 8d shows the other SiO x -based threshold switching structure formed by V/SiO x single-layer stacking.The nonlinearity and selectivity of this device is about 102, which can stably operate at 1.2 V, where the device threshold voltage is 1.1 V.Note that an electroforming process is necessary before any operation. [38,56]ne of the promising features of these two threshold switching devices is that they are able to demonstrate the all-or-none principle in neuronal behavior.It means that, if a neuron responds at all, then it must respond completely.Also, it means that a greater intensity of stimulation does not produce stronger spiking but can increase firing frequency. [58,59]Figure 8e shows the schematic of the simple 1R-1R test circuit and its actual experimental setup to realize the all-or-none principle b,d,e) Reproduced under the terms of the CC BY license. [53]opyright 2016, The Authors, published by Springer Nature. biomimetically.By applying the green input pulse shape as shown in Figure 8e and measuring the voltage between the resistance and the measurement instrument (Agilent DSO9254A), we can distinguish the all-or-none principle, as shown in Figure 8f in blue color.When the applied voltage (CH2, 2 V) is below the threshold voltage (note to the voltage divider setting, which is 0.4 V for the SiO x /HfO x stacked structure and 1.1 V for V/SiO x structure), there are no spikes firing.However, when the applied voltage is at or above the threshold voltage (3.7 V), a sequential and repeatable 1.2 V output spike (peak-to-peak voltage) with 25 μs period is generated. The above memristive neurons only replicate the thresholdbased spiking IF behavior of neurons.However, biophysical neurons are considered to provide richer dynamical behavior and more computational complexity in comparison with their bioplausible counterparts such as IF and LIF neurons.These demonstrate that spiking occurs for the smaller number of pulses if the separation between input current pulses is shorter.Adapted under the terms of the CC BY license. [55]Copyright 2016.The Authors, Published by Frontiers Media. HH neuron [54] and its simplified version such as Morris-Lecar (ML) [60] are members of the biophysical neuron family and due to their neurocomputational properties, nonbiomimetic CMOS circuits are not capable of reproducing their dynamics efficiently in terms of energy and area.The introduction of memristor devices has transformed the design of HH neuron circuits, [61,62] whose design progress slowed due to the lack of built-in stochasticity in CMOS circuits for building more realistic brain-plausible neuromorphic platforms. [63,64]e have previously shown that the known functionalities of HH [65] and ML [66] neuron models can be simulated using behavioral memristive models.In the last two sections, we presented a number of designs implementing synaptic and neuronal functionalities such as LTP, LTD, and STDP for memristive synapses and all-or-none and threshold spiking behavior for memristive neurons.Except for the simulation-based HH and ML memristive neuron models discussed, all other designs explained using SiO x structures.In addition to the design presented earlier, SiO x -based devices have been extensively used in other neuromorphic systems, specifically for synaptic behavior demonstration.Here, we provide a comparison of various SiO x -based memristive synaptic devices discussing their functionalities and physical/ switching structures, as shown in Table 1.Pulses with 100 μs width are used here.When CH1 is below the firing threshold (e.g., at 2 V), there is no firing observed, whereas if threshold switching occurs (e.g., input pulse amplitude > 3.7 V), the neuron firing begins on CH2 (damping Vp 1.2 V). a,b) Adapted with permission. [38]Copyright 2018, The Royal Society of Chemistry. As Table 1 shows, various techniques have been used to implement different devices with diverse layer stacks, deposition methods, switching polarity, switching type, and SiO x thicknesses. These designs that operate at different set/reset voltages, as low as 0.5 V and as high as 10-15 V, offer various degrees of functionality and on/off ratios.Some of these devices also present a Figure 9. Bioinspired memristive neuron circuit diagrams, memristive channel characteristics, and bifurcation behaviors at different frequencies, for HH (top panel) and ML (bottom panel) neuron models.Adapted with permission. [65,66]Copyright 2016 and 2017, IEEE.multilevel structure, which is useful in implementing neuromorphic computing systems.It is worth noting that, not all designs, except for two of our previous works [51,53] are able to implement the three investigated synaptic plasticity functionalities, i.e., LTP and LTD, and STDP.Note that in Gao's study, [67] a porous silicon oxide structure has been used.The main function of SiO x here is to facilitate lithium-ion migration in forming a conductive path, with wellcontrolled lithium ions in the SiO x layer.These features enhance neuromorphic computing performance, i.e., LTP and LTD, similar to those observed in the SiO x device developed in a previous study. [68]In the work by Wang et al., [69] an opposite SiO x functionality is used to enhance the resistive switching characteristics for analog resistance modulation.The SiO x here was found to have a denser microstructure compared with the TaO x layer.This microstructural attribute could lead to the retardation of oxygen diffusion in the SiO x layer.Also, the diffusion constant is 1-6 orders of magnitude lower than that in the substoichiometric TaO x .Therefore, in their proposed device, the diffusion rate of oxygen ions is expected to decrease in SiO x .As a result, the resistive switching in the cell is likely to take place in the more stoichiometric and insulating SiO x layer that is in series with the substoichiometric and more conductive TaO x layer (valence change-type memristor).Similarly, in Acevedo's study, [70] it is reported that SiO x drives the observed resistive switching behaviors by taking and releasing oxygen ions to and from the nearby manganite layer.These results confirm that the combination of two active oxides (stacked structure) improves the neuromorphic functionalities compared with the single-oxide structures, as shown in Table 1.However, some materials (such as LiCoO 2 or LCMO) may not be fully CMOS compatible. Hybrid CMOS-Memristive Neuromorphic Designs Although neuronal and synaptic behaviors have been replicated using memristors, as discussed in the previous sections, silicon and CMOS-based neuron designs have shown more convenient implementation processes and better functionalities. [22]Unlike neurons, synaptic behaviors and functionalities are more conveniently implemented using memristive devices.Therefore, one may choose to use memristive devices to implement synaptic learning in a neuromorphic system, whereas all other components of the system are implemented using analog and digital CMOS. [15]This requires seamless integration of CMOS and memristive technologies in a hybrid design, so the benefits of both domains are achieved in the final system. ][73] In most of these systems, the neuron and interfacing circuitry are designed in CMOS, whereas synapses implementing targeted synaptic plasticity rules, such as STDP, are realized using one or a few memristors, which are programmed through shared or individual CMOS circuits.Here, we show a hybrid CMOS-memristor neuromorphic synapse that implements rate-based synaptic plasticity in the form of the Bienenstock Cooper Munro (BCM) [74] rule, as a neuromorphic component.In addition, in Section 3.3, we show a hybrid system implementing spike-based image sensing. [16]n a previous work, [15] we proposed a hybrid CMOSmemristor neuromorphic synapse that was shown to be capable of reproducing a number of biological experiments including pair-based, triplet, and quadruplet, similar to the CMOS circuit shown in Figure 2a.The hybrid synapse circuit that is connected to a presynaptic and a postsynaptic neuron is shown in Figure 10a.Here, we show for the first time that this circuit can generate BCM-like behavior, which is observed in biological experiments. [75]Figure 10b shows the weight modifications achieved using the circuit in (a), driven by random pre-and post-synaptic Poissonian spike trains.This experiment is composed of ten trials.In each trial, a random presynaptic spike train with the rate ρ pre along with a random postsynaptic spike train with the rate ρ post , both of a 10 s duration, are applied to the bimemristive hybrid synapse.The rates of pre-and postsynaptic spike trains are considered equal and are swept over the range Figure 10.a) A hybrid CMOS-memristor neuromorphic synapse connected to CMOS pre-and postsynaptic devices (Adapted with permission. [15]opyright 2017, IEEE).The crossed square in the synapse circuit is representative of a multiplication/rectification circuit.In addition, S 1 is a digital signal that activates two switches in the synapse and one in the neuron, to implement the correct timings required for the STDP rule. [15]b) The hybrid synapse is able to reproduce a BCM-like weight modification behavior, [74,75] by modifying A þ 3 , a triplet STDP potentiation parameter, which dictates the amplitude of potentiation due to the interaction between a postsynaptic spike, with its two immediate previous postsynaptic and presynaptic spikes. [32]f 0-50 Hz. Figure 10b shows the average weight change and their standard deviations over the ten trials.This figure shows the main characteristic of the BCM rule, which is turning LTD to LTP at a specific spike rate, i.e., the BCM threshold.Additionally, this figure shows the sliding threshold feature of the BCM rule, which can alter the frequency at which LTD turns to LTP.This turning threshold as shown in a previous study [32] can be controlled using the STDP parameters.Here we have utilized the triplet potentiation amplitude, A þ 3 , to slide the threshold toward lower or higher depression. [15]lthough previous CMOS circuits implementing triplet STDP have also shown the ability to realize BCM behavior, they occupy a large silicon real estate, mainly due to the large capacitances required to store the synaptic weight and provide time constants required for triplet STDP dynamics.However, the proposed memristor-based hybrid circuit, compared with its CMOS counterparts, occupies up to a ten times smaller chip area. [15]This feature makes it a promising component for large-scale neuromorphic systems with spike-based learning capabilities and encourages designers to benefit from hybrid designs in more applications, such as neuromorphic image sensing. Neuromorphic Systems Design In Section 2, we discussed the design and implementation of various CMOS, memristive, and hybrid CMOS-memristive neuromorphic circuits that were designed and implemented to realize neuronal or synaptic behaviors.We showed that the devices and circuits designed are able to replicate some known functionalities and behaviors of biological systems, such as LTP, LTD, STDP, triplet STDP, all-or-none spiking, threshold spiking, HH and ML neuron channel characteristics, and bifurcation behaviors.In this section, we utilize several of the designed circuits developed and explained earlier to implement neuromorphic computing systems applied to spike-based as well as artificial neural network learning.We also discuss the design and implementation of a hybrid CMOS-memristor neuromorphic sensing system. CMOS Neuromorphic Computing A neuromorphic system can be implemented completely using digital [76,77] or using a mix of analog and digital CMOS technology. [18,27]If the system is implemented in digital, the behaviors of neurons and synapses are approximated and the spiking and AER structure of the system is realized in digital architectures. [8,76,78]However, if the system is designed in mixed analog-digital domain, the synaptic and neuronal behaviors are replicated closely in analog, whereas AER and interfacing are realized using digital technology. [9]ost of the previous CMOS-based neuromorphic systems include fixed learning and neuron circuits, where an array of analog synaptic cells implementing a fixed learning rule, such as STDP [28] or SDSP, [25] is connected to an array of IF neurons.These implementations offer great biomimicing properties because their components closely mimic biological experiments; however, they can only be used with the learning rules implemented in their hardware.Some neuromorphic systems, such as those described in a previous study, [18] provide more flexibility by giving the user the ability to describe the network structure as well as the required learning rule in software, whereas the neuron and synapse components are implemented in analog hardware.The AER and spike transmission in these systems use digital technology and are usually implemented on field programmable gate arrays (FPGAs) [79] that facilitate programmability and reproduction. [76]ere, we describe the operation and use of such a hardwaresoftware system [18] that uses CMOS circuits providing programmable synaptic learning.This programmability gives the user the freedom to explore any arbitrary spike-based learning rule.The design that is shown in Figure 11a is composed of a) an asynchronous static random access memory (SRAM) array that can be programmed as virtual synapses, connected to b) an array of CMOS synapses, which integrate a current proportional to the weight stored in their corresponding SRAM cells, to feed into c) IF neurons similar to the neurons explained in Figure 3, which receive/transmit spikes from/to other neurons/synapses in a set network, through d) asynchronous control and interfacing circuits that manage the AER communication.All analog components on the chip are tuned by biases received from e) a bias generator circuit.Figure 11b shows an example SNN that has been implemented on the chip shown in (a).The chip receives AER input spikes, which contain information on a) the address of the postsynaptic neuron, b) the address of the programmable SRAM-based virtual synapse connected to this neuron, c) the type and address of the physical integrator synapse, and d) the new five-bit weight value that will be written to the addressed virtual synapse (SRAM cell).The AER output spikes, in contrast, only show the address of the postsynaptic neuron that generated them. Using the network shown in Figure 11b, two UP and DOWN rate-based spike patterns (shown at the top of Figure 11c), with 20% correlation (6 fixed common input spike trains shown with red circles, from the total 30), are learned through triplet STDP, [32] to be classified unsupervised.The four panels in the figure show the output neuron firing rate during learning.In the first trial, learning has not happened yet; hence, the output neuron fires with a similar rate for both UP and DOWN patterns.As the learning progresses, at each trial either the UP or DOWN spike-based pattern is randomly selected to be applied to the network.Also, a new Poissonian spike train is generated for each of the 18 active synaptic inputs for either UP or DOWN.It is seen that after 20 trials, the output neuron successfully distinguishes between the two patterns, showing a higher firing rate for UP patterns.The figure shows the results for 20 runs, each including 20 trials.For each run, the initial synaptic weights, the order in which UP and DOWN patterns are applied to the network over 20 trials, as well as the definition (spike timing intervals and distribution) of UP and DOWN patterns are randomized.Note that the learning rule used for this classification, implemented in software to program the SRAM virtual synapses on the chip, provides the same functionality as the circuit shown in Figure 2a.We have shown that this classification task can achieve 100% accuracy, even in the presence of 87% (i.e., 26 common input spike trains) correlation between the two patterns. SiO x Memristive Neuromorphic Computing ][82][83] Here, we mainly focus on neuromorphic systems that utilize SiO x memristive devices for learning, where memristors are used as weight elements.We also show simulation results of neuromorphic learning using spike-based learning and STDP, realized using ideal memristor models.However, there exist challenges in the use of memristive devices as learning components, which should be considered in neuromorphic learning systems design.Here, we show and discuss some of these challenges that mainly arise from the unavoidable device nonidealities. In addition to the ability to replicate STDP-like behavior for spike-based learning (shown in Figure 5), memristive and RRAM crossbars offer the execution of vector-matrix multiplication in the analog domain. [19]When voltage pulses are applied on one side of the programmed RRAM crossbar, and current is sensed on the orthogonal terminals, this system provides approximate vector matrix multiplications (multiply and accumulate [MAC]) operations in constant time steps using Kirchhoff 's and Ohm's laws. [84]This approach promises speed and power efficiency improvements of many orders of magnitude compared with conventional CMOS systems. [19]However, there exist obstacles on the way of utilizing memristive devices as STDPenabling synaptic devices or MAC operation facilitators. The main obstacle to realizing the full potential of RRAM crossbars is the number of both device-level and system-level nonidealities.These include device-to-device and cycle-to-cycle variabilities, a limited number of resistance states or a narrow operational range of resistance modulation, nonlinearity of both voltage-current characteristics and pulse response of devices, and high line resistances in crossbar arrays.Despite these, SiO x -RRAM devices have been used to implement weights in physical neural networks, in which multiple weight values (memristive resistance states) are required. [51]igure 12a,b shows the feasibility of achieving multiple resistance states in intrinsic SiO x -RRAM devices by controlling the reset process (i.e., varying the reset voltage) during voltage sweeps.Stable resistance states are achieved as devices reset gradually from the LRS to the HRS.However, for practical applications, pulse operation is preferred.In this case, the gradual reset dynamics is highly dependent on voltage amplitude and pulse width, as clearly shown in Figure 12c,d.Programming curves that gradually switch devices from the LRS to the HRS (see for instance Figure 12d) are typically called LTD and are akin to synaptic functionality.As these figures show, the memristive devices could be challenging for implementation of distinct resistance levels required for learning and inference in artificial neural networks.In addition, other nonidealities such as device failure and fabrication yield are present that can affect the network performance. From these figures, it is evident that higher-device nonidealities result in lower inference accuracy in the implemented classification task for MNIST.The figure also suggests that small-device nonidealities (<10%) do not significantly affect the performance of the implemented SiO x -based neuromorphic system, which uses memristors as programmable weights, whereas learning happens through supervised backpropagation (BP). [51] a similar study to the one explained earlier, we developed another SiO x -based memristor neuromorphic learning platform, in which a three-layer neural network (shown in Figure 15a) was designed and trained for the classification of MNIST dataset (784 Â 64 Â 10) and topology patterns (TP) of the atmosphere effect (256 Â 64 Â 7). [38]Note that the experimental LTP and LTD property of the synaptic devices was used to devise a Reproduced under the terms of the CC BY license. [51]Copyright 2019.The Authors, published by Frontiers Media.function of synaptic weight (W ) with respect to the input pulse numbers. [85]During the training process, the feedback error of network weights obtained from the BP algorithm was modulated using the function.Then, the deviation between the output and the target signal was minimized in the learning process.We found that the network learning performance relied heavily on the linearity and accuracy of the weight resistance tuning process that was developed for the memristive network.Here, the network has two hidden layers.Reproduced under the terms of the CC BY license. [51]Copyright 2019.The Authors, published by Frontiers Media.c) devices failed at LRS. Adapted under the terms of the CC BY license. [51]Copyright 2019.The Authors, published by Frontiers Media. RRAM crossbar arrays Figure 15b shows that the best learning accuracies achieved for MNIST and TP are around 89% and 80%, respectively.The figure also shows that there is no clear difference between various deviceto-device variation cases.Furthermore, the accuracy correlation with LTP and LTD nonlinearity (as defined in a previous study [85] ) is shown in Figure 15c.It is shown that the implemented neuromorphic learning system that uses SiO x -based weight reaches a simulated accuracy of up to 90%, but the accuracy decreases with the increase in nonlinearity by a slope of 6. The promising neural network learning results shown in Figure 15 were achieved using the simple SiO x -based metalinsulator-metal (MIM) structure in place of the network weight elements, in a crossbar structure.In addition, it has been shown [86,87] that such MIM devices simplify memory array design if used in a crossbar architectures.However, the leakage through the sneak paths inevitably induced while accessing MIM crossbar networks may cause weight variation issues detrimental to the development of reliable memristor-based neuromorphic learning and computation. [88]To mitigate the sneak paths currents, a diode or a selector device is usually positioned in series with a memristor cell to form a 1D-1R or 1S-1R structure. [89,90]hese configurations considerably increase fabrication and circuit design complexity and cost. To address this problem, in Figure 16, we have developed a selectorless memristor, which does not require a 1D or 1S series connection.This is achieved using simple high-k/low-k bilayer stacks (as shown in Figure 16b).The intrinsic selectorless property (or the nonlinear I-V characteristics, as shown in Figure 16a) can be realized by inserting an ultra low-k layer (e.g., SiO x layer or graphite oxide layer). [91]The main benefit here is that the ultra low dielectric constant layer provides nonlinearity intrinsically by carrier transport formulation design, specifically in Poole-Frenkel defect cases. [92]ere we use this selectorless memristor to demonstrate neuromorphic learning.Figure 16a shows typical bipolar resistive switching I-V characteristics during DC voltage sweeps for HfO x single layer (H11) and HfO x (7 nm)/graphite (5 nm) stacked (H7G5) bilayer selectorless memristive devices.The sneak path current in H7G5 device can be avoided by taking advantage of nonlinearity in the self-rectifying I-V characteristics of this device.The I-V characteristics shows that the H11 has a higher sneak path current at LRS.Therefore, the H7G5 structure can be used that shows the self-rectifying behavior, where the LRS current is suppressed and sneak paths current can be eliminated. Figure 16b shows the TEM image of the H7G5 stacked selectorless memristor device.The device structure and fabrication processes have been fully described in a previous study. [93]he immunity to the sneak path currents introduced through the nonlinearity of the selectorless devices introduced here mitigates the potential weight variability in neuromorphic learning and computing applications.Moreover, the selectorless device introduced can result in larger crossbar sizes, as shown in the figure.This results in a larger number of wordlines, which in turn means a bigger network suitable for extended computing demands. Figure 16c shows LTP (top) and LTD (bottom) behaviors using identical pulses method to examine the conductance flexibility and modulation in H7G5 devices.For depression, the pulse height is À0.7 V with 50 μs pulse width; for potentiation, the pulse height is 1.54 V lasting for 100 μs. Figure 16d shows the simulation accuracy results obtained using H11 and H7G5 selectorless devices mapped into the three-layer neural network shown in Figure 15a for classifying the handwritten digits MNIST dataset.As the figure shows, the best classification accuracy achieved using the network composed of the H7G5 devices is around 85%, which is far better than that of the network using H11 devices (30%).This network uses selectorless RRAM devices as their weight elements, which suppress the sneak path current without the need for additional transistors compared with previous designs. In all aforementioned learning experiments performed using SiO x devices, neural networks were simulated with realistic device models, where learning occured through machine learning approaches such as BP.In these networks, the memristive devices were mainly used as weight elements, which are programmed to represent a certain resistance state.Therefore, no unsupervised learning through neural-inspired algorithms such as STDP, similar to that shown in Figure 11c, occured.However, it has been shown that memristive devices can be used to perform unsupervised learning through synaptic plasticity mechanisms used in neuromorphic systems.In the next section, we provide simulation results of a memristive neuromorphic system that uses STDP for unsupervised learning of simple patterns. Hybrid CMOS-Memristive Neuromorphic Sensing The use of memristors as a processor of input sensory information has rapidly gained traction but has only had limited success in the frontend generation of sensory data.For example, in previous studies, [16,94] both works use memristive meshes for spatiotemporal smoothing of information as a way to reduce noise through averaging.The resistive power dissipation and lack of equal set/reset processing speeds have made it too difficult to justify the additional fabrication steps required to implement thin-film metal-oxide memristors in the back-end-of-the-line (BEOL) for CMOS integration. In the circuit shown in Figure 17a, we present a novel memristor-CMOS image circuit that utilizes temporal storage of information in RRAM to block signals in the absence of temporal change in light intensity.This circuit is an extension and improvement on what is presented in the image sensing circuit from a previous study, [16] which relied on the use of a separate match line for voltage comparisons to a constant reference.A constant reference means that an output was generated at the source of a transistor M3 when input intensity exceeded some threshold; that is to say, there was no detection of temporal difference of intensity, and therefore light adaptation was a mechanism of DC thresholding.We draw inspiration from dynamic vision sensors here, but rather than using a capacitance to block DC content, we rely on the identical signals generated from the application of a read pulse to a pair of identically programmed memristors at two consecutive points in time to block the output.The voltage and memristor state X read-out are shown in Figure 17b, and the use of the match line alone in a spike-based programming scheme is shown in Figure 17c.The output signal from the image-sensing circuit is passed through a subthreshold amplitude-to-frequency converter which generates voltage spikes, in a manner similar to that of the retinal ganglion cells, [95,96] the schematic of which is shown in Figure 17d, and array-based architecture of image sensor interfacing to subthreshold cell circuits in Figure 17e.The final spiking output results are shown in Figure 17f for a given input of light intensity change.The chip is fabricated in the SK Hynix Figure 16.a) I-V characteristics of bipolar resistive switching operation in HfO x single-layer memristor (H11) and selectorless HfO x (7 nm)/graphite (5 nm) stacked memristor (H7G5) (Adapted under the terms of the CC BY license. [91]Copyright 2019.The Authors, published by Springer Nature).b) TEM image of a sample H7G5 stacked device is shown at the top (Adapted with permission. [93]Copyright 2019, The Royal Society of Chemistry.)and crossbar array size calculation based on measured nonlinearity and memory window with various device sizes (0.4, 0.6, 0.8, 1 μm) on H11 and H7G5 devices are shown at the bottom.c) The LTP/LTD behaviors using identical pulses are shown for H7G5 devices.d) Neural network simulation accuracy using BP algorithm and supervised training is shown.The accuracy is around 85% for H7G5% and 30% for H11.A similar network to that shown in Figure 15a was used. 180 nm process, V DD ¼ 1.8 V and V th ¼ 0.4 V. Here, V G OUT is the voltage-converted current output, whereas V A ON and V G IN are the voltage-converted inputs from the preceding stage.Note that the response is shifted negatively to replicate a biologically plausible resting membrane potential. Simulation of Memristive Neuromorphic Computing Biophysical neuron models, such as those developed for HH and ML neurons, have shown memristive behavior in their footprint. [97,98]There exist only a few experimental implementations [62,99] of memristor-based biophysical neurons due to the high complexity of the neuronal dynamics and bifurcation behaviors.To the best of our knowledge, there is no memristive biophysical neuron-based network, which has been tested for any type of application.To address this challenge, one approach is to develop and utilize simulation frameworks for memristive neuromorphic networks.Significant research [64,[100][101][102] has been conducted on simulating memristive networks, where experimental implementation cannot be conducted due to the network [16] Copyright 2018, IEEE.size, complexity, technology limitations, etc.The simulation analysis can provide insight into the dynamical behavior of the equivalent memristive circuit model of various neuron models and the learning behavior and performance of their network. We have developed and simulated simple crossbar structured memristive networks to validate the functionality of the biologically-inspired ML and HH memrisitive neurons (that were shown in Figure 9), in a simple pattern classification task.Figure 18a shows the STDP learning mechanism for two coupled HH neurons with memristive synapses in a small-scale crossbar structure.Figure 18b shows the membrane voltage of pre-and postsynaptic HH neurons connected by a memristive synapse for Figure 18.Simple patterns are classified by HH and ML memristive neurons, connected through crossbar structures of memristive synapses, following STDP learning, through overlapped input spikes.Adapted with permission. [65,66]Copyright 2016 and 2017, IEEE. a 15 μA stimulus current. Figure 18c shows a 2 Â 2 two-layer perceptron network with HH neurons and memristor synapses, where the inputs are two classes of two-pixel images.The classification results of the two-pixel input patterns performed by the 2 Â 2 memristive network are shown in Figure 18d.Here, the membrane voltages of the presynaptic (black) and postsynaptic (blue) memristive HH neurons are shown.Initially, postsynaptic neurons spike without any specific pattern, and it takes some times for them to follow the input patterns.The learning is unsupervised and the winner neuron follows one of the patterns based on its initial weight vector.When the class 2 image is assigned to postsynaptic neuron 1, the weight of memristive synapse 1 increases due to STDP.As post-synaptic neuron 1 spikes after presynaptic neuron 1 (a black pixel makes a neuron spike), the weight of memristive synapse 1 increases.Also, the weight of memristive synapse 2 decreases as postsynaptic neuron 2 is inactive whereas presynaptic neuron 1 spikes. Figure 18e shows a 4 Â 2 two-layer perceptron network with ML memristive neurons and memristive synapses.In this case, inputs are two classes of four-pixel images.Four presynaptic neurons' membrane voltages are shown in Figure 18f, which show their spiking activity, after applying the input spike trains.LTD and LTP phenomena are shown in Figure 18g during the classification in the memristive network on the synaptic devices.Finally, Figure 18h shows postsynaptic neurons' membrane voltages and the results show successful classification of the two classes of input patterns, where after 5 s, neuron 1 fires for one pattern, whereas neuron 2 fires for the other pattern. Discussion and Conclusions Neuromorphic computing is a far-reaching field that encompasses the hardware implementation of basic artificial neural networks, through to biologically plausible spike-based systems.In both cases, learning and inference are the cornerstones of building functional large-scale networks that facilitate tasks that are fundamentally tied to neural cognition. We have shown how CMOS and memristive systems have become highly pervasive in neuromorphic computing as they mimic the functional primitives of SNNs and artificial neural networks alike and reproduce neuronal and synaptic models at the device/physics level.While various RRAM switching mechanisms are available, we have focused on the use of filamentary-based switching and joule-heating modulation in SiO x memristors to construct substantially simplified neurons and synaptic circuits when compared with their purely CMOS counterparts.In essence, the integration of nanoscale nonvolatile devices in the BEOL has completely removed reliance on physically disparate volatile DRAM or SRAM arrays, where information transfer is thwarted by restricted bus sizes. The von Neumann bottleneck in conventional computing systems is alleviated by leveraging analog domain in-memory computing in memristive crossbar systems capable of spike-based and nonspiking training and inference, both supervised and unsupervised.This was demonstrated in both oxygen migration of hafnium-oxide devices and filamentary growth within SiO x memristors, which may be integrated with active CMOS arrays or passive arrays by exploiting highly nonlinear characteristics that exhibit diode-like suppression of sneak paths currents.As cognitive neuroscientists better understand that learning mechanisms that occur within our neural systems, having a fundamental physical device-level primitive enables an optimized mode of implementation that can be used to successfully demonstrate more complex learning rules and higher-order behaviors such as triplet-based, quadruplet-based STDP, and beyond. In-memory processing has also been demonstrated in similar analog domain form by implementing multibit flash cells, but these are bottlenecked by the lower-limit technology nodes that floating-gate transistors are fabricated in, and memristive devices appear to be the prime candidates for moving beyond such limitations.An alternative in both array-based computing and biological spike generation has been shown in the form of subthreshold analog circuits such as those shown in this article, which take advantage of ultralow power consumption but suffer from extremely high RC delays which are introduced from high drain-to-source equivalent resistance values when the channel has not been fully inverted due to low voltages applied at the gate. Neuromorphic engineering is not limited to drawing inspiration from the processing that occurs within the brain and can be broadened to any biological process.We have demonstrated neuromorphic vision sensing by developing a RRAM-based adaptive image sensor, which is equally important as a method to optimize the frontend procurement of information that is transmitted to the processor.The notion that event-based processing strips input data of redundancy and that the retina implements ultralow power processing through adaptive vision are replicated in our sensory neuromorphic RRAM-CMOS integrated system.The realization of fully optimized systems may require similar approaches in integrating neuromorphic processing with frontend neuromorphic sensing. The advantages of RRAM-based neuromorphic computing are naturally counterbalanced by the requirement to convert read-out currents into digital domain information for interfacing with computing systems, and this requires the use of analogto-digital converters (ADCs) which partially offset the power, speed, and area advantages of purely RRAM neuromorphic systems.Current integration techniques use active resistive switching layers above higher-level metal layers for any technology process, which enables us to take advantage of vertical real estate of a chip and relax the burden of planar topological area.The same methodology has been applied in using FinFET technology to reduce the impact of electron traps in creating channels with a larger cross-sectional area for CMOS scaling below 5 nm feature sizes.There is a continued need to explore the effect of noise associated with analog computing and quantization effects on precision but with the silver lining that neural networks can be designed to be robust to minor errors, signal fluctuations and nonidealities, making them optimal candidates for applications in soft computing. In addition, retention degradation (i.e., time-dependent RRAM state decay) and its impact should be considered in neuromorphic computing, as it is done in previous works such as a study by Wu et al. [103] At a temperature of 85 C, stacked TaO x devices have shown retention times of over 10 years. [104]Though when baking at higher temperatures, a 12% degradation in classification on the MNIST dataset is observed, and most degradation occurs after 10 4 s. [105]As a result, a refresh operation is required in practical applications to maintain the accuracy. To further improve retention, electrical design at the state level (e.g., compliance current limiters) should be fine tuned.From a structural and architectural perspective, oxygen exchange layer (OEL) and one transistor-one resistor (1T-1R) cells are needed to mitigate stochastic oxygen vacancy generation, annihilation, and overshooting pulses.The use of stacked-layer structures and thin-film plasma treatment are also helpful to eliminate the defective states recovery.At the board level, a passive heat sink is a common practical solution to prevent thermal throttling during heavy workload cycles. It should also be noted that semi-volatile devices, such as WO x from previous studies, [106,107] which display millisecond retention characteristics, may also be leveraged as LIF neurons.This enables devices to reproduce time-dependent spiking adaptations which have been observed in pyramidal neurons and is useful in reservoir computing, thus efficiently processing temporal information. [108]t this stage, researchers have a strong idea of the architecture of biological cells within sensory systems such as the retina and, to some extent, the brain as well.However, what is lacking is a unified account of the computation that takes place within biological sensors and how they process, filter, and store information before transmitting them to the brain.As neuroscientists converge toward a better understanding of the biological processing that takes place across the nervous system, CMOS and memristive researchers will be able to continue to cooperate in building large-scale systems to both better understand the cognitive circuits in performing functional and behavioral tasks such as pattern recognition and higher-order interpretation and introduce the low-power and highly adaptive advantages of biological processes to conventional computing. Figure 1 . Figure 1.The structure of the article at a glance.a) The article is composed of discussions on CMOS, memristive, and hybrid CMOS-memristive neuromorphic components and systems.b) The tree structure of the article outline is shown. 28 Figure 2 . Figure 2. A synapse circuit implemented in CMOS to realize triplet-based STDP a) can modify synaptic weight based on pair-based STDP (top traces in [b,c]) when only its pair-based (red) part is activated, whereas it shows triplet-based weight change dynamics (bottom traces in [b,c]) when all its parts, i.e., the triplet depression (left blue) and triplet potentiation (right blue), work in conjunction with the pair-based (red) part.Note that a pre-post-pre spike triplet b) can result in a higher synaptic depression in triplet STDP interactions (bottom trace), whereas it shows a lower depression (top trace) in the pairbased spike interaction, due to considering only the post-pre pair, and not the pre-post-pre triplet.Similarly, in the case of post-pre-post spiking in (c), higher potentiation is expected when considering the triplet combination (bottom trace), whereas lower potentiation is elicited when only a pre-post pair is considered (top trace). Figure 4 . Figure 4. Taxonomy of resistance switching in silicon oxide.Upper LHS: Schematic of extrinsic resistance switching.Upper RHS: Schematic of intrinsic resistance switching.Lower LHS: Air-sensitive resistance switching: typically, electroforming and resistance switching occur only in devices with an exposed oxide surface and not in bulk devices.This is attributed to re-oxidation of surface-based silicon filaments by an oxidizing ambient.Lower RHS: Air-stable resistance switching.This type of switching occurs in ambient (oxidizing) conditions and is defined by the microstructure of the oxide material.Switching voltages are typically lower for air-sensitive switching.Reproduced with permission.[39]Copyright 2018, John Wiley and Sons. Figure 5 . Figure 5. a) Illustration of the experimental setup to test STDP-like behavior: Non-identical pre-and postvoltage pulses and the resulting overlapping voltage across the device.b) The plot demonstrates successful device operations (conductance increase or decrease depending on time delays).Plots demonstrate STDP-like behavior for c) mean and d) median change in device conductance.Dotted lines serve as a visual guide.Adapted under the terms of the CC BY license.[50]Copyright 2018, The Authors, published by Frontiers Media. Figure 6 . Figure 6.SiO x -based 1 R-1 D synaptic device.The device structure is shown in SEM images shown in a) for top view and b) cross-sectional view.c) The device shows great controllability to increase and decrease its conductance in response to pulse amplitude modulation.d) The device is used to demonstrate ten different conductance change behaviors as a result of an STDP experiment, where positive and negative spike timing difference as well as spike widths are used.Here, (e) shows the same data as (d) but in the reversed order.a,b,d,e) Reproduced under the terms of the CC BY license.[53]Copyright 2016, The Authors, published by Springer Nature. Figure 7 . Figure 7. a) Voltage transients (spikes) do not occur until the constant current of 5 mA is applied (current threshold).b) Schematic of the LIF neuronal model.The lower figure shows the integration of input current pulses over time.Theta is the voltage threshold for spiking.c) Excitatory current pulses (4 mA) are applied to the device, and small current pulses (1 μA) are used for sensing the voltage across the device.Device voltage response is shown for pulse time separations of d) 640 ms, e) 215 ms, and f ) 65 ms.These demonstrate that spiking occurs for the smaller number of pulses if the separation between input current pulses is shorter.Adapted under the terms of the CC BY license.[55]Copyright 2016.The Authors, Published by Frontiers Media. Figure 9 shows two different panels, each describing one bioinspired memristive neuron circuit diagram, memristive channel characteristics, and bifurcation behaviors at different frequencies, for a different neuron model.In the top panel of Figure 9, (a) shows the basic circuit schematic for the HH neuron memristor-based circuit model with memristive behavior of sodium and potassium channels.A voltage-gated potassium (sodium) channel is emulated by positively (negatively) biased memristor devices coupled with a membrane capacitor.In this panel, (b) shows the tonic spiking behavior of memristive HH neuron and potassium (K þ ) and sodium (Na þ ) channels for HH model.In the bottom panel, (c) shows the ML neuron model circuit diagram and the behavior of potassium and calcium channels for the ML memristive circuit model in response to a 2.5 V sinusoidal input voltage at different frequencies.The otassium channel memristor I-V curve is shown at 5 Hz, 50 Hz, and 5 kHz (from left to right).The calcium channel memristor I-V is displayed at 100 Hz, 500 Hz, and 50 kHz (from left to right).The scaled ML neuron bifurcation behavior for different current stimulus is shown in Figure 9d.The membrane voltage, calcium channel's current, potassium channel's current, and state variables (M, N) have been also displayed for the stimulus currents of 60 and 100 μA. Figure 8 . Figure 8. SiO x -based 1R-1R neuron device.a) TEM cross-section image of metal-insulator-semiconductor (MIS) structure: TaN/30 nm-SiO x /1 nm-HfO x /N þ Si substrate.b) 100 times I-V threshold switching of MIS device in air.c) Threshold switching in the air for selectivity and nonlinearity of the stacking bilayer structure shown in (a).d) Typical I-V threshold switching of vanadium electrode/6 nm-SiO x /TiN structure.e) Experimental setup for a simple neuron circuit by the SiO x -based threshold switching selector.f ) The screenshot of measurement instrument (Agilent DSO9254A) and measured voltage results in CH1 (green line) and CH2 (blue line).Pulses with 100 μs width are used here.When CH1 is below the firing threshold (e.g., at 2 V), there is no firing observed, whereas if threshold switching occurs (e.g., input pulse amplitude > 3.7 V), the neuron firing begins on CH2 (damping Vp 1.2 V). a,b) Adapted with permission.[38]Copyright 2018, The Royal Society of Chemistry. Figure 12 . Figure12.a) I-V curves demonstrate a typical bipolar resistance switching.b) Adjustment of reset voltage shows a gradual reset process achieving multiple stable resistance states.Dynamics of resistance increase (LTD) is highly dependent on both pulse amplitude and pulse width.This is demonstrated in (c) and (d) receptively.e) Three very different LTD curves are obtained from the same RRAM device by changing the pulse amplitude and width.Reproduced under the terms of the CC BY license.[51]Copyright 2019.The Authors, published by Frontiers Media. I 1 -Figure 13 . Figure 13.a) The structure of the implemented ANN for MNIST classification task.b) The network weights at each layer are mapped to a two-xbar structure.Here, the network has two hidden layers.Reproduced under the terms of the CC BY license.[51]Copyright 2019.The Authors, published by Frontiers Media. Figure 14 . Figure 14.The decrease in inference accuracy of RRAM-based ANNs from a) the effect of un-electroformed devices, b) devices failed at HRS, andc) devices failed at LRS. Adapted under the terms of the CC BY license.[51]Copyright 2019.The Authors, published by Frontiers Media. Figure 15 . Figure 15.a) The structure of the simulated neural network is shown.It learns through BP algorithm and supervised training.b) The actual network sizes are 256 Â 64 Â 10 and 256 Â 64 Â 7, which achieved different accuracy levels after 100 learning epochs for various device-to-device variation levels (each curve), averaged around 89% and 80%, for the two network sizes for MNIST and TP classification.Note that the convergence speed was similar in both network sizes and for various device variation levels.c) Correlation between LTP and LTD nonlinearity effect and the simulated accuracy is shown. Figure 17 . Figure 17.a) The schematic structure of the CMOS-RRAM photoreceptor.b) Photoreceptor cell output and state response.c) Photoreceptor cell pulsing.d) Circuit schematic of a ganglion cell-inspired subthreshold CMOS amplitude-to-frequency converter.The ganglion cell circuit accepts current as input from the previous image-sensing stage and will only generate an output where there has been a change in intensity, to achieve retina-like power optimization.e) Retinal network CMOS chip layout.The full flow of photocurrent commencing at a 128 Â 128 array of photoreceptor cells, which are averaged to a 64 Â 64 bipolar cell network to produce a graded action potential and are subsequently converged to a 16 Â 16 retinal ganglion cell network for fast spike generation.f ) Experimental results of the RRAM-CMOS retinomorphic architecture.e,f ) Reproduced with permission.[16]Copyright 2018, IEEE. Table 1 . Comparison of switching characteristics and parameters of SiO x -based resistive switching devices.	2020-03-12T10:16:29.736Z	2020-04-14T00:00:00.000	{ "year": 2020, "sha1": "a200a40b985670b4695e8ee3e4cb42c167d45bd9", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/aisy.201900189", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "b6b7e789fcae1050cf99da842b8f7473b0fa4cd2", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
3737173	pes2o/s2orc	v3-fos-license	Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma Background In the present study, we aimed to investigate the role of epidermal growth factor receptor (EGFR) pathway in the up-regulation of programmed death ligand-1 (PD-L1) caused by radiotherapy (RT). Materials and Methods Tissue microarrays (TMA) consisting of glioma cancer specimens from 64 patients were used to examine the correlation between PD-L1 and EGFR levels. Furthermore, we performed in vitro experiments to assess the role of EGFR pathway in RT-upregulated PD-L1 expression using human glioma cell lines U87 and U251. Results Our data demonstrated that the PD-L1 expression was significantly correlated with EGFR expression in glioma specimens (χ2 = 5.00, P = 0.025). The expressions of PD-L1 at the protein and mRNA levels were both significantly up-regulated by RT (P < 0.05). The expressions of phosphorylated EGFR and janus kinase 2 (JAK2) were also induced by RT (P < 0.05). Besides, inhibition of EGFR pathway could abrogate the RT-triggered PD-L1 up-regulation (P > 0.05). The combination of RT with EGFR inhibitor exhibited the same effect on antitumor immune response compared with the combination of RT with PD-L1 neutralizing antibody (Ab). Conclusions RT could up-regulate the PD-L1 expression through the pathways downstream of EGFR in glioma. Introduction Malignant glioma is a highly lethal and common central nervous system tumor with poor 5-year survival rate (Wu et al., 2015). According to the histological classification, glioma can be divided into three types as follows: anaplastic oligodendroglioma, anaplastic astrocytoma and glioblastoma (Wang and Jiang, 2013). At the moment, the main therapeutic strategy for glioma is multimodal therapy, which consists of radiotherapy (RT), surgical resection and systemic treatment with alkylating agents (Stupp et al., 2005;Wen and Kesari, 2008;DeAngelis, 2005). Previous studies have shown that radiotherapy can improve the median survival of glioma patients from 6 months to 1 year (Walker et al., 1978;Stupp et al., 2005). However, it is still urgently necessary to develop more effective treatments since most patients with glioma will eventually die of disease relapse (Vatner et al., 2014). In recent years, immunotherapy targeting inhibitory checkpoint molecules has become a new treatment strategy for glioma (Song et al., 2016). Programmed death ligand-1 (PD-L1) is a representative inhibitory checkpoint, which is expressed in many types of cancers (Pardoll, 2012). When PD-L1 binds to its receptor named programmed death-1 (PD-1) which is expressed in CD8 + cytotoxic T lymphocytes (CTLs), the function of activated CTLs is suppressed (Jie et al., 2013;Pardoll, 2012). Indeed, blocking the PD-L1/PD-1 pathway using antibodies could reduce the inhibition effect on the activated CTLs. Researchers have found that patients with high PD-L1 expression have better treatment response (Topalian et al., 2012b;Brahmer et al., 2012;Topalian et al., 2012a;Taube et al., 2014). Benavente et al. have found that the PD-L1 expression in tumor cells is regulated by two major mechanisms (Concha-Benavente et al., 2016). First, an 'extrinsic' mechanism relies on interferon gamma (IFN-γ) produced by natural killer (NK) cells and CD8 + CTLs, in which IFN-γ not only activates the antitumor cellular immune response but also in turn induces PD-L1 expression in tumor cells. Second, an 'intrinsic' mechanism independent of IFN-γ exists, in which epidermal growth factor receptor/janus kinase 2 (EGFR/JAK2) signaling pathways within the tumor cells lead to PD-L1 over-expression. A previous study has confirmed that radiotherapy can induce PD-L1 expression in tumor cells through the 'extrinsic' mechanism, but the effect of 'intrinsic' mechanism in this process remains ambiguous . Park et al. have found that radiotherapy can up-regulate the expression of phosphorylated EGFR to activate pathways downstream in glioblastoma . Therefore, we hypothesized that the 'intrinsic' mechanism, especially pathways downstream of EGFR, also played an important role in up-regulating PD-L1 expression by radiotherapy in glioma. Moreover, we further investigated the effects of EGFR and PD-L1 inhibitors on tumor immune response in irradiated glioma cells. Cell Culture and Reagents Human glioma cell lines (U251 and U87) were obtained from the Shanghai Institutes of Biological Sciences Cell Bank and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO 2 . Antibodies (Ab) against EGFR, JAK2, PD-L1 and beta-tubulin as well as phosphorylated forms of EGFR (Y1173) and JAK2 (Y1007 + Y1008) were purchased from Abcam (UK). Specific inhibitor of EGFR (AG490) was also supplied from Abcam. The PD-L1-blocking antibody (avelumab) was purchased from EMD Serono (Germany). Bound antibodies were detected with horseradish peroxidase-linked antibody against mouse (Abcam) or rabbit (Santa Cruz Technology, USA) immunoglobulin G, followed by enhanced chemiluminescence (ECL) detection (Amersham, USA). Lymphocyte separation medium and the CellTrace™ CFSE kit were purchased from Sigma (USA). AntiCD3/CD28 stimulation beads were purchased from Thermo Fisher Scientific Pierce (USA). CD8 + T cell immunomagnetic beads positive selection kit was purchased from Stem-Cell Tech (USA). Annexin V/PI kit was purchased from Abcam. Immunohistochemistry (IHC) and Staining Evaluation Approved by the Ethics Committee of Soochow University, cancer specimens from 64 patients with glioma were selected to build tissue microarrays (TMA). The following clinical parameters of patients were collected: gender, age, histopathology and grade. The quality of the TMA slides was confirmed by the pathologist using HE-stained slides. The tissue section slides were deparaffinized and rehydrated, and then washed in phosphatebuffered saline (PBS) solution three times. For antigen retrieval, slides were immersed in 10 mM sodium citrate (pH 6.0) and autoclaved at 120°C for 15 min. The sections were incubated in 0.3% hydrogen peroxidase in absolute methanol for 30 min to deactivate endogenous peroxidases. After nonspecific binding was blocked with 3% bovine serum albumin (Cell Signaling Technology, USA) in PBS, the specimens were incubated with primary antibodies at 4°C overnight. Then the specimens were incubated with anti-mouse/rabbit secondary antibody at room temperature for 30 min (Abcam). Staining was carried out using diaminobenzidine (DAB) kit (Sigma). The intensity of staining was evaluated according to the following scale: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining (Ikeda et al., 2016). The proportion of all staining tumor cells was determined and then multiplied by the staining intensity score to obtain a final semi-quantitative H score (maximum value of 300 corresponding to 100% of staining tumor cells with an overall staining intensity score of 3). The scores exhibiting b 100 were classified Fig. 1. IHC staining for PD-L1 and EGFR in glioma specimens. Representative images of HE staining, positive PD-L1 and EGFR staining on slides from a selected tumor specimen, and representative HE staining, negative PD-L1 and EGFR staining on slides from another tumor specimen, are shown. Three consecutive slices from the same patient were stained, and one was chosen as representative image for shown. as low expression and the remainder as high expression. All IHC images were blindly evaluated by two experienced observers (J.X. and D.Z.), and the mean of the two determinations was used for further analysis. Cell Treatments and RT Cells were plated for 2 days until 70% to 80% confluence and then serum-starved overnight (16-18 h, DMEM/0.5% FBS). Stock solution of AG490 was stored in aliquots at −20°C. Before RT, cells were treated with 10 μM AG490 at 37°C for 1 h. Subsequently, cells were exposed to 6-MV x-rays from an Elekta Synergy linear accelerator (Elekta Instrument AB, Sweden) at a dose rate of 0.5 Gy/min and then incubated at 37°C . Western Blotting Analysis Cells were lysed and protein was extracted using mammalian protein extraction agent (Thermo Fisher Scientific Pierce) plus halt protease inhibitor cocktail (Thermo Fisher Scientific Pierce). Protein concentrations were determined using a bicinchoninic acid assay (Thermo Fisher Scientific Pierce). Equal amounts of proteins were loaded onto 8% or 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocked with 5% bovine serum albumin in PBST (0.1% Tween 20 in PBS) for 1 h, membranes were hybridized with primary antibodies to human EGFR, JAK2, PD-L1 and betatubulin as well as phosphorylated forms of EGFR (Y1173) and JAK2 (Y1007 + Y1008) at 4°C overnight. Then the membranes were washed three times with PBST for 10 min. Subsequently, membranes were hybridized with the corresponding secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature. After being washed three times with PBST for 10 min, immunoreactive proteins were detected using ECL assay. Membranes were exposed to X-ray film (Kodak China Investment, China) to visualize the bands. The band quantification was conducted using ImageJ (National Institutes of Health, USA). Beta-tubulin was used as loading control. Reverse Transcription PCR (RT-PCR) Total RNA was isolated from irradiated glioma cells using the Trizol reagent (Takara Bio., Japan). Purified RNA was reversely transcribed into cDNA with the M-MLV First Strand kit (Invitrogen, USA) according to the manufacturer's instructions. RT-PCR reactions were carried out using the SYBR Premix Ex Tag II (Takara Bio.) on an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, USA). Briefly, after an initial denaturation step at 95°C for 30 s, amplifications were conducted with 40 cycles at a melting temperature of 95°C for 5 s, and an annealing temperature of 60°C for 34 s. Human GAPDH was used as the housekeeping gene to normalize the expression level of target gene. Primers used for amplifications were as follows: 5´-ACTGGCATTTGCTGAACG-3′ (forward) and 5´-TCCTCCATTTCCCAATAGAC-3′ (reverse) for PD-L1; and 5´-TGACTTCAACAGCGACACCCA-3′ (forward) and 5´-CACCCTGTTG CTGTAGCCAAA-3′ (reverse) for GAPDH. 2.6. Co-culture Experiments 10 mL fresh morning fasting human venous blood treated with anticoagulant was collected, then diluted with equal parts of PBS. 10 mL lymphocyte separation medium was slowly added in the blood. We centrifuged the mixture at 3000 r/min for 20 min then pipetted the monocytes (cloud-like) with capillary. Subsequently, the monocytes were washed with 10 mL PBS. Then the supernatant was discarded after centrifugation, and the monocytes were suspended in buffer. CD8 + T cells were isolated using immunomagnetic beads positive selection kit, and the isolating process was strictly in accordance with the product manual. For co-culture assays, CD8 + T cells were seeded into 96-well plates (1 × 10 5 cells/well) with tumor cells at a ratio of 1:1. Before co-culture, CD8 + T cell proliferation was induced by anti-CD3/CD28 beads (Invitrogen) and the tumor cells were treated with vehicle control, RT, PD-L1 Ab, RT + PD-L1 Ab, AG490, RT + AG490, or RT + AG490 + PD-L1 Ab. The dosage of RT was 5 Gy. The concentrations of PD-L1 Ab and AG490 were 20 μg/mL and 10 μM, respectively. After co-culture for 48 h, the CD8 + T cells were isolated using immunomagnetic beads positive selection kit again. The CD8 + T cell proliferation and tumor cell apoptosis were detected by flow cytometry (FCM). For proliferation assays, the isolated CD8 + T cells were CFSE-labeled (3 μM). For apoptosis assays, U251 and U87 cells were stained with antibodies against PI and annexin V after removal of CD8 + cells. Statistical Analysis Statistical analysis was performed using SPSS v21.0 (SPSS Inc., USA) and GraphPad Prism v5.0 software (GraphPad Software, USA). The relationship between PD-L1 expression and clinical parameters of patients was evaluated by the chi-squared (χ 2 ) test. Difference between two groups was assessed using Student's t-test. When comparing more than two groups, the statistical significances were determined by oneway analysis of variance (ANOVA) followed by Dunnett's test. Data were presented as the means ± standard error of the mean (SEM). All experiments, consisting of three replicates, were performed at least twice independently. A P b 0.05 was considered as statistically significant, unless otherwise stated. Correlation Between PD-L1 Expression and Clinical Parameters of Patients The expressions of PD-L1 and EGFR in 64 tissue specimens obtained from patients with glioma were assessed by IHC staining. Interobserver agreement in the assessment of IHC findings was excellent. The results from IHC staining indicated that positive staining of PD-L1 and EGFR was predominantly observed on the membrane and in cytoplasm of glioma cells (Fig. 1). High PD-L1 expression was identified in 28 (43.8%) of 64 glioma specimens, and high EGFR expression was evident in 31 (48.4%) of 64 glioma specimens (Fig. 1). Table 1 shows that there was a significant correlation between PD-L1 and EGFR protein levels in glioma specimens (χ 2 = 5.00, P = 0.025). Furthermore, high PD-L1 expression was significantly associated with advanced clinical parameters, such as age ≥ 55 (χ 2 = 11.46, P = 0.001) and grade III/IV (χ 2 = 7.11, P = 0.008). RT Activates the EGFR Signaling and Up-regulates the PD-L1 Expression in Glioma Cells To investigate the impact of RT on the PD-L1 expression in glioma cells, we irradiated human glioma cell lines (U87, U251), and then the expression of PD-L1 at the protein and mRNA levels was examined after 24, 48 and 72 h by Western blotting and RT-PCR, respectively. We found that the expression of PD-L1 at the protein and mRNA levels in both cell lines was all significantly up-regulated (P b 0.05; Figs. 2 and 3). Under the condition of 5 Gy, the promotive effect of RT on the PD-L1 Fig. 3. EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; **P b 0.001; ns, nonsignificant; versus DMSO group. expression at the protein level in both cell lines was in a time-dependent manner (Fig. 2). However, an opposite result was observed under the condition of 10 Gy, under which the PD-L1 expression was increased first and then decreased, and the peak appeared at 24 h for U87 cells and at 48 h for U251 (Fig. 2). EGFR Signaling Regulates RT-Induced PD-L1 Expression in Glioma Based on the significant correlation between the expressions of PD-L1 and EGFR in glioma cancer specimens, we hypothesized that the PD-L1 expression was regulated by EGFR signaling in glioma cells. Therefore, we examined the expressions of EGFR, p-EGFR and downstream signals (JAk2 and p-JAK2) under the same RT conditions. The result showed that the expressions of EGFR and JAK2 were not altered by RT (P N 0.05; Fig. 2). However, the levels of activated molecules, p-EGFR and p-JAK2, were significantly up-regulated (P b 0.05; Fig. 2). After EGFR-JAK2 pathway was blocked by specific inhibitor AG490, Western blotting and RT-PCR analyses revealed that the expression of PD-L1 was not altered by RT (P N 0.05; Figs. 3 and 4). Role of EGFR Signaling in Tumor Immune Escape after RT for Glioma As a co-stimulatory molecule, PD-L1 plays an important role in tumor immune escape. Therefore, we co-cultured tumor cells (U87 and U251) with T cells to compare the regulatory effects of EGFR and PD-L1 on the immune responses under RT. The result showed that ability of tumor cells to suppress T cell (anti-CD3/CD28 antibody stimuli) proliferation was strengthened by RT (P b 0.001; Fig. 5). This RTstrengthened ability was inhibited when PD-L1 Ab was added to the co-culture system, and AG490 exhibited the same effect in this process (P b 0.001; Fig. 5). However, the combined application of PD-L1 Ab and AG490 was not able to further suppress such RT-strengthened ability compared with PD-L1 Ab or AG490 alone (P N 0.05; Fig. 5). Moreover, compared with RT or PD-L1 Ab alone, the combined application of RT and PD-L1 Ab could promote tumor cell apoptosis when tumor cells (U87 and U251) were co-cultured with human CD8 + T cells, and AG490 also exhibited the same effect as PD-L1 Ab in this process (P b 0.001; Fig. 6). However, RT in combination with PD-L1 Ab and AG490 did not further enhance the tumor cell apoptosis (P N 0.05; Fig. 6). Discussion Radiotherapy is one of main therapeutic strategies for tumor patients, but several studies have proved that RT can induce PD-L1 expression in various human cancers (Gong et al., 2017;Zhang et al., 2017;Hecht et al., 2016). As an immune checkpoint, PD-L1 is generally expressed in various human cancers and helps tumor cells escape host immune responses Cho et al., 2017). Previous study has shown that PD-L1 can activate the functions of PD-1 to inhibit the proliferation, survival and effects of CD8 + CTLs (Barber et al., 2006). In our study, irradiated glioma cells with up-regulated PD-L1 expression Fig. 4. EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37°C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; P b 0.05; P b 0.01; *P b 0.001; versus 0 Gy group. could effectively inhibit the proliferation and cytotoxicity of CD8 + T cells. Interestingly, the inhibitory ability of irradiated glioma cells to CD8 + T cells disappeared once the PD-L1 expression was blocked. In the co-culture experiments, when phosphorylated EGFR was blocked, the inhibitory ability of irradiated glioma cells to CD8 + T cells was also abolished, further implying that EGFR signaling pathway was responsible for the RT-triggered PD-L1 up-regulation. EGFR plays an important role in the proliferation, invasion and metastasis regulation of tumor cells (Scaltriti and Baselga, 2006). There are many tyrosine residues in the intracellular domain of EGFR (Shelton et al., 2005). The tyrosine phosphorylation can specifically bind to downstream proteins, activating the EGFR signaling pathway to complete the cell signal transduction from the cell outward to the cell (Hubbard and Miller, 2007). Therefore, there may be more pronounced changes in the expression of p-EGFR than that of EGFR during the process of signal transduction, which is consistent with our findings. The Western blotting analysis showed that only the expression of phosphorylated EGFR was up-regulated by RT. This observation is also found in the study of Chang-Min Park et al. . In addition, we found that RT triggered the same reaction of JAK2 and p-JAK2. However, it remains largely unexplored how RT facilitates the tyrosine phosphorylation. We hypothesized that there was a self-repair mechanism of tumor cells to deal with RT-induced damages, especially the sublethal damage (Zaider and Wuu, 1995). In our future work, we will investigate the underlying mechanism. In recent years, many EGFR tyrosine kinase inhibitors (TKIs), represented by nimotuzumab, panitumumab, cetuximab and mAb806, have been applied to the clinical trials of glioma (Yang et al., 2015;Greenall et al., 2015;Chakraborty et al., 2016;Meng et al., 2015;Hong et al., 2012). However, the drug-resistance is a huge challenge for targeted therapy. Several studies have demonstrated that the PD-L1 expression of patients with EGFR mutations is usually increased (Akbay et al., 2013;Azuma et al., 2014;Tang et al., 2015), suggesting that PD-1/PD-L1 inhibitor may be a more efficient approach for patients with EGFR mutations who are resistant to EGFR-TKIs. Moreover, it may not be a good strategy to use EGFR-TKIs and PD-1/PD-L1 inhibitor at the same time, as EGFR-TKI can inhibit the PD-L1 expression in tumor cells, which in turn significantly reduces the efficiency of PD-1/PD-L1 inhibitor (Zhang et al., 2016). However, several studies have also reported that RT in combination with PD-1/PD-L1 or EGFR-TKI inhibitor has better effect in glioma than single treatment (Zeng et al., 2013;Solomon et al., 2013). Zeng et al. have demonstrated that RT in combination with anti-PD-1 therapy improves the survival compared with either modality alone (Zeng et al., 2013). Cuban researchers have confirmed that nimotuzumab in combination with RT can achieve better curative effect than RT alone in the treatment of high-grade glioma (Solomon et al., 2013). In the present study, we confirmed that the up-regulation of PD-L1 caused by RT could be restrained by EGFR-TKI in glioma cells. Although we did not observe better results from the combination of three treatments according to the proliferation and apoptosis experiments, it is too early to conclude that RT in combination with EGFR-TKI and PD-1/PD-L1 inhibitor is an unnecessary treatment strategy. More in vivo experiments are necessary to clarify the effect of this new combination strategy, because EGFR is a key molecule not only in the proliferation but also in the invasion and metastasis regulation of tumor cells (Scaltriti and Baselga, 2006). There are certain limitations in our study. First, we only selected wild-type glioma cells and confirmed that the EGFR/JAK2 pathway was involved in the up-regulation of PD-L1 caused by RT. Although studies conducted on the EGFR wild-type cells may be more universally applicable, experiments using EGFR-mutated cells can still provide valuable insights. Moreover, the effect of combination therapy should be validated using EGFR-mutated animal model. Second, besides EGFR/ JAK2, some other molecules may also be involved, such as interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3). A previous report has shown that activation of EGFR induces IL-6 secretion from cancer cells, leading to subsequent activation of JAK/STAT3 (Zhang et al., 2016). Then activated STAT3 binds to PD-L1 promoter and eventually promotes transcription of PD-L1 (Wolfle et al., 2011). Therefore, IL-6 and STAT3 may also play an important role in the regulation of PD-L1 after RT. More studies are required to confirm the interactions between these molecules. In conclusion, our findings indicated that RT could up-regulate the PD-L1 expression through the pathways downstream of EGFR in glioma. RT in combination with PD-1/PD-L1 inhibitor and/or EGFR-TKIs might be a potential novel treatment strategy for patients with glioma. Funding Sources National Natural Science Foundation of China (31570877, 31570908); National Science and Technology Support Project (2015BAI12B12); Cooperative Research Foundation of National Natural Science Foundation for Hong Kong and Macao Scholars (31729001); Changzhou Science and Technology Project (CJ20160021). These funding sources primarily provided financial help for our purchase of reagents. We have not been paid to write this article by any pharmaceutical company or other agency. Conflicts of Interests There are no conflicts of interest in this study. Authors' Contributions X.S. detected the changes of glioma cells after radiation, and was a major contributor in writing the manuscript. Y.S. collected the tissues and clinical data of patients. T.J. analyzed the experimental data. Y.D. and B.X. draw the figures. X.Z. and Q.W. searched the literatures. X.C. and W.G. did the data interpretation. C.W. and J.J. designed this study. All authors read and approved the final manuscript.	2018-04-03T04:41:03.571Z	2018-01-31T00:00:00.000	{ "year": 2018, "sha1": "b0808ea487b9a4fce18c51eecfa91354385f75a8", "oa_license": "CCBYNCND", "oa_url": "http://www.thelancet.com/article/S2352396418300318/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b0808ea487b9a4fce18c51eecfa91354385f75a8", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
12626582	pes2o/s2orc	v3-fos-license	Five Questions Critical Care Educators Should Ask About Simulation-Based Medical Education Simulation is now commonly used in health care education, and a growing body of evidence supports its positive impact on learning. However, simulation-based medical education (SBME) involves a range of modalities, instructional methods, and presentations associated with different advantages and limitations. This review aims at better understanding the nature of SBME, its theoretic and proven benefits, its delivery, and the challenges posed by SBME. Areas requiring further research and development are also discussed. INTRODUCTION Simulation is rapidly permeating into every sphere of medical education, including teaching, assessment, and research. 1 Publications about SBME have grown exponentially over the past 10 years. 1,2 In addition, the quality of the studies published on SBME has consistently increased, as illustrated by a series of reviews published by Issenberg, McGaghie and colleagues. [3][4][5] This impressive body of evidence now includes original studies, narrative and systematic reviews, opinion and position papers in different clinical domains, as well as a fewer number of publications specifically related to critical care medicine. These studies combined with the research findings in other clinical areas demonstrate the relevance of SBME for critical care health professionals. [6][7][8][9][10][11] In the presence of such an extensive literature, it seemed superfluous to undertake another systematic review on the topic of simulation in critical care medicine. However, this abundance of information on SBME can be overwhelming for critical care educators who attempt, in the midst of many other professional responsibilities, to design, implement, and evaluate sound educational innovations or curricula for their trainees. The focus of this review on SBME is therefore to summarize the evidence relevant for frontline educators in critical care medicine. The authors briefly examine 5 practical questions aimed at better understanding the nature of SBME, its theoretic and proven benefits, its delivery, as well as the challenges posed by SBME. The term SBME is used broadly to include education of all health care professionals. Although SBME has traditionally been predominantly focused on physicians, simulation studies are increasingly directed toward other health care professionals, such as nurses, pharmacists, and dieticians. [12][13][14] WHAT IS SIMULATION-BASED MEDICAL EDUCATION? Simulation has been broadly defined as "an instructional medium used for education, assessment, and research, which includes several modalities that have in common the reproduction of certain characteristics of the clinical reality." 15 Simulation modalities typically include part-task trainers (interactions with a physical or virtual model requiring the use of specific psychomotor skills to complete procedures), human simulation (interactions with a simulated or standardized patient), computer-based simulation (interaction with a screen-based interface), and simulated clinical immersion (interaction with a physical or virtual work environment including team members, computerdriven manikin patient, and equipment). 9,10,[15][16][17] More recently, simulation modalities have been combined into hybrid simulations to facilitate the simultaneous and integrative practice of complementary skills (eg, part-task trainer on a simulated patient to practice suturing and communication skills). 18 In addition, simulators have been placed in real clinical environments to create in situ simulations. 19 In the face of such variety of simulation modalities, medical educators need to choose a modality that best aligns with the learning objectives of their training programs and depending upon the benefits and limitations of each modality. 9,15 WHY SHOULD SIMULATION-BASED MEDICAL EDUCATION BE USED? If simulation has ever elicited doubts among health care educators about its roles in medical education, many think that we have now moved beyond the need to justify its use. 13,20,21 Based on a growing body of evidence, the use of simulation seems judicious for certain aspects of health care training. The need to better define how to use SBME optimally and cost effectively has been identified as the next question to be answered by the medical education community. 7,13,20 It is worthwhile to briefly consider the context in which SBME rapidly gained popularity over the recent years, after initial delays in the uptake of a technology developed more than three decades ago. 7,9 As medical education evolved, educators developed a better understanding of the learning processes involved in health care education, such as deliberate practice, 22,23 reflection, [24][25][26] and feedback. [27][28][29] Meanwhile, the traditional apprenticeship model, based on the prolonged and repeated interactions between junior and senior health care professionals, was increasingly under threat because of numerous changes in the health care system: increased clinical workload; rapid turnover of patients and health care professionals in a given clinical unit; competing academic roles of faculty as clinicians, educators, and researchers; and so on. 9,30-32 Such changes led to perceived inadequacies of the medical training system: the key processes thought to benefit clinical learning seemed increasingly hard to experience in acute care environments. 7,32 Furthermore, growing concerns about patient safety made the idea of inexperienced trainees practicing their skills on real patients morally inacceptable. 17,31,33 Working hour limitations and increased patient supervision were implemented to increase the quality of care provided in teaching hospitals. 9,17,[32][33][34][35] The educational shortcomings of this new form of time-limited, autonomyrestricted clinical experience needed to be compensated. 31,36 In addition to individual competencies, human factors and teamwork were identified as a common source of medical errors. 9,37 Specific training for interdisciplinary teams was strongly recommended to further improve the quality of care. 37 Simulation represented a natural response to these problems for several reasons. 17,38 First, simulation can provide a safe learning environment where mistakes can be made, reviewed, corrected, and reflected upon. 9,17 Second, simulation offers the opportunity to practice clinical skills and to achieve a certain level of proficiency or mastery before caring of real patients. 7 Third, simulation offers greater control and predictability of the learning experience in terms of type, order, number, and length of the sessions; type of feedback provided; number and level of participants; and so on. 17 In theory, SBME therefore presents desirable characteristics to meet the educational needs of health care professionals in training, as well as the moral obligation to prioritize patient safety in real clinical situations. 9,17 The medical education literature provides clear evidence that SBME can fulfill an important role within health care professional training. At least 3 systematic reviews have now demonstrated that SBME, as an instructional medium, can positively affect learning and can translate into benefits for the patients. 13,14,39 However, the heterogeneity of the literature on SBME justifies taking a closer look at the potential educational benefits of SBME. This topic is the focus of the following section. WHAT CAN BE TAUGHT AND ASSESSED WITH SIMULATION-BASED MEDICAL EDUCATION? Simulation-based education has been used to teach and study a broad range of knowledge, skills, and attitudes. Multiple reviews have attempted to summarize the current evidence supporting the use of SBME. Some of these reviews are specific for critical care medicine, 6,[9][10][11]40 whereas others are related to different clinical specialties, such as anesthesia, 1,41 obstetrics, 42 emergency medicine, 17,43 surgery, 44 or pediatrics. 45 Specific modalities or groups of modalities have also been the object of literature reviews, including simulated patients, 46 virtual simulation, 47 technology-enhanced simulation, 13 computerassisted learning, 48 and in situ simulation. 19 The following conclusions can be derived from the examination of these reviews: 1. Simulation is generally well accepted by learners as a teaching strategy, as illustrated by the positive ratings consistently reported in trainee satisfaction surveys. 9 2. The types of learning outcomes measured to demonstrate the benefits of SBME are not equally represented in the literature: studies showing short-term gain in knowledge and skills in the simulation environment (levels 1 and 2 of Kirkpatrick's model 49 presented in Box 1) are overrepresented when compared with studies assessing long-term (eg, 6-12 months) gain of knowledge and skills, or changes in behaviors transferred to real clinical environments and benefiting patients (levels 3 and 4 of Kirkpatrick's model). 50 3. The quality of the evidence on SBME is generally limited, as indicated by small sample sizes, lack of control group or randomization, scarcity of multicenter studies, and poor reporting. 13,51 4. Improvement of knowledge, technical and nontechnical tasks, 10 teamwork, communication skills, and system issues has been achieved by SBME in certain areas. SBME has shown promising results in many areas relevant for critical care educators. Specific examples related to technical and nontechnical skills, system issues, and assessment tools are presented in Box 2. When appropriate, the Kirkpatrick's level of learning outcomes measured by individual studies is indicated. Simulation is also being increasingly considered in 2 other educational domains: high-stakes assessment and mass casualty training. Simulation-based assessment is not a new phenomenon and has been incorporated in licensure and certification examination at the undergraduate and postgraduate level for many years. 52 More recently, simulation has also been used in regulatory programs for practicing physicians in the fields of anesthesia, internal medicine, and family medicine. 52 In addition to part-task trainers and simulated patients, full-body manikin simulators are now increasingly used for high-stakes assessment. 53 As the caveats of simulation-based assessment are better understood and slowly overcome (eg, psychometric properties and technological limitations), 53 simulation will likely play an important role in high-stakes assessment in critical care. Finally, SBME has been shown to be useful for mass casualty training 54 and may represent a useful tool for health care professional training in response to specific threats, such as the recent Ebola epidemic. 55 There are also studies in the domains of neonatal resuscitation, 56,57 teamwork during cardiac arrests, 58 airway management, 59 and Advanced Trauma Life Support (ATLS) skills 60 that have failed to demonstrate a clear benefit of SBME. Most of these studies have compared SBME with other types of educational interventions such as video training, case-based or problem-based discussions, and traditional teaching. Limitations in the methodological rigor of many studies supporting the role of SBME, as well as inconsistencies regarding its efficacy 111,112 Family Conference OSCE for professionalism and communication skills 113 Checklist for professionalism during ethical dilemma 114 Piquette & LeBlanc when compared with other learning strategies, call for thoughtful reflection as to when simulation should, or should not, be used for critical care instruction. In this regard, another type of literature on SBME, described in the following section, helps to further inform the judicious use of SBME in critical care medicine. HOW SHOULD SIMULATION-BASED MEDICAL EDUCATION BE USED? SBME is a time-consuming, potentially costly enterprise and must therefore be carefully planned to maximize its educational benefits and minimize its resource requirements. Thankfully, the body of literature dedicated to the understanding of the features characterizing effective SBME is slowly growing. 5 Box 3 summarizes the types of questions that critical care educators should consider when planning a simulation-based curricular activity. 4,5 Chiniara and colleagues 15 have also nicely summarized many issues related to the instructional design of SBME. The investigators discussed the learning objectives for which simulation may be the most appropriate medium, the choice of a simulation modality, the choice of an instructional method (self-directed or instructor-based method), and the simulation presentation (including feedback, fidelity, type of simulator, scenarios, and team composition). 15 Studies assessing the best ways to deliver SBME have been increasingly conducted in the critical care setting. For example, Springer and colleagues 61 concluded that multiple 30-min simulation sessions held over 3 consecutive days were more effective than one 90-min session to improve resident knowledge regarding recognition and management of septic shock. Ali and colleagues 62 reported that both students and instructors perceived the use of mechanical simulators and of simulated patients as equally satisfactory for ATLS training. Such studies can contribute to the improvement of SBME by increasing its efficacy or reducing its costs. However, there are still a large number of unanswered questions regarding the best ways to optimize the use of SBME. This is an area that requires further high-quality research. Individual technical or mixed skills Interdisciplinary management of septic shock 115 Integrated Procedural Performance Instrument for technical and communication skills 116 Scenario-specific performance checklist for pediatric scenarios, 117 undergraduates, 118 and acute care scenarios 119,120 IPETT for emergency technical and nontechnical skills 121 Comparison between written examination, simulation-based, and oral viva examinations for procedural skills 122 Abbreviations: ACLS, Advanced Cardiac Life Support; ATLS, Advanced Trauma Life Support; CRM, crisis resource management; ICU, intensive care unit; IPETT, Imperial Pediatric Emergency Training Toolkit; KL, Kirkpatrick's level; MET, medical emergency team; OSCE, Objective Structured Clinical Examination; SARS, severe acute respiratory syndrome; TEAM, Team Emergency Assessment Measure. Box 3 Important questions to consider when planning a simulation-based educational intervention How is this intervention integrated with other aspects of the curriculum? Based on the learning objectives, should this training be interdisciplinary and/or focused on team training? How will the facilitator/instructor be chosen and trained (clinical, educational, and interpersonal skills)? How should the right level of simulation fidelity be chosen (physical, psychological, and sociocultural)? How much and what type of practice will be required of the participants (repetitive, deliberate, massed, or distributed)? How will the feedback be provided (by whom, when, how often, how)? How will each practice session differ from the other (progressive difficulty, variety of cases, adapted to individual learners)? When will the intervention end (achievement of mastery learning)? How and where will the outcomes of this intervention be measured (in the simulation setting or in real clinical environments)? How can it be ensured that the knowledge and skills acquired will be maintained? WHAT ARE THE CHALLENGES RELATED TO SIMULATION-BASED MEDICAL EDUCATION? As described earlier, the rising popularity of SBME has emerged in a specific context where traditional clinical-based training is increasingly challenging because of complex and interdependent societal and organizational changes. Based on the current evidence, the use of SBME to prevent medical errors and adverse events seems totally legitimate. However, the authors foresee important challenges that critical care educators should carefully consider before engaging in SBME activities. First, the authors feel the need to address the aspects of health care professional training that SBME will not address. Clinical training is challenging in part because of the lack of availability of dedicated clinical teachers (who struggle to fulfill other professional responsibilities) and motivated trainees (overwhelming workload, working hours limitation, etc.). Health care resources are globally limited. The lack of time and scarcity of human and financial resources identified as barriers for clinical learning also apply to SBME. Clinically competent, properly trained simulation instructors are a rarity in most institutions. Training faculty to fulfill these responsibilities is time and resource consuming. Furthermore, the authors do not share the enthusiasm of others regarding the potential to add hours of SBME to the working hours of medical trainees. The educational value of any training completed beyond 80 hours of clinical work is questionable. Although the mistakes committed in a simulated environment will not harm any patient, tired trainees are unlikely to efficiently learn and to positively process their simulation experience. Furthermore, the time and energy invested in SBME necessarily redirect part of the energy and resources from clinical-based training toward SBME. Such unilateral shift could potentially represent additional impediments to the improvement of clinical-based learning, still recognized, even among the strongest proponents of SBME, as a core component of health care education. The authors believe that clinical-based learning and SBME can inform and complement each other in many aspects of Box 4 Development and curricular integration of SBME Identification of an educational problem Which educational need is not adequately addressed by the current curriculum? How is this problem currently addressed? Could SBME help address this problem? Targeted need assessment Which learners should be targeted for this program/activity? What are the learners' specific needs? Is a simulated learning environment appropriate to address these needs? Goals and objectives What are the general goals of this program/activity? What are the measurable objectives that will be achieved? Educational strategies Which simulation modality is the most appropriate to achieve these goals? Which instructional method will be the most helpful? How should the simulation activity/program be presented (timing, duration, feedback, etc.)? Implementation Which kind of support/resources will be required? Which barriers to implementation can be expected? Evaluation and feedback How will feedback be obtained from individual learners? How will this activity be assessed at the program level? Piquette & LeBlanc training. In their opinion, recent calls for better curricular integration of SBME are an encouraging step in the right direction. SBME is not a panacea that will fix all the medical education problems, and clinical-based training should continue to be a high priority for medical educators and researchers. The second challenge faced by SBME is the significant gap between the theoretic understanding of how SBME should be delivered and the way it is currently delivered in most institutions. With the exception of a few programs led by groups of committed and trained educators, SBME is frequently delivered in an ad hoc and unsystematic manner, separate from the broader curriculum. Box 4 suggests an approach to SBME based on general principles of curriculum development. 63 Far from wanting to blame educators, their intention is rather to highlight how difficult it can be for an educational intervention to present most of the features of effective SBME. In an attempt to be pragmatic about the implementation of SBME, educators must often select one of 2 features on which to focus their time and energy. The real benefits of SBME as currently applied may therefore be less than the ones presented in the literature. SUMMARY SBME has come a long way since the introduction of the first simulator more than 30 years ago. The authors have many reasons to be optimistic about SBME: the role of SBME is expanding (identification of gaps in clinical training and practice; undergraduate, postgraduate, and continuing education; formative and high-stakes assessment), the quality of evidence to support its use is increasing, and the strategies to implement SBME effectively and efficiently are better understood. There seems to be a consensus in the literature that SBME has an important role to play in the improvement of the safety of the care delivered in health care institutions; patient outcomes can be affected by our educational choices. These conclusions likely apply to the critical care environment in which patient outcomes critically depend on timely, complex, and highly coordinated care. However, significant research is still needed to further explore the advantages and limitations of SBME for specific clinical activities completed in particular clinical contexts. Such efforts should be coordinated with larger educational initiatives aimed at providing the best and most comprehensive educational experience for the critical care trainees.	2018-04-03T00:32:24.703Z	2015-06-26T00:00:00.000	{ "year": 2015, "sha1": "a0771fe53c58180f74d7e0f50ff88af251876062", "oa_license": null, "oa_url": "https://doi.org/10.1016/j.ccm.2015.05.003", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "1a944b3869bbceb713b2a9d9f8746c50189781f5", "s2fieldsofstudy": [ "Education", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
266512242	pes2o/s2orc	v3-fos-license	Epileptic brain network mechanisms and neuroimaging techniques for the brain network Epilepsy can be defined as a dysfunction of the brain network, and each type of epilepsy involves different brain-network changes that are implicated differently in the control and propagation of interictal or ictal discharges. Gaining more detailed information on brain network alterations can help us to further understand the mechanisms of epilepsy and pave the way for brain network-based precise therapeutic approaches in clinical practice. An increasing number of advanced neuroimaging techniques and electrophysiological techniques such as diffusion tensor imaging-based fiber tractography, diffusion kurtosis imaging-based fiber tractography, fiber ball imaging-based tractography, electroencephalography, functional magnetic resonance imaging, magnetoencephalography, positron emission tomography, molecular imaging, and functional ultrasound imaging have been extensively used to delineate epileptic networks. In this review, we summarize the relevant neuroimaging and neuroelectrophysiological techniques for assessing structural and functional brain networks in patients with epilepsy, and extensively analyze the imaging mechanisms, advantages, limitations, and clinical application ranges of each technique. A greater focus on emerging advanced technologies, new data analysis software, a combination of multiple techniques, and the construction of personalized virtual epilepsy models can provide a theoretical basis to better understand the brain network mechanisms of epilepsy and make surgical decisions. Introduction Epilepsy is defined as a serious chronic brain dysfunction characterized by transient, recurrent, spontaneous epileptic seizures, and it affects nearly 1% of the global population (Fisher et al., 2017;GBD 2016Epilepsy Collaborators, 2019).Recurrent seizures can lead to local or global brain network dysfunction, resulting in drug resistance, mental disorders, cognitive impairment, and an increased risk of sudden unexplained death in epilepsy (Wang and Chen, 2019;Bensken et al., 2021;Kløvgaard et al., 2021;Martinez and Peplow, 2023). Brain network dysfunction is an important mechanism for epileptogenesis because this process affects both specific brain areas and the interaction between different brain regions (Ciolac et al., 2023;Goldberg and Coulter, 2013;Kalra, 2023).Numerous studies have found brain network changes in the process of epileptogenesis in patients and animal models, such as the corticothalamic circuit in patients with absence epilepsy and discrete subicular circuits and depolarized γ-aminobutyric acid-ergic (GABAergic) subicular microcircuits in a kainic acid-induced epilepsy animal model (Wang et al., 2017;Fei et al., 2022).Moreover, obtaining more knowledge about these network changes could help to detect new hubs or epileptogenic zones for therapeutic strategies in neurosurgical decision making.Some researchers have even proposed "connectome-and hub-based epileptic network neurosurgery" (Wei et al., 2022).Furthermore, it has been Review recently hypothesized that alterations in the brain network are associated with the severity and clinical outcomes of epilepsy and may be an excellent biomarker in the diagnosis of epilepsy (Park et al., 2018;Tavakol et al., 2019;Dai et al., 2021). For these reasons, the analysis of structural or functional brain network properties is thought to be an alternative way to investigate the pathophysiology of epilepsy and may serve as a phenotypic marker for epilepsy.Thus, brain network analysis is gaining increasing attention, and connectivity analysis is being increasingly used to explore the integrity of brain networks.Many studies have explored the brain network or circuits in animal models by a number of techniques such as chemogenetics, optogenetics, viral tracing, and two-photon microscopy (Desloovere et al., 2019;Wang et al., 2019;Bao et al., 2022;Adhikari et al., 2023); however, these techniques have not been approved for evaluating brain network in the human brain.Many neuroimaging and neurophysiological techniques have successively been applied to clinical practice to evaluate the brain network in the human brain.Thus far, an increasing number of advanced techniques such as diffusion tensor imaging (DTI)-based fiber tractography, diffusion kurtosis imaging (DKI)-based fiber tractography, fiber ball imaging (FBI)-based fiber tractography, electroencephalography (EEG), functional magnetic resonance imaging (fMRI), magnetoencephalography (MEG), positron emission tomography (PET), PET/MRI-based molecular imaging, and functional ultrasound imaging (fUSI) have been used to evaluate brain networks in patients and healthy individuals.In this paper, we mainly introduce these techniques used to explore brain networks in patients with epilepsy (PWE), elucidate the advantages and limitations of each technique, and propose the future direction of evaluating the epilepsy network. Search Strategy We searched the PubMed, Embase, and MEDLINE databases for articles published from 2000 to 2023, and the searches included Medical Subject Headings (MeSH) terms and free text as follows with the following term: "network" OR "circuit" OR "connectivity" OR "functional network" OR "structural network" and combination of "epilepsy" OR "epileptogenesis" OR "seizure."The literature search and papers were evaluated by three reviewers.Only papers published in English and those with human subjects were included. Structural Brain Network Evaluation in Patients with Epilepsy It has long been known that epileptogenesis involves brain dysfunction at the molecular, cellular, and network levels (Li et al., 2023).Generally speaking, the brain network comprises both a structural and a functional network, and the structural network is the direct anatomic connection between separate nuclei or separate brain areas, and action potential can spread through this connection (Bourel-Ponchel et al., 2019).Axonal and dendritic sprouting, reorganization of synapses, disrupted neurogenesis and abnormal gliosis are the basis of the structural epileptic network (Althaus et al., 2016;Loewen et al., 2016;Aloi et al., 2022).In the 1940s, Edward Mills Purcell first discovered the phenomenon of the magnetic resonance of atoms (Keiji, 2019) DTI-based fiber tractography DTI-based fiber tractography is considered a standard neuroimaging method that enables the evaluation of fibertract trajectories within the brain white matter and other fibrous tissue based on water diffusion (Mori et al., 1999;Bopp et al., 2021).Diffusion tensor MRI (DT-MRI) was first conducted in the human body by Le Bihan et al. (1986).DTI assumes that water diffusion follows Gaussian diffusion. On the basis of single exponential diffusion imaging, it uses the anisotropic diffusion tensor to reconstruct the tissue microstructure.DT-MRI is an excellent approach to detect microstructural alterations in white matter and provide a model of neural connections in the brain, and has been extensively used to explore the projection of white matter fibers between multiple brain regions or nuclei and has the potential to be a gold standard for detecting disorders in white matter fiber tracts. Review To date, DT-MRI has been widely applied in the investigation of brain networks in traumatic brain injury (Nakuci et al., 2022), cognitive disorders (Aben et al., 2021), brain development (Corroenne et al., 2022;Kar et al., 2022), and brain aging (Taha et al., 2022).Robinson et al. (2010) collected data from DT-MRI and analyzed the brain structural connectivity between different age groups by machine learning, their results showed that these different age groups can be classified successfully with 87.46% accuracy and that the characteristics extracted from the discriminant analysis are in accordance with the current consensus on the neurological impact of aging. Another study also assessed structural brain connectivity by DTI-based fiber tractography in subjects at high resolution and found that the organization of the structural network includes strong interrelations among regions (Robinson et al., 2010).A large number of researchers have evaluated structural brain networks in epilepsy by DTI-based fiber tractography, and their results have shown extensive and bilateral alterations in white matter fiber, including increased diffusivity, decreased fractional anisotropy, decreased global/local efficiency, and structural connectivity (Bonilha et al., 2012;Kamiya et al., 2016;Christiaen et al., 2022).Bernhard et al. ( 2019) analyzed high-resolution anatomical and DT-MRI data from 44 patients with temporal lobe epilepsy (TLE) (25 patients with a histopathologic diagnosis of hippocampal sclerosis and 19 patients with isolated gliosis) and 25 healthy individuals, and they found that global and local efficiency were significantly decreased in TLE patients, indicating a decreased connectivity in temporo-limbic networks that are strongly connected to the hippocampus.Another study also evaluated the anatomical network in mesial TLE (mTLE) patients with hippocampal sclerosis and revealed different connectivity patterns in the cortico-limbic network and cortico-cerebellar networks between the right and left mTLE, suggesting that the difference in the cortico-limbic/cortico-cerebellar networks may be an excellent marker to guide personalized diagnosis of right and left mTLE (Fang et al., 2015). The metrics measured by DTI are not sensitive to changes in microstructure or pathology, which is the main limitation (Jones et al., 2013).The signals of DTI fiber tractography emanate from both intra-axonal and extra-axonal water diffusion; thus, the accuracy and sensitivity of DTI fiber tractography are hampered by many factors such as the complex fiber projection of white matter, fiber crossings, abnormal neuronal regeneration and neuronal loss, diverse periaxonal micromilieus, and tissue edema (Savadjiev et al., 2008;Yeh et al., 2013).Moreover, the smaller fiber bundles are poorly displayed or cannot be displayed by DTI-based fiber tractography. DKI-based fiber tractography The basis of DTI is the Gaussian distribution of water in the brain tissue, but many factors affect water diffusion and result in a non-Gaussian distribution in the brain.As a mathematical extension of DTI, the diffusion kurtosis model was first introduced by Jenson et al. (2005).DKI is a method described as a dimensionless approach that quantifies water diffusion which follows non-Gaussian distribution in a voxel (Arab et al., 2018).The metrics of DKI include diffusion tensor and kurtosis metrics, and detailed descriptions of each parameter and their interpretations are provided in Table 1. Given its advantages, DKI can detect both anisotropic and isotropic water diffusion; moreover, it sensitively detects microstructural abnormalities in both white matter and gray matter.There have been numerous studies showing that DKI can detect the microstructure alterations of white matter in patients with active neurodegenerative diseases (Blockx et al., 2012;Falangola et al., 2013;Arab et al., 2018;Sun et al., 2021).Limited evidence has also confirmed that DKI is a potential neuroimaging tool to explore the brain network and localize the epileptogenic zone in PWE.Del Gaizo et al. (2017) performed a study to analyze data from diffusion MRI (including DTI and DKI) in TLE patients by machine learning and found that the DKI metric of MK (with an accuracy of 0.82) achieved higher accuracy than the DTI metrics of fractional anisotropy and MD (with accuracy values of 0.68 and 0.51, respectively) in every iteration, indicating that DKI is more sensitive than DTI in measuring microstructure abnormalities in patients with TLE.Another study also identified epilepsy using DKI-based fiber tractography on a convolutional epileptic brain network, and their results showed that DKI-based fiber tractography can act as a vital biomarker for the diagnosis of epilepsy in clinical practice (Huang et al., 2020).Based on these advantages of DKI in clinical practice, we propose that DKI-based fiber tractography is a potential method to evaluate epilepsy networks in both white and gray matter. FBI-based fiber tractography To overcome these limitations of DTI-and DKI-based fiber tractography, a high-b-value diffusion imaging technique that combines advanced MRI equipment, sequence engineering, and data processing modes was also used to achieve higher accuracy in the measurement of fiber tractography in white matter (Jensen et al., 2016).This imaging technique can be specifically used to reflect intra-axonal water diffusion, without contamination from the mobile water in the extraaxonal microenvironment.FBI-and fiber ball white matter (FBWM)-based tractography are examples of high-bvalue diffusion imaging techniques, and the details of the parameters and interpretation of FBI and FBWM are provided in Table 1.Although few studies have demonstrated that FBI-or FBWM-based tractography are clinically feasible alternative techniques to evaluate structural brain networks in PWE (Bryant et al., 2021;Gleichgerrcht et al., 2022), these studies recruited patients with various epilepsy syndromes in small sample sizes.Further studies with more homogeneous epilepsy cohorts in larger sample sizes will be critical to detect brain networks with FBI-or FBWM-based fiber tractography. There are several limitations to FBI-or FBWM-based tractography in clinical practice.The major limitation is that these techniques require high-b-value (over 4000 s/mm 2 ) diffusion MRI to absolutely suppress the extra-axonal water diffusion signal, which results in an increased signal-to-noise ratio compared with methods using lower b-values (e.g., DTI-and DKI-based tractography).Second, FBI-based fiber tractography requires more than 60 diffusion directions, which is significantly more than required for DTI (6 directions)-or DKI-based tractography (15 directions); thus, the minimal scan times are obviously longer in FBI-based fiber tractography (Moss et al., 2019).Third, FBI-based tractography typically necessitates the use of somewhat larger voxels than either DTI-or DKI-based tractography and must be performed on 3-tesla (3 T) MRI scanners. Functional Brain Network Evaluation in Patients with Epilepsy The brain functional network can be thought of as the temporal correlation or statistical dependence of the neural activities of spatially separated neurons; it is an intuitive description of the dynamic interaction between different neurons, nuclei, or brain regions in the brain structure network.Stephan et al. (2000) evaluated the functional brain network between spatial regions of the primate cerebral cortex using an invasive neural tracing method in monkeys; their findings were consistent with corresponding analyses of structural data of the mammalian cerebral cortex and provided initial empirical proof of the small-scale brain network structure in the primate.This study was the prelude to the utilization of complex network theory in examining functional brain networks.Previously, EEG, fMRI, MEG, and PET were widely used to generate functional networks in epilepsy, and it was thought that PET/MRI-based molecular imaging and FUSI also have the potential to detect brain networks in the future.The characteristics of each advanced technique for evaluating functional networks in epilepsy are shown in Table 2. Review in 2020, another study provided evidence that the STN receives firing from the motor cortex and that the STN-cortical circuit is engaged as a propagation network of focal motoronset seizures by SEEG recording (Ren et al., 2020).SEEG can detect neuronal firing and the activity of neural circuits deep in the brain and can be used to detect discharges in larger brain areas, even bilaterally.However, this method is invasive, and implanting intracranial electrodes carries the risks of bleeding, infection, and neurological deficits. fMRI Functional MRI is considered a noninvasive method for detecting brain metabolic activity and functional networks in the brain for many years.fMRI measures the synchronous fluctuations of blood oxygen level-dependent (BOLD) signals to indirectly reflect neural activity in the brain, and oxygen consumption was found to be increased when the neurons were active, thus accompanied by an increased amount of deoxyhemoglobin.This is followed by vasodilation and increased cerebral blood flow, accompanied by a large amount of oxygenated hemoglobin and resulting in a decrease in the local concentration of deoxygenated hemoglobin.Oxyhemoglobin is a diamagnetic substance, and deoxyhemoglobin is a paramagnetic substance (Cheng et al., 2019;Biondetti et al., 2023).These two substances have different responses to magnetic fields, a decrease in local deoxyhemoglobin concentration manifests as an increase in the BOLD signal on T2-weighted images (Cerdonio et al., 1981)., 2007).Task-based fMRI was used to investigate the different functions in different brain regions and can be performed when subjects execute specifically designed cognitive tasks; this approach can be used to evaluate expressive language, motor, emotion, memory, and sensory-motor processing.To evaluate brain functional connectivity in different states, multiple state fMRI has been extensively used to detect functional brain connectivity in PWE by measuring the segregation and integration properties of functional brain networks (Integration and segregation are the manifestation of the brain network, functional integration considers the brain as a large interacting network, and it integrates different networks in the brain to work together to perform specific functions, while separation means connectivity within the brain's networks). As fMRI reliably identifies various functional connectivity patterns in the brain, it is invaluable for the noninvasive assessment of pathological changes in functional networks in varied types of epilepsy and syndromes.Narasimhan et al. (2022) evaluated the connections by resting-state networks in mTLE, and their results showed that right mTLE patients have a lower level of nondirected connectivity between the right hippocampus and default-mode network than left mTLE patients and healthy controls, indicating that assessing the fMRI connectivity between mesial temporal structures and the default-mode network can likely assist in determining the lateralization of mTLE and provide guidance for surgical decision-making.Researchers investigated the subcorticalcortical circuit in idiopathic generalized epilepsy by using resting-state fMRI, and their results revealed that the thalamus and precuneus were key nodes with abnormal functional connectivity in the subcortical-cortical circuit, and the increased connectivity between the precuneus and adjacent regions had a causal effect on the widespread decrease in connectivity in the thalamocortical circuit (Qin et al., 2022).In addition, fMRI has also been used to evaluate brain networks in other epilepsy syndromes, including juvenile myoclonic epilepsy (Yang et al., 2022), rolandic epilepsy (Kwon et al., 2022), benign epilepsy with centrotemporal spikes (Narasimhan et al., 2022), frontal lobe epilepsy (Dong et al., 2016;Klugah-Brown et al., 2019), and childhood absence epilepsy (Tenney et al., 2018).Moreover, fMRI has also helped understand the cognitive and language networks affected by epilepsy or the ways these techniques are being used for the prediction of treatment outcomes from surgery, electrical stimulation, or medication (Table 1). fMRI has three main advantages in evaluating functional networks in epilepsy.1) fMRI can be performed without injection of a signal-boosting agent, thus reducing the rate of allergic reaction; 2) fMRI can detect neural activity deep in the brain; and 3) fMRI is noninvasive and safer than PET scans.However, many researchers readily admit that fMRI also has several limitations: 1) fMRI merely detects neuronal activity indirectly; 2) detailed information on neuronal activity is not clear, such as the number of activated neurons or whether the activation of neurons in one area of the brain facilitates or inhibits the firing of neurons in neighboring regions; and 3) extraction of the signal from the regular fluctuations in blood flow can be challenging, leading to an inevitable misunderstanding and misuse of statistical methods. MEG Magnetoencephalography is a noninvasive tool used to detect brain electric activity without signal deterioration from the skull and scalp.In 1819, Hans Christian Ørsted first detected a magnetic field around the electric conductor, and then in 1972, Choen measured the brain magnetic field by superconducting quantum interference device-based sensor devices for the first time (Cohen, 1972), and this highlighted the clinical use of MEG.For its ability to measure the magnetic field generated by electrical currents in neurons, several researchers started paying attention to the use of MEG in brain network research, such as for neurodegenerative disease (Stam, 2010), traumatic brain injury (Antonakakis et al., 2017), and stroke (Kielar et al., 2016).In addition, MEG has been extensively utilized for presurgical localization in drug-refractory epilepsy (Juárez- Martinez et al., 2018), and it is also an accessible way to evaluate brain networks in epilepsy (Sun et al., 2023). MEG has several advantages. 1) MEG can record the whole brain activity with groups of sensors positioned in over 100 locations, and the combination of MEG with superimposition of MRI (this image is named magnetic source imaging, MSI) data can significantly improve the localization of interictal Review discharges.2) MEG possesses high temporal and spatial resolution (spatial resolution can reach 1 mm).3) MEG has ideal sensitivity in detecting epileptiform firing parallel to the skull, allowing the identification of epileptic discharge deep within a sulcus, which compensates for the shortcomings of an EEG.4) The current source detected by MEG comes from the intracellular synaptic current, and the electromagnetic field is not affected by the skull; in addition, the underlying muscle or eye movement during MEG monitoring also does not confound MEG (Hamandi et al., 2016). However, MEG also has several limitations in clinical practice.MEG is typically prone to detecting interictal discharge rather than ictal discharge.In addition, some researchers argue that MEG-based source localization is difficult in detecting neuronal firming within deeper regions (Shigeto et al., 2002;Leijten et al., 2003), and believe that the rapid decay of the magnetic field is likely to be the main reason for this limited sensitivity to detect discharges originating deep in the brain (Shigeto et al., 2002).In addition, MEG is not readily accessible to patients in the majority of epileptic centers due to the high cost, which may also be a major obstacle to its use in clinical practice. PET Fluorodeoxyglucose-PET (FDG-PET) has been widely used for over 40 years to detect the metabolism of the human brain, and this neuroimaging method has also been applied to evaluate metabolic brain networks in drug-resistant TLE. 18 F-FDG-PET is proven to be highly sensitive in the detection of focal cortical dysplasia type 2 (especially when negative on MRI) and for presurgical evaluation and assessment of postsurgical outcomes in drug-resistant epilepsy.A study retrospectively recruited 64 drug-resistant TLE patients and divided them into two groups according to outcome (32 patients with Engel class Ia and 32 without).The connectivity between the contralateral fusiform gyrus and contralateral lingual gyrus was significantly increased in patients with non-Engel class Ia, while the connectivity between the bilateral frontal, temporal, and parietal cortices was decreased in patients with Engel class Ia.These differences in the brain metabolic network can also predict surgical outcomes in TLE (Ren et al., 2021).Another study evaluated the global and local brain metabolic networks in TLE with hippocampal sclerosis and found widespread regional differences in local metabolic networks according to surgical outcomes (Cho et al., 2022). FDG-PET has several advantages in evaluating brain function networks: 1) FDG-PET reflects functional brain networks directly by evaluating neuronal consumption (Hyder et al., 2013); 2) the data obtained from FDG-PET have lower signalto-noise ratios, variance concentrations, and out-of-sample replication (Yakushev et al., 2017); and 3) FDG-PET scans evaluating the functional brain network are unaffected by neurovascular coupling inherent in BOLD MRI (Yakushev et al., 2017).The main drawback of FDG-PET is that it requires the injection of a signal-boosting radioactive compound. In addition, neuronal loss caused by epilepsy, diaschisis, decreased density of synapses, and interictal inhibitory processes were found to account for the hypometabolic region, but the hypometabolic region observed by PET was much larger than the epileptogenic foci. PET/MRI-based molecular imaging The concept of molecular imaging was proposed by Ralph Weissleder for the first time (Rudin and Weissleder, 2003).Molecular imaging mainly applies imaging methods to conduct qualitative and quantitative research on organisms at the cellular and molecular levels.PET or MRI-based molecular imaging is another potential technique to evaluate functional brain networks, and flumazenil PET or MRI and alpha-[ 11 C]-methyl-L-tryptophan PET are considered pioneering techniques in epilepsy characterization. Flumazenil is a radioactive ligand of the benzodiazepine receptor sites and specifically binds to the α subunit of the GABA receptor complex.Flumazenil PET has been shown to have the ability to detect the density of γ-aminobutyric acid receptors in the brain cortex, and some studies have also revealed that flumazenil PET is a useful way to identify epileptic foci with higher accuracy (Hammers et al., 2008;Komoto et al., 2015;Ergün et al., 2016).Nørgaard et al. (2021) presented a detailed atlas of the benzodiazepine binding site of postsynaptic GABAA receptors in healthy human brains by flumazenil PET.Similarly, α-[ 11 C]-methyl-L-tryptophan PET has also been used in preclinical practice (Krasikova et al., 2020).In addition, a large number of studies have focused on the preclinical and clinical investigation of radioactive ligands binding acetylcholine, serotonin (5-hydroxytryptamine), opioid-based PET or MRI for epilepsy (Billard et al., 2014;Komoto et al., 2015;Kim et al., 2020). fUSI Doppler ultrasound has been widely used to assess cerebral flow in clinical practice for several decades and is thought to be a potential method to directly reflect brain metabolism, but it cannot be applied to evaluate cerebral blood velocity in smaller vessels or cerebral metabolism maps owing to its limited temporal and spatial resolution.To overcome these limitations, Macé et al. (2011) introduced a Doppler ultrasound-based imaging technique known as fUSI.fUSI is an excellent technique to detect cerebral blood flow in each blood vessel in the brain and can be applied to image propagation of discharge by detecting epileptic seizureinduced alterations in blood flow (Macé et al., 2011), and an increasing number of studies have applied fUSI to explore the physiological and disease-related brain networks or connections in animal models.The excellent spatiotemporal resolution (< 100 µm in spatial resolution and < 10 Hz in temporal resolution) of fUSI is the main advantage and is thought to be an alternative method to image functional brain network behavioral tasks and transient, episodic physiological or disease-related situations in a dynamic manner.The attenuation of ultrasound by the skull limits the application of fUSI in clinical practice.However, in research applications, fUSI has been used to image functional intrinsic brain network organization in primates and rodents (Dizeux et al., 2019;Sans-Dublanc et al., 2021). Review Given its excellent resolution and sensitivity, some researchers have also tried to assess human brain networks by fUSI.Imbault et al. (2017) first applied fUSI to image brain activity in clinical practice; they assessed functional activity deep in the cortex and differentiated regions according to the correlation of task-evoked activity (e.g., in response to slight motion of a finger) between neighboring cortical areas in awake or anesthetized patients.Later, another study enrolled 10 patients with tumors in the frontal and temporal lobes and revealed that fUSI can not only capture a wide range of functional responses but also precisely mark the regions of the tumor (Soloukey et al., 2019).Both these studies applied fUSI intraoperatively when the skull was absent.To date, researchers have also identified functional brain networks in human neonates with seizures or cortical malformations.Demene et al. (2017) first detected brain activity by fUSI in two different sleep states and then revealed dynamic brain circuit alterations in two human newborns with seizures related to congenital cortical malformation.Another study also revealed interhemispheric connectivity and disconnection of thalamocortical networks in premature newborns (Baranger et al., 2021).These results highlighted the clinical utilization of fUSI to image brain networks in PWE.The main limitations of fUSI are potential head or body motion artifacts and the attenuation of the signal by the skull; hence, the potential use of fUSI in adult humans needs further study. Future Directions Brain network research for epilepsy is a promising direction to understand the mechanism of epilepsy and develop precise drug therapy.At present, an increasing number of studies have revealed local or whole brain network alteration, but these results have merely been used for preoperative evaluation in PWE.Thus, we believe that the network research of epilepsy still needs further and more in-depth research.Therefore, we thought that paying more attention to new advanced techniques, new software for data analysis, combinations of multiple techniques, and the construction of personalized virtual epileptic models could help us to further evaluate brain networks in the future (Figure 2).Paying more attention to new advanced techniques, new software for data analysis, combinations of multiple techniques, and the construction of personalized virtual epileptic models could help us to further evaluate brain networks in the future.Created with WPS (V12.1).3DCNN: Three-dimensional convolutional neural network; EEG: electroencephalography; MEG: magnetoencephalography; MRI: magnetic resonance imaging; PET: positron emission tomography. New advanced techniques Each technique has specific strengths and limitations (the characteristics of each neuroimaging technique for evaluating brain networks in epilepsy are shown in Table 3, and an increasing number of research studies are dedicated to improving these advanced techniques to refine their superiority and remedy these flaws.Some new advanced techniques have been used in preclinical or clinical practice to evaluate brain activity or the localization of epileptic foci, and these techniques have considerable potential for evaluating epileptic networks.For example, optically pumped magnetometer (OPM)-based MEG, 7.0 T ultrahigh-field MRI, and functional near-infrared spectroscopy (FNIRS) may have future clinical application. MEG has several limitations that make it inaccessible to patients in specific situations, including that it cannot be used to assess brain networks during ictal events.Recently, a new OPM-based moving MEG was used in preclinical practice (Mellor et al., 2022).Unlike a traditional superconducting quantum interference device-based MEG, the advanced OPM-based MEG can be carried out at room temperature without using a large Dewar flask of liquid helium for cooling (Sander et al., 2012;Boto et al., 2018).However, the helmet is lighter (< 1 kg) and is customized so that the sensors are directly adjacent to the scalp surface.This new OPM-based MEG also has an excellent signal-to-noise ratio, with improved sensitivity and lower operating costs (Sander et al., 2012;Boto et al., 2018).The advanced OPM-based MEG system opens new possibilities for some special individuals (such as older patients and infants).Thus, these advantages of OPM-based MEG make the technique particularly attractive for localizing sources of brain activity or evaluating brain networks in PWE. Another advanced technique is high-and ultrahigh-field MRI.MRI with a field of 3 T has rapidly gained popularity in clinical practice for more than 10 years, and 3 T MRI-based DTI/ DKI/FBI fiber tractography is irreplaceable in the research of epileptic networks.In this section, we mainly address the preclinical use of ultrahigh-field MRI.In general, ultrahighfield MRI is defined as a magnetic field over 7 T.The utilization of 7 T MRI in neuroimaging is becoming increasingly popular following its approval for clinical application in Europe and the United States.A 7 T MRI can provide excellent contrast and has higher resolution than lower magnetic field MRI owing to its better signal-to-noise ratio and contrast-to-noise ratio. Based on this advantage, Parekh et al. (2015) investigated the ultrastructure of the hippocampus using the balanced steady-state free precession sequence on 7.0 T MRI.Their results described the ultrastructure of the hippocampus and identified the endfolial pathway in vivo.In addition, molecular imaging can be performed in 7 T MRI for its capability to measure neurochemical levels with higher spectral resolution. A study detected the concentrations of GABA using 7.0 T MRI-based molecular imaging, and their results found that the concentrations of GABA and glutamate and the average strength of the GABA network were increased in PWE, and the increased GABA network may reflect the disrupted balance in excitatory and inhibitory neurotransmitters (van Veenendaal et al., 2018).In addition to 7 T MRI, more advanced 9.4 T Review human MRI systems for scientific research have been built or are under construction worldwide, and this ultrahigh-field MRI is a candidate neuroimaging technique to investigate brain networks in PWE.Compared with 3 T MRI, the main limitations of ultrahigh-field MRI are more obvious artifacts, increased magnetic field inhomogeneity, increased magnetic susceptibility effects and potential safety risks (mainly including the danger caused by magnetic substances being placed in the scanning cavity, and adverse reactions of tissue heating). Functional near-infrared spectroscopy is another advanced tool to detect functional brain activity with a similar mechanism to fMRI.The difference is that fMRI is not suitable for measuring brain functional activity in children (especially infants) or in advanced-age individuals, and it also cannot be used to assess functional brain activity during ictal events or natural situations (such as in daily life and work).However, FNIRS is considered a promising technique that can meet the above requirements.Owing to its excellent temporal resolution, portability, convenient operating procedure, painless experience, applicability to a wide range of individuals, and long-term uninterrupted monitoring at any time and place, it has been widely used to explore functional networks in psychologic disease (Wang et al., 2020;Gong et al., 2022).Limited studies have revealed the value of FNIRS and FNIRS-EEG in the detection of interictal epileptiform discharges et al., 2019;Tung et al., 2021).In terms of its limited spatial resolution, FNIRS was also combined with EEG and fMRI to evaluate brain network, and it was reported that FNIRS may be a potential method to evaluate brain network in the future (Arun et al., 2018;Sirpal et al., 2022). New software for data analysis Methods for analyzing the raw data obtained from these neuroimaging techniques need to be further identified. Because of the large amount of these raw data, the means to analyze and obtain proper conclusions should be addressed.Machine learning is a new method used to analyze raw data.Machine learning is a type of artificial intelligence that utilizes data exposure instead of explicit instructions to create models.It has been widely used in many aspects of epilepsy evaluation, such as the prediction of surgical outcomes and the identification of epileptic foci (Luckett et al., 2022).A study analyzed resting-state fMRI data by machine learning to identify the epileptic foci in TLE patients, and the results showed that the hemisphere of epileptic foci can be detected with 90.6% accuracy by a machine learning-based threedimensional convolutional neural network, suggesting that deep machine learning is a useful way to identify the lateralization of epileptic foci in TLE patients (Luckett et al., 2022).Another study also demonstrated that a machine learning model suggested that individual variations in the contralateral hippocampal network based on PET/MRI could be used to predict seizure recurrence after TLE surgery (Wang et al., 2022).An increasing number of studies have proposed different computation methods to identify the epileptic foci and brain networks in different types of epilepsy (Pardoe et al., 2021;Ren et al., 2021;Zhang et al., 2021Zhang et al., , 2022;;Aparicio et al., 2022;Qin et al., 2022). Combination of multiple techniques Each neuroimaging technique has specific advantages and disadvantages.Some of them may have excellent temporal resolution, while others have excellent spatial resolution.To Review overcome these obstacles and refine their use, a combination of multiple neuroimaging techniques has been widely used in presurgical evaluation, and these are also ideal methods to detect epileptic foci and brain networks.These methods include simultaneous EEG/fMRI, EEG/MEG, PET/MRI, PET/ MEG, PET/MRI/MEG, and PET/MRI/EEG recordings. Simultaneous EEG/fMRI plays a vital role both in epilepsy research and the presurgical evaluation of epilepsy.Neuronal discharges result in increased regional metabolism and blood flow, which can be detected by fMRI.Simultaneous EEG/ fMRI recording can be a reliable method to noninvasively investigate the brain region involved at the time of epileptic discharges on scalp EEG (Laufs and Duncan, 2007;Gotman and Pittau, 2011;An et al., 2013).Kay et al. (2013) examined resting-state network functional connectivity in patients with drug-resistant idiopathic generalized epilepsy by simultaneous EEG/fMRI, and their results showed that default-mode network connectivity was significantly reduced in patients with drug-resistant idiopathic generalized epilepsy. In addition, simultaneous EEG/MEG (Plummer et al., 2019) or even SEEG/MEG recording (Plummer et al., 2019) may be another method, and combined EEG/MEG would be an alternative method to combine the strengths of both approaches, with SEEG providing information on deep sources with a high signal-to-noise ratio and MEG providing wholebrain information (Vivekananda, 2020); however, this method is challenging to perform. PET/MRI is another advanced neuroimaging technique that acquires data from both PET and MRI scans together in one session, and it can be used to simultaneously acquire functional and structural images.It is critical for capturing dynamically changing brain activity and brain networks.PET/ MRI imaging combines the advantages of PET and MRI to provide molecular, morphological, and functional information in the lesion tissue.PET-MRI can significantly improve the accuracy of epileptogenic focus localization, thereby guiding the surgical resection of epileptic foci.PET-MRI can be used to detect potential epileptogenic foci not detected by PET or MRI alone.A recent study evaluated the association between surgical outcome and brain networks obtained from simultaneous PET/MRI in TLE patients, and their results showed that PET and fractional amplitude of low-frequency fluctuation obtained from simultaneous PET/MRI gained the maximum area under the receiver operating characteristic curve of 0.905 for evaluating seizure recurrence, which was higher than that achieved using PET (0.762) or fractional amplitude of low-frequency fluctuation alone (0.810) (Wang et al., 2022).An increasing number of studies have confirmed that hybrid PET/MRI can significantly improve the detection of epileptic foci in MRI-negative drug-resistant epilepsy (Poirier et al., 2021;Guo et al., 2022a) and nigro-putaminal functional connectivity in Parkinson's disease (Zang et al., 2022).PET/ MRI acquired data from PET and MRI scans together in one session, but the specificity of this approach is extremely limited; if combined with MEG or electroclinical data, this approach may be a useful strategy to compensate for this flaw.A study assessed the clinical value of PET/MRI, MEG, and PET/MRI/MEG among patients with localized TLE and found that 94.5% subjects showed definite structural abnormalities in accordance with surgical resection by PET/MRI/MEG, which was significantly higher than MRI, PET, and PET/MRI alone (46.6%, 67.1%, and 82.2%, respectively) (Guo et al., 2022b).These results demonstrated that PET/MRI/MEG may be a useful method to guide surgical decision making.Another study also assessed the localized value of PET in focal cortical dysplasia type-related drug-resistant epilepsy and found that positive localization increased to 83% after co-registration of PET/MRI and electroclinical data (44% with PET alone, 71% with PET and electroclinical data) (Desarnaud et al., 2018). Construction of personalized virtual epileptic models Epileptic seizure generation and propagation depend on structural and functional networks, and patients with different seizure types vary with respect to different networks or how they evolve across multiple cortico-subcortical pathways (Spencer et al., 2018;Jirsa et al., 2023).The personalized virtual epileptic brain model based on high-resolution imaging is another way to study epileptic networks (Guye et al., 2019;Makhalova et al., 2022).The virtual epileptic brain model has the following three advantages: (1) the personalized virtual epileptic brain model provides a noninvasive approach to evaluate optimal placement for SEEG electrodes; (2) the personalized virtual epileptic brain model can improve and verify the detection of the epileptogenic zone or hubs of the epileptic network following invasive SEEG; and (3) the personalized virtual epileptic brain model can systematically test multiple surgical strategies and predict the surgical prognosis. The data collected from high-resolution neuroimaging will significantly improve the construction of the brain network model and its specificity to PWE.These advanced neuroimaging data can be obtained in vivo (such as 7 T MRI) or ex vivo (postmortem connectivity data).More importantly, postmortem data can provide additional high-resolution information for the construction of epileptic networks.Thus, the combination of more advanced multimode neuroimaging with a new algorithm will significantly improve the understanding of the epileptic network. Limitation There are some limitations of this review should be addressed.First, the majority of studies cited in this review are conducted on human patients; thus, the techniques used in preclinical experiments that have the potential to evaluate brain network in patients will be ignored.Second, all the included articles were English publications, which may have resulted in less relevant international data.Third, the advantages of combination with multiple techniques and the networkbased precise therapeutic neurosurgery need to be further elucidated. Conclusion . Thus far, structural magnetic resonance imaging (MRI; especially high-resolution MRI) is considered a vital neuroimaging technique to detect structural abnormities in PWE.DTI-based fiber tractography, DKI-based fiber tractography, and FBI-based fiber tractography are noninvasive neuroimaging techniques that enable the evaluation of fibertract trajectories in the brain.In this section, we address the use of these structural MRI-based tractography techniques in brain network research.The clinical use and mechanisms of each structural MRI-based fiber tractography are shown in Figure 1. Figure 1 ｜ Figure 1 ｜ The clinical use and mechanisms of each structural MRI-based fiber tractography imaging tool.DTI-based fiber tractography is used to detect water diffusion that follows Gaussian distribution in both intra-and extra-axonal regions of the white matter; the DKIbased fiber tractography is used to detect water diffusion that follows non-Gaussian distribution in both intra-and extra-axonal regions of the gray and white matter; and the FBI-based fiber tractography is used to detect water diffusion that follows non-Gaussian distribution in the intra-axonal region of the white matter.Created with WPS (V12.1).DKI: Diffusion kurtosis imaging; DTI: diffusion tensor imaging; FBI: fiber ball imaging-based; MRI: magnetic resonance imaging. Figure 2 ｜ Figure 2 ｜ Future directions of advanced techniques for evaluating brain networks in PWE.Paying more attention to new advanced techniques, new software for data analysis, combinations of multiple techniques, and the construction of personalized virtual epileptic models could help us to further evaluate brain networks in the future.Created with WPS (V12.1).3DCNN: Three-dimensional convolutional neural network; EEG: electroencephalography; MEG: magnetoencephalography; MRI: magnetic resonance imaging; PET: positron emission tomography. Table 1 ｜ Main parameters evaluated by diffusion tensor MRI and their interpretations Parameter Abbreviation Interpretation DKI Electroencephalography is the major method used to evaluate brain activity in epilepsy.Currently, EEG plays an irreplaceable role in the diagnosis of epilepsy and localization of the (Yu et al., 2018)al., 2022) 2022;Jaseja, 2023)o-Villablanca et al., 2022;Jaseja, 2023).EEG can be divided into scalp videoassisted EEG, electrocorticography (ECoG), and stereotactic EEG (SEEG).Scalp EEG and long-duration scalp video-assisted EEG have been used to evaluate brain activity on the scalp in clinical work.Easy accessibility is the main advantage of scalp EEG, but noise from electromyography artifacts (i.e., prefrontal myoelectric activity with eyes open, mouth and throat muscle contractions during swallowing, temporal muscle activity during chewing, muscle spasms or tremors, eye movements, tongue movements, blinks, and cardiac or cardiac duct fluctuations) disrupts EEG signals(Xu et al., 2022).The electrodes used in ECoG are placed directly on the cerebral cortex, and ECoG can directly record the synchronous current generated by neuronal firing without being abated by the skull and scalp, but ECoG cannot be used to record epileptic firing deep in the brain(Zweiphenning et al., 2022).To overcome the noise from scalp EEG, ECoG and SEEG are frequently utilized in presurgical evaluation for drug-resistant epilepsy, and these techniques are effective methods to study the functional brain network in epilepsy.Researchers have used SEEG to explore brain networks in TLE and have demonstrated that the subthalamic nucleus (STN) is a key hub in TLE.High-frequency stimulation of the STN can also decouple large-scale neural activity involving the hippocampus and distributed cerebral cortex(Yu et al., 2018).Subsequently, Table 3 ｜ The specific clinical features of each neuroimaging technique DKI: Diffusion kurtosis imaging; DTI: diffusion tensor imaging; ECoG: electrocorticography; EEG: electroencephalography; FBI: fiber ball imaging-based; MEG: magnetoencephalography; fMRI: functional magnetic resonance imaging; PET: positron emission tomography; SEEG: stereotactic electroencephalography. www.nrronline.org DTI-based tractography, DKI-based tractography, FBI-based tractography, EEG, fMRI, MEG, and PET are candidate neuroimaging techniques to evaluate brain networks in epilepsy.While each technique has specific advantages and Review flaws, further attention to new advanced neuroimaging techniques, new software for data analysis, combination of multiple techniques, and the construction of personalized virtual epileptic models can help us to evaluate brain networks in the future.	2023-12-24T16:06:18.672Z	2023-12-21T00:00:00.000	{ "year": 2023, "sha1": "27f57b903b4c2d30cd6a96524abbf66579291ec4", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/1673-5374.391307", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "32474c3f3d33b9ba773928c18f34cd7cf61b78e7", "s2fieldsofstudy": [ "Medicine", "Engineering" ], "extfieldsofstudy": [] }
9612483	pes2o/s2orc	v3-fos-license	The Evolution of Human Cells in Terms of Protein Innovation Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. Introduction Multicellular life relies on the interplay of different specialized cell types within a single organism. As such, the understanding of these components of multicellularity is at the center of much of modern biology. However, little is known about the origin of these cell types and how they emerged and diversified from a single-celled ancestor (Arendt 2008). As cell types from the same organism are constrained to using an identical genome sequence, the difference between two cell types emerges through differential expression of genes and the proteins that they encode (Barbosa-Morais et al. 2012). Understanding the evolutionary story behind these differences in expression can help identify the order in which cell types evolved (Ponting 2008;Mukhopadhyay et al. 2012); grouping cell types identifies those that have a shared evolutionary past as well as highlighting genes that are critical for modern cell phenotypes. A typical approach for studying cell-type evolution in an organism is to take a set of features from cells of interest in one organism and then look for similarities between these features and those from other organisms across the tree of life (Arendt 2003). This reveals the branching points where an ancestral form of a cell type might have existed. This however requires a large number of outgroup organisms for a high resolution of branching. Classical studies of this form used features based on cell morphology (Arendt 2003(Arendt , 2008. The advent of sequencing technology has now enabled the study of biomolecular features. Genetic sequences can be compared using BLAST searches across other genomes which extract evolutionary relation via sequence homology (Mukhopadhyay et al. 2012). Such an approach is called phylostratigraphy (Domazet-Loso et al. 2007). These studies are however affected by the inability of BLAST to resolve distant evolutionary relations between homologs. It is well described that protein structure is better conserved than sequence over evolution (Dayhoff et al. 1975;Russell et al. 1994;Illergård et al. 2009); hence, in this analysis, our features of evolution are protein structures, which are in turn comprised of modular units-protein structure domains (listed as an architecture; fig. 1). A structurally oriented approach that, unlike BLAST, incorporates protein domain architectures will allow for more sensitive detection of homologous proteins in other organisms (Geer et al. 2002;Madera and Gough 2002). In turn, this will increase the quality of resolving the epoch when more ancient proteins were created, not visible through pure sequence homology alone. We study cell-type, cell-line, and tissue-specific expression data from the human FANTOM5 (Forrest et al. 2014) set and investigate how different cell types utilize domain architectures created at different epochs in evolutionary history. This analysis comes in two parts: first, looking at domain architectures that are unique to particular cell types, to set upper and lower bounds for when these cell types could have arisen in evolutionary time; second, looking at how the usage of shared domain architectures differs between all cell types, tissues, and samples. In doing this, we create protein innovation usage (PIU) profiles for each cell type over the human evolutionary lineage, and a novel measure of the distance between these histories of protein innovation is presented. Each profile identifies key taxonomic points that were critical in the development of a cell type. By combining all cell-type profiles, we are able to group those that are evolutionarily similar, highlighting cell types that might have evolved in concert. Profiles are then used to create an evolutionary timeline of human cells, enabling a discussion about the relationship between the emergence of cellular phenotypes and the evolution of the underlying components (proteins). Characterization of FANTOM Data The human libraries from the FANTOM5 (Forrest et al. 2014) data set were collapsed to 492 unique tissues, primary cell types, and cell lines by combining replicas. In the analyses presented later, we specify if only primary cell types were used (156 in total) or if all 492 samples are used. The primary cell-type samples each contain only a single cell type rather than a mixture of cell types, as is the case with tissue samples. Human primary cells are taken from anonymous donors and as a result are not modified in any way, as is the case with the cell-line samples. In the second part of the analysis, we use the whole set of FANTOM5 samples together but find that tissue and cell-line samples naturally cluster apart from primary cells. Using pooled expression for each gene in each sample, we chose a binary threshold of ln (Tags Per Million) more than 2 to determine whether a gene was expressed. For each sample, we then identified the set of structural domain architectures that are annotated to the longest transcript of each expressed gene. The total number of distinct domain architectures expressed in the union of all cell types is 4,204, annotated to 16,259 distinct genes. This is out of a possible 4,608 distinct domain architectures in all the longest transcripts in ENSEMBL Homo sapiens (build 37). As the genes that are expressed in each cell type are different, the protein (and hence domain architecture) usage also varies. For instance, profiling the "clonetech universal reference RNA," which is a sample made up of RNA from a mixture of sources, detected the greatest number of distinct domain architectures (3,609) whilst the "tongue epidermis" sample had the fewest (578). The average number of distinct domain architectures for a given sample is 2,652 (see supplementary figure S4, Supplementary Material online, for more detail). It has been shown in other studies that the effect of alternative splicing is important for both the protein structure and regulatory network (Yura et al. 2006;Barbosa-Morais et al. 2012;Buljan et al. 2012). As this study uses CAGE data and not RNAseq, we have chosen to abstract each transcript from a given gene to a single longest transcript (see Materials and Methods). The most recent common ancestor (MRCA) of this longest transcript represents the lower bound (i.e., most recent) in terms of the introduction of any possible splice variants of a gene. As we are interested in studying evolution in terms of genes and not the evolution of splice variation, we consider this a suitable level of abstraction. left, to C 0 , right) ENSP00000002596 and ENSP00000209728, alongside mouse sequence ENSMUSP00000091469. This sequence of domains constitutes an architecture. Domain architectures can be assigned in this fashion to every protein sequence for every gene in every full-sequenced cellular genome using a library of several thousand domain sequence models that represent over 2,000 SCOP structural superfamilies (Murzin et al. 1995). Two genes in the same genome can readily possess identical architectures. Possession of the same domain architecture in two or more proteins is a sensitive and reliable indicator of evolutionary relation. In this example, the architecture of ENSP00000002596 is considered an evolutionarily distinct object to that of ENSP00000209728 and ENSMUSP00000091469, which share the same architecture. Addition/loss of a domain or a long stretch of unannotated sequence within a protein is considered as the creation of a new architecture. Convergent evolution of these architectures has been shown to be rare (Gough 2005). Figures produced using D 2 P 2 (Oates et al. 2013). The MRCA of a domain architecture represents the point in evolution at which it is thought to have come into existence. In this study, a domain architecture MRCA can be in 1 of 13 epochs, spanning from H. sapiens back to the last universal common ancestor (LUCA). The distribution of expressed domain architecture MRCAs is not homogeneous; older epochs contain more domain architecture MRCAs than newer ones (supplementary fig. S1, Supplementary Material online). This work suggests that exaptation of existing domain architectures into new contexts is the dominant source of cell-type-specific domain architectures. There is a trend from LUCA to H. sapiens (supplementary fig. S2, Supplementary Material online) of domain addition to existing architectures being the predominant creation event driving domain architecture innovation. However, few of these domain addition events are specific to one functional role in the cell which is evident as that there are few domain architectures solely unique to one primary cell type (supplementary fig. S4, Supplementary Material online). One explanation is that the increase in functional specialization has occurred as a result of more complex networks of regulation within the cell, as previously suggested (Buljan et al. 2012;Habib et al. 2012), and facilitated by the reuse of functional modules (domains) in different molecular contexts (Moore et al. 2008;Wang and Caetano-Anollés 2009;Moore et al. 2013). Cell-Type-Specific Domain Architectures An intuitive question to ask of cellular evolution is at what point in time did each human cell evolve. One way to attempt to answer this question for a given cell type is to identify the earliest point in evolution when all of the proteins which it expresses exist. The result of doing this however points to almost all cell types having appeared, in their current observed form, very recently, that is, since Primates or even Great apes. This is because once a new cell type appears, it continues to evolve indefinitely, and so most cell types express some proteins that have evolved very recently. Accepting that ancestral cells will be of a slightly different form to modern human cells, the issue arises as to how different two cells must be before they can no longer be considered as the same cell type. As measured by overlap in domain architectures, this is approximately 95% (supplementary fig. S8, Supplementary Material online) for the matrix of existing primary cells. Asking again the original question, but looking for the earliest point in time when only 95% of expressed proteins existed, merely points to a slightly earlier point in time when the form of the ancestral cells would be recognizably similar to the modern cell. The fact that most cell types appear at a similar point in time according to this criterion (data not shown) suggests that the rates of evolution (as measured by protein innovation) are not wildly different for most cell types. Thus, to successfully examine cellular evolution on a historical timeline, cell types must be grouped to represent a common ancestral type of origin. To group related cell types, we make use of the Cell Ontology (Forrest et al. 2014) to group together the 156 primary cell-type samples at two levels: first, we group fibroblast, embryonic, epithelial, mesenchymal, and hematopoietic cells; then break them down into 6, 3, and 12 subgroups, respectively. Even when grouped, extracting an evolutionary history of an ancestral cell is a challenge. Unlike organisms, which speciate and follow independent paths of molecular innovation, cell types are not independent. By example, a very recently evolved protein may be expressed in many related cell types. We overcome this by locating the epoch in evolution when a protein first appears by comparing its domain architecture to those in all other completely sequenced genomes (see Materials and Methods). The groupings of cell types and epochs of protein creation are combined to produce figure 2. This kind of approach is the best possible in the absence of cell-specific expression data for all other genomes as well (which would allow the reconstruction of a full evolutionary tree of cell types). Major Features of Cell-Specific Evolution We observe that fibroblast cells do not contain many unique protein domain architectures as compared with other cell type groupings. From the 16 fibroblast samples taken from various parts of the body, only three unique architectures are found, and all were already present in the LUCA. These three architectures are comprised of a single repeated domain (Fibronectin type 3, Galactose oxidase central domain, and the PKD domain). Each of these domains appears in other domain architectures adding to the evidence that structural innovation since the last universal ancestor has not been important for fibroblast cells. Each of the four remaining groupings possesses domain architectures exclusive to just them, with epochs spread across epoch ranges. It is surprising however, that the majority of cell-type-specific domain architectures in the remaining groupings are also already present at the LUCA, before the rise of multicellularity. This means that these domain architectures have undergone specialization over the course of evolution, now only being required in a single cell-specific phenotype. "Embryonic stem cell" samples contain a unique domain architecture derived from the IRS4 (insulin response substrate 4) gene that came about in H. sapiens. It is a gene whose expression has not been detectable in mice (Qu et al. 1999) and is also linked with tumor growth and proliferation (Cuevas et al. 2009;Mardilovich et al. 2009). However, little is known about the function of this gene, with previous attempts failing to identify a tissue type containing detectable levels of expression (Schreyer 2003). In the FANTOM5 data, this domain architecture (annotated to the IRS4 gene) is reliably detected in each of the "H9 Embryonic stem cell" samples. The human-specific innovation of this protein results from the addition of a Formin homology 2 domain. This domain has previously been identified as a promiscuous protein domain playing a role in lineage-specific structural and signaling interactions in a number of proteins (Cvrcková et al. 2004). dcGO (Fang and Gough 2013) assigns GO terms such as cellular component organization of biogenesis and organelle organization to this domain, meaning that this gene would be a strong target for investigations into H. sapiensspecific embryonic development. Epithelial cells contain the highest number of domain architectures unique to that grouping in the cell ontology (149). As there is a bias toward epithelial cells in the FANTOM5 data set, this result may not on first inspection seem surprising. Looking more deeply, these epithelial-specific domain architectures are not shared by all of the epithelial cells; in fact, they seem disparate in their usage of domain architecture innovation. This suggests that whilst they come from the same class of cell-type, the location in the body in which they are located also plays a large role in the domain content of their expressed genes. For instance, the nephron epithelial cells (e.g., renal mesangial, renal proximal, and renal glomerular endothelial cells) only contain one nephron-specific domain architecture whilst the columnar epithelial cells (e.g., melanocytes, lens, and ciliary epithelial cells) contain seven unique domain architectures. Within the hematopoietic cells, there is a large bias toward cell-specific domain architectures occurring early in evolutionary time. The only recent innovation specific to hematopoietic cells has occurred specifically in T-cells, mast cells, and reticulocytes (in the adaptive immune system and blood). There are six domain architectures that arise in T-cells only (especially CD8 + , CD4 + CD25 + CD45RA + , and CD4 + T-cells). These domain architectures arise from TNFRSF4, SPEF2, ZMYND12, UMODL1, and IL12RB2 genes (domain architecture annotation is shown in supplementary fig. S9, Supplementary Material online). From these six, UMODL1 and NKRF arose at Euteleostomi and Euarchontoglires, respectively. NKRF or NF-kappa-B-repressing factor is a transcription factor that mediates transcriptional repression of certain NF-kappa-B-responsive genes. UMODL1 or Uromodulin-like-1 gene has a domain architecture comprising of four protein domains (Fibronectin type 3, Elafin-like, Growth factor receptor domain, and EGF/Laminin). Uromodulin-like 1 protein has been shown to demonstrate a prompt and robust response to CD3/CD28 antibodies in proliferating CD4 + T cells, implicating it in immune response to pathogens (Wang et al. 2012). As this innovation took place in the ancestral Euarchontoglate, T-cells found in organisms more distant from human than Euarchontoglires (Rodents, Rabbits, and Primates) cannot posses the same structural content in their T-cells as we do. Conversely, monocytes appear to be older, with the last monocyte-specific protein innovations taking place at Coelomata (species containing fluid-filled body cavities). This is also echoed by Langerhan cells, another innate component of the immune system. The last point of innovation specific to this group is at the ancestral Euteleostome (ancestor of all bony vertebrates). For instance, a monocyte-specific domain architecture can be found in NOD2, which is an intracellular sensor for bacteria (Ogura et al. 2001). When compared with other cellular types the cells involved in the innate immune system appear to be the oldest. Patterns in Usage of Protein Innovation We have seen above that it is not uncommon for groups of phenotypically similar cell type to posses several domain architectures that are mutually unique to just them. The number of these exclusive architectures across samples, however, is insufficient to study function solely in terms of uniqueness. Instead, we look now toward studying patterns in cell-type usage of all expressed domain architectures, created at different evolutionary epochs. Over evolutionary time, an evolving cell type has available to it all protein domain architectures within the genome of that era. Some of these might only be expressed in one modern cell type (as investigated earlier), but many more will be expressed in multiple other samples (as seen in sample pairwise domain content overlaps in supplementary fig. S8, Supplementary Material online). These architectures will be accumulated over the evolutionary history of that cell. A single cell-type history is defined by the points in evolutionary time where new protein content used by that cell appeared in the genome. We anticipate modern versions of that cell type to express more architectures from an age crucial to the development of its core internal components. To numerically capture such historic information about the protein innovation in a cell type, we construct a novel measure: the PIU, see Materials and Methods. This relative measure describes how far above or below the average protein innovation at a given epoch those proteins expressed in a given cell type are. Combining the PIU for each of 13 key phyletic divisions in the NCBI taxonomy (Federhen 2012), we create an evolutionary profile, giving an insight into the trend of protein innovation over time. An example profile can be found in figure 3 for the cell type sample CD8 + T-cells. The evolutionary profiles we present identify periods of evolutionary time where prolonged above or below average use of protein innovation has occurred. To group samples with similar evolutionary histories, we use a self-organizing map (SOM) clustering algorithm (presented in supplementary fig. S3, Supplementary Material online). Samples in the same cluster have similar patterns in PIU over human ancestry and hence have similar evolutionary histories. This does not necessarily mean that they are expressing identical proteins created at those epochs simply that they express a similar proportion of their total number of distinct architectures to those dated to that period. Figure 4 details the results of the SOM clustering over all evolutionary profiles for all 492 unique tissues, cell types, and cell lines. Displayed are stylized profiles of above average, high, and very high (increasing line thickness) usage of protein innovation across epochs, alongside annotation of distinct and enriched architectures. We identified ten clusters, each containing between 3 and 20 subclusters (units). The profiles shown are those for representative samples of the cluster in which they sit. SOM cluster and unit membership, alongside sample evolutionary profiles, can also be explored through an associated webpage located at http://supfam.cs.bris.ac.uk/ SUPERFAMILY/trap/, last accessed March 17, 2014). The clusters qualitatively compare well with groupings determined by gene coexpression in the core FANTOM5 article (Forrest et al. 2014); smooth muscle cells are close to epithelial samples and immune system components are all grouped nearby. However, the data here has only 13 dimensions (the ancestral phylogenetic epochs), as opposed to the many thousands of promoters in the genome. Cluster divisions also broadly group samples into biologically meaningful categories. Clusters 1 and 2 group similar evolutionary profiles for many white blood cell types (leukocytes), with a significant division between the adaptive and innate immune system (Fischer's exact P value < 0.05; supplementary fig. S5, Supplementary Material online). It is interesting to note that in general, cancer cell lines and tissues each separately cluster with their counterparts and not with their primary cell components (clusters 8 and 10). This suggests that phylostratigraphic evolutionary analysis between different classes of biological sample (tissue, cell type, and cell line) is not possible as the ancestral trends in expressed genes are not similar. There are new domain architectures created at every major division point in the NCBI taxonomy (supplementary figs. S1 and S2, Supplementary Material online); however, tissues and cell types have not made uniform usage of this protein innovation. Evolutionary profiles reveal punctuated evolution of samples; "chondrocytes" (cluster 6) makes use of architectures from Metazoa, Chordata, Euteleostomi, and H. sapiens whilst "whole blood" (cluster 2) draws heavily from proteins created before Bileteria and then again at Mammalia. This reiterates the earlier point that it is not possible to unambiguously date the emergence of a particular cell or tissue type to a single taxonomic era by the majority age of the components that they express. To do so would require equally high quality expression data from many organisms across the full breadth of the tree of life. Using our evolutionary profile information, we can still gain insights into the progression of cell type and tissue evolution, as discussed later. Evolution of the Human Immune System: The Components Are Old As discussed earlier, the innate immune system shows strong evidence for being an ancient cell type, with all hematopoietic innovation taking place within adaptive immune cell types. An example profile for T-cells from cluster 2 can be seen in figure 3. To summarize, these cell types contain four periods of above average use of domain-architecture innovation at Metazoa, Chordata, Amniota, and Mammalia ( fig. 4). During the expansion of the metazoans, it is known that the first immune system cells evolved to cope with the large number of single-celled pathogens that attempted to infect the newly formed multicellular life forms. The enriched domain architectures that are associated with cluster 1 include Toll-like receptors and leucine-rich repeats, both of which are thought to have played an important role in these early immune cells (Hoffmann 1999;Janeway and Medzhitov 2002)-see supplementary materials (Supplementary Material online) for a full list of superfamilies, many of which have creation points before Chordata. Furthermore, the use of structural innovation in Chordata, Amniota, and Mammalia concurs with what is known about the evolution of the adaptive immune system at these times. Enriched domain architectures for this cluster include the SH2 domain, which is known to regulate the signaling events in the adaptive immune system of Eukarya (Liu et al. 2011) as well as the ADP-ribosylation domain that is known to modulate immune response (Corda and Di Girolamo 2002). This pattern of use of protein innovation (high at Chordata and again at Mammalia) lends evidence to a hypothesis discussed by Cooper and Herrin (2010) that an ancestral immune system existed around the time of the ancestral chordate, with newer cell types developed around the time of the rise of mammals. Evolution of the Spleen and Thymus: Concerted Evolution with the Brain Cluster 5 groups tissue samples of the brain together. It is homogenous in this respect, with the exception of "spleen" and "thymus" tissue samples being placed in a subcluster alongside "spinal cord," "pineal gland," and "adult globus pallidus" tissue samples. The spleen and thymus are both lymphoid organs that modulate immunity and their presence within a cluster of brain-specific samples suggests concerted evolution in their structural innovation over time. Specifically, whenever the brain has undergone innovation due to selective pressure the same has been true for the spleen and thymus. Both organs are heavily innervated and reflex control of immunity by the central nervous system has been recently demonstrated (Rosas-Ballina et al. 2008;Rosas-Ballina and Tracey 2009). Here, we add new molecular-expression evidence to support such a hypothesis. This is in contrast to the idea that the spleen is solely part of the circulatory system, as was previously hypothesized by anatomic descriptions (Tischendorf 1985). Conclusions We have used phylogenetic stratification of the structural domain architectures of proteins expressed in FANTOM5 human cell samples to chart their histories in terms of protein innovation. The results show that human cell types, in the form that they currently exist in, are the product of continuous and ongoing evolution. This means that we do not see a clear point in time when a human cell came into being, because the ancestral cell type would have been of a different form from that which they now exhibit. By considering groupings of related cells, however, we can see the points in evolution when proteins uniquely expressed in the cells of that type were created. Without equivalent data for a large number of genomes, it is not possible to reconstruct an evolutionary tree of cell types; the best possible picture we can achieve is a timeline of cellular evolution. This is however sufficient to allow corroboration, at a molecular level, of some current theories on the evolution of specific cell types and tissues. It has been suggested previously that species-specific adaptive processes are enabled by de novo creation of genes (Toll-Riera et al. 2009;Bornberg-Bauer et al. 2010) or through domain shuffling/gene fusion (Moore et al. 2008;Buljan et al. 2010;Bornberg-Bauer and Albà 2013). We propose that adaptive processes are the result of reuse of existing domain architectures, homologs of which might not be detectable through use of BLAST (Mistry et al. 2013), rather than de novo creation of the entire gene sequence. This is supported by a search using sensitive indicators of homology across all completely sequenced genomes and demonstration that many different niches are filled by homologs of the same domain architecture. This said we should remain mindful of the fact that of these many structural homologs, some might fall into more or less evolutionarily related groupings; a resolution that we miss here and should be considered for further study. The findings we present have been made possible for the first time by the cell-type-specific digital gene expression from the FANTOM5 project. A future extension of this work to all cell types in many species, to the same high quality as in FANTOM5, is required to complete the picture of cellular evolution in nature and reconstruct the full evolutionary tree of cell types. Materials and Methods There are four core components to our methodology ( fig. 5): 1) identification of expressed genes, 2) assignment of domain architectures, 3) creation of evolutionary profiles, and 4) clustering of evolutionary profiles. An enrichment analysis of the clustered domain architectures and associated dcGO ontological terms was also conducted. The details of these processes are explained in the following sections. Identification of Expressed Genes The FANTOM5 CAGE human data set is a collection of tissue and cell samples (Forrest et al. 2014), providing the genomic transcription start site (TSS), a mapping to the closest Entrez GeneID and expression level of the gene products transcribed. A binary value was set for each gene expressed in each distinct tissue sample above a tags per million (TPM) intensity threshold of ln½TPM > 2 (TPM !~7:4). This cutoff was in line with previous characterization of the CAGE methodology (Balwierz et al. 2009), which showed a roughly linear relationship, after a tag count of just under ten, between lnðTPMÞ and the number of TSSs expressing at that magnitude. In cases, where replicates were present, a further stipulation was made that the average binary expression value of a gene amongst replicate samples must exceed 0.75. Assignment of Domain Architectures and Epochs We used a mapping from Entrez GeneID to gene-product protein sequences, provided by UniProt (UniProt The profiles can then be clustered to find cell types which share a common evolutionary past. These clusters can then be analyzed to discover domain architectures key to bifurcations in cell phenotype. from EntrezID to UniprotID is one-to-many. At the expense of not studying at the resolution of per-sample splice-isoform products, we selected the longest transcript from the mapped UniprotIDs. As we are interested in studying evolution in terms of when a gene first appeared in its current form, and not the evolution of splicing, we consider this level of abstraction appropriate. SUPERFAMILY provides domain architecture assignments to all proteins within 1,559 fully sequenced cellular genomes; 373 eukaryotes at varying taxonomic divisions alongside 1,175 archaea & bacteria (at an outgroup from human at cellular organisms). It also details taxonomic placement of these genomes in accordance with the NCBI taxonomy (Federhen 2012). Dollo parsimony was used to determine the MRCA of the related gene transcript homologs, which was set as the creation point or "epoch" of the domain architecture. The epochs for each of the domain architectures expressed in human fall into 1 of 13 evolutionary eras, matching key taxonomic division points, stretching from H. sapiens to cellular organisms. These were chosen so as to ensure that there was sufficient variability in the number of domain architectures expressed from that age, necessary for the evolutionary profiles (discussed later). A domain architecture that is ubiquitous and found in all kingdoms of life would have an epoch at cellular organisms, whereas a domain architecture found only in four-limbed vertebrates would have an epoch at Tetrapoda. Further studies that might be more focused on functional characterization of cell transcriptomes, and less on evolutionary analysis, might be interested in considering the effect of splice isoforms. Previous studies have shown that alternative splicing can affect protein structure (Yura et al. 2006); however, this effect is not as strong at the domain architecture level. The point at which an existing gene undergoes domain shuffling, acquiring a new architecture, is a reasonable point at which to declare that the protein no longer exists in its previous form. Creation of Evolutionary Profiles Each cell type expresses a collection of domain architectures, created at various evolutionary epochs and with differing abundance. So, as to compare different phyletic profiles of domain architecture usage between samples, we created a per-sample Z-score of the proportion of distinct expressed domain architectures of each epoch, as compared against all other samples (supplementary figs. S6 and S7, Supplementary Material online). This value is also called the PIU in the Discussion section. This assignment across all epochs provides an evolutionary profile, detailing eras of high and low usage of structural domains created at points in time over our evolutionary past. These relate to evolutionary eras of high usage of structural innovation and low usage of structural innovation, respectively. For a set of samples S and set of evolutionary epochs e, the number of unique domain architecture expressed in a given sample S at epoch e is X e s . This means that the total number of expressed architectures for a given sample s is P j E j i¼1 X i s or T s . To calculate the z-value for a sample S at an epoch e, we used the following equation: X e s =T s À Á À À Á = À Á , where is mean and is the standard deviation at each evolutionary epoch over all samples. Clustering of Evolutionary Profiles A coarse-grained overview of the data was achieved by clustering all experimental samples from the FANTOM data set (i.e., primary cells and cell lines of different tissue origins) by their evolutionary profiles. The clustering was performed using SOM methods (Vesanto 1999). The SOM and its derivatives have been used extensively to cluster and visualize highdimensional biological data (evolutionary profiles in this setting). We chose distance matrix-based clustering of the SOM to obtain a total of 110 subclusters (units) that were further grouped into ten major clusters in a topology-preserving manner. A crucial feature of this clustering is that they optimize the number of clusters and division criteria to fully respect the inherent structure of the input data (without a priori assumption of the data structure). The clusters and units were visualized using a component plane presentation integrated SOM (Xiao et al. 2003), displaying evolutionary epoch-specific changes of clustered cell types (presented in supplementary fig. S3, Supplementary Material online). Unit and cluster membership can be fully explored using the publically accessible website (see Public Accessibility of Data). Assignment of Ontological Terms Domain architecture and gene enrichment was performed by identifying unique items to groupings of samples, whether by a cell ontology (Forrest et al. 2014) or through use of the evolutionary profile SOM clusters. The dcGO resource (Fang and Gough 2013) was used to provide domain-centric Gene Ontology (GO) for these enriched architectures. The most specific (highest information) terms in dcGO were used. Public Accessibility of Data All of the data described in this manuscript, including protein domain architecture assignments to each and every gene transcript of the FANTOM5 human data set and details of cell type placement in the SOM clustering are available as part of a mySQL compatible dump available at http://supfam.cs. bris.ac.uk/SUPERFAMILY/trap/ (last accessed March 17, 2014). Also provided are evolutionary epoch profiles, such as that presented in figure 3 for T-cells, for every distinct cell type. Finally, scripts used to generate these data are available from GitHub (https://github.com/Scriven/TraP, last accessed March 17, 2014) for reuse or inspection under an open source license.	2018-04-03T01:16:05.002Z	2014-03-29T00:00:00.000	{ "year": 2014, "sha1": "0a18c6cc8893578844832493d2eb53d80cd767b8", "oa_license": "CCBYNC", "oa_url": "https://academic.oup.com/mbe/article-pdf/31/6/1364/13166926/mst139.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "e8f7f721fa8e3fc9aecfc7c3be41bbf9ef544a98", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }

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