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232293063 | pes2o/s2orc | v3-fos-license | New Zealand Shellfish Detection, Recognition and Counting: A Deep Learning Approach on Mobile Devices
New Zealand maintains excessive effort to organise the sustainable development of its marine resources, wildlife, and ecological environment. New Zealand has stringent rules to control fishing and to protect the continued growth of marine inhabitants. Fishing inspections, such as identifying and counting shellfish, are part of the daily routine of many New Zealand Fisheries officers. It is however considered labour-intensive and time-consuming work. This project, thus, develops a touch-less shellfish detection and counting web/mobile application on handheld devices using Mask R-CNN to assist New Zealand Fisheries officers in recognising and totalling shellfish automatically and accurately. New Zealand shellfish species are different from other places in the World. Thus, this study firstly investigates the best deep learning model to use for New Zealand shellfish recognition and detection. Selected shellfish dataset is collected from a local fish market in Auckland and trained by using the chosen artificial neural network. At last, a portable system is built to support Fisheries officers to count shellfish quickly and accurately. At this current stage, a web-based application has been successfully deployed at a local server (cvreact.aut.ac.nz) in which users can upload target objects to get results related to three major shellfish species including cockle, tuatua, and mussel. In the near future, this proposed model is scaled up to recognise more species to cover the popular shellfish species in New Zealand, thus benefiting the aquaculture as well.
Introduction and Backgrounds
New Zealand is well-known for its beautiful natural scenery and abundant natural resources. New Zealand has managed to organise the sustainable development of marine resources, wildlife and ecological environment. New Zealand, therefore, has legislated out for fishing different sea creatures such as finfish and shellfish in different regions and seasons. For instance, each fisherman can only gather 50 cockles per day in Auckland Coromandel area [1]. New Zealand Fisheries officers work hard to ensure the sustainability of New Zealand's fishery. Checking and counting shellfish manually is one of the daily work of New Zealand Fisheries officers; however, it is very time-consuming (Fig. 1).
Recently, actual applications using deep neural networks (DNNs) technologies have been widely applied and achieved remarkable successes in both of the research and industrial fields. Object detection, classification, and recognition are some of the most frequently used techniques that efficiently extract specific objects from images or video stream by pre-trained models. Artificial intelligence (AI) applications have taken over the roles of Fisheries officers in the time-consuming. It is useful to have a portable system to support Fisheries officers to recognise and count shellfish quickly, thereby reducing the workload of Fisheries officers. This research aims to develop an application to recognise and count shellfish based on input images or videos from a handheld camera. Its approach also figures out whether shellfish species being caught meet New Zealand different fishing rules [1].
Visual Object counting (VOC) as a useful application of object recognition has been widely applied in many areas in the real-world to measure the number of target objects with the input from images or videos. For instance, using cell counting system to count cells in microscopic images provides a faster and affordable disease diagnose solutions [11]. Image processing techniques, such as neural network, Hough transform, clustering, and shape matching, are adopted to recognise and detect patterns or objects [9]. However, big concerns when using VOC are the frequent overlap between objects, occlusion, and complex background environment. The object counting process in image processing can be performed by object detection, regression, and segmentation techniques. All of these approaches require a machine learning process on labelled data to build a detection model, thereby predicting the number of target objects in an image [13]. Global regression-based VOC (GR-VOC) and density estimation based VOC (DE-VOC) are two significant techniques using supervised approaches [17]. DE-VOC method counts object instances by a density function from dense local features of the image [5]. Both these two techniques can perform efficiently with sufficient training images. Convolutional neural networks (CNN) and hardwareaccelerated optimisation can improve the performance of regression-based counting approach significantly [11]. An innovative framework to count objects without any preliminary training step was proposed and can count multiple object types [13]. To minimize the data preparation costs introduced by labelling training data of regression-based approaches, an unsupervised approach was introduced to count objects without object recognition [7].
Object detection has been a significant part of image processing and CV fields. It is a technique that localises and classifies an object in an input image by predicting the bounding box location that contains the object [4]. Geometrybased and appearance-based are two ways of object representation [14]. Object detection can be achieved using traditional machine vision approaches such as SVM (Support Vector Machines). The traditional method uses a sliding window to generate candidate regions on an input image, then extracts features and classifies the regions using a trained classifier [20]. Time-complexity is one of the main drawbacks of the traditional method. Compared to traditional methods, deep learning algorithms, such as CNN, are able to accept raw data as input and automatically learn features [10].
Object detection and counting techniques have been successfully implemented in agriculture fields. A model to detect and count plant seedling in the field using Faster R-CNN with Inception ResNet v2 was implemented [6]. Their model successfully achieved an F1 score of 0.969 at the IoU threshold of 0.5. Another model implemented a real-time corn kernel detection and counting to help farmers to estimate corn harvest and make marketing decisions [8]. The results indicated that the CNN model has higher accuracy (0.947) than HOG+SVM, since it can extract features automatically from an input. A fruit detection model was developed for strawberry harvesting check based on Mask R-CNN [18]. From their experiment results, the average precision rate is 95.78% out of 100 test images. A comparison of fruit detection and counting performance between Faster R-CNN with inception V2 and SSD with MobileNet on a fruit dataset was conducted [15]. Experiments have shown that Faster R-CNN has higher performance (94%) than SSD (90%). Furthermore, they pointed out that the detection performance of CNN architectures is relied on experimental dataset quality.
Apart from the agriculture fields, the use of deep learning methods for object detection and counting has been used in other areas in daily human life. A realtime vehicle counting system using SSD algorithm to monitor traffic was designed [2]. The SSD based network was replaced by ResNet-34, which is more accurate than the original based network (VGG-16). From the experiment results that the vehicle detection accuracy of the system can reach to 99.3% and the classification accuracy can achieve to 98.9%. A model to detect and count threatening objects from images using TensorFlow object detection API was built [12]. Their model was implemented by Faster R-CNN algorithm, and it is able to detect and classify two classes of threatening objects: knife and gun. The experiment results show relatively good accuracy and counting results.
This paper proposes the portable model based on Mask R-CNN, which recognises and counts target objects from an image or video to figure out a legal shellfish grabbing. The target objects are uploaded to a processing server, and then its results are popped up on the officer's handheld device. The notices or recommendations are also presented to make proper decisions.
Overall System Design
A web application is easy to be used on a wide variety of operating systems of laptops and mobile devices. To facilitate the use of the detection system by New Zealand fisheries officers both in their offices and at beaches, this system is designed as a web application that allows users to upload images or videos to check the shellfish species and quantity. The entire recognition system with the shellfish detection model is hosted on a processing server. As shown in Fig. 2, the system is deployed on a processing server to detect and count the target shellfish objects from an uploaded image. The detection result that includes labelled shellfish and count numbers of each shellfish species is finally returned to the end-user.
Dataset Preparation
Cockle, tuatua are major shellfish species that New Zealanders can gather from beaches both in South Island and North Island. Cockles are plump, round shells with fine ridges, and they are widespread in New Zealand harbours. Tuatua is endemic to New Zealand which can be found when low tide and has a more irregular shell shape. Therefore, this study selects cockle and tuatua as target shellfish species to conduct the shellfish detection and counting task. To extend the application of this detection model, mussel, another common and popular shellfish, is included in this research.
The shellfish used in this study were collected randomly. The appearance of the same shellfish species varies among different regions. Therefore, the cockles used in this study are gathered initially from both South Island and North Island New Zealand, as shown in Fig. 3. Due to the resource constraints, the tuatua and mussel are originally gathered from North Island New Zealand. In this research study, 700 images that include 200 images of each shellfish species and 100 images of combined shellfish species are selected for the further detection experiment. The sample images of cockle, tuatua, and mussel are shown in Fig. 4. Figure 5 describes the data pre-processing process that includes data annotation, data format conversion. Data augmentation is an essential technique for expanding dataset in deep learning tasks.
Detection Model Design
Since the purpose of this shellfish detection system is to accurately recognise and count shellfish for New Zealand fishery control, this paper focuses more on detection accuracy rather than detection speed. Faster R-CNN and Mask R-CNN have better performance than YOLO and SSD [19]. Furthermore, the results of general object detection experiments on the MS COCO dataset shows that Mask R-CNN has higher AP than other deep learning object detection methods. Due to the advantage in accuracy, Faster R-CNN and Mask R-CNN are considered for this purpose.
Bearing in mind the model comparison and results of previous researches above, this paper adopts Mask R-CNN to train the shellfish detection model. Figure 6 demonstrates the structure design of the Mask R-CNN shellfish detection model that is used in this paper. This paper uses ResNet101 and FPN as the backbone network to extract feature maps from an input image.
Web Application Deployment Design
Once the detection model is trained, the final model and a user interface will be deployed as a web application. This application is written in Python. The web user interface is developed using Streamlit, which is an app framework designed for data analysis and machine learning. Moreover, the application deployment uses Docker which is a platform to help automatic software delivery using containers. Docker builds a lightweight image which can be deployed on various cloud platforms such as AWS and Azure [3]. Figure 7 demonstrates the workflow of the system deployment. First, the system settings and package requirements are configured within a Docker file. Then the container image that contains the system libraries and application code is pushed and deployed on a cloud server. Finally, the cloud server will host the application and provide a website for users. The system provides the users with simple GUI application, to keep the page straightforward so that users with different knowledge backgrounds can easily use the application. Users can upload and preview an image by clicking 'browse files' on the main page. Once the image is uploaded, users can use this application to detect and view the predicted results by clicking 'Detect and Count' buttons on the web page.
Data Pre-processing
Since this research aims to conduct the object detection task, the target objects on each image need to be labelled for training purpose. This project uses Labelme, which is an easy-to-use image annotation tool to label the shellfish objects on each image. As shown in Fig. 8, all target objects in the image are selected and labelled within a polygon that covers on each target object. Labelme generates a JSON file for each image to store the image information and annotations.
This project trains the detection model based on COCO pre-trained weight and uses COCO evaluation metrics. The shellfish data should be converted to COCO format dataset. A Python script randomly allocates 80% of images in the shellfish dataset to train dataset and the other 20% to the validation dataset. The detection model is trained with the training dataset and evaluated with the validation dataset. The size of training dataset for deep learning is critical for the performance of a model. The more data are involved, the more robust the model is, so data augmentation techniques that automatically enlarge the dataset with annotations are very crucial for dealing with small dataset in a CNN model training process. Data augmentation includes colour operations and [16]. Data augmentation is able to increase detection accuracy over +3.2 mAP on COCO dataset [21] (Figs. 9 and 10).
Model Implementation
Due to the limitation of hardware, the detection model training process is performed on Google Colaboratory. Additionally, this research uses TensorBoard to visualise the summary output of the model training process, such as AP and loss. Transfer learning is also adopted to train the detector. It is faster than training a model from scratch and can help the model achieve better performance in training on a small dataset. Table 1, the inference results of Mask R-CNN ResNet101+FPN model with learning rate 0.001 has the highest bounding box prediction AP (86.826%) and segmentation AP (87.191%). The AP in the table is averaged over three categories on 10 IoU thresholds from 0.5 to 0.95. Additionally, the model with learning rate 0.001 has an acceptable inference speed which is around 0.1 s per image. Therefore, the learning rate is set to 0.001 to train the detection model. The experiment evaluates the model on the validation dataset using COCO evaluation metrics during the training process. The evaluation performs every 500 iterations. Additionally, the experiment metrics like loss and average precision are visualised and monitored within TensorBoard during the training process. Finally, an inference is performed on the validation dataset using the final trained model. To evaluate the performance of the final detection model, models of Mask R-CNN with ResNet50+FPN, Mask R-CNN with ResNeXt101+FPN, and Faster R-CNN with ResNet101+FPN have experimented with the same settings as a comparison.
Analysis and Discussion
Since the main propose of this research is to recognize and locate the target shellfish objects. This research mainly analyses and discusses the results of the bounding box prediction accuracy. As Shown in the table, the average precision (AP) is averaged over 3 categories at 10 IoU thresholds which are 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95. The AP of the bounding box prediction is 87.414%, while the AP of segmentation prediction is 87.032%. Furthermore, AP50, which is similar to PASCAL VOC evaluation metrics, is calculated at 0.5 IoU threshold. The AP at 0.5 IoU threshold is 99.592% for both bounding box and segmentation prediction. The prediction results of AP50 show a good performance of the final prediction model. The detection results for each shellfish category that the model has better detection capabilities for mussel and tuatua, and the AP of bounding box prediction can reach 91.084% and 90.576% respectively.
Because the samples of cockle are collected from both South Island and North Island of New Zealand, their shapes and colors are different. Therefore, the average precision is a bit lower than that of mussel and tuatua, which is 80.584% for bounding box prediction and 81.142 for segmentation prediction. The training speed of the detection model is 0.3776 s for each iteration. The inference time is 0.1006 s for each image on one device. The testing results on random validation images show good detection performance, and the classification score for each object can reach over 95%. Figure 11a shows the accuracy of classification during the training process. Accuracy indicates the percentage of the correctly predicted results in the total predictions. After around 500 steps, the accuracy of object classification remains at between 98% to 99%.
During the training process, a COCO model evaluation inferences on the validation dataset every 500 iterations over 1050 images. Figure 11b demonstrates the total loss of final Mask R-CNN model on training and validation dataset respectively. The total loss is obtained by the loss of classification, bounding box, and mask. As shown in Fig. 11b, the total loss stops dramatically decreasing and shows a smooth delay after 500 iterations. The total loss of both training and validation shows a slightly downward trend as expected.
Results Demonstration
The evaluation results that are computed from the validation dataset is not enough to determine the model performance. Because the dataset used in the experiments is small, overfitting problem may be introduced by training. The final model should be tested using images that are not in the experimental dataset. Figure 12 shows the test result on a new image from the web interface. As can be seen, every tuatua is detected and counted from the test image. Figure 13 demonstrates the detection results of an image that are downloaded from the Internet using the final model. The model can detect almost all shellfish objects even on the under layers of other objects. But the model classifies 2 cockles which are partly covered by other objects as tuatua. The object overlap makes the detection more difficult. Making the shellfish objects spread instead of overlapping with each other will increase the detection results. Additionally, the detection will be improved by increasing the dataset size and including tuatua and cockle from multiple locations both of the North Island and South Island of New Zealand.
Conclusion and Future Work
Recently, deep learning technology is developed strongly; it has been becoming beneficial for object detection techniques in order to reduce repetitive tasks. This study designs and develops a web/mobile application to recognize and count New Zealand shellfish from images or videos. This is a tool potentially built for helping New Zealand Fisheries officers to control shellfish gathering. At present, there are no studies that develop models to detect and count New Zealand shellfish species. This study can reduce not only the workload of New Zealand Fisheries officers but also fill the gap in the literature review of shellfish detection field. This research demonstrates a procedure of object detection implementation, which includes data pre-processing, model training, and evaluation. From the evaluation results of COCO evaluate metrics, the final Mask R-CNN detection model can recognize and count three major shellfish species, for example, cockle, tuatua, mussel. In terms of bounding box prediction, the average precision is 87.4% when the IoU threshold is calculated from 0.5 to 0.95. The AP at 0.5 IoU threshold can reach 99.6%. The proposed model has proven its potential application in fishery, and other fields to reduce the workload and improve the efficiency. | 2021-03-22T17:34:44.008Z | 2021-03-18T00:00:00.000 | {
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121160747 | pes2o/s2orc | v3-fos-license | A pool-adjacent-violators-algorithm approach to detect infinite parameter estimates in one-regressor dose–response models with asymptotes
Binary response models are often applied in dose–response settings where the number of dose levels is limited. Commonly, one can find cases where the maximum likelihood estimation process for these models produces infinite values for at least one of the parameters, often corresponding to the ‘separated data’ issue. Algorithms for detecting such data have been proposed, but are usually incorporated directly into in the parameter estimation. Additionally, they do not consider the use of asymptotes in the model formulation. In order to study this phenomenon in greater detail, we define the class of specifiably degenerate functions where this can occur (including the popular logistic and Weibull models) that allows for asymptotes in the dose–response specification. We demonstrate for this class that the well-known pool-adjacent-violators algorithm can efficiently pre-screen for non-estimable data. A simulation study demonstrates the frequency with which this problem can occur for various response models and conditions.
Introduction
When fitting a quantal dose-response model to observed data, it is common to use maximum likelihood for estimating the model parameters. [1,Section 7.2] In many cases, however, anomalies can arise in obtaining the maximum likelihood estimator (mle) when the model is highly nonlinear. To illustrate, consider the following Monte Carlo-based scenario taken from Buckley and Piegorsch [2]: to study the small-sample behaviour of the mles under an Abbott-adjusted [i.e. positive lower asymptote; 3] Weibull dose-response model, ∼ Bin(n, π(x i )) with π(x i ) = γ 0 + (1 − γ 0 )(1 − exp(−e β 0 x β 1 )), we simulated 2000 data sets with n = 50. Four dose levels of x = 0, 0.25, 0.5, 1 were chosen, to match a common single-exposure design in cancer risk experimentation. [4] The true parameter values of γ 0 = 0.01, β 0 = −2.35, and β 1 = 1.63 were taken to correspond with a known *Corresponding author. Email: rcdeutsc@uncg.edu dose-response pattern having π(0) = 0.01, π( 1 2 ) = 0.04, and π(1) = 0.1. The mles of the three parameters were estimated using the function optim in R 2.15. [5] Investigating the fitted parameter values for all these 2000 replications, we found that a number of unusually large point estimates were evidenced for the parameter β 1 (ranging up to 3.219453 × 10 13 ) comprising about 17% of all the 2000 generated data sets. Clearly, when applied to real data any consequent inferences based on such inflated estimates could lead to improper or incorrect conclusions on the scientific phenomenon under study.
As discussed below, such anomalies in the model estimation process are affected by the underlying functional form of the dose-response function, the sample size, and the limited number of doses. Furthermore, the mle of the parameters in these cases actually contains infinite values that drive the estimated dose-response function to a degenerate structure: it is a step function and impossible to find using standard parameterizations.
The issue of infinite maximum likelihood estimates in quantal dose-response modelling is well known, and has received considerable attention in the literature. Wedderburn [6] and Haberman [7] provided a series of conditions for existence (i.e. finiteness) and uniqueness of the mle which apply to the class of generalized linear models. However, these authors gave no explicit means of detecting cases that could lead to infinite parameter estimates. Towards that end Silvapulle [8] demonstrated that finite parameter estimates in binomial response models require overlap to exist in the observed data, i.e. for any p regressor variables there does not exist an x 0 for which the entire data set is separated into responses only and non-responses only, with a possible 'quasi-separated' mixture of responses and non-responses at x 0 . This phenomenon is known as the separated data issue and was further generalized by Albert and Anderson, [9] who considered separated data effects for a wide class of logistic regression models. They also mentioned that their result applied in principle to many other quantal regression models, and they gave a linear programming algorithm for detecting separated data. Santner and Duffy [10] expanded on the Albert-Anderson approach, and discussed further the concept of quasiseparated data. Lesaffre and Albert [11] provided a non-existence theorem for the mle with partially separated data. Also, these authors looked at how to distinguish separated data from cases of multi-collinearity. Clarkson and Jennrich [12] considered new algorithms for detecting possible infinite parameter estimates, while Kolassa [13] presented an advanced algorithm to identify separated data. Numerous other works have since appeared, expanding on these earlier articles.
However, none of these results apply to an additional issue that can occur for binomial response models using asymptotes in the model formulation (such as anAbbott-adjustment).A further drawback of the references discussed above is that the detection of data leading to infinite parameter estimates is performed during the actual estimation procedure and/or requires a linear programming problem to be solved. When considering only one predictor variable this process may be inefficient and computationally expensive. As part of her unpublished dissertation, Buckley [14] provides preliminary work for this finding in the case of four design points and a lower asymptote -introducing the concept of specifiable degenerate functions (calling them specifiably separable), and proposing an ad hoc detection method.
Below, we generalize Buckley's results and relate this problem to the easily implemented pool-adjacent-violators (PAV) algorithm [15,Section 2.4] in order to detect both separated data and asymptote induced non-convergence with a single predictor for the dose, x i . This result applies to an arbitrary number of design points and a very broad class of non-decreasing doseresponse functions. We also allow for both lower and upper asymptotes in the dose-response model specification for π(x). Our results can inform sample size and model choice decisions, and may help explain seemingly odd parameter estimates that can occur in practice. They also allow for efficient pre-screening of such problematic data sets when conducting computer simulations, thus saving valuable computing time, especially in complex Monte Carlo scenarios.
In Section 2, we introduce the family of specifiably degenerate functions (sdfs) under which separated data may occur. In Section 3, we study their impact on fitting quantal dose-response models and also present necessary conditions to avoid degenerate fits. We further examine the impact of sdfs in a short simulation study in Section 4, and end with a discussion of these results.
The limit of the parameter sequence {β i : i ∈ N} can be a boundary value of the parameter space B and thus represents a non-permissible value of the parameter vector β. Also note that the limiting function, which we describe as degenerate or as a single-step function, will not be a cdf since it violates right-continuity. This definition applies to a wide range of cdfs which are commonly employed in the specification of response functions. In particular, it applies to all location-scale families: Let G((x − μ)/σ ) be a cdf and define β 1 = −μ/σ and β 2 = σ −1 . Then, for any x * ∈ R and g * ∈ (0, 1), let where G −1 (·) is the quantile function associated with G(·). In this case, the sequence of parameter vectors β i = (β 1,i , β 2,i ), i = 1, 2, . . . satisfies the conditions from Definition 2.1. This result includes the family of normal, logistic, and extreme value distributions, which are related to the popular probit, logit, and complementary log-log links in generalized linear modelling.
[1, Section 8.4.3] By substituting log(x) for x this also applies to the Weibull distribution.
Necessary conditions for a non-degenerate fit
To see how Definition 2.1 affects the process of fitting quantal dose-response models as in Equation (1), first note that our model specification orders the observed stimulus levels π 1 < π 2 < · · · < π q . Consider maximizing the log-likelihood function, which can be reduced to the quantity Where needed for simplicity, we write is required only to be non-decreasing, the non-parametric mle satisfying this restriction is given by the sequencẽ [16] This can be found using the well-known PAV algorithm: Weibull Logistic (2) Initializeπ i =π i (this will be modified to become the restricted mle).
Note that when no asymptotes are specified, a sequence of PAV-estimators containing no values distinct from 0 or 1 corresponds to the case of completely separated data, whereas a sequence with only a single estimate separate from 0 or 1 corresponds to the case of quasi-separated data. Theorem 3.1 can immediately be restated as: (2), where G(x; β) is sdf and where the PAV-estimators satisfyπ 1 ≤π 2 ≤ · · · ≤π q . Then, a necessary condition for a non-degenerate mle is that at least two of the PAV-estimates of π i must be distinct fromγ 0 andγ 1 . In particular, if the conditions of the corollary are not met, any estimation attempt using the logistic, probit, complementary log-log or Weibull response functions in the parametric form (3) will fail by diverging. Also note that the above condition is necessary but need not be sufficient. There may be cases where estimation still fails for other reasons.
Simulation study
To examine how often this sort of degeneracy may result, we expanded upon the simulation mentioned in Section 1 and simulated data via Equations (1) and (2) β 1 ln x))). We studied a model's susceptibility in two aspects: number of specified asymptotes and number of dose levels used. For each base-model specification, we generated data using no asymptotes, only a lower asymptote, only an upper asymptote, and using both asymptotes. The dose levels were studied over four designs: x = (0, 1 4 , 1 2 , 1), x = (0, 1 3 , 2 3 , 1), x = (0, 0.25, 0.5, 0.75, 1), and x = (0, 0.2, 0.4, 0.6, 0.8, 1). The parameterizations for β were then determined by fixing the response rates at x = 1 2 and at x = 1 for all models. The response rates represent variations on dose-response functions studied previously by Bailer and Smith [17] and Buckley and Piegorsch.
[2] All parameterizations chosen for the simulation models are summarized in Table 1.
For each of the 48 specified models and at each specified sample size n, N sim = 2000 data sets were generated. For every data set, the corresponding sequence of PAV-estimates was computed and it was determined whether the sequence violates the necessary conditions specified in Corollary 3.2. From the 2000 replications, the proportion of violating PAV-sequences (resulting in degenerative fits) was determined as an estimate of the probability of a degenerate fit given the current per-dose sample size and a specified model. (For full results, refer to the Supplementary Materials of this paper online.) Examining the results from the six model configurations, we observed that model configuration A (comprising models A1-A4; see Figures 1 and 2) was most prone to producing data sets resulting in degenerate fits. When no asymptote was used in the model, the proportion of degenerate fits could be as high as 0.75 at smaller sample sizes. This proportion dropped rapidly as n increased, however, and at n ≥ 50 it became negligible for practical purposes over all four dose spacings. The proportions tended to be slightly higher for the Weibull model than for the logistic model. Increasing the number of dose levels also had a favourable impact, reducing the proportion of degenerate fits down to slightly above 40% at small n for q = 6 dose levels. The pattern was similar in the presence of a lower asymptote, but the decrease was a bit slower; n > 100 may be required for the Weibull model, with n > 300 required for the logistic model. Introducing an upper asymptote or two asymptotes had a much more severe impact, with more than 95% degeneracy at small sample sizes. The differences to no or one asymptote were most pronounced for n between 5 and 300. For small sample sizes, introducing two asymptotes had a similar effect as with the upper asymptote alone. However as n increased, the occurrence of degenerate fits consistently remained higher for two asymptotes. Depending on the number of dose levels, one could require as many as n = 3000 observations per dose to avoid degenerate fits altogether. The results for model configuration B were roughly similar to model configuration A; however, the proportions of degenerate fits tended to be slightly smaller. For model configuration C, the proportions of degenerate tended to be even smaller. For only q = 4 dose levels, however, introducing two asymptotes had a particularly severe effect: even at per-dose sample sizes between 50 and 1000 the incidence of degenerate fits was still 40% or higher (both spacings). Model configurations D, E and F were slightly less problematic (we display model configuration E in In these cases having no or only one asymptote barely produced degenerate fits, especially when n ≥ 20. In these cases, the only real impact arose when two asymptotes were introduced. Also, an upper asymptote had more impact in the Weibull models than a lower asymptote. For the logistic models, the difference was a bit more ambiguous. Examining the differences between use of lower and upper asymptotes, it should be noted that (for the response probabilities in Table 1) introducing a lower asymptote tends to produce a doseresponse curve that is more similar to a dose-response curve without any asymptote. Conversely, introducing an upper asymptote produces a dose-response curve much more similar to a doseresponse curve with two asymptotes (see Figures 5 and 6 for an illustration with configuration A). We see this more as a particular characteristic of our simulation studies and not as a generalizable result.
Discussion
Herein, we have demonstrated that non-convergence issues apply to many popular non-decreasing quantal dose-response functions, including both separated data issues and asymptote induced degeneracy. We also provided a necessary condition that allows a researcher to quickly identify dose-response data resulting in degeneracy prior to starting any iterative parameter estimation routine and without solving linear programming problems. A simulation study was conducted to examine the susceptibility of a variety of dose-response models to producing degenerate fits. From these simulations, we can draw the following conclusions: • The incidence of degenerate fits shrinks as sample size increases.
• The incidence of degenerate fit also depends on the model; for flatter responses such as configurations A, B and C in Table 1) larger sample sizes may be required to avoid degenerate fits. • Increasing the number of dose levels has a greater impact on steeper models (such as configurations D, E and F) than on flatter ones. • Introducing asymptotes to a model greatly increases the occurrence of a degenerate fit (especially at lower sample sizes). Introducing two asymptotes has the most severe impact, especially on flat models. The impact of only one asymptote is more ambiguous and can range from a minor impact to an impact almost as severe as having two asymptotes.
For a researcher investigating new or existing statistical methodology based on dose-response functions, these results may prove quite valuable, especially when studying the operating characteristics of these methods via Monte Carlo simulation. Early filtering of data sets resulting in degenerate fits can save valuable simulation time and avoid unusual and undesired results. Our Corollary 3.2 proves particularly useful in this regard, since the calculation of the PAV-estimates is computationally inexpensive and quickly accomplished. To illustrate, our entire simulation study ran in less than 5 min on a laptop computer running Windows XP with a 2.5 GHz CPU and 3.5 GB of RAM. Due to the widespread use of the class of dose-response models we study here, we also expect that these results will be useful for applied researchers. Clear impacts on study design and model formulation are apparent: with the former, our simulation studies call for (not very surprisingly) either larger sample sizes or a greater number of dose levels, especially when a shallow dose-response curve is anticipated. The results also emphasize that any model formulation using response asymptotes should be considered carefully when only limited sample sizes are available. | 2019-04-19T13:04:23.690Z | 2014-08-20T00:00:00.000 | {
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12070187 | pes2o/s2orc | v3-fos-license | Correlation of early murine leukemia virus titer and H-2 type with spontaneous leukemia in mice of the BALB/c times AKR cross: a genetic analysis.
Tissue extracts from 6-wk old mice of the AKR strain (H-K) show high levels of infectious murine leukemia virus, and these mice show a near 100% incidence of spontaneous leukemia. In F1 mice of the cross, BALB/c times AKR (H-2K/H-2K), both the occurrence of virus and the incidence of spontaneous leukemia are suppressed to very low values, due largely to the presence of the FV-1b allele inherited from the BALB/c parent. Mice of the (BALB/c times AKR) F-1 times AKR backcross generation were observed for possible correlations between virus expressions at 6 wk of age, H-2 type and leukemia incidence. H-2 type showed at most a weak influence on the occurrence of infectious virus, but there was a very strong correlation between the level of virus expression and the occurrence of leukemia and a strong correlation between H-2 type and leukemia. In addition, there was a highly significant nonrandom distribution of virus-negative mice among the backcross litters, suggesting a maternal effect on virus expression.
The AKR mouse strain was developed by selective breeding for a high incidence of spontaneously occurring leukemia, starting from a randombred stock in which the incidence of the disease was low to moderate (1) . The success of the breeding program strongly suggested that genetic factors play a role in the occurrence of the disease . This implication was amply confirmed in studies of crosses between high-and low-leukemia mouse strains (2) .
After the discovery by Gross (3) that the tissues of AKR mice yield a virus capable of transmitting the disease, it soon became clear that great variations exist from one strain of mice to another in the degree of susceptibility to the leukemogenicity of the virus . The analysis of the complex genetic control of this phenomenon has proceeded rapidly in recent years (4) .
A major advance in this genetic analysis came from the development of a rapid assay for the infectivity in vitro of murine leukemia virus (MuLV), the XC cell plaque assay (5) . With this assay it could be demonstrated that AKR mice first showed detectable levels of MuLV from the perinatal period and attained high adult levels by 6 wk of age (6) . Mice of low-leukemia strains, by contrast, were generally devoid of detectable infectious MuLV, at least until relatively late in life. From studies of crosses of AKR with low-leukemia, low-MuLV mouse strains, it appears that two autosomal loci, Aku-1 and Aku-2, are the determi nants of MuLV expression in AKR mice (7)(8)(9)(10) . Recent studies lend support to the idea that these prototypic "V" genes of the mouse consist of chromosomally integrated DNA copies of the complete RNA MuLV genome (11) .
The presence of these Aku genes is strongly associated with spontaneous tumors in segregating generations of the cross, AKR x C57L (12) . Since in this cross no major regulatory genes exist which can suppress MuLV expression, the presence of an Aku gene is tantamount to the presence of the virus . The studies we now report investigated the relation between the expression of endogenous MuLV and spontaneous leukemia in a segregating population of mice all of which * This work was supported in part by a contract from the Virus Cancer Program of the National Cancer Institute and by grant CA 07160-09 from the National Cancer Institute.
882
THE JOURNAL OF EXPERIMENTAL MEDICINE -VOLUME 141,1975 possess two or more copies of these Aku genes but which vary genotypically in respect to regulatory genes capable of interfering with the expression of infectious MuLV . The findings indicate that about half of the mice were MuLV-positive (titer range: 10°. 3 -103 8) at 6 wk of age and that the level of virus expression at this age strongly correlates with the occurrence of spontaneous leukemia much later in life .
Materials and Methods
Experimental Design . Table I summarizes the relevant characteristics of mice of the AKR and BALB/c parental inbred strains and their F, progeny. AKR mice show a near 100% incidence of spontaneous leukemia, whereas BALB/c mice show only a very low and much later incidence of the disease; the low BALB/c level of the disease is dominant in F, hybrids. Similarly, AKR mice express high levels of N-tropic MuLV in their tissues at 6 wk of age, whereas both BALB/c and F, mice show at most very low levels of the virus (8,9) . We therefore bred and observed the backcross generation, (BALB/c x AKR) F, x AKR, for a possible correlation between MuLV expression and spontaneous leukemia .
MuLV expression
Aku-1 and Aku-2 Both parents of this backcross generation possess the Aku-1 and Aku-2 genes, the AKR parent being homozygous and the F, heterozygous for these genes. Since the genes are dominant, this difference in zygosity is not a significant factor in the phenotypic differences noted between the parents or among the backcross mice in relation to MuLV expression or the development of leukemia . On the other hand, differences among these mice at the Fu-1 and H-2 loci do influence their phenotypes . The AKR parents are homozygous Fu-1^lFu-1" and H-2'/H-2', whereas the F, parents are heterozygous Fu-1"lFu-1°and H-2'/H-2 . The Fu-1°allele confers relative resistance to cellular infection with N-tropic MuLV, and the H-2d haplotype is less favorable for viral leukemogenesis than Mice of the backcross generation, which should include half Fu-1"IFu-1" homozygotes and half Fu-1"lFu-1°heterozygotes, were not typed directly for this difference, which in our standard procedure would require challenging them with exogenous MuLV . In addition, the parental strains do not differ at the Gpd-1 locus, which is closely linked to Fu-1 and could thus serve as a marker for it (13) . Rather, the mice were tested individually for the presence of endogenous N-tropic MuLV in their tissues at 6 wk of age. The major genetic factor influencing this trait in this cross is the presence or absence of the Fu-1°allele . H-2 typing of the backcross mice was carried out directly .
Mice . Backcross progeny of (BALB/c x AKR) F, 44 and AKR as were bred and maintained at Albert Einstein College of Medicine from parental strain mice originally obtained from The Jackson Laboratories, Bar Harbor, Maine. The majority of the backcross mice were from first litters, and they were housed three to four mice per cage during the observation period . The mice were observed for the development of leukemia until the youngest were 600 days old; the experiment was then terminated, since the disease had become a rare event among the survivors . When in extremis the mice were sacrificed and atuopsied. The leukemias were generally characterized by extreme enlargement of the thymus often associated with pronounced enlargement of the lymph nodes and occasionally with moderate enlargement of spleen and liver.
Virus Testing. At 6 wk of age a 1 cm terminal segment of the tail was cut off each mouse; the segments were immediately frozen, stored at -75°C and packed in dry ice and shipped to the National Institutes of Health for virus testing. Tail segments were rinsed with ether, ground with a chilled mortar and pestle, and suspended in cold Eagle's basal medium containing 20% veal infusion broth. The extracts were centrifuged, and the supernates were tested for infectious MuLV by the XC cell method (5) using secondary cultures of NIH Swiss mouse embryo cells. Titers are expressed as the number of plaque-forming units per 0.4 ml of approximately 2% extract. It is probable that the storage and shipping of the tail segments before extraction and virus testing had some effect of lowering the observed virus titers, by comparison with what one would expect from fresh tissues examined immediately.
H-2 Typing. The hemagglutination method of Gorer and Mikulska (14) with minor modifications was used to determine the H-2 type of the mice . Serum from (AKR x C57BL/6) F, mice hyperimmunized with cells of the chemically induced BALB/c sarcoma, Meth A, detects antigens of the H-2°haplotype, transmitted from the BALB/c grandparent to about 50% of the backcross population studied. To one drop of this antiserum, diluted 1 :100 and 1:400 in a saline solution of 1.8% dextran (mol wt 115,000), was added one drop of a dilute suspension of washed erythrocytes in 50% heat-inactivated fetal calf serum in saline . The mixture was agitated and incubated at 37°C for 90 min without further agitation, and the sedimented erythrocytes were examined for agglutination by low-power microscopy .
Results
Leukemia vs . MuLV . Of the 335 (BALB/c x AKR) F, x AKR backcross mice for which complete MuLV and H-2 typing results were obtained, 111 (33 .1%) developed leukemia during the 19-21 mo observation period (Table 11) . All but 12 of these leukemic mice showed pronounced enlargement of the thymus, and their spleens and/or lymph nodes were usually involved as well . 31 mice (9 .3%) died of causes other than leukemia, and 16 mice (4 .8%) died of unknown causes since they were not autopsied . Of the 111 leukemic deaths, 96 occurred among the 173 mice which had shown detectable levels of MuLV in their tissues at 6 wk of age ; 15 leukemic deaths occurred among the 162 MuLV-negative animals. This difference in leukemia incidence-55 .5% vs . 8 .7%, respectively-is highly significant (X I = 80 .7 ; P << 0.001) .
Among the MuLV-positive mice the virus titers observed ranged from 10 1-9 to 10°3 . Table III and Fig . 1 show that there was a marked correlation between MuLV titer and leukemia incidence, the animals with the highest virus titers showing the highest and earliest incidence of the disease. Fig. 2 depicts this relationship graphically, illustrating the remarkable similarity of these results to a dose-response titration pattern.
Leukemia vs . H-2 Type . The data in Tables II and III also demonstrate that the H-2 type of the backcross mice was a further factor of importance in the occurrence of leukemia among these mice . 71 of 166 H-2'/H-2" homozygotes (42.8%) developed the disease, whereas only 40 of 169 H-2a/H-2d heterozygotes (23.7%) did so . This influence of H-2 type was highly significant (X I = 13 .8 ; P < 0.001), although it was a less important factor than the level of expression of MuLV .
Leukemia vs . Sex. Since leukemia is known to occur somewhat more frequently among females than among males of the AKR strain, the backcross population was also analyzed for a possible sex difference in the occurrence of the disease. However, the difference observed-34 .8% in females vs . 29 .3% in males-was not statistically significant . MuLV vs . H-2 Type . Since both the presence of MuLV at 6 wk of age and H-2 type were important factors in the occurrence of leukemia, these two factors were analyzed for possible interaction between themselves . 94 of 166 H-2k/H-2k homozygotes (56.6%) and 79 of 169 H-2k/H-2d heterozygotes (46 .7%) were MuLV-positive . This difference is of only marginal statistical significance (X z 3.3 ; P = 0.08), and it appears that H-2 type had little influence on the early expression of MuLV in this population .
Discussion
The experiments of Gross (3) demonstrated that a virus isolated from the tissues of AKR mice is capable of inducing the leukemia typical of this strain . This endogenous viral agent (MuLV) proved to be an RNA virus of C-type morphology which matures by budding from cell membranes . A virus present in AKR tissues and possessing these same properties has been detected by the XC cell assay in vitro (6) . Our experiments demonstrate a strong association between the presence of endogenous, XC-detectable virus and the occurrence of leukemia . This association is particularly striking in terms of the correlation observed (Table III and Fig . 2) between the titer of the virus and the incidence of leukemia . Such a dose-response titration of naturally occurring MuLV is unique in tumor virus studies to date .
The capacity of AKR mice to produce infectious MuLV appears to be a function of the dominant Aku-1 and Aku-2 genes (8,9) which seem to consist of chromosomally integrated DNA copies of the RNA viral genome (11) . Meier et al . (12) demonstrated that, in a segregating cross of AKR with the Fu-1 compatible strain C57L, the inheritance of the Aku genes, as detected by the presence of gs antigen and infectious MuLV, was associated with the occurrence of spontaneous lymphoma and possibly other tumors . The results of our experiment go further, demonstrating that the presence of Aku genes does not lead to neoplasia unless these genes are expressed in terms of mature, infectious virus .
The suppression of MuLV replication in the cross described here is largely a function of the Fu-1°allele. This dominant allele, which is absent in AKR mice, is present in (BALB/ c x AKR) F, hybrid mice. Thus, although these two types of mice carry the Aku genes, they differ in that the hybrids, unlike the AKR parent, are restrictive for MuLV expression and rarely develop leukemia . Studies in vitro (9) indicate that embryo cells from AKR and (BALB/c x AKR) F, mice exhibit a similar frequency of induction of N-tropic virus production following treatment with 5-iododeoxyuridine . In cultures of AKR cells, which are Fu-1"/Fu-1" homozygous, the induced virus spreads rapidly to other cells, in which it replicates freely . However, in cell cultures from the hybrid mice, the induced virus spreads little if at all, largely because of the presence ofthe dominant Fu-1°allele which blocks the spread of infection (15) . This mechanism is throught to be responsible also for the low levels or absence of MuLV in hybrids between AKR and Fu-1°mice (9), which in turn appears to be responsible for the absence of leukemia observed here.
An additional factor resulting in suppression of infectious MuLV was observed in the experiment described here: a maternal factor, presumably representing antiviral antibody . This effect was noted previously (9) with (BALB/c x AKR) x AKR mice, as well as with other backcrosses involving AKR and Fu-1°mice, and was seen when the mother was the virus-positive Fu-1 heterozygote . In the present study, of 44 litters of (BALB/c x AKR) F, x AKR, 8, totalling 61 progeny, contained either no virus-positive mice or had at most one positive mouse with only a trace (titer < 10') of MuLV; this is significantly different from a random distribution (P << 0 .0001 by chi-square testing for heterogeneity) . This maternal effect may have resulted in excess numbers of mice with low titers in other litters as well. Analysis of the data with these eight litters excluded did not alter the patterns of results .
Thus we envision two mechanisms contributing to the suppression of MuLV in this study: resistance due to the Fu-1°allele, and the maternal resistance factor . Both of these operate by suppressing cell-to-cell spread of virus, and there is no reason to postulate that they inhibit viral genome functions (induction of virus or synthesis of viral gene products) in cells expressing their endogenous viral information. These considerations strongly indicate that spontaneous lymphoma . in the AKR hybrids studied here results primarily from the consequences of extensive viral replication and spread .
It seems worth emphasizing that the virus testing in our experiment was carried out on tissues taken at 6 wk of age, whereas the leukemias observed occurred much later in life . It is doubtful that virus testing of tissues taken at 6 mo of age would have produced such a clear correlation . The difference in magnitude of MuLV expression between AKR and hybrids of AKR with mice carrying the Fv-1°allele is much more marked in tests at 2 wk than at 6-8 wk of age (9) ; also, in mice whose early viral expression is inhibited by the maternal resistance factor, virus reaches high titers during adulthood (W . P . Rowe, unpublished) . It thus appears that early virus expression is a critical factor in leukemogenesis in AKR mice .
Our experiment also confirms previous studies showing that H-2 type exerts a significant influence on the occurrence of virus-induced leukemia (16) . However, it is clear that H-2, unlike Fu-1, has little effect on early MuLV expression . Thus H-2 must exert its influence by a different means from Fv-1, presumably by an effect on a defense mechanism which controls the outgrowth of transformed cells, perhaps by an immunological mechanism . It is of interest that this effect of H-2 manifests itself in different titration curves for the viral induction of leukemia (see Table III) . In backcross mice with high MuLV titers ( > 10 3), H-2 type was not a significant factor in the occurrence of the disease, since virtually all such mice developed it . Conversely, mice showing no detectable MuLV showed minimal incidences of leukemia regardless of H-2 type . Among mice with moderate MuLV levels, however, the presence of the H-29' haplotype was associated with a markedly lower incidence of the disease by comparison with mice homozygous for the H-2k haplotype.
Summary
Tissue extracts from 6-wk old mice of the AKR strain (H-2k) show high levels of infectious murine leukemia virus, and these mice show a near 100% incidence of spontaneous leukemia . In F, mice of the cross, BALB/c x AKR (H-2d/H-2k), both the occurrence of virus and the incidence of spontaneous leukemia are suppressed to very low values, due largely to the presence of the Fu-11' allele inherited from the BALB/c parent . Mice of the (BALB/c x AKR) F, x AKR backcross generation were observed for possible correlations between virus expressions at 6 wk of age, H-2 type and leukemia incidence . H-2 type showed at most a weak influence on the occurrence of infectious virus, but there was a very strong correlation between the level of virus expression and the occurrence of leukemia and a strong correlation between H-2 type and leukemia . In addition, there was a highly significant nonrandom distribution of virus-negative mice among the backcross litters, suggesting a maternal effect on virus expression .
We are grateful to Ms . Christine Cook for her careful technical assistance in the course of these studies.
Received for publication 20 December 1974 . | 2014-10-01T00:00:00.000Z | 1975-04-01T00:00:00.000 | {
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7683192 | pes2o/s2orc | v3-fos-license | Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation
Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.
Background
Tumour cell growth is driven by active biosynthetic and glycolytic pathways [1] fuelling interest in finding anti-cancer uses for drugs which interfere with these processes [2][3][4][5]. Mechanistic/mammalian target of rapamycin (mTOR) is an element of the mTORC1 signalling complex which drives energy generation, macromolecule synthesis and cell growth [6][7][8]. Constitutive activation of mTOR is commonly found in tumour cells, but in quiescent normal cells mTOR activity and biosynthetic pathways are suppressed [1,5]. This may happen in an energy-rich and nutrient-replete environment, such as in the case of circulating lymphocytes [9,10], or may be a homeostatic response to nutrient or energy depletion in which AMPK is activated and mTOR subsequently inactivated to promote conservation of essential cell functions [1,4,11]. What remains unclear in these scenarios is the behaviour of the dormant cancer cell. Reversible exit from the cell cycle into the quiescent, G0 state is well described in somatic cells, and is characterised by small size and low RNA and protein synthesis [12,13]. The mitogenic factors driving malignant transformation might be thought not to permit a state of true (G0) quiescence in cancer cells [13]. Nevertheless, in acute myeloid leukaemia, dormant (apparently quiescent) cells which retain proliferative potential have been described [14,15]. A high proportion of circulating and bone marrow blasts in AML also have phenotypic features of dormancy, as measured by lack of Ki-67 [16]. Ki -67 is expressed in all active phases of the cell cycle including G1 [17].
Standard chemotherapy for AML tends to spare dormant leukaemia cells [16,18], so it would be useful to characterise this subset in order to establish how best to target it. Do dormant leukaemia cells better resemble normal dormant cells or proliferating cancer cells? To further our understanding of Ki-67leukaemia cells, particularly with regard to their metabolic activity and hence potential susceptibility to therapeutic inhibition of this activity, we have measured biomarkers of mTOR activation status in presentation samples, using flow cytometry. This technique has enabled us to examine mTOR activation concurrently with proliferation status at the single cell level. We have measured activation-related epitopes of mTOR, 4E-BP1 and ribosomal protein S6, in conjunction with Ki-67 or the transferrin receptor CD71 and maturation markers, in primary cells of pre-treatment samples from patients with AML. MTOR phosphorylation was measured at serine 2448. This phospho-epitope is lost when raptor is depleted, indicating its specificity for mTORC1 [19]. MTOR is phosphorylated at serine 2448 by p70S6 kinase: whereas the phosphorylation is not thought to be intrinsically "activating", it can be used as an indicator of the level of mTOR signalling because p70S6 kinase activity is, in turn, mTOR-dependent [20,21]. S6 kinase also phosphorylates ribosomal protein S6 (rpS6) [22]. Antibodies to ribosomal protein S6 (rpS6) phosphorylated at serine 235/236 have been optimised for flow cytometry, where they are well-established as biomarkers for mTORC1 activity [23,24]. A second major target of mTOR is 4E-BP1, which is directly phosphorylated by mTOR at T36/T45 [25]. 4E-BPs control protein synthesis [5,26] and mediate mTORC1-dependent cell proliferation [27].
Conditional deletion of raptor from AML cells has revealed that mTORC1 deficiency increases the proportion of undifferentiated and self-renewing haematopoietic cells and decreases the proportion of differentiated cells in vivo [28], suggesting that mTOR might be most activated in the context of maturation rather than self-renewal. This would have implications for mTOR as a therapeutic target, and we therefore also measured mTOR activity in undifferentiated (CD34+CD38-) AML cells and those with monocytoid maturation compared to blast cells.
with 10% foetal calf serum (FCS; First Link, Birmingham, UK) and 2mM L-glutamine. KG1a experiments were performed with cell lines in log phase and were authenticated by genetic fingerprinting.
Antibody labelling
In order to allow metabolic activity to resume after processing, thawed or freshly isolated mononuclear cell preparations were rested for 90 minutes in medium consisting of 20% foetal calf serum (First Link) in RPMI. They were fixed using paraformaldehyde to a final 2% concentration. After 15 minutes' incubation at room temperature (RT), neutralising buffer (1.7 M Tris base, 1.25 M Glycine, pH 9.1) was added at twice the volume of paraformaldehyde solution and the cells were incubated for a further 15 minutes. Cell membranes were permeabilized with intracellular staining buffer containing 0.1% saponin. Cells were labelled with Alexa Fluor 647-conjugated antibodies to mTOR p-serS2448 (BD#564242), 4E-BP1 p-thr36/45) (BD#560286) or rpS6 p-ser235/236 (CST#4851), and with antibodies to Ki-67 (BD556026) or CD71 (BD #555537). The phenotype of the cells was determined using CD45, CD34 and CD38 antibodies. In the stem cell harvest analysis CD38 was replaced by CD2. Labelling took place overnight at 4°C. Preliminary experiments indicated that the cell surface phenotype was not affected by this procedure, whilst sensitivity for the Ki-67 and mTOR pathway epitopes was improved compared to a short incubation (data not shown).
Flow cytometry and standardisation
Fluorescence overlap was compensated and data were collected on a FACSCanto II flow cytometer (Becton Dickinson). Data were analysed with FACS Diva software (Becton Dickinson). Additional graphics were produced using Flowing software (www.flowingsoftware.com). In order to be able to compare expression levels obtained on different occasions, all mTOR, rpS6, 4E-BP1 and their isotype control mean fluorescence intensity values were converted to standardised values using standard curves obtained with Rainbow Calibration Beads (BD#559123), before subtracting control from test values. The resulting values termed standardised relative fluorescence intensity (SRFI) units were used throughout. In samples with <1% events in the CD34 + CD38subset, this subset was not included, but otherwise sufficient events were counted (10,000-200,000) to allow a reliable coefficient of variation, as described previously [29].
Statistics. Comparisons between paired subsets were made using the Wilcoxon signed rank test and between blasts and normal CD34 + cells by Mann Whitney analysis, with SPSS software.
Methodology
We measured mTOR phosphorylation at serine 2448 (hereafter termed p-mTOR), which is dependent on mTOR kinase activity [20]. We also measured ribosomal protein S6 phosphorylated at serine 235/236 (hereafter termed p-rpS6) [22,30], a downstream target of mTORC1. Previous studies have shown p-rpS6 to be a sensitive marker for mTORC1 activation in flow cytometric studies [23,24,31]. However, although ERK activation can stimulate mTOR activity through TSC2 [32], rpS6 can be additionally phosphorylated at S235/236 in an mTORC1 independent/ERK dependent manner by p90S6 kinase [33]. To confirm the mTORC1 dependence of rpS6 S235/236 phosphorylation in AML cells, we first incubated cells with rapamycin with and without the MEK (ERK pathway) inhibitor U0126 in KG1a cells and measured p-mTOR and p-rpS6. At a concentration where U0126 induced 32% inhibition of both phosphoepitopes rapamycin inhibited both epitopes by 99%, confirming that MEK-mediated activation of rpS6 is upstream of mTOR in these cells and that p-rpS6 is a reasonable surrogate for rapamycin-sensitive mTORC1 activation ( Fig 1A). In patient blasts, p-rpS6 and p-mTOR were also totally or mostly inhibited by rapamycin (80-100%, median 95%, Fig 1B). Fig 1C indicates a rationale for why p-rpS6, with a much stronger signal than p-mTOR, is a useful surrogate for mTORC1 activity. AML samples were selected to provide a wide range of cytogenetic and molecular abnormalities ( Table 1). The gating strategy for comparing Ki-67 positive and negative blasts is indicated (Fig 1D). The use of CD45/side scatter (SSC) gating to distinguish blasts from lymphocytes and from leukaemia cells with cytoplasmic maturation and monocytoid differentiation (SSC high /CD45 high ) is well described in the literature [34][35][36][37].
2. The p-rpS6ser235/236 signal is amplified in AML blasts compared to mobilised normal CD34 + cells Total p-mTOR-ser2448 expression was compared in blasts from presentation AML samples and mobilised SCH CD34 + and CD2 + cells. P-mTOR was negligible in the lymphocytes. Using a mean +/-2 standard deviation range for the five stem cell harvest CD34 + subsets, we calculated that no AML sample had p-mTOR greater than the normal CD34 + cells (Fig 2A). In contrast, AML blast rpS6 phosphorylation was higher than that of CD34 + SCH cells in 11/13 samples ( Fig 2B, P = 0.002). Marked heterogeneity was noted both between and within patient samples (Fig 2C).
The mTOR-RPS6 pathway remains active in dormant AML cells
We reasoned that p-mTOR would be lowest in dormant cells and therefore compared Ki-67 positive and negative blasts. Contrary to expectation, we found no overall difference in mTOR activation between these subsets ( Fig 3A). However, we also found 70% of normal SCH CD34 + cells to be Ki-67 + , also with no difference in mTOR phosphorylation between the Ki-67and Ki-67 + subsets, indicating that the Ki-67subset with active mTOR is not a leukaemia-specific phenotype. SCH CD2 + (lymphocyte subset) cells were 99% Ki-67and these had negligible p-mTOR, as expected [17]. Since rpS6 S235/236 phosphorylation is blocked by rapamycin, we also expected this to be low in dormant cells. However, p-rpS6 tended to be higher in Ki-67 than Ki-67 + subsets, and using Wilcoxon signed rank test, the difference was significant (Fig 3B, P = 0.03). Heterogeneity both between and within samples was marked ( Fig 3C). CD71, the transferrin receptor, is expressed on all proliferative cells, and its absence is characteristic of dormant populations such as resting lymphocytes [38]. In a previous publication, we showed that AML cells can express the transferrin receptor CD71 whilst remaining Ki-67 - [18] and therefore we wondered whether a CD71 + population might include a subset metabolically primed to enter G1 but without Ki-67 expression. No consistent pattern emerged from our analysis (Fig 3D and 3E). Notably, sample A2 expressed high levels of p-rpS6, although it had <1% CD71blasts. However, in samples A8 and A13, in both of which expression of CD71 was high (see Table 1), the relative overexpression of p-rpS6 in the Ki-67subset was reversed in the CD71subset.
4. Ribosomal protein S6 is phosphorylated in CD34 + CD38subsets and in cells with myeloid maturation 8/13 samples had sufficient CD34 + CD38cells (primitive subset) for analysis. The CD34 + CD38subset had higher p-rpS6 than the total blast population (Fig 4, P = 0.02). A Fig 1. Methodology. (A) Inhibition of p-mTOR and p-rpS6 fluorescence in KG1a cells (mean + standard deviation of three independent assays) following a 90 minute incubation with 100nM rapamycin or 3 μM U0126. The percentage inhibition was established by also incubating cells with 100nM rapamycin statistically significant difference in p-mTOR was not observed. MTOR activation features in the maturation and differentiation of normal myeloid cells [8]. Maturing leukaemia cells have an increased cytoplasmic complexity which is shown flow cytometrically as increased side scatter, and furthermore CD45 expression increases with monocytoid maturation [34][35][36]. To examine mTOR activation in maturing cells, we re-gated our samples on a subset with increased side scatter and high CD45 (illustrated in Fig 1D as region 4). 6/13 samples, all with M4 or M5 FAB types, had distinct CD45 high SSC high subpopulations. MTOR and RpS6 were more highly phosphorylated in this subset (P = 0.05, 0.03 respectively).
5. 4E-BP1 phosphorylation is decreased in Ki-67and CD34+CD38 subsets, contrasting with S6 phosphorylation 4-EBP1 thr36/45 phosphorylation is mediated directly by mTOR [25] and is thus an additional indicator of mTOR activity. We evaluated a directly conjugated antibody to this epitope and were able to document reproducible measurements in SCH CD34+ cells. P-4E-BP1 was documented in these mobilised, normal CD34+ cells but not lymphocytes from stem cell harvests + 3 μM U0126 to generate a functional fluorescence baseline. (B) Inhibition of -rpS6 and p-mTOR fluorescence in five primary samples following a 90 minute incubation with 100nM rapamycin. P-mTOR modulation was not measured in the two samples with a negligible basal signal (A3, A8). A fluorescence baseline was generated as for (A) above. (C) Scatterplot and trendline of p-rpS6 against p-mTOR in patient blasts, Spearman's rho = 0.54, P = 0.05). SRFI (standardised relative fluorescence intensity) values were calculated as detailed in Materials and Methods. The two values shown in red correspond to samples A3 and A8 used in Fig 1B, such that taken together the plots illustrate that rpS6 phosphorylation is rapamycin-sensitive (plot 1B) despite the low measured values for mTORser2448 phosphorylation in these two samples (Plot 1C) and is thus p-rpS6 is a useful surrogate when the p-mTOR antibody signal is negligible. (D) Gating strategy; (i) and (ii) Scatter gates to exclude doublets and debris; (iii) CD45/side scatter gate to select leukaemic blasts (P3) or cells with monocytoid maturation (P4); (iv) gating of CD34 + /CD38blast subset; (v) Fluorescence minus one tube analysis to set accurate isotype control fluorescence for Ki-67 (FITC) and p-mTOR or p-rpS6 (APC) in P3-gated blasts; (vi) Ki-67 and antigen of interest co-expression in P3-gated blasts. Quadrants and fluorescence baselines were set using isotype controls. Mean APC fluorescence intensity for (Q1-1+Q3-1) dormant and (Q2-1 + Q4-1) cycling cells is recorded.
Discussion
In this study, we have measured the phosphorylation status of mTOR pathway molecules in leukaemia cells in conjunction with their proliferation status. CD34 + and CD2 + cells from mobilised SCH provided normal controls. Absence of Ki-67 has long been used to distinguish cells that have exited from the cell cycle, and indeed the CD2 + lymphocytes had the expected Ki-67phenotype [17] with negligible p-mTOR, 4E-BP1 or p-rpS6. In contrast, the normal SCH CD34 + cells expressed phosphorylated mTOR and 4E-BP1. These mobilised SCH CD34 + cells were mostly (70%) Ki-67 + , showing that the majority of these were cycling, and we demonstrated in Figs 3 and 5 that p-mTORser2448 and p-4E-BP1thr36/thr45, biomarkers of mTORC1 activation, were no higher in the cycling than the dormant fraction of these normal cells. As mTOR signalling is a central mediator of metabolic activity, driving energy generation, The latter were gated for subsets expressing CD34 or CD2. The shaded area indicates ± 2 standard deviations of the mean for the normal CD34 + cells. C. Flow cytometric distributions of p-rpS6 for one representative stem cell harvest (CD34 + gated cells) and three patient samples (P3 blast-gated cells-see Fig 1D). Flow cytometric histograms illustrate fluorescence signals from test antibody (shaded) compared to isotype control (unshaded).
doi:10.1371/journal.pone.0151480.g002 macromolecule synthesis and cell growth [6][7][8], our results suggest that metabolic activity can be as marked in Ki-67as in Ki-67 + cells. Likewise, AML samples failed to show upregulation of mTOR and rpS6 phosphorylations in the Ki-67 + subset. Only 4E-BP1 phosphorylation was higher in the the Ki-67 + subset, in keeping with its specialised role in proliferation [27], but the increase, although statistically significant, was a modest 15%. On balance, the data indicate that Ki-67mobilised, normal CD34+ cells as well as Ki-67leukaemic blasts are metabolically active. Dormant cells do not all behave in the same way: genes activated and repressed vary with the length of time since the last mitosis and the nature of the quiescence-inducing stimulus [39]. Moreover, dormant short-term haematopoietic stem cells (HSCs) express higher CDK6 than long-term HSCs [40]. It is highly probable that normal, mobilised CD34 + cells as well as leukaemia cells, even where exit from cycle is indicated by Ki-67 or CD71 negativity, tend to cycle much more frequently than unstimulated lymphocytes, and maybe this is associated with the ability to maintain a high level of metabolic priming. Dormant AML cells selected by absence of Hoechst33342 and pyronin Y staining have been found to re-enter the cell cycle without exogenous stimulation [14]. Our data suggest that phenotypically dormant leukaemia cells would not be resistant to metabolic targeting such as the synthetic lethal co-activation of AMPK and mTORC1 recently described in AML [41]. Our second unexpected finding was that, when compared to normal CD34 + cells from stem cell harvests, p-mTOR was low, 4E-BP1 was 2.6-fold elevated in AML blasts, and p-rpS6 was increased by 22-fold in the blasts, with an additional 95% elevation in leukaemic CD34 + CD38cells. What could account for this discordance, especially taking into account that rpS6 phosphorylation is downstream of mTOR activation, as indicated by rapamycin inhibition in Fig 1? Differential phosphorylation of rpS6 and 4E-BP1 by mTORC1 is well-described in the literature, particularly with regard to the variable rapamycin sensitivity of 4E-BP1, which may be attributable to the influence of PIM kinases [5,[42][43][44]. Differences in total rpS6 expression levels between the cell types are likely to contribute to the high phosphorylation of this protein that we have documented in AML blasts, as shown in S1 Fig. Differences in subcellular localisation may be a contributory factor, as may be relative expression and activation of endogenous inhibitors of rpS6 phosphorylation such as protein phosphatase 1, or activators such as p70S6 kinases and casein kinase [45][46][47][48]. RpS6 has additional binding partners which may be involved in its regulation, including the protective chaperone HSP90 [49]. Examination of the 200 AML patient mRNAs in the Cancer Genome Atlas database revealed a close correlation between rpS6 and HSP90 at the level of mRNA expression in primary samples (dataset available from cbioportal.org) [50,51].
The issue of whether malignant myeloid cells might be more dependent on rpS6 activity than normal dividing CD34+ cells is also raised by our findings. RpS6 phosphorylation-mutant mice are characterised by small cell size, decreased ribosomal biogenesis and muscle weakness, associated with low ATP levels despite normal mitochondrial oxygen consumption [52][53][54]. The mice have low protein levels of mitochondrial enzymes NDUFS4, and UQCRH [53]. The possible relevance of this latter observation to AML is supported by our examination of the TCGA database, in which these enzymes correlate with rpS6 at the mRNA expression level (S1 Table). The expectation of aerobic glycolysis in all cancer cells has been unravelling over the past few years [3], and leukaemia initiating cells have been shown to use oxidative phosphorylation [55]. By stimulating synthesis of respiratory chain enzymes, rpS6 may contribute to this.
MTOR activity is essential for normal multi-lineage haematopoiesis [56]. The finding that both mTOR and rpS6 are highly phosphorylated in monocytoid AML cells supports a previous finding of high p-rpS6 in these cells [23]. The literature on this topic is complex: whilst mTOR inhibition has been shown to induce differentiation in myeloid leukaemic cell lines, possibly through prolonging PU1 effects, it has also been shown to preserve an undifferentiated phenotype and long term survival in AML cells in vivo and in vitro [28]. It would also seem logical for differentiated cells to require mTOR pathway activation to meet the metabolic needs of specialisation, and in this respect the monocytoid cells would appear to follow a normal maturation pattern. This contrasts with the hyper-phosphorylation of rpS6, but not of mTOR, in AML blasts, especially in CD34 + CD38cells. Our data indicate that differences between cell subsets need to be taken into account in predicting and monitoring cellular responses to therapeutics.
A couple of technical discussion points may be useful to some. MTOR pathway phosphorylation is highly dependent on the environment, and our protocol included a 90 minute preincubation in serum-containing medium. We abandoned initial protocols of positive selection for CD34 + cells from stem cell harvests and of culture in media without serum, as these diminished the signal. The decision to use fluorescence intensity rather than percentage of positive cells was taken based on observations such as those illustrated in Fig 4E, where the whole population's fluorescence may shift compared to the isotype control, but a highly fluorescent subpopulation may also be seen. Mean fluorescence changes better encompass these two types of change than percentage positivity.
In conclusion, our results indicate that biomarkers of metabolic activity are highly expressed in the Ki-67subsets of both normal SCH CD34 + cells and AML blasts. Moreover, although rpS6 ser235/236 phosphorylation is commonly thought of as a surrogate for upstream mTOR activity, our results suggest a hyper-activation of p-rpS6 in the malignant cells only that raises new questions. Firstly, is the hyper-phosphorylation of rpS6 simply an epiphenomenon of oncogene-driven activity, or is it metabolically important (and thus potentially druggable)? Secondly, given the strength of the p-rpS6 signal in AML cells, is dephosphorylation a suitable predictive biomarker for therapeutic responses? AML modalities, such as DNA damaging agents and receptor kinase inhibitors, converge to dephosphorylate rpS6 through their actions on the akt/mTOR, ATM/AMPK/mTOR and ERK pathways, suggesting that further research in this area could be fruitful.
Supporting Information S1 Table. RPS6 correlations from the Cancer Genome Atlas AML samples. RpS6 phosphorylation is specifically associated with expression of NDUFS4, and UQCRH respiratory chain genes in vivo [53]. MTOR activation is associated with transcriptional activation of a wide range of genes, notably HIF1, PFPK and PDK1 (glycolysis), G6PD and RPIA (pentose phosphate pathway), MVK and SC5D (lipid and sterol biosynthesis, see additional reference). We determined the relationship between mRNA expression levels of rpS6 and these genes in the TCGA data of 200 AML samples. Only NDUFS4, and UQCRH were in the top 1% of genes associated with RPS6 (datasets available via cbioportal.org, refs [50,51]. | 2016-05-12T22:15:10.714Z | 2016-03-17T00:00:00.000 | {
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28302856 | pes2o/s2orc | v3-fos-license | Special Editorial
The Medical Council of India (MCI) must be commended for its efforts to introduce definitive criteria for appointments and promotions for teachers in medical institutions. On June 8, 2017, the MCI issued a circular to amend the Minimum Qualifications for Teachers in Medical Institutions Regulations, 1998 (henceforth Regulations, 1998). The amendment clarifies the minimum qualifications required for various postgraduate teaching positions in medical colleges. It indicates MCI's sustained engagement with qualifications of teachers in medical colleges, with the aim of enhancing the quality of teaching and thereby the quality of medical professionals passing out. However, we believe that these efforts continue to be inadequate in addressing the varied issues that face medical education and the educators in India.
T he Medical Council of India (MCI) must be commended for its efforts to introduce definitive criteria for appointments and promotions for teachers in medical institutions. On June 8, 2017, the MCI issued a circular [1] to amend the Minimum Qualifications for Teachers in Medical Institutions Regulations, 1998(henceforth Regulations, 1998 [2]. The amendment clarifies the minimum qualifications required for various postgraduate teaching positions in medical colleges. It indicates MCI's sustained engagement with qualifications of teachers in medical colleges, with the aim of enhancing the quality of teaching and thereby the quality of medical professionals passing out. However, we believe that these efforts continue to be inadequate in addressing the varied issues that face medical education and the educators in India. Some of these issues are: (i) the lack of transparency in the manner in which new medical colleges are approved, (ii) the variation in the proportion of private and public medical colleges across states, (iii) the lack of change and innovation in the undergraduate and postgraduate medical curricula to keep up with changing needs, (iv) the poor uptake of newer teaching-learning methods, (v) the poor quality of teachers in several medical colleges, (vi) methods used to assess teachers during selection and promotions, and (vii) failure to assess the impact of policy changes (such as a recent increase in the number of postgraduate seats) on the quality of medical education and training.
In this editorial, we focus on one of these issues, ie, the appointment and promotion of teachers in medical colleges. The MCI had on September 3, 2015 [3] stated its requirements with regard to research publications for eligibility for promotion of faculty members in medical colleges. This had been critiqued [4,5] mainly on four counts: exclusion of publications in 'electronic-only' journals from consideration for assessment of performance; awarding points only to original research articles or papers; awarding points only to first or second authors; and, the choice of indexing services for assessing the quality of a journal. While lauding the MCI's efforts towards improving the standards for teaching faculty at medical colleges in India, these critiques argued that an ill-informed framework for determining eligibility for promotion is likely be self-defeating and even harmful to the profession.
A few steps forwards and a few steps backwards
The 2017 Amendment [1] is noteworthy for it states that in order to be eligible for assessment, a paper must be published in "indexed" journals. This appears to be a step in the right direction because indexing indicates that a journal meets certain standards and quality within a specialty, specific to a particular index. However, in this amendment, the MCI has not specified any particular index(es). Thus, possibly the list of indexes previously specified in its 2015 order will continue to apply. Let us look at this issue more closely. Accepting only the MEDLINE-indexed journals would exclude some research in valuable allied fields, such as medical humanities, basic sciences and social sciences as applied VOLUME 55 __ FEBRUARY 15, 2018 SPECIAL EDITORIAL to medicine. Thus, inclusion of other indexes may be useful for recognizing these diverse related disciplines beyond the pure health sciences, although with due diligence. For example, some indexes have little credibility as they are known to include pseudojournals (also known as 'predatory journals') in their listings. Hence, it is important that MCI specifies only those indexes which are reputed to have quality journals. The 2015 MCI list of eligible indexes has been faulted on this score [5]. Aggarwal and colleagues [5] had suggested the following list of acceptable databases: Medline, PubMed Central, Science Citation Index, Embase/Excerpta Medica, Scopus and IndMED. The latest amendment missed out on an opportunity to revise the list of eligible indexes.
The amendment does not specify whether or not papers published in "e-only" indexed journals are acceptable for assessment. Here too, possibly the stipulation in the 2015 order, that e-journals, are not included, will continue to apply. Currently, many e-only journals (e.g. PLoS group, BioMed Central, etc) are comparable in quality to, and at times even better than, those published as hard copy. Their inclusion would allow a much wider range of journals for the faculty to choose from for publishing their work.
Unfortunately, as with Regulations, 1998 [2], the 2017 Amendment [1] also limits the credit for authorship to only some of those listed on the author byline. Unlike the first version in which those listed as the first and the second authors were eligible, the amendment gives the credit for a paper to only the first author and the corresponding author. As critiqued earlier, this approach inhibits collaborative research and could be counterproductive by undermining the advancement of knowledge. Some of the best research today is multidisciplinary and multi-author.
Problems untouched
The MCI regulations, even after the recent amendment [1], are problematic in other aspects too. These lay down two criteria to assess a candidate's eligibility for a particular position: duration of service and number of research publications. One would expect the parameters assessed during appointment and promotion to be aligned with the responsibilities of teachers in medical colleges, with a strong convergence suggesting appropriateness and sufficiency of the criteria. In clinical or paraclinical departments, medical teachers have three primary activities; providing clinical or laboratory/imaging service, teaching, and doing research; which vary for different specialties However, in most medical colleges, irrespective of specialty, the research activity forms a small part of the total work of a medical teacher. Hence, any assessment of only research output without an assessment of the other two domains does not appear to be reasonable. What about the other two activities? Provision of clinical or laboratory services and teaching are integral to the core work of a medical faculty member. The assessment, if any, of these domains is only by the years of service put in. This appears unfair. The MCI regulations should address the issue of assessing medical faculty in all the three activity domains. Undue focus on research and not on the other two domains might prove to be detrimental both to the training of medical students as well as to clinical work.
Failure of the faculty to do research is a welldocumented problem in Indian medical colleges. It has been argued [6] that this is due to commercialization of medical education in the country. However, we believe that this phenomenon is multifactorial. One major reason may be a lack of interest and training to do research on part of the teachers, or of lack of infrastructure to facilitate research on part of the institutions. Also, good research requires financial resources -and most of the institutions, whether funded publicly-or privately have no or little funds dedicated to this activity. These factors may need to be corrected first, before we can expect research to be an important criterion for assessing eligibility for appointment and promotion of medical teachers.
Lack of adequate funding too discourages the MCI's approach. India has nearly 450 medical colleges. Let us assume that each college has around 100 teachers, and that each of the nearly 45,000 teachers needs to publish a research paper every three years. This translates to around 15,000 research papers a year. In addition, around 20,000 students join a medical postgraduate course every year in the country, and each of them has to write a thesis. Let us assume the "most optimum" case scenario -that each thesis results in a paper with a student and his teacher-guide as the two eligible authors. Even with this unlikely scenario, we would require to generate at least 20,000 new research ideas every year -a formidable task. For these research works to be novel and publishable, a large proportion of these ideas would need fundingwhich is currently not available.
Another important consideration is whether there are sufficient peer-reviewed, "indexed" journals to publish this large body of work. The requirement to publish by teachers in Indian medical colleges and universities has seen a proliferation of "predatory journals" in India [7][8][9].We are not arguing that research may be altogether abolished as a criterion for eligibility and assessment of VOLUME 55 __ FEBRUARY 15, 2018 SPECIAL EDITORIAL medical faculty. It is known that good research institutions globally and in India are sought after by students and patients alike, as these are considered better centres for learning and providing a better quality of care. However, whether the quality of teaching and patient care can necessarily be improved by mandating research of whatever quality remains uncertain.
Thus, it is not reasonable to make an assessment for promotion of a medical teacher solely on the basis of research activity -that too by counting the number of publications.
The way forward
It is evident from the above that the assessment of medical teachers must encompass all the three domains of their activities. Furthermore, the assessment should focus not on quantity, as is done currently by counting only years of service or number of papers, but on the quality of work in each sphere. Unfortunately, we will be told that "assessment of quality" would not be objective, and would be liable to bias and manipulation. However, this is an excuse for not doing what seems to us the right thing to do. Around the world, as in many fields in our country, employees are assessed using the so-called "subjective" criteria, with sufficient reliability. Setting up such systems -though admittedly hard -is not impossible. These will surely take time, effort and commitment to set up. But if we unquestioningly accept the simplistic tools such as publication count, we will never move to a higher plane. Hence, as a profession, we need to initiate debate for moving towards better systems of assessing quality. Such an assessment system would necessarily mean a multi-pronged evaluation -by peers, students as well as administrators.
Variation is an important rule of nature and all medical teachers cannot be expected to have exactly the same skill set. Thus, one of them may be an excellent researcher, but not a particularly good teacher. Similarly, someone else might be better at providing a laboratory service than doing research. This is in fact desirable since it allows some persons to excel in one specific area beyond the average skill level expected, and should be encouraged. This requires that individuals with different skill sets and inclinations be provided the opportunity to do more of what they are good at and less of what they may not be so skilled at. The proportion of time spent on the three core activities referred to above could thus vary between different teachers. Thus, it would be reasonable that the assessment of medical teachers for the quality of work would be a weighted average of the quality of work in the three domains, with the weights decided by the predefined proportion of time spent by each teacher on activities in these domains. Clearly defining each faculty member's job description at the time of appointment or during the course of service will facilitate and/or enable such an approach to assessment.
Each core activity could be assessed using different parameters. Teaching should be assessed by the end user; ie, the student and the performance of students in an assessment should be part of the assessment of a teacher. Similarly, peers should sit in on teaching activities and provide a peer evaluation. These suggestions are neither exhaustive nor necessarily tested to be appropriate for our milieu. Hence, a constant evaluation and evolution of these methods would be essential.
Research should be evaluated but not by the number of publications. The quality of a medical faculty's research output should be assessed. This would include a peer evaluation of the individual's select few publications -a smaller number at the time of selection and an increasing number with each step in the academic ladder. For example, two best papers at the time of initial selection as a faculty member, five best at the next level, and seven and ten in the further steps. As almost all medical faculty positions in India are tenured, there are few who would make the effort to write a grant application and obtain funding. Those who do so should be assessed on the quality of their grant applications or the amount of funding obtained.
How does one assess the service component of the medical faculty? This could be difficult to do but an effort should be made to use laboratory and clinical audits, and peer and patient assessment and feedback.
All this must be done transparently. The assessors, the method and process of assessment and the final evaluation must all be transparent. Anybody can make errors and hence there must be a transparent system of appeals and evaluations of appeals. Questioning a decision with sound reasoning must be permitted but the process must be free of corruption. | 2018-11-10T06:17:23.830Z | 2018-01-01T00:00:00.000 | {
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228995723 | pes2o/s2orc | v3-fos-license | Confusion, difficulty concentrating, and slow response induced by ropinirole therapy in an elderly patient with secondary Parkinsonism: a case report
Background: Ropinirole is a therapy widely used in patients with Parkinson's disease or Restless Leg Syndrome (RLS). A number of research journals report on the long-term use of ropinirole causing side effects to patients. Unfortunately, there are no published studies from Indonesia that document the side effects of ropinirole, especially in elderly subjects. Case Presentation: This case study reports a number of side effects, such as confusion, difficulty concentrating, and slow response, experienced by a 75-year-old elderly patient who consumed ropinirole 2 mg as a therapy for secondary Parkinsonism. Prior to using ropinirole, the patient had taken levodopa-benserazide, trihexyphenidyl, and pramipexole to overcome Parkinson's for more than 6 years during which no side effects occurred. Side effects appeared after ropinirole therapy was given to the patient, thereby strongly suggesting the administration of ropinirole as the underlying cause of such effects. The assessment methods involved time series data collection followed by causality analysis using the Naranjo Scale, resulting in a score of 6, or Probable. According to some literature, such side effects are probably triggered by the presence of neurotransmitter imbalance, one of which is due to the activation of dopaminergic receptors. Ropinirole is a dopamine agonist drug normally working on dopamine receptors, especially on D2-receptors. An excessive activation of dopaminergic receptors can lead to neuropsychiatric symptoms. In addition, the activation of D2-receptor causes an inhibition of acetylcholine synthesis, resulting in cholinergic deficiency that also becomes one of the triggers for neuropsychiatric symptoms. Conclusion: The analysis using the Naranjo Scale shows a score of 6, indicating a probable association of confusion, difficulty concentrating, and slow response with ropinirole in this elderly patient with secondary Parkinsonism.
Introduction
Parkinson's is one of the most common neurodegenerative diseases in elderly patients (Willis, 2013). Discovered in 1960, Levodopa has since become the main therapy widely prescribed for Parkinson's patients. However, as the world of knowledge develops, a large number of other drugs begin to be used for Parkinson's as both primary therapy and adjunctive therapy, and one of them is in the dopamine agonist class.
In practice, dopamine agonists play an important role in Parkinson's therapy. Literature suggests that adding dopamine agonists to the regimen can reduce the symptoms of dyskinesia in patients (Konta & Frank, 2008). In general, dopamine agonists are classified into two main groups: dopamine agonists with ergotic structure and dopamine agonists with non-ergotic structure.
Since its approval by the FDA in 1997 and Badan Pengawas Obat dan Makanan (BPOM) in 2012, ropinirole, a non-ergoline dopamine agonist drug, has been widely used as a treatment for Parkinson's and Restless Leg Syndrome (RLS). The effectiveness of ropinirole has been proven through various published studies, but the side effects were seldom reported.
Until this article was written, only two case reports documenting the side effects of ropinirole were found globally (Grover & Ghosh, 2010;Pal et al., 2012) while there were no reports of such adverse events in Indonesia. Documentation and publication of the side effects of drugs are important to increase pharmacist's or other healthcare professional's awareness of patients with especially long-term use of drugs.
Methodology
We report on a case of ropinirole side effects and describe the pharmacological background.
The assessment methods used were time series data collection followed by causality analysis using the Naranjo Adverse Drug Probability Scale.
Case report
Mr. U is a 75-year-old man diagnosed with Parkinson's since approximately three years ago.
During this period, the patient was treated by Doctor F and prescribed the following drugs: trihexyphenidyl 2mg (3x daily 1 tablet), pramipexole 0.125mg (3x daily 1 tablet), and a combination of levodopa 100mg -benserazide 25mg (3x daily 1 tablet). While consuming the medicines, his Parkinson's symptoms were controlled, and he did not experience any side effects. Based on the medical history, the patient also had other diseases, including Benign Prostatic Hyperplasia (BPH) treated with mirabegron, tamsulosin, silodosin, and dutasteride. In addition, he also routinely used other drugs: candesartan 8mg (1x daily 1 tablet), coenzyme 100 (1x daily 1 tablet), bisoprolol 2.5mg (1x daily 1tablet), and clopidogrel 75mg (1x daily 1 tablet). The only laboratory examination performed on the patient was serum creatinine carried out on November 22 nd , 2017 and resulting in normal serum creatinine (1.1 mg/dL).
In September 2018, the patient came to other doctors (doctor B and doctor S) because he had such symptoms as tremors, limb rigidity, bradykinesia, postural instability, and retropulsion. After a number of examinations, both doctors gave the same diagnosis as the previous one, thereby allowing the patient to continue the treatment from Dr. F without any additional new regimen. In to secondary Parkinsonism, resulting in addition of ropinirole 2mg (1x daily 1 tablet) to the ongoing treatment.
In June 2019, the patient's family came to the pharmacy to report the discontinuation of ropinirole use as he developed some symptoms, including confusion, difficulty concentrating, and slow response. The patient then tried to take the medicine every 3 days and once a week, thereby resulting in reduction but persistence of symptoms which led to a stop of ropinirole consumption.
Having received such report, the pharmacist then analyzed the possibility of side effects using the Naranjo Adverse Drug Probability Scale, thus obtaining a score of 6 which indicated Probable. The pharmacist then discussed with the doctor who eventually agreed to stop administering ropinirole.
Discussion
Parkinson's is a long-term degenerative disease of which the causes remain unknown but are believed to involve both genetic and environmental factors. Some literature stated that one of the suspected causes of Parkinson's is the loss of cells containing melanin in the substantia nigra located in the brain, leading to degenerative loss of neurons in the corpus striatum of mesencephalon. This structure also functions as a producer of neurotransmitters, including dopamine. Due to such cell disappearance, dopamine is not sufficiently produced in the brain, resulting in movement disorders that are commonly found in Parkinson's patients (Meder et al., 2019).
In the case reported herein, a 75-year-old male patient had been diagnosed with Parkinson's for more than three years, which then developed into secondary Parkinsonism. Based on literature studies, patients with secondary parkinsonism have the same symptoms as those with Parkinson's disease, but the causes vary from drugs or exposure to hazardous chemicals to other diseases (Falvo & Holland, 2018). In this case, however, the cause of secondary Parkinsonism in the patient was not explicitly mentioned.
During the treatment for Parkinson's and secondary Parkinsonism, the patient was given a combination of trihexyphenidyl 2mg (3x daily 1 tablet), pramipexole 0.125mg (3x daily 1 tab), and levodopa 100mg-benserazide 25mg combination (3x daily 1 tab). The regimen has been used for more than three years during which the patient's condition was controlled and no drug side effects developed. When ropinirole 2 mg was added to the therapeutic regimen, however, the patient started experiencing side effects, such as confusion, difficulty concentrating, and slow response.
Literature suggests that the side effects of these drugs may be categorized into neuropsychiatric symptoms. Neuropsychiatric is a term that refers to a variety of medical conditions involving neurology and psychiatry (Sachdev & Mohan, 2013). The hypothesis of drug side effects was strengthened through an analysis using the Naranjo Adverse Drug Probability Scale, which yielded a score of 6 indicating Probable (Table 1). Probable means the reaction follows a reasonable temporal sequence after a drug, follows a recognized response to the suspected drug, is confirmed by withdrawal but not by exposure to the drug, and cannot be reasonably explained by the known characteristics of the patient's clinical state.
References suggest that neuropsychiatric side effects are likely to arise due to various factors.
One approach that can be used in this case is through the mechanism of delirium. In this report, the patient experienced some symptoms similar to those found in delirium patients, but they were not necessarily identified as delirium. Further examination and analysis are required to diagnose delirium in patients.
Some of the mechanisms contributing to delirium include cholinergic deficiency, disruption in the synthesis of acetylcholine, or neurotransmitter imbalance (Hshieh et al., 2008).
Neurotransmitter imbalance involves acetylcholine, dopamine, or gamma aminobutyric acid (GABA) (Alagiakrishnan & Wiens, 2004). Some neurotransmitters in the brain such as dopamine, norepinephrine, and serotonin play a role in arousal and sleep-wake cycle as well as in mediating psychological responses to the presence of stimuli. Excessive stimulation of dopaminergic receptors results in decreased acetylcholine, thus leading to a variety of neuropsychiatric symptoms. This phenomenon occurs due to an overlap of the dopaminergic and cholinergic pathways in the brain, thereby suggesting a close relationship between acetylcholine and dopamine activities (Hshieh et al., 2008).
Ropinirole is a group of dopamine agonist drugs that acts primarily on Dopamine D2 receptors. Activation of dopamine receptors decreases acetylcholine synthesis, triggering obstacles in the cholinergic pathway and resulting in neuropsychiatric symptoms found in the patient concerned. In addition, physiological changes in the body also play a role in the existence of neuropsychiatric symptoms. In elderly patients, the response to drugs can be influenced by the physiological changes due to aging. Some factors that can affect include increased body fat, decreased lean body mass and water, decreased albumin, and decreased Glomerular Filtration Rate (GFR) (Alagiakrishnan & Wiens, 2004).
Several other co-morbidities also affect the action of drugs in the elderly. For example, in patients with liver disorders, albumin synthesis usually decreases, thereby reducing protein binding and increasing the volume of distribution of some drugs. A decrease in kidney function also reduces drug elimination, which eventually increases the concentration of some drugs. Increased blood concentration of drugs can escalate drug effects thus triggering neuropsychiatric symptoms such as those found in delirium patients (Alagiakrishnan & Wiens, 2004). In this case, such reasons can also affect the occurrence of side effects as mentioned in Naranjo Scale point 5. In addition, administration of other drugs and other diseases can contribute to the development of side effects.
Therefore, further research is required to investigate the exact cause of the side effects experienced by the patient. Interpretation of Scores: Total score ≤0: doubtful, total score 1-4: possible, total score 5-8: probable, total score >9: definite The influence of neurotransmitter imbalance on neuropsychiatric symptoms can be strengthened by the presence of Serum Anticholinergic Activity (SAA) levels. However, in this study, laboratory tests have not been conducted to determine the quantitative levels of the neurotransmitters in the body. Therefore, it is recommended to perform a more in-depth examination related to SAA level to confirm the existence of these side effects due to the use of ropinirole.
Conclusion
Ropinirole causes various neuropsychiatric side effects, such as confusion, difficulty concentrating, and slow response. The monitoring of drug therapy needs to be performed by health professionals with regard to the use of Parkinson's drugs, especially in elderly patients. Changes in the physiological profile, pharmacokinetics, and pharmacodynamics of the body will influence the effects of drugs in the body. In addition, adjustment of individual doses according to each patient's characteristics is extremely essential. | 2020-12-14T21:03:47.405Z | 2020-10-29T00:00:00.000 | {
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244280458 | pes2o/s2orc | v3-fos-license | The unit log–log distribution: a new unit distribution with alternative quantile regression modeling and educational measurements applications
In this paper, we propose a new distribution, named unit log–log distribution, defined on the bounded (0,1) interval. Basic distributional properties such as model shapes, stochastic ordering, quantile function, moments, and order statistics of the newly defined unit distribution are studied. The maximum likelihood estimation method has been pointed out to estimate its model parameters. The new quantile regression model based on the proposed distribution is introduced and it has been derived estimations of its model parameters also. The Monte Carlo simulation studies have been given to see the performance of the estimation method based on the new unit distribution and its regression modeling. Applications of the newly defined distribution and its quantile regression model to real data sets show that the proposed models have better modeling abilities than competitive models. The proposed unit quantile regression model has targeted to explain linear relation between educational measurements of both OECD (Organization for Economic Co-operation and Development) countries and some non-members of OECD countries, and their Better Life Index. The existence of the significant covariates has been seen on the real data applications for the unit median response.
Introduction
In educational measurement, it should be considered that the measurement process is done for an educational purpose. Educational measurement is the tool in order to obtain information about the characteristics of students and the educational politics of the countries as well as it provides information about the development levels of the countries. Among these educational measurements, the results obtained from international exams, educational attainment percentage of countries, education and school living conditions, data about education and training can be given as examples. These measurements of countries are easily accessible by the OECD. OECD has examined the metrics of countries related to education on two themes. The first of these is the education and learning theme, and the other is the Social Protection and Well-being theme. Using the data sets from OECD countries, there are many papers in the literature. It can be seen [1,2,4,7,13,14,18,21,28,30,52] for some of them. These data sets, which make up the educational measurements, vary according to the development status of the countries. Therefore, some countries' measurements may appear as outliers in the sets. For such situations, using statistically robust methods would be more appropriate for inferences. For example, quantile regression, introduced by [34], models can explain the linear relationship between response variable and independent variable with more robust results than ordinary regression models. One may see [2,13,14,23,37,54] for applications of the educational measurements based on the regression modeling.
On the other hand, modeling the real data sets by the proposed probability models is a very important issue in terms of statistical inferences. This modeling process draws attention to the importance of the probability distributions and it has received great attention by many statisticians. Especially, the modeling approaches on the unit interval have increased recently since they related to specific issues such as the recovery rate, mortality rate, proportion of the educational measurements etc. So, it is needed a unit distribution defined on the (0, 1) interval to model random variable (rv) belonging to the above important issues. When the unit modeling is wanted to apply, no doubt that the beta distribution is firstly remembered by the researchers. Although the beta distribution has different density shapes to fit data on the unit support, in order to obtain better results based on inferences from data sets, the new alternative models have been defined on the unit interval in the statistical distribution literature. These alternative models generally have been obtained via transformations of the well-known rv. For example, the Johnson S B distribution, introduced by [32], has been obtained via the inverse function of the logit transformation of the standard normal rv. Further, the Johnson S B [33], unit logistic [57], logit slash, [9] and unit Johnson S U [25] distributions have been proposed with the same transformation method via standard Laplace, logistic, slash, and unbounded Johnson S U rvs respectively. The unit gamma [15], log-Lindley [29], log-xgamma [5], unit Weibull [43], unit Birnbaum-Saunders [40], unit inverse Gaussian [26], unit Gompertz [41], unit generalized half-normal [10] and log-weighted exponential [2] distributions have been defined by the transformation of the negative exponential function of the baseline rvs. These proposed unit distributions have more flexible density shapes on the (0,1) interval than those in their baseline distribution. This result emphasizes that the suitable transformations, which transform the baseline rvs to the unit interval, propose the distinguishing features in the new density on the (0,1) interval. For example, the ordinary slash distribution has the heavy-tailed and symmetric bell-shaped density on the real support. So, this distribution models only the data sets with the unimodal heavy-tailed density shape on the unit interval. If the data set has different properties, it does not model this data. However, the logit slash distribution [9], which has been defined on the unit interval by the inverse function of the logit transformation of the ordinary slash distribution, has very flexible density shapes such as the w-shaped, N-shaped, inverse N-shaped, bimodal, unimodal, increasing and decreasing. This transformation has brought into the open the different density properties that the ordinary slash distribution does not have on the unit interval. This situation has been seen for both the transformation distribution of the negative exponential function of the baseline rv and the above all proposed distributions. The Topp-Leone [58], Kumaraswamy [35], generalized beta [44], simplex [6], standard two-sided power [59], unit Lindley [39], unit improved Lindley [3] distributions have been suggested as the other alternative models to beta distribution. It is noted that the Kumaraswamy distribution can be obtained with the transformation of the negative exponential function with respect to the exponentiated exponential distribution [27]. Further, for the above some distributions, the alternative regression models such as the beta regression [24], Kumaraswamy quantile regression [46], log-Lindley regression [29], unit-Lindley regression [41], log-weighted exponential regression [2], improved Lindley regression [3] and unit-Weibull quantile regression [42] models have been proposed.
On the other hand, the log-log (LL) distribution has been proposed by Pham [48]. If T rv is the LL rv then its cumulative distribution function (cdf), probability density function (pdf) and hazard rate function (hrf) are respectively given by and where t > 0, α > 0 and β > 1. The pdf can be decreasing and unimodal. The author has also shown that the hrf is the decreasing if t ≤ t 0 and it is the Hence, the hrf of the model has the bathtub shape. Some statistical properties of the LL distribution such as hrf shapes, mean residual lifetime and estimation of the parameters have been studied by the author. Pham [48] has used this model for evaluating the reliability of several helicopter parts as well as it has been modeled for the mortality rate with the data year 2005 of the United States by Pham [49,50]. The [56] has derived the Bayes estimations of the model parameters via the Markov chain Monte Carlo technique. This distribution is also known as Pham distribution in the literature. Under the above information, the goal of this study is to propose a new unit alternative distribution and its quantile regression modeling to model the percentages and proportions. A new alternative bounded statistical model, which has a very flexible density in terms of the data modeling, to well-known bounded unit models such as beta and Kumaraswamy has been aimed by the current paper. Further, it is to show its modeling ability with their applications based on the proportion of the educational measurements' OECD countries. To obtain a new unit distribution, we use the negative exponential transformation of the LL distribution. In this way, we will transport its applicability and work-ability to the unit interval. Hence, we will bring into the different density and hrf properties that the LL distribution has not on the unit interval.
The paper has been planned as follows. The proposed distribution has been defined in Section 2. Its basic distributional properties and procedures of the maximum likelihood estimation (MLE) for model parameters have been introduced by Supplemental File.
The new quantile regression model based on the newly defined distribution, its MLE processes for the model coefficients, and its residual analysis are introduced in Section 3. The simulation studies are given to see the performance of the MLE estimators of the model parameters in Section 4 based on the new unit distribution and new quantile regression model. Three real data illustrations, one is related to the univariate data modeling and others are the quantile regression modeling, have been illustrated in Section 5. Finally, the paper is ended with concluding in Section 6.
Definition of the new unit distribution
The new unit distribution is defined as follows: Let T ∼ LL(α, β) and define the X = exp(−T) rv. Then, the corresponding cdf and pdf are respectively given by and where x ∈ (0, 1), β > 1 and α > 0 are the model parameters. These equations can be obtained with the transformation method. We denote call it the unit log-log distribution and denote it with the ULL(α, β). The shapes of the pdf and hrf are given by Supplemental File in detail. In addition, for the newly defined unit distribution, Supplemental File provides the basic distributional properties such as stochastic ordering, quantile function, moments, and order statistics as well as the MLE processes for the model parameters. Related references are [55] and [45] for the stochastic ordering and moments subsections respectively.
The ULL quantile regression model
If the support of the response variable is defined on the unit interval, using a unit regression model based on the unit distribution can be used for modeling the conditional mean or quantiles of the response variable via independent variables (covariates). Ferrari and Cribari-Neto [24] proposed the beta regression model for continuous variates that assume values in the standard unit interval, e.g. rates, or proportions. When the response variable has the outliers in the measurements, robust estimation results based on the regression model are needed for model inference. The quantile regression model is a good robust alternative model to the ordinary LSE and beta regression models. Whereas the method of least squares and beta regression models estimate the conditional mean of the response variable, the quantile regression estimates the conditional median or other quantiles of the response variable. With this approach, [11][12][13][14]38,42,46] have introduced the Kumaraswamy, unit Weibull, exponentiated arcsecant normal, unit Birnbaum-Saunders, unit Burr-XII, transmuted unit Rayleigh, and arcsecant Weibull quantile regression models respectively. Now, we introduce an alternative quantile regression based on the ULL distribution. The quantile function of the ULL distribution is given by where u ∈ (0, 1). Now, the pdf of the ULL distribution can be given with re-parameterized based on its quantile function. Let μ = x u (α, β) and β = (1 − log u) (− log μ) −α . Then, the pdf and cdf of the re-parametrized distribution is given by and respectively, where α > 0 is the shape parameter, the parameter μ ∈ (0, 1) represents the quantile parameter, and u is known. We call it as quantile unit log-log distribution and denote it with Y ∼ QULL(α, μ, u). Some possible shapes of the QULL distribution are shown in Figure 1. We see that the QULL distribution has the skewed shapes as well as bathtub shaped, inverse N-shaped, decreasing, increasing and unimodal. Now, it can be focused on the quantile regression model based on the QULL distribution with pdf (3). Let y 1 , y 2 , . . . , y n random observations from QULL distribution with unknown parameters, i , and α. Note that the parameter u is known. The QULL quantile regression model is defined as are the unknown regression parameter vector and known ith vector of the covariates. The function g(x) is the link function that is used to link the covariates to the conditional quantile of the response variable. For instance, when the parameter u = 0.5, the covariates are linked to the conditional median of the response variable. Since the QULL distribution is defined on the unit interval, we use the logit-link function which is given by
Parameter estimation via MLE method
We point out the unknown parameters of the QULL quantile regression model to obtain them via the MLE method. Consider below link function From (5), the following equation is obtained Inserting the equations (6) in (3), the log-likelihood function of the QULL quantile regression model is given by where = (α, δ) is the unknown parameter vector. Since (7) includes nonlinear function according to model parameters, this log-likelihood function can be maximized directly by the software such as R, S-Plus, and Mathematica. It can be noticed that when u = 0.5, it is equivalent to model the conditional median. Under mild regularity conditions, the asymptotic distribution of (ˆ − ) is multivariate normal N p+2 (0, I −1 (ˆ )), where I −1 (ˆ ) is the expected information matrix. One may use the (p + 2 × p + 2) observed information matrix for the I −1 (ˆ ). The elements of observed information matrix are evaluated numerically by the software. We use the maxLik function [31] of R software to maximize (7). This function also gives asymptotic standard errors numerically, which are obtained by the observed information matrix.
Residual analysis
The residual analysis can be needed to check whether the regression model is suitable. In order to see this, we will point out the randomized quantile residuals [22] and the Cox-Snell residuals [16].
Randomized quantile residuals
The randomized quantile residuals are defined bŷ where the G(y i ,α,δδδ) is the cdf of the QULL distribution, which is given by (4). If the fitted model successfully deals with the data set, the distribution of the randomized quantile residuals will distribute standard normal distribution.
Cox-Snell residuals
Cox and Snell residuals are given bŷ If the model fits data accordingly, the distribution of theê i 's will distribute exponential distribution with scale parameter 1.
Simulation studies
In this Section, we separately point out the simulations studies about the MLE method for ULL distribution and proposed quantile regression modeling. All calculations based on the estimation procedures have been obtained by optimization functions in the R software.
Empirical results for the MLE methods of the ULL distribution
In this section, we perform the graphical simulation studies to see the performance of the MLEs of the ULL distribution with respect to varying sample size n. We generate N = 1000 samples of size n = 20, 25, . . . , 1000 from the ULL distribution based on the actual parameter values α = 1 and β = 2. The random numbers from the ULL model are obtained by its quantile function. All estimations based on the estimation methods have been obtained by using constrOptim routine in the R program. Further, we calculate the empirical mean, bias and mean square error (MSE) of the estimations for comparisons between estimation methods. The bias and MSE are calculated by (for = α, β) respectively. We expect that the empirical means are close to true values when the MSEs and biases are near zero. The results of this simulation study are shown in Figure 2. This figure indicates that MLEs are consistent since the MSE and biasedness decrease to zero with increasing sample size as expected. Hence, MLEs are asymptotic unbiased.
Furthermore, based on the above MLEs, the simulation study has been given to see the performance efficiency of the 95% confidence intervals. In order to see this, the empirical coverage probability (CP) and coverage length (CL) have been used as criteria. The estimated CPs and CLs are respectively given by and where the I(·, ·) is the indicator function and sˆ is the standard errors of the MLEs, for i = 1, . . . , N, are evaluated by inverting the observed information matrix I. Figure 3 shows the performance of this simulation results. As seen from these Figures, the CPs are near to the nominal value as well as the CL decreases for each parameter when the sample size increases for each parameter. The simulation results verify the consistency property of MLEs. Table 1. The empirical biases, MSE, 95% CP, and 95% CL for the QULL regression model with α = 0.5, δ 0 = −0.5 and δ 1 = 1. Table 2. The empirical biases, MSE, 95% CP, and 95% CL for the QULL regression model with α = 2, δ 0 = 0.5 and δ 1 = −1.
Simulation studies for the proposed regression model
In this subsection, it has been given the simulation studies in order to see behaviors of the MLEs belonging to the QULL regression model based on the estimated bias, MSE, CP and CL measurements. For the varying sample size n, known u = 0.5, true α, and generated covariates values, the values of the unit response variable have been obtained with For different three simulation scenarios, it has been considered as the true parameters values are (α = 0.5, δ 0 = 0.5, δ 1 = 1), (α = 0.5, δ 0 = 0.5, δ 1 = 1) and (α = 0.5, δ 0 = 0.5, δ 1 = 1) with the following regression structure N(0, 1). In all simulation studies, the replication number is N = 1000, sample sizes are n = 25, 50, 100, 250, 500, 1000 and known quantile level is u = 0.5.
The simulation results for the QULL regression model have been shown in Tables 1-3. As seen from these Tables, the empirical CLs decrease while the sample size increases as well as the empirical CPs are around the 0.95 value. All biases are close to zero value as well as all MSEs tend to zero value at the same time as expected.
Data analysis
In this section, three data applications have been given to see the applicability of the proposed distribution model. The first application is about univariate modeling and others applications are about regression modeling for the ULL model. We obtain all data sets Table 3. The empirical biases, MSE, 95% CP, and 95% CL for the QULL regression model with α = 1.5, δ 0 = −1 and δ 1 = −0.5. Each theme is divided into topics. All used data sets are related to the educational measurements of the above countries. We also compare performances of the real data fittings of the proposed distribution under the MLE method with well know unit distributions in the literature for the first application. These distributions are the beta, Kumaraswamy (Kw) [35], exponentiated Topp Leone (ETL) [51], unit Gompertz (UGom) [41], log-weighted exponential (LWE) [2], unit gamma (UG) [15], and unit Birnbaum-Saunders (UBS) [40] distributions. Their pdfs have been given by Supplemental File. The second and third applications have been given in order to see the applicability of the newly defined regression model. Two competitor regression models, which are wellknown literature, also have been considered. They are beta regression [24] models as well as Kumaraswamy quantile regression [46] models. The used pdfs of the beta regression and Kumaraswamy quantile regression are where μ ∈ (0, 1) is the mean and α > 0 and where μ ∈ (0, 1) is the median and α > 0 respectively. For univariate data modeling application, theˆ values, Akaike Information Criteria (AIC), Bayesian information criterion (BIC), Kolmogorov-Smirnov (KS), Cramer-von-Mises, (W * ) and Anderson-Darling (A * ) goodness-of-fit statistics have been obtained based on all distribution models to determine the optimum model. In general, it can be chosen as the optimum model the one which has the smaller the values of the AIC, BIC, KS, W * and A * statistics and the larger the values ofˆ and p-value of the goodnessof-statistics. For regression modeling applications, the Vuong likelihood ratio test (see [60]) has been used for comparison of the non-nested two regression models whether there is any significant difference in the fit models. The Vuong statistic is defined as where λ))} 2 , g(y, θ) and f (y, λ) are the corresponding competitor pdfs calculated at the MLEs. It is noticed that, while n → ∞, the asymptotic distribution behind V is the standard normal distribution. Therefore, at a significance level of υ% distribution equivalence is rejected if |V| < z υ/2 . The calculations have been obtained by the maxLik function [31] of the R software. In addition, the betareg function of the R software for results of the beta regression model has been used. The details are the following.
Univariate real data modeling
This subsection introduces analyzing of the real data set to show the modeling ability of the proposed ULL model. We use the variable novice teachers who have an assigned a mentor at the current school of the Teachers' in the initial and induction training indicator of the TALIS (Teaching and Learning International Survey) in the Education and Training theme of the OECD.Stat as data set. This variable is defined by proportions of the novice teachers with mentor at the school and it has been given as percentage value for 48 countries which are the OECD countries and selected non-member of OECD economies. The reference year is 2018 for this data set. Some summary statistics of the data set have been given in Table 4. The data is rightskewed and has normal kurtosis.
We compare performance of the real data fitting of the proposed distribution under the MLE method with well know unit distribution in the literature. We give the data analysis results belong to other competitor models in Table 5. Table 5 shows that the ULL distribution has the lowest values of AIC and BIC statistics. Also, the ULL distribution has the lowest values of A * and W * and K-S statistics with a higher p-value. These results show that the ULL distribution can be chosen as the best model for the data set. The ULL distribution has captured skewness and kurtosis of the data set successfully as well as the estimated cdf is near to empirical cdf. The plotted lines of the probability-probability (PP) are close to the diagonal line which indicates that the performance of the ULL distribution is acceptable for the modeled data. We note that to show the likelihood equations have a unique solution, we plot the profile log-likelihood (PLL) functions of the α and β parameters for the data set in Figure 5. From this figure, we see that the likelihood equations have a unique solution for the MLEs.
Quantile regression modeling for the educational attainment data set
In this subsection, we use the educational attainment, homicide rate and life satisfaction variables of the Better Life Index indicator in the Social Protection and Well-being theme of the OECD.Stat as data set. The reference year is 2017 for this data set. The data set can be found in https://stats.oecd.org/index.aspx?DataSetCode=BLI. In this application, the data set deals with the educational attainment values' percentage of the 35 OECD countries and those of Brazil, Russia, and South Africa. Educational attainment is defined as the highest grade completed within the most advanced level attended in the educational system of the country where the education was received. Educational attainment considers the number of adults aged 25-64 holding at least an upper secondary degree over the population of the same age.
The aim is to associate these educational attainment values (y) with covariates. The covariates associated with this response variable are: • x 1 : homicide rate (ratio); • x 2 : life satisfaction (average score of the Cantril Ladder known also as the Self-Anchoring Striving Scale).
The regression model based on μ i is given by where the μ i is the mean for the beta model whereas it denotes the median for the Kumaraswamy and QULL models. We give the results of the regression analysis in Table 6. Table, the δ 1 and δ 2 parameters have been seen statistically significant at usual level for the QULL regression model. The δ 0 is the positive value for the beta and Kumaraswamy models however, it is negative for the QULL model. The δ 1 parameter has been found statistically significant at the usual level and has affected negatively for all regression models. Whereas, the δ 2 parameter has not been found statistically significant at the usual level for the beta and Kumaraswamy regression models. It is obvious that all covariates have affected educational attainment based on the QULL model and they are significant statistically. It is also noticed that there is a positive relationship between the median response (educational attainment) and the life satisfaction index of the countries whereas there is a negative relationship between the median response and the homicide rate index of the countries. This means that big values of life satisfaction tend to have more percentage of the educational attainment and its opposite is true for the homicide rate. It is interesting that the regression coefficient δ 2 of the median models has been affected by the distribution of the response variables. It has been obtained as positive signed for QULL and beta models whereas it has been obtained as negative signed for the Kumaraswamy model. This situation can emphasize better goodness of fit because the related coefficient is not statistically significant at any usual significance level for the Kumaraswamy and beta regression models. The fit of the errors also supports better goodness of fit for the QULL model. It has been pointed out fit of the errors following plots. One may see [42] for a similar situation. Further, the Vuong test shows that the QULL regression model is not equivalent to either the Kumaraswamy or the Beta regression models, considering a level of 6.5% of significance. Based on the estimated log-likelihood value and results of the Vuong test, the QULL regression model can be chosen as the best model among application models in terms of better modeling ability than other regression models. Figures 6 and 7 respectively display the Quantile-Quantile (QQ) plot of the randomized quantile residuals and PP plot of the Cox-Snell residuals for all regression models. We can say that the fit of the errors of the QULL regression model to related distributions are better than those of the beta and Kumaraswamy regression models. In addition, these figures support the above results and are a good fit of the QULL regression model to the educational attainment data set.
Quantile regression modeling for the adolescents, who want top grades at school, data set
In this subsection, we use the variables, which are the proportion of the Adolescents (15-year-olds) who want top grades at school, sciences performance at age 15 (PISA), proportion of the Adolescents (15-year-olds) with a desk and a quiet place to study at home, and proportion Adolescents (15-year-olds) who do paid work before or after school, of the education and school life sub-indicator of the Child Well-Being indicator in the Social Protection and Well-being theme of the OECD.Stat as data set. The reference year is 2015 for all variables. In this application, we use the proportion of the Adolescents (15-year-olds) who want top grades at school percentage of the 37 OECD countries as the response variable. The response variable is that the percent of 15-year-old students who report that they agree or strongly agree with the statement I want top grades in most or all of my courses. The aim is to associate these response values (y) with covariates. The covariates associated with this response variable are: • x 1 : sciences performance at age 15 (PISA); • x 2 : Adolescents (15-year-olds) with a desk and a quiet place to study at home; • x 3 : Adolescents (15-year-olds) who do paid work, before or after school.
The regression model based on μ i is given by where the μ i is the mean for the beta model whereas it denotes the median for the unit-Weibull, Kumaraswamy and QULL models. A similar data set has been analyzed by [14].
We give the results of the regression analysis in Table 7. From this Table, the δ 1 parameter has not been obtained statistically significant at the usual level for all models. The δ 2 and δ 3 parameters have been seen statistically significant at the usual level only for the QULL regression model whereas these parameters are statistically significant at 10% level for the beta regression model. The δ 0 is the positive value for all models. The δ 2 parameter has affected negatively to the QULL regression model whereas, the δ 3 parameter has affected positively to this model. It is also noticed that there is a positive relationship between the median response (percentage of the desire of the top grades) and the adolescents, who do paid work before or after school. There is a negative relationship between the median response and the adolescents, who have a desk and a quiet place for studying at home. This means that the percentage of the adolescents, who want the top grades, increases when percentage of the adolescents, who do paid work before or after school, percentage increases. However, it decreases when the percentage of the adolescents, who have a desk and a quiet place for studying at home, increases. The result obtained from all regression models shows that x 1 covariate (sciences performance) is not statistically significant to explain the median response variable. Further, the Vuong test shows that the QULL regression model is not equivalent to either the Kumaraswamy or the Beta regression models. Based on the estimated log-likelihood value and results of the Vuong test, the QULL regression model can be chosen as the best model among application models in terms of better modeling ability than other regression models. Figure 8 displays the QQ plot of the randomized quantile residuals and PP plot of the Cox-Snell residuals for the QULL regression model. We can say that the fit of the errors of the QULL regression model to related distributions has a good fit.
Conclusion
We define a new unit model, called unit log-log distribution, in order to model the proportion of educational measurements as well as other data sets defined on the unit interval. We investigate the general structural properties of the new distribution. The model parameters are estimated by the six different methods. The simulation study is performed to see the performances of these estimates. The data sets with proportions of some educational measurements belonging to OECD countries are analyzed to point out its applicability, flexibility and comparability.
For the univariate real data modeling, we used to model the proportions of the novice teachers with a mentor at the school for 48 countries according to the data set of TALIS.
According to the results, the proposed distribution has been chosen as the best model under model selection criteria.
For applications of the proposed quantile regression model, we have targeted to explain linear relation between educational measurements of both OECD countries and some nonmembers of OECD countries, and their well-being index. The unit response variable has been considered as the conditional median of the covariates in the proposed regression models. According to the first regression application, it has been seen that there is a positive relationship between the median response (educational attainment) and the life satisfaction index of the countries whereas there is a negative relationship between the median response and the homicide rate index of the countries. This means that big values of life satisfaction tend to have more percentage of the educational attainment and its opposite is true for the homicide rate. Via a unit mean response modeling, Altun [2] has related the educational attainment values of the OECD countries with Labor market insecurity and homicide rate variables and has concluded that the Labor market insecurity and homicide rate affect the educational attainment negatively. Similar results have been also supported by [4]. According to Salinas-Jimenez et al. [19], a positive relationship between education level and life satisfaction has been observed based on data from some OECD countries. Salinas-Jimenez et al. [20] have reported that a positive correlation between education and life satisfaction is found according to data which comes from the World Values Survey of more than 50 countries. Botha [8] has explored various dynamics in the relationship between life satisfaction and education in South Africa using the 2008 National Income Dynamics Survey and his report has indicated a strong positive association between educational attainment and individual satisfaction with life. Nadanovsky and Cunha-Cruz [47] have shown that countries with higher education levels have lower homicide rates, based on a new index called impunity.
For the second regression application, it has been seen that there is a positive relationship between the median response (percentage of the desire of the top grades) and the adolescents, who do paid work before or after school. There is a negative relationship between the median response and the adolescents, who have a desk and a quiet place for studying at home. This means that percentage of the adolescents, who want the top grades, increases in the country when the percentage of the adolescents, who do paid work before or after school, percentage increases in the country. By using the Career Maturity Inventory data, based on the high school students, Creed and Pattan [17] have concluded that students with paid work experience have reported higher levels of Career Development Attitude than those with no paid work experience. Further, they have reported that gender differences have also occurred, with females with paid work experience generally reporting higher levels of career maturity than males with paid work experience. However, the unit median response variable decreases when the percentage of the adolescents, who have a desk and a quiet place for studying at home, increases in the country. The sciences performance covariate has not been found statistically significant to explain the median response variable. One may see [36,53] for some results about socio-economic status and student performance. The existence of the significant covariates has been seen on both real data applications for the unit median regression modeling. Based on another quantile regression modeling, similar results have been also observed by [14].
All empirical findings, which are about modeling of the educational measurements, show that the proposed model provides better fits than the well-known unit probability distribution in the literature for both its univariate data modeling and its regression modelings. It is hoped that the new distribution will attract attention in the other disciplines.
Disclosure statement
No potential conflict of interest was reported by the author(s). | 2021-11-18T16:15:17.863Z | 2021-11-16T00:00:00.000 | {
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256038182 | pes2o/s2orc | v3-fos-license | Precision Drell-Yan measurements at the LHC and implications for the diphoton excess
Precision measurements of the Drell-Yan (DY) cross sections at the LHC constrain new physics scenarios that involve new states with electroweak (EW) charges. We analyze these constraints and apply them to models that can address the LHC diphoton excess at 750 GeV. We confront these findings with LEP EW precision tests and show that DY provides stronger constraints than the LEP data. While 8 TeV data can already probe some parts of the interesting region of parameter space, LHC14 results are expected to cover a substantial part of the relevant terrain. We derive the bounds from the existing data, estimate LHC14 reach and compare them to the bounds one gets from LEP and future FCC-ee precision measurements.
Introduction
Recently both ATLAS and CMS reported an excess in the search for diphoton resonances around M γγ ∼ 750 GeV with 3.2 fb −1 and 2.6 fb −1 of integrated luminosity at √ s = 13 TeV, respectively. The local (global) significance of the excess is 3.9 σ (2.3 σ) for the ATLAS data, with the best-fit value for the width of the resonance Γ ∼ 45 GeV [1]. CMS reported a local excess with significance of 2.6 σ at a mass compatible with ATLAS, assuming a narrow width. This significance goes down to 2 σ if Γ = 45 GeV is assumed [2]. The findings are compatible with searches at √ s = 8 TeV, given that the production cross section of the potential resonance S increases by about a factor of 5 for the larger center-of-mass energy. This is, for example, realized if the resonance with the mass around 750 GeV is produced in gluon-gluon or bb fusion [3]. Although these hints are by no means decisive and it is possible, that the origin of both is in somewhat unlikely fluctuations of the background, it is very interesting to understand the consequences of interpreting this excess as a true new physics resonance.
The tentative large width, however, poses challenges for model building, hinting to a large number of new states with sizable charges mediating the decay S → γγ in a weakly JHEP07(2016)136 coupled framework [4][5][6][7][8][9]. This width implies that also the partial width into photons should be sizable, at least of order 2 × 10 −4 M S , because there are considerable constraints on the relative size of other possible decay channels of S from the 8 TeV data.
The most popular and reasonable realization of the large number of the new states would be vector-like fermions, charged under the electroweak (EW) force. 1 Since these new states have EW production cross section (and, possibly, very difficult decay modes for experimental detection) they can relatively easily evade the direct LHC searches and still be sufficiently light, well below the TeV scale. It has been noticed in several works before, that these new vector like states significantly modify the running of the hypercharge coupling [11][12][13]. This in turn leads to new constraints, originating from the consistency of these models, perturbativity and the scale of the Landau pole, which all have been discussed in detail in the above mentioned references.
Interestingly, on top of these constraints, one can put experimental constraints on the existence of such large number of new vector-like EW states. As we will show in detail, the presence of such vector-like fermions can be indirectly tested by measuring the neutral Drell-Yan (DY) process at the LHC, considering their impact on the running of the hypercharge coupling. We will demonstrate that large portions of the parameter space, relevant for the 750 GeV diphoton resonance, can be probed in a relatively model independent way via the LHC DY production far away from the Z-pole.
The idea to explore the running of the EW couplings to probe new EW states was carefully elaborated on in ref. [14]. This paper has shown that the running of EW couplings, α 2 and α Y , can be successfully probed at the LHC14 and at a future 100 TeV collider, putting new limits that are much stronger than one gets from LEP measurements. For example, it is claimed that the high-luminosity LHC will be sensitive to deviations in α 2 of less than 10% from the SM value at a scale of 2.5 TeV. In this work we take this idea one step further, and show that precisely the same measurements at LHC8 already put meaningful constraints on the models that are explaining the 750 GeV resonance. We show, for example, that for the EW states around 400 GeV, values of N Q 2 ∼ 60 (with N being a total multiplicity number and Q the hypercharge) are in tension with the DY data, already reducing the parameter space for the large width interpretation of the 750 GeV resonance. Future measurements at LHC14 will further shed light on the parameter space of the S, which will be particularly powerful in the latter case.
Our paper in organized as follow. In section 2 we present the perturbative model, which might address the 750 GeV LHC diphoton excess. We also discuss the necessity of the new EW states, explain how they affect the LEP measurements (via changing the Y-parameter) and show the LEP bounds. We will see that these bounds are fairly weak. In section 3 we briefly overview the theory of DY production at the LHC, with an emphasis on the possible change in the cross sections due to new physics which affects the hypercharge coupling running. In section 4 we describe our statistical procedure, show the bounds that we get from LHC8 and provide the sensitivity projection for LHC14. Here, we also 1 Any other solution could potentially introduce difficult conceptual problems, like flavor-changing neutral currents in the case of chiral representations under the SM. Scalars will also have difficulties, which were discussed in detail in ref. [10]. We comment more on the scalar case in section 4.3.
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discuss NLO, PDF and other systematic uncertainties. We summarize the implications of our findings on the 750 GeV diphoton resonance and discuss some future developments in section 5. Finally, we briefly comment on direct searches for the new EW states in section 6 and conclude. The technical details concerning the simulation of the SM prediction for the DY process are relegated to appendix A.
2 Toy model for 750 GeV excess and EW precision tests
Overview of data and interpretation
If we interpret the LHC data as a signal for a new resonance in the γγ channel, the suggested production cross sections are [3] σ(pp → S) × BR(S → γγ) = (10 ± 3) fb/(6 ± 3) fb (2.1) at ATLAS / CMS, respectively. ATLAS data prefers a relatively wide resonance, Γ S /M S ≈ 0.06. It is important to mention that CMS data does not prefer large width, and it is unclear whether the wide resonance assumptions notably improves the overall fit, when the LHC8 and LHC13 data of both experiments is taken into account. For example, it is claimed in ref. [15] that narrow width of the resonance is more likely if the Run II information of both ATLAS and CMS is considered, while the preference to large width is merely marginal, when it is combined with the Run I data. 2 For concreteness we consider the following effective theory for S, assuming that S is a scalar and its couplings do not violate CP: If S is a pseudoscalar, one essentially gets the same couplings with the obvious replacements F µν F µν → F µνF µν and G µν G µν → G µνG µν . Here we also assumed that the dominant production channel for S is via gluon fusion. Of course this is not the only option, and production from heavy flavors can be even slightly favored by data if LHC8 is taken into account [3].
In a perturbative model, the couplings in eq. (2.2) are induced by new states, charged under the EM and strong force, respectively. As we have alluded, we will further study the model, in which these couplings are induced by new vector-like fermions. In principle, one can induce the coupling of S to photons either via introducing new vector like representations under SU(2) L , or under hypercharge (or both). For simplicity, we will focus on the latter case and consider N X new vector-like fermions X i , i = 1, . . . , N X , only charged under hypercharge. Moreover, we assume that they all share the same mass M X i ≡ M X , JHEP07(2016)136 quantum numbers, and couplings. In this case, one easily matches the scale Λ γ as 3 Here, Q X denotes the common hypercharge of the X i , τ X ≡ 4M 2 X /M 2 S and, assuming S is a scalar particle, while y is the Yukawa coupling between the new scalar resonance and the vector-like states, L ⊃ ySXX. The pseudo-scalar case corresponds to the replacement S(τ ) → P(τ ) ≡ arctan 2 1/ √ τ − 1 together with trading y for the pseudo-scalar Yukawa coupling. What should the number N X Q 2 X be to match the data? This again depends on our assumptions. If we assume that S is a narrow resonance, one can end up with a fairly small number of exotic vector-like fermions, which will have a relatively limited impact on the DY precision measurements. In this case the only constraint that the data imposes is where we use the short-hand notations, Γ γγ ≡ Γ(S → γγ), Γ gg ≡ Γ(S → gg). Dijet searches constrain the width into gluons relatively weakly compared to the observed width into photons, Γ gg 1200 Γ γγ . This essentially means that if we assume narrow width, we can saturate Γ Γ gg and it can be sufficient to reproduce Γ γγ /M S ∼ 10 −6 . This goal is easy to achieve with only N X Q 2 X ∼ O(5-10). The situation dramatically changes if we try to reproduce the 6% width of the resonance, which is preferred by ATLAS. As the width (into some other particles) grows substantially, one needs to increase the width to photons and is easily driven to Γ γγ /M S 10 −3 , depending on the dominant decay products f of S, due to 8 TeV upper bounds on Γ(S → f )/Γ γγ , scaled to 13 TeV [3]. In any case, in the wide width scenario, the generic constraint from 8 TeV data, Γ 1500 × Γ γγ (considering standard decay modes), suggests that Γ γγ /M S cannot be lower than ∼ 10 −4 . It is important to mention that this requirement is not unique for gluon fusion production and one gets roughly the same requirements of the partial width into diphotons if heavy flavor production is assumed.
The goal Γ γγ /M S ∼ 10 −3 is notoriously hard to achieve and it demands sufficiently large N X Q 2 X ∼ O(100-500), depending on the masses of the new fermions. These numbers are already big enough to significantly deflect the running of α Y from the SM trajectory, such that the effects are clearly visible in DY production. Figure 1. Left panel. One-parameter fit of LEP data in terms of the Y oblique parameter. All the remaining oblique parameters are set to zero. Right panel. Two-parameter fit of LEP data (allowed region in green) in term of the mass M X of the new vector-like states and the combination d X N X Q 2 X , where d X is the number of degrees of freedom (e.g. d X = 3 for a color charge). We superimpose (region shaded in gray) the parameter space in which the U(1) Y Landau pole is lowered below 3 TeV [13].
LEP constraints
Before analyzing in detail the bounds that we get from the LHC, we first show the bounds from LEP. The SU(2) L -singlet vector-like fermions X with non-zero hypercharge Y ≡ Q X and mass M X contribute to the two-point function, Π µν BB (q 2 ). Below the mass scale of the heavy fermions, it generates an effective operator (∂ µ B µν ) 2 which maps on the Y parameter [17], (2. 6) where d X N X denotes the total number of degrees of freedom. The one-loop β function 4 of the hypercharge coupling is also encoded in the same two-point function, leading to the same parametric dependence d X N X Q 2 X , and is given by where the modification due to the new vector-like states alters the SM running above the threshold µ > M X . Below the threshold, the heavy fermions X are integrated out and the resulting one-loop β function reduces to the SM one.
The best-fit of the Y parameter from LEP data and the recasted bounds on new vectorlike fermions are illustrated in figure 1. As one would naively expect, these bounds are very weak.
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3 Drell-Yan at the LHC In this section we show how one can estimate the DY double differential cross section in the presence of the α Y running due to the new physics. The computation of the DY cross section at the LHC proceeds in the following steps.
• At the parton level and in the center of mass partonic reference frame the DY scattering process qq → Z * /γ → l + l − is described by the following double-differential cross section where z ≡ cos θ, being θ is the scattering angle in the partonic center of mass frame. The factor a and the rescaling function in eq. (3.1), following [14], are defined as (3. 2) The dependence of the scattering cross section on the running gauge couplings is encoded in a 2 f qq (s 2 W , M ll , z) by means of the relations At this stage it is important to stress that the cross section in eq. (3.1) is derived in full generality with respect to the running of the gauge couplings g Y and g L . Since the energy scale of the process is set by √ŝ = M ll , one has to use the running couplings evaluated at µ = M ll . The impact of new vector-like states with mass M X is captured by solving eq. (2.7) with specific values of d X , N X , Q X , and using the corresponding expression for g Y (µ = M ll ) in eqs. (3.1)-(3.3). The SM prediction corresponds to N X = 0.
The coefficients c (i) qq , i = 0, . . . , 4 depend on dilepton invariant mass, quantum numbers of the initial quark-antiquark pair, and scattering angle. We find 5 5 Notice that, comparing our results with [14], we found a factor 3 discrepancy in the explicit expression of the coefficients c The scattering cross section in eq. (3.1) is averaged over the initial spin and color. At the weak scale we use the numerical matching values g L (µ = m Z ) = 0.649, g 1 (µ = m Z ) = 0.459.
• The hadronic differential cross section is obtained by convoluting with the PDFs withŝ = x 1 x 2 s, being √ s the total energy in the hadronic center of mass frame. We introduce the variable τ ≡ x 1 x 2 which corresponds to the fraction of the energy transferred to the partonic system. The rapidity of the lepton is defined as The integration over x 1 , x 2 can be converted to the one over τ , y via the relations, Using the identity δ(τ s − M 2 ll ) = δ(τ − M 2 ll /s)/s we get rid of the integration over τ , and we find where z max ≡ 1 − 4p 2 T /M 2 ll and y max = min{log(s/M 2 ll )/2, y cut }; the detector acceptance sets the value y cut = 2.5. The nominal cut on the transverse momentum of the leptons is p T = 25 GeV. We use the MSTW parton distribution functions at NNLO [18].
• In eq. (3.12) the PDF are evaluated setting the renormalization scale at the dynamical value µ = M ll . Furthermore, we make use of the central PDF set. Fixing these two values implies the introduction of both scale and PDF uncertainties in the computation of the theoretical cross section. We estimate the impact of the scale uncertainty by varying the renormalization scale in µ = [1/2 M ll , 2M ll ], and we find that it introduces at most ∼ 1% error (see [14] for the related discussion). We therefore neglect such correction in our analysis. PDF uncertainty is larger, and can be estimated JHEP07(2016)136 evaluating the DY cross section over a statistical sample obtained by changing the PDF eigenvector set in eq. (3.12). We include the PDF uncertainty in our analysis, and we refer the reader to section 4 for a detailed discussion.
• Eq. (3.12) is defined at the LO in the hard scattering process. We include NNLO corrections by properly rescaling -bin by bin in the invariant mass spectrum range -the LO cross section with respect to the NNLO cross section, relying on the numerical evaluation of ref. [19].
We are now ready to compare the theoretical cross section in eq. (3.12) with experimental data extracted from the measurement of the differential DY cross section in the di-electron and di-muon channels.
Analysis and results
We extract our bounds by means of a simple χ 2 analysis derived comparing theory and data. We start defining the χ 2 function (4.1) where the sum runs over the invariant mass bins. The theoretical cross section in eq. (3.12) depends on the many free parameters, d X , N X , Q X , M X . To simplify the notation we further define N eff ≡ d X N X Q 2 X . We allow for a free overall normalization N of the theoretical cross section -over which we marginalize -in order to include possible unknown correlated systematic uncertainties. Denoting with χ 2 min the minimum of the function χ 2 (N , N eff , M X ), and introducing the marginalized distribution χ 2 marg (N eff , M X ) = dN χ 2 (N , N eff , M X ), we derive confidence level contours by requiring χ 2 marg (N eff , M X ) > χ 2 min + κ, with κ = 2.30, 6.18, 11.83 at, respectively, 1-, 2-and 3 σ level. In eq. (4.1) dσ/dM ll | exp represents the experimentally measured differential cross section in the dilepton invariant mass while Σ is the covariance error matrix. Before we proceed further, we have to distinguish between the analysis at √ s = 8 TeV and √ s = 14 TeV. The reason is that at √ s = 8 TeV we can use data and errors from the DY analysis carried out by CMS in [19]. At √ s = 14 TeV, on the contrary, we shall rely on a projection.
Drell-Yan at √ s = 8 TeV
We use the results of the analysis presented by the CMS collaboration in [19] where the differential cross section in the dilepton invariant mass range M ll = [15,2000] GeV was measured using proton-proton collisions at √ s = 8 TeV with an integrated luminosity of 19.7 fb −1 . Data and errors are publicly available at the website of the Durham HepData Project [20].
The covariance error matrix entering in eq. (4.1) is given by Σ = Σ exp + Σ PDF . Σ exp encodes experimental errors (both statistical errors and systematic uncertainties, as discussed in [19]) and correlations related to the pre-FSR invariant mass distribution in the JHEP07(2016)136 combined dilepton channel [19] while Σ PDF takes into account the impact of PDF uncertainties in the computation of the differential DY cross section. We take Σ exp from [20]. The knowledge of the covariance matrix is of fundamental importance since it gives us the possibility to compute the correlation matrix describing correlations between different bins in the invariant mass distribution. The correlation matrix can be written as In the left panel of figure 2 we show the comparison between experimental data and theoretical cross section at large dilepton invariant mass. We simulated the SM theoretical prediction of the DY fiducial cross section with FEWZ [21] and we explain all the details of this simulation, its expected accuracy and comparison with the experimental CMS data in the appendix. The dashed blue line corresponds to the NNLO SM cross section, while the blue lines include the extra vector-like fermions. The NNLO SM cross section is in excellent agreement with the measured values in the whole range of dilepton invariant mass. Let us now discuss the impact of the new vector-like fermions. For illustrative purposes we fix M X = 400 GeV, and, to better visualize the impact of running couplings, we show two specific cases with d X N X Q 2 X = 150 (lighter blue) and d X N X Q 2 X = 200 (darker blue). In the dilepton invariant mass range 500 M ll 1000 GeV the differential cross section is measured with a 10% accuracy. For M ll M X the vector-like fermions actively participate to the hypercharge running, and their impact on the differential cross section may easily overshoot the data points, as qualitatively shown in figure 2, for sufficiently large d X N X Q 2 X . In the right panel of figure 2 we show the correlation matrix derived in eq. (4.2).
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As expected, the plot highlights the presence of strong correlations between adjacent bins. The inclusion of the correlation matrix in the χ 2 fit plays an important role since it allows to constraint -in addition to the absolute deviation from the observed values in each individual bin -also the slope of theoretical cross section.
Let us now discuss the size of PDF uncertainties. The differential cross section in eq. (3.12) was obtained considering the central PDF set (corresponding to the PDF bestfit). In order to assess the impact of PDF uncertainties we need to statistically quantify -using all the remaining eigenvector PDF sets -the relative change in the cross section. Let us discuss this point in more detail. In order to construct the covariance error matrix Σ PDF we need two ingredients • PDF uncertainties in individual bins; • Correlation matrix among different bins.
In the following we denote with σ (k) ± (M ll ) the differential cross section dσ/dM ll evaluated at dilepton invariant mass M ll using the k th PDF eigenvector pair. We start computing the PDF uncertainty in individual bins. We follow the standard treatment in [18,22]. The PDF uncertainty corresponds to Correlations among different bins can be computed using standard statistics, and we find where i and j denote two invariant mass bins. Equipped by these results, we can compute the covariance error matrix. We have where we defined the diagonal matrix S ≡ diag(S i ), with S i ≡ S(i). We show our results in the left column of figure 3. In the upper plot, we show the relative PDF errors per individual bin (red circles), and we compare them with the experimental errors quoted in [19,20] (blue squares). As clear from this plot, at √ s = 8 TeV the impact of PDF uncertainties is sub-leading if compared with the experimental errors. For completeness, in the lower panel we show the PDF correlation extracted according to eq. (4.4).
Let us now discuss our findings, shown in the left panel of figure 4 where we plot the 1-, 2-and 3 σ bound. First of all, it is interesting to compare the DY bound with the constraint placed by LEP in figure 1. The net result is that the measurement of the DY differential cross section at large invariant mass at the LHC with √ s = 8 TeV already provides a bound stronger than the one obtained at LEP. This is a conceptually remarkable JHEP07(2016)136 result, given the penalizing price unavoidably paid by an hadronic machine like the LHC in performing precision measurements. Notwithstanding this important observation, it is also clear from figure 4 that the DY bound extracted at √ s = 8 TeV does not rule out any relevant portion of the parameter space since it requires at least d X N X Q 2 X 50, a value objectively too large for any realistic model. However, encouraged by the promising result obtained at √ s = 8 TeV, we move now to explore future prospects at √ s = 14 TeV.
Drell-Yan at √ s = 14 TeV
At √ s = 14 TeV we have to rely on a projection. We generate mock data assuming that the observed invariant mass distribution agrees with the NNLO QCD SM prediction. We include the effect of running couplings according to eq. tribution as in eq. (4.1) -thus including an overall free normalization N as nuisance parameter to account for correlated unknown systematic uncertainties. We write the covariance error matrix as Σ = Σ stat + Σ uncorr syst + Σ PDF . Statistical errors are obtained assuming an integrated luminosity L = 300 fb −1 , and converting the cross section in terms of number of events per bin. The corresponding covariance error matrix Σ stat is diagonal, with entries equal to the square of the statistical errors. The PDF uncertainties are estimated as discussed before (see eqs. (4.3), (4.4)). We show our results in the right column of figure 3. Finally, Σ uncorr syst is built assuming a flat 1% (2.5%) uncertainty across all invariant mass bins in order to simulate the presence of uncorrelated systematic errors. At √ s = 14 TeV the simulated data extend up to M ll 5 TeV; the statistical error, assuming L = 300 fb −1 , does not exceed the 10% level up to M ll 2 TeV as shown in the upper-right panel of figure 3. At invariant mass M ll 1 TeV the statistical error stays below 1%, and the PDF error, as well as the uncorrelated systematic error, starts to become important. From this simple estimate it is clear that the constraining power of the DY differential cross section at √ s = 14 TeV will lead to much stronger bounds w.r.t. those obtained in section 4.1 using data at √ s = 8 TeV. We show our findings in the right panel of figure 4 where we plot the 1-, 2-and 3 σ bound. The solid (dashed) contours in red (black) were obtained considering 1% (2.5%) uncorrelated systematic uncertainties. The impact of these uncorrelated systematic uncertainties is particularly important for light vector-like fermions and small values of d X N X Q 2 X , where the deviation due to running couplings is smaller and thus it can be more easily hidden in the uncertainty accompanying the measured cross section. Despite this, it is clear that the constraining power (or, said differently, the discovery potential) of the DY
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process at the LHC with √ s = 14 TeV and 300 fb −1 starts to bite into an interesting region of the parameter space where, in particular for light vector-like fermions, the effective coupling d X N X Q 2 X is lowered down to phenomenologically realistic values. In section 5 we will discuss the implication of the DY process for the diphoton excess at 750 GeV.
It is possible to speculate even further about the role of the DY process as precision observable at the LHC. As clear from the upper right panel of figure 3, the dominant error at large dilepton invariant mass comes from limited statistics. The High Luminosity LHC (HL-LHC) program has the scope of attaining the threshold of 3000 fb −1 of integrated luminosity. In this case, reducing the statistical error by roughly a factor 10, it would be possible to largely improve the constraining power/discovery potential of the DY channel even for vector-like states with TeV-scale mass.
New charged scalars
Before proceeding, let us briefly discuss the case in which the SM is extended by adding N X complex singlet scalars X with mass M X and hypercharge Y = Q X . For a more general discussion we refer the reader to [10]. At one loop, the contributions to the Y parameter and the hypercharge one-loop β function are where, in parallel with the fermionic case, the number of effective degrees of freedom d X N X accounts for possible color multiplicity. If compared to the fermionic case, the scalar contribution to the Y parameter turns out to be eight times smaller, and the bound from LEP becomes irrelevant even for very light scalar particles. In the absence of any constraint from LEP, it is important to assess the constraining power of the DY analysis at the LHC. Using the fermionic case as basis for comparison, from eq. (4.7) we see that the impact of a charged scalar particle on the g 1 running is four times smaller. We therefore expect a weaker bound from the DY analysis. To fix ideas, at √ s = 8 TeV and for M X = 400 GeV we find that the running induced by new charged scalar particles produces a 10% correction to the differential cross section at M ll = 800 GeV only for d X N X Q 2 X 270. Contrarily, at √ s = 14 TeV and for M X = 400 GeV a 2% deviation at M ll = 1000 GeV can be obtained with d X N X Q 2 X 50. We therefore conclude that the constraining power of the DY process in the scalar case is only marginally relevant even considering collisions at √ s = 14 TeV with large integrated luminosity.
FCC-ee
The FCC-ee is a high-luminosity, high-precision e + e − circular collider envisioned in a new 80-100 km tunnel in the Geneva area with a centre-of-mass energy from 90 to 400 GeV [27]. Thanks to its clean experimental environment, the FCC-ee collider could explore the EW physics with unprecedented accuracy, allowing for a careful scrutiny of new physics models predicting new particles at the TeV scale and beyond. In the following, we base our discussion on [23]. To give an idea of the constraining power of the FCC-ee collider, it is possible to recast the LEP results. For instance, according to the estimates presented Figure 5. Left panel. One-parameter fit of LEP data in terms of the Y parameter. We rescale the errors of the EW precision measurements according to what expected in [23] for a future circular e + e − collider in order to provide a flavor of its constraining power (see text for details). We set the precision on the effective squared mixing angle to δ sin 2 θ eff W = 10 −5 . The central value of Y is arbitrarily set to zero. Right panel. 1-and 3-sigma exclusion regions in terms of the parameter space (d X N X Q 2 X , M X ) (yellow). We also show the corresponding bounds if δ sin 2 θ eff W = 10 −6 (orange).
in [23], at the FCC-ee it will be possible to measure the pole mass of the Z-boson with a precision twenty times smaller than the one reached at LEP, δm Z | LEP = 0.0021 GeV. One can fit the LEP data implementing all the upgraded precisions quoted in [23]. Following this logic, we show in the left panel of figure 5 the bound obtained considering a one-parameter fit made in terms of the Y parameter. The most important measurement controlling the precision on Y is the square of the effective weak mixing angle. Assuming δ sin 2 θ eff W = 10 −5 , we find δY = 0.035 × 10 −3 (0.1 × 10 −3 ) at 1-σ (3-σ). In the right panel of figure 5 we translate these confidence regions in the parameter space (d X N X Q 2 X , M X ) (regions shaded in yellow). As expected the constraining power of the FCC-ee collider turns out to be much stronger than the capabilities of present and future LHC searches in the DY channel. If δ sin 2 θ eff W = 10 −6 (the value envisaged in [23]) the constraint becomes even stronger, and we find δY = 0.0036 × 10 −3 (0.01 × 10 −3 ) (orange lines in the right panel of figure 5).
Implications for the diphoton excess at 750 GeV
We are now in the position to comment about the importance of the DY process at the LHC for the diphoton excess discussed in section 2. The Yukawa interactions between the (pseudo-) scalar resonance S and the vector-like fermions X are encoded in the Lagrangian where L SM is the SM Lagrangian and where we assumed that the N X fermions have the same mass and couplings. The Yukawa coupling y (ỹ) is present if S is a scalar (pseudo-JHEP07(2016)136 H) is not important for the purposes of our discussion, and we refer to [10] for a more general analysis including vacuum stability. As pointed out in [11][12][13], it is important to keep track of the RG evolution of the Yukawa coupling since it can easily exceed the perturbative regime. The Yukawa couplings y X andỹ X have the same RGE, and we find 6 The CP-odd couplingỹ X contributes to the diphoton decay width of S more than the CP-even coupling y X . For the latter, we have with τ X ≡ 4M 2 X /M 2 S . The pseudo-scalar case corresponds to the substitutions y X →ỹ X , S(τ X ) → P(τ X ).
In figure 6 we show the maximal diphoton decay width of the (pseudo-) scalar resonance S as a function of the scale at which the theory becomes non-perturbative. In more detail, our logic goes as follows. i) For a given value of d X N X , Q X and y X (ỹ X ) we compute the scale at which the theory becomes non-perturbative by solving the RGEs for g 1 and y X (ỹ X ). The x-axes in figure 6 is therefore formally defined as the scale µ max at which either JHEP07(2016)136 y X (ỹ X ) = 4π (perturbativity of the Yukawa coupling) or g 1 = ∞ (hypercharge Landau pole) is realized along the RG flow. ii) By scanning over d X N X , Q X and y X (ỹ X ), and using eq. (5.3), we can compute the maximal diphoton decay width that the (pseudo-) scalar resonance S can obtain for a given µ max . To give an even more clear understanding, we also show in figure 6 the maximal Γ γγ corresponding to fixed values of d X N X , Q X . For a fixed value of d X N X , Q X one has the freedom to move on the corresponding line by changing the Yukawa coupling. Large Yukawa couplings correspond to the left-most part of the plot, where the diphoton width Γ γγ rapidly increases (being proportional to y 2 X (ỹ 2 X )) at the prize to lower the cut-off scale of the theory down to the TeV range. iii) Finally, we impose -at each point d X N X , Q X -the LEP and LHC constraint derived in section 2 and section 4. In the left (right) panel of figure 6 we include the impact of the 1-and 2-σ bounds derived from the analysis at √ s = 8 TeV ( √ s = 14 TeV): every point in the scan violating such bound is discarded.
Following the logic explained above, in figure 6 we show the maximal diphoton decay width for a scalar (dashed lines) and pseudo-scalar (solid lines) resonance S as a function of the cut-off scale µ max . For simplicity, we fix M X = M S /2 since this value maximizes the diphoton width. Gray lines include only the bound from LEP, while the black lines include the bound extracted by the DY analysis at the LHC. The cases with d X N X = 3, Q X = 1 and d X N X = 1, Q X = 3 are shown in blue and magenta. We are interested in values 10 −6 Γ γγ /M S 10 −3 , where the left (right) part of the disequality corresponds to narrow (large) width, as discussed in section 2. In this respect, the bound from LEP plays non role in constraining phenomenologically interesting values of Γ γγ /M S . At √ s = 8 TeV, the DY bound bites into a small corner of the parameter space, as clear from the comparison between the gray and the black lines in the left panel of figure 6. However, it does not have any relevant implications w.r.t. the diphoton excess. At √ s = 14 TeV, the story changes. The DY bound starts to become relevant. In the scalar case the black line is lowered down to Γ γγ /M S 10 −4 -10 −5 (at, respectively, 2-and 1-σ), thus affecting the whole region of the parameter space favored by the large width assumption. The pseudo-scalar case gives a similar result, with the maximal diphoton width lowered down to Γ γγ /M S 10 −3 -10 −4 .
To conclude, we argue that the new physics involved in the explanation of the diphoton excess at 750 GeV could leave -especially if the indications in favor of a large width will be confirmed -a footprint in the differential cross section of the neutral DY process at large dilepton invariant mass.
Comments on direct searches for the new states
In this section let us shortly comment on the direct detection of the new states. Of course, direct detection reach strongly depends on the particular decay modes of the vector like fermions. Comparing these searches to the indirect searches via DY production is by no mean straightforward. Surveying all various possibilities in terms of hypercharge and decay chains is well beyond the scope of this paper. Therefore, we will just emphasize several points, which are generic to the EW production. By drawing analogy to the existing JHEP07(2016)136 σ(�� → ψψ) [��] searches for the EW pair-produced states we will merely try to give a flavor of what might the ballpark of the direct detection bounds.
At the partonic level, the cross section for the on-shell production of a XX pair is for, respectively, up-and down-type quarks in the initial partonic state. The cross section pp → XX for producing a vector-like fermion pair with charge Q X at the LHC with √ s = 8, 14 TeV is shown in figure 7. Naively one would expect that the production cross sections are EW, not that different, for example, from SUSY EW-ino production cross sections. In general it is right, however the cross sections that we get for a single state are generally smaller than typical EW cross sections. The pair-production cross section of a single, charge-one state, is smaller than the production cross sections of the SUSY charged winos (cf. figure 7). The explanation is very simple: our production is proportional to the α 2 Y rather than α 2 2 which suppresses the cross sections by more than order of magnitude. On the other hand, if all the new states lie at the same mass scale, we expect the total cross sections to be increased by factor of d X N X Q 2 X . 7 Therefore, when both these factors are taken into account we end up with the cross sections, which are slightly bigger (by order-one factor) than the standard EW cross sections. 7 The same dX NX Q 2 X dependence originates from the optical theorem.
JHEP07(2016)136
Most of the searches for the EW states at the LHC for now, are motivated by the SUSY EW-ino. This usually ends up in final states leptons (including, possibly, taus) and with / E T . Although we do not know, what would be exactly the bounds on every single scenario one would consider, the direct detection bounds are very unlikely to exceed the bounds on the SUSY wino-chargino particles. The bound on chargino is around 475 GeV if it is assumed to decay to the electrons or muons [24], and it is around 360 GeV if we are considering decays into taus [25].
Conclusions
In this paper we made one of the first attempts to confront some theoretical explanations of the diphoton access with LHC SM precision measurements. Phenomenological models which try to fit the large width of diphoton excess, suggested by ATLAS, suggest large multiplicity of the EW-charged states below the TeV scale that have a significant impact on the hypercharge coupling running. We point out that this running can be probed via DY production at the LHC, estimate the bounds from LHC8 and project the bounds from the LHC14. We show, that contrary to the direct detection our method is robust and it can clearly exclude or confirm such new states at the EW scale.
Interestingly the bounds that we derive from the LHC8, although still relatively weak, are already much stronger that the bounds one get from LEP. Moreover, LHC14 DY measurements will significantly improve the reach, pushing the bounds (or maybe making discoveries) deep into the parameter space relevant for the wide 750 GeV resonance.
We also briefly comment on the possibilities of the direct detection of the new EW states at the LHC. Unfortunately this question is much more model dependent, and lacks the robustness of the precision measurement approach. While it is relatively easy to hide the new states from the direct detection by assuming complicated decay chains and large multiplicity state, it would be interesting to survey more carefully various decay modes. as described in [20]. We employ the code FEWZ [21], version 3.1b2, which includes NNLO QCD and NLO EW corrections to the process. The cross section is considered over the full phase space, i.e., no cuts are applied -beyond the standard p T > 10 GeV (p T > 20 GeV, η < 4.5) cuts on real photons (jets). We use CTEQ12NNLO pdfs and chose a dynamical scale of µ R = µ F = m + − . As CMS presents their data [20] after unfolding actually both initial state and final state radiation (employing Monte Carlo simulation), we set EW control = 7 in FEWZ. 8 This removes the main source of difference between final state electrons and muons.
JHEP07(2016)136
Moreover, we turn off the photon-induced channel via setting Alpha QED(0)=0, since it has been removed in the results as given in [19,20]. The other physical parameters are kept as in the FEWZ default, in particular we employ the G µ input scheme and use the Z-pole focus.
It turns out that the accuracy of the results in the large m + − bins could be improved by running each bin individually -allowing to generate sufficient Monte Carlo statistic also for high-mass bins (which have a limited impact on the total cross section). We show the resulting errors quoted by FEWZ (not including pdf uncertainties or scale variation) for √ s = 8 TeV in the left panel of figure 8 -demonstrating that the Monte Carlo error seems under good control in all bins, i.e., Finally, we present our √ s = 8 TeV results for the differential cross section in table 1, including the FEWZ errors. We also provide in the right panel of figure 8 a simultaneous plot of our predictions (blue, joined -neglecting the small δ th ) and the experimental results as given in [20] (orange -including the quoted error bars). Moreover, in the left and right panels of figure 9, we present for completeness the ratio σ th DY /σ exp DY and the difference between theory and experiment in standard deviations, adding the corresponding errors in quadrature. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. | 2023-01-21T14:14:10.120Z | 2016-07-01T00:00:00.000 | {
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52939731 | pes2o/s2orc | v3-fos-license | Efficacy of temperature-guided cryoballoon ablation without using real-time recordings – 12-Month follow-up☆
Background We sought to evaluate a temperature-guided approach of cryoballoon (CB) ablation without visualization of real-time recordings. Methods and results We analysed 166 patients (34.9% female, 60 ± 11 years) with paroxysmal or short-term persistent atrial fibrillation (AF). Comorbidities included diabetes mellitus (n = 28), coronary artery disease (n = 24), hypertension (n = 122), previous stroke or TIA > 3 months (n = 12). Cryoablation of the pulmonary veins (PV) was performed using first-generation (n = 78) and second-generation CB (n = 88). Two 5-minute freezes were performed for the first-generation and two 4-minute freezes for the second-generation CB with the intention to achieve a temperature drop below −40 °C. At 12-month follow-up, we observed overall freedom from AF in 92 patients (56.6%, mean time to AF recurrence 3.4 ± 2.9 months). There was a significant difference in freedom from AF between first-generation CB (45%) and second-generation CB (67%; p < 0.005). Complications were groin hematoma (4.8%) and phrenic nerve palsy (PVP) (2.4%). PVP disappeared after 12 months in all patients. Three patients developed cardiac tamponade (1.8%) that resolved without further sequelae after pericardiocentesis. Multivariate analysis revealed that only the achieved temperature in the right inferior PV (RIPV) was a predictor of long-term freedom from AF (OR 0.9; p = 0.014). Female gender was a predictor of AF recurrence (OR 6.1; p = 0.022). Conclusion Temperature-guided CB ablation without real-time recordings is feasible and safe without reducing the efficacy if second-generation CB is used. Deep nadir temperatures especially in the RIPV are necessary for long term-success.
Introduction
Atrial fibrillation (AF) is the most important tachyarrhythmia in the developed world. It is associated with increased morbidity and mortality due to thromboembolism, heart failure and impaired cognitive functioning [1]. The prevalence and incidence of AF is increasing with age [2]. Data from randomized multicentre studies indicate that catheter ablation of AF is superior to antiarrhythmic drug therapy [3]. Current guidelines recommend catheter ablation of symptomatic paroxysmal AF in patients in whom antiarrhythmic drug therapy is ineffective and who prefer further rhythm control strategy [1]. The cornerstone of conventional AF ablation is pulmonary vein isolation (PVI). In clinical praxis, it is the preferred technique for patients with paroxysmal and persistent AF [4][5][6][7]. Over the last years, radiofrequency (RF) current ablation in a point-by-point mode around the pulmonary vein ostia/antra was the most common method [4]. Using RF current, the cellular necrosis is achieved by tissue heating. More recently, it could be shown that cryoballoon (CB) ablation for PVI is an effective alternative to RF ablation in paroxysmal AF [8,9]. The CB ablation of the pulmonary veins (PV) is a simple, single-step approach that leads to necrosis by freezing. Before the Achieve Mapping Catheter (AC) was available for the CB, ablation was carried out "blindly" during the freezing application. A recently published study found that a temperatureguided CB ablation without using real-time recordings was effective in producing PVI and freedom from AF in 85% of patients at 12-month follow-up (FU) [10]. In our study with a larger sample size, we aimed at investigating the feasibility and efficacy of a simplified mode of CB ablation, using a solely temperature-guided approach in patients with IJC Heart & Vasculature 21 (2018) 50-55 ☆ All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.
Contents lists available at ScienceDirect
IJC Heart & Vasculature j o u r n a l h o m e p a g e : h t t p : / / w w w . j o u r n a l s . e l s e v i e r . c o m / i j c -h e a r t -a n d -v a s c u l a t u r e paroxysmal and short-term persistent AF and further attempted to identify predictors of AF recurrence at 12-month FU.
Patient cohort
Patients ranging in age from 30 to 80 years, who had received the first PVI with a temperature-guided CB ablation between January 2011 and December 2014 at our centre, were consecutively included in our analysis. We included patients with symptomatic paroxysmal or short-term persistent AF. Eligibility criteria for PVI were at least one episode of AF documented by electrocardiogram (ECG) despite treatment with antiarrhythmic drugs including beta-blockers. We defined short-term persistent AF if the duration of arrhythmia was ≤12 months. Patients were excluded if they had previously undergone ablation in the left atrium (LA), if their LA was N55 mm in diameter, or if there was evidence of LA thrombus. Furthermore, patients were excluded if they had unstable angina, myocardial infarction, percutaneous transluminal coronary angioplasty or cardiac surgery within the previous three months, heart failure grade IV (New York Heart association criteria), stroke or transient ischemic attack within the previous 6 months, pregnancy or life expectancy bone year. All patients provided written informed consent prior to the intervention.
Study procedure
Baseline evaluation of patients comprised clinical history, review of medication, physical examination, blood chemistry, and 12-lead ECG. In order to exclude LA thrombi, transesophageal echocardiogram was performed in all patients at the day of intervention. Procedures were carried out under moderate sedation with midazolam and fentanyl. Single transseptal puncture was performed using a SL1 introducer sheath (ST Jude Medical, Minnetonka MN, USA). Immediately after having positioned the transseptal sheath across the interatrial septum, therapeutic anticoagulation was initiated with boluses of heparin. During the procedure, activated clotting time was maintained 300-350 seconds (s). Thereafter, the SL1 sheath was exchanged for a 12 french (F) FlexCath sheath (CryoCath, Medtronic Inc., Minneapolis, MN, USA). After PV angiography, CB was positioned in the left atrium. Cryoablation of the PVs was then performed using the first-generation CB (Arctic Front, Medtronic Inc.) for the first 78 procedures and the second-generation CB (Arctic Front Advance, Medtronic Inc.) for the last 88 procedures. The selection of CB size (23 mm vs. 28 mm) was left to the discretion of the operator. After the CB was applied to the PV ostium, contrast dye was injected through the central lumen of the CB to ensure an adequate "seal" and an occluded PV ostium. Optimal vessel occlusion was achieved when contrast dye injection showed complete contrast retention without backflow to the left atrium (vessel occlusion was evaluated in a semi quantitative manner ranging from grade 0 (very poor) and 4 (perfect)). With the intention to achieve at least a temperature drop below −40°C, at least two 5-minute freezes were performed for the first-generation and two 4-minute freezes were performed for the second-generation CB according to the manufacturer's recommendation. Additional applications could be used if deemed necessary. During CB ablation of the right PVs phrenic nerve injury was monitored using phrenic nerve pacing or breathing manoeuvres. If signs of phrenic nerve injury occurred, CB ablation was stopped immediately.
All patients received oral anticoagulation therapy during the first three months after the procedure, and thereafter according to the CHA 2 DS 2 -VASc-Score. After three months, anticoagulation was only discontinued in patients with a CHA 2 DS 2 -VASc-Score of ≤1 for men and ≤ 2 for women. Follow-up (FU) assessments were performed at three, six and 12 months during visits in our outpatient clinic. Antiarrhythmic drug therapy was stopped in all patients at least at 3-months FU. Additional visits were performed if patients were symptomatic or AF has been documented (12-lead-ECG or Holter ECG). Clinical and biochemical evaluation, as well as Holter ECG were carried out at baseline and at each FU visit. Each documented episode of ≥30 s after the three months blanking period was considered a failure. Furthermore procedural data, total radiation exposure, and occurrence of adverse events including phrenic nerve palsy (PNP), pericardial effusion, stroke, and vascular complications were reviewed.
Statistical analysis
Continuous variables are shown as mean ± standard deviation (SD). Categorical data are summarized as frequencies and percentages. Differences in baseline characteristics among patients were analysed with an unpaired Student's t-test for continuous variables and with χ 2 or Fisher's exact test for proportions, respectively. Event curves were determined according to the Kaplan-Meier method, with comparisons of cumulative event rates by the log-rank test. A multivariate logistic regression model was estimated to adjust for potential confounding factors for arrhythmic events, such as age, gender, aetiology, New York Heart Association (NYHA) functional class, left atrial size, left ventricular ejection fraction (LVEF), or beta-blocker treatment. In the multivariate model, continuous covariates were categorized according to age (in 10-year steps) and LVEF (in 5% steps). The multivariate analysis was also repeated without categorizing covariates. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated as an estimate of recurrent arrhythmic events.
All tests of statistical significance were 2-tailed, and a p value b 0.05 was considered statistically significant. The analyses were computed with SPSS software version 19 (SPSS Inc., Chicago, IL, USA) statistical software.
Clinical outcomes
During the 12 months of FU, we observed freedom from AF in 92 patients (56.6%). There was a significant difference in freedom from AF between first-generation CB (45%; 43 out of 78 patients) and secondgeneration CB (67%; 29 out of 88 patients; p b 0.005). The mean time to AF recurrence was 3.4 ± 2.9 months. The results are shown in Fig. 1. The most common complications were groin-site complications that occurred in 4.8% of the cases (8 patients with hematoma) and phrenic nerve palsy (PNP) that occurred in 2.4% of the cases (4 patients). PNP disappeared after 12 months in all patients. Three patients developed cardiac tamponade (1.8%) that resolved without further sequelae after uncomplicated pericardiocentesis. No stroke, arterioesophageal fistula, pulmonary vein stenosis or death occurred.
Four patients experienced cavotricuspid isthmus (CTI)-dependent atrial flutter during the blanking period. Ablation of the CTI was performed with nonirrigated RF energy catheter in all four patients within the blanking period. In our analysis, these cases were not classified as a failure of cryoballoon ablation.
Predictors of long-term freedom of AF
Univariate analysis showed that those patients with better NYHA functional class, better LVEF (65.2 ± 11.2 vs. 60.4 ± 11.1) and patients in whom a deeper mean temperature in the PVs was achieved had significant lower recurrence rates of AF. We found no differences in medical treatment between patients with and without recurrence of AF (Tables 1 and 2). However, multivariate analysis found that only the achieved temperature in the right inferior PV (RIPV), but not in the left superior PV (LSPV), left inferior PV (LIPV), and right superior PV (RSPV) was a predictor of long-term freedom from AF (OR 0.9, 95% CI 0.8-1.0; p = 0.014). Co-morbidities like NYHA functional class, coronary artery disease (CAD), diabetes mellitus, or hypertension as well as echocardiographic parameters like LAD and IVSd were not associated with AF recurrence following CB ablation. Female gender was a predictor of AF recurrence after CB ablation of the PVs (OR 6.1, 95% CI 1.3-29.5; p = 0.022). The results are shown in Table 3.
Discussion
To our knowledge, this is the largest real-life study of a simplified combined anatomical-and temperature-guided CB ablation of the PV without real-time recordings. The main findings of our study are the following: A temperature-guided CB ablation of the PVs without the use of real-time recordings in patients with paroxysmal or short-term persistent AF is feasible, safe, and effective during 12 months of FU. The overall success rate in our patient group defined as freedom from AF at 12-month FU after the procedure was lower in comparison to recent randomized trials. However, the success rate between firstand second-generation CB differed substantially. The long-term success rate by using the second-generation CB was comparable with recent randomized CB trials [9]. The achieved temperature in the RIPV was a predictor for long-term freedom from AF. Female gender was associated with an increased risk for AF recurrence.
At the beginning of the CB ablation for PVI in AF, no inner-lumen mapping-catheter was available. Ablation was done without real-time monitoring of PV potentials during the procedure and PV potentials could only be mapped after CB ablation. During that time, the CB was technically modified with implementation of a higher number of injection ports that are positioned more distally and further, the inner-lumen AC mapping catheter was introduced into clinical praxis.
The use of the second-generation CB in line with the inner-lumen AC mapping catheter led to a significant improvement of acute PVI [11]. Currently, the single catheter CB ablation in combination with the AC is the standard approach in CB ablation. The AC is a CB guidewire that allows simultaneous mapping of PV potentials during the freezing.
Ablation and mapping of the PVs can be done with only one transseptal puncture and without the need of catheter changes during the procedure. However, the verification of ostial PVIs using the AC might not always be sufficient to confirm ostial PVI in an accurate manner [12]. Recently Miyazaki et al. reported that the accuracy in verification of ostial PVIs with the AC ranged between 75% and 89%, depending on the location of the PV [12]. Meissner et al. compared the number of PV potentials recorded either by 32-pole high-density mash mapper (HDMM) or by 8-pole AC. Despite a significant difference in the identification of PV potentials before and after the CB ablation, the outcome between the standard group (AC mapping only) and the study group (additional HDMM mapping) by means of freedom from AF was similar at 3 (82% vs 83%) and at 6 months (79 vs 76%) [13]. This refers to the question whether a simplified method of a solely combined anatomical and temperature-guided PVI without measurement of real-time recordings might be feasible and effective during long-term FU.
The use of a J-tip guidewire instead of the AC might have some advantages. The J-tip guidewire is more flexible in comparison to the AC. Thus, it seems to be far easier to negotiate smaller PV branches that might lead to a better CB positioning in the PV ostium. Furthermore, the use of the AC in smaller PV branches could be more dangerous, since few cases of fracture of the AC in a PV branch during PVI and intramural PV hematoma following PVI have been reported [14,15]. It is further noteworthy that CB ablation by using a J-tip guidewire is much cheaper than CB ablation with AC given the need of reducing health care costs. This point should not be underestimated. Fig. 1. Kaplan-Meier estimates of time to first AF recurrence in the study. The first 3 months after ablation was the so-called "blanking period". Events during this time were not counted in the determination of AF recurrence, except in those patients who did not maintain sustained sinus rhythm after medical treatment or electrical cardioversion. Panel A shows the overall success rate of freedom from AF. Freedom from AF was observed in 56.6% of the patients. The mean time to AF recurrence was 3.4 ± 2.9 months. Panel B shows the subgroup test of success rate among firstand second-generation CB. There was a significant difference in freedom from AF between first-generation CB (45%; 43 out of 78 patients) and second-generation CB (67%; 29 out of 88 patients; p b 0.005). Recently, Iacopino et al. published the results of a temperature-guided strategy to CB ablation [10]. The authors compared the long-term efficacy of PVI in patients with paroxysmal AF (n = 52) who received secondgeneration CB ablation with a temperature-guided approach in comparison with a propensity score matched group of patients (n = 52), in whom the CB ablation was performed with the inner-lumen AC. With the goal to achieve a temperature drop below −40°C within 60 s, the authors found acute PVI in 99% of the cases. Despite of the fact that we did not check the PVs with a mapping catheter for PV potentials, the need of a temperature drop below −40°for successful ablation is in agreement with the long-term results in our study population. Patients without recurrent AF had a mean temperature drop below −40°C, whereas patients with recurrent AF had not. In the study of Iacopino et al. the long-term success rate after a median of 12 months was 85% in the wire group and 88% in the AC group [10]. This success rate was higher in comparison to our overall results with firstand second-generation CB together (AF freedom for all patients was 57% after 12 months), but also much higher as in recent multicentre randomized trials with larger patient samples, in which 65.4% of patients were free of AF at 12 months (FIRE AND ICE), respectively 67% of patients (The Cryo versus RF Trial) [9,16]. The results of these trials are, however, comparable to our results in those patients, who received ablation with second-generation CB (AF freedom in patients treated with the second-generation CB was 67%). In contrast to our study, Iacopino et al. checked PVs for remaining PV potentials, even in the temperature-guided group. Patients with remaining PV potentials received further cryoapplications [10]. Because of this, the results of this study are not really only temperature-guided and the long-term results of freedom from AF are therefore not completely comparable with our results. According to the baseline characteristics, our patients had also higher rates of hypertension, diabetes mellitus, CAD, and chronic kidney disease in comparison to the patients in the studies of Kuck et al. and Iacopino et al. [9,10]. In addition, we included patients with paroxysmal AF and also with short-term persistent AF in our analysis that might have led to the lower long-term success rate, as shown formerly [17,18]. In recent trials, success rate at 12-month FU in persistent AF patients was 50% by using first-generation CB [19] and 69% by using secondgeneration CB [20]. Only few data exist about CB-based PVI in a mixed cohort of patients with paroxysmal and persistent AF. Recently, three-year clinical outcome data after second-generation CB-based PVI have been published. The study included patients with paroxysmal (70%) and persistent AF (30%). After a median FU of 38 months 60% of patients remained in stable sinus rhythm. However, in comparison to our results with 38% of patients with persistent AF, the success rate in this trial was higher at 12 months (nearly 80%). But it was also much higher than the success rate at 12 months FU in the multicentre randomized "FIRE AND ICE" trial [9,21].
The temperature drop during CB ablation depends on anatomical conditions, the operator dexterity, and experience. Recent studies found that the achieved nadir temperature during CB ablation is of major importance for the success rate of CB ablation in patients with AF [11,22]. Our results support these findings. Univariate analyses demonstrated that the achieved temperatures in the LSPV, LIPV, RSPV, and RIPV were predictors of freedom from AF following CB ablation. However, multivariate analysis found that only the achieved temperature in the RIPV was significant. The fact that especially the achieved temperature in the RIPV predicts freedom from AF is a new and interesting finding. A perfect closure, especially of the RIPV, is for anatomic reasons sometimes difficult to achieve. According to our results, it seems necessary to make intensive efforts to reach deep nadir temperatures, especially in the RIPV.
Since PVI is expensive and associated with small but definite risk of serious complications, the prediction of the outcome of patients before PVI is of particular interest. In patients with AF, cardiovascular risk factors such as diabetes mellitus, CAD, hypertension, or obesity often occur simultaneously. Consequently, we additionally investigated the impact of these co-existing risk factors on the success-rate of CB ablation in paroxysmal and short-term persistent AF. In the multivariate analysis, only female gender predicted AF recurrence, whereas the above-mentioned confounding factors as well as left atrial diameter or interventricular septum thickness did not. This is in contrast to the results of the study of Winkle et al., who found that patients with pre-existing diabetes, hypertension, CAD, LA diameter or persistent AF were strong predictors of AF recurrence following AF ablation [23]. However, current guidelines do not recommend exclusion of patients with the aforementioned risk factors [1].
Our study is limited by the fact that it is a small non-randomized trial with a heterogeneous patient cohort (persistent and paroxysmal AF). But it shows real-life data with the largest patient sample ever studied, with a simplified combined anatomical-and temperature-guided CB ablation of the PVs with 12 months of FU. Our results clearly show that PVI with CB without real-time monitoring of PVPs is feasible and effective with comparable results of AF freedom in long term FU, if the second-generation CB is used. This approach of CB-ablation reduces procedural costs and simplifies the procedure, without reducing the efficacy. Further randomized trials should evaluate whether there is a nadir temperature that should be necessarily reached to achieve a definite acute PVI. Intensive efforts should be undertaken to reach deep nadir temperatures and the best possible occlusion, especially in the RIPV. | 2018-10-23T02:26:41.283Z | 2018-10-04T00:00:00.000 | {
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36870218 | pes2o/s2orc | v3-fos-license | Anticancer Therapy for Breast Cancer Patients with Skin Metastases Refractory to Conventional Treatments
Skin metastases of breast cancer are usually late events in the course of tumor progression and signify a poor prognosis. They may remain as a therapeutic challenge especially after failure of standard treatments. Topical interventions, together with or without radiotherapy, may only palliate the symptoms temporarily. However, there may be alternative treatment modalities for unresectable breast cancer skin metastases resistant to chemotherapy and radiotherapy. There are various genetic alterations in tumors and therapeutic potential of expression patterns for factors like epidermal growth factor receptor may have important clinical implications in case of disease refractory to the conventional treatments. Here, we clarified the therapeutic options and genetic alterations in skin metastatic breast cancer patients refractory to standard chemotherapeutics.
Introduction
In women, skin metastases are seen more commonly in breast cancer than in any other malignancy. The presence of skin metastases signifies disseminated disease and a poor prognosis. For this reason, aim of the treatment is usually to delay disease progression, control symptoms, and maintain quality of life. However, breast cancer is a genetically heterogeneous disease. Even after refractory to conventional treatments, there might be subgroups of breast cancer patients with cutaneous metastases who could already response to certain anticancer therapies.
Effective Chemotherapeutics in Refractory Patients
After progression on standard chemotherapy regimens, few treatments have shown to be effective for regression of the skin metastases from breast cancer. In fact, the increasing use of anthracycline and taxane-based chemotherapies in the neoadjuvant and/or adjuvant settings has led to investigate new cytotoxic therapies. The primary tumor and metastasis of skin and lymph nodes showed a significantly better response to capecitabine treatment than other metastases such as lung, liver and bone (Tsuyuki, 2010). This could be explained by the high penetration capacity of capecitabine and its metabolites to malignant and healthy tissue (Mader et al., 2003). In two patients who received capecitabine showed a dramatic response with resolution of their skin lesions (Sideras, 2008). In another two published case reports of breast cancer patients, oral
Anticancer Therapy for Breast Cancer Patients with Skin Metastases Refractory to Conventional Treatments
Umut Varol 1 *, Ibrahim Yildiz 1 , Ahmet Alacacioglu 1 , Ruchan Uslu 2 agent S-1 showed its efficacy for the skin metastases developed or progressed after major chemotherapy combinations (Tokugawa, 2009;Hirao et al., 2011). So, current data potentially encourage documentation of cutaneous specific responses from fluorouracil (5-FU) derivatives in breast cancer patients with multiple skin metastases. Cisplatin either in combination with 5-FU or alone was active in breast cancer patients with skin metastases refractory to both anthracyclines and taxanes because of the observation of remarkable objective responses more frequently in skin metastatic cases (Coşkun, 2011). Intratumoral administration of cisplatin is also effective in the local treatment of cutaneous tumor lesions of breast cancer (Rebersek, 2004). In addition, Franchina et al. reported two cases of disseminated TN breast cancer with extensive cutaneous metastases and a favorable response to pegylated liposomal doxorubicin in combination with gemcitabine (Franchina et al., 2012).
Efficiency of Anti-EGFR Agents
Breast cancer subtypes based on the hormonal receptor (HR) and human epidermal growth factor receptor 2 (HER2) status may reflect skin changes and soft tissue infiltration patterns at the time of skin metastasis. The presence of skin ulceration was shown predominantly in the HR+ group than in the others and erythematous infiltrations were more commonly found in the triplenegative (TN) group (Kong et al., 2011). On CT scans, soft tissue infiltration appeared to be more common in the HER2-enriched and TN groups than in the HR+ group and erythematous infiltrative lesions were more commonly established in patients with epidermal growth factor receptor (EGFR) overexpression (Kong et al., 2011). EGFR, a transmembrane receptor tyrosine kinase of the erbB family, is expressed in 15-30% of all breast cancers. Since squamous cell carcinoma (SCC) has been shown to express the EGFR, there is an increasing evidence of cetuximab efficacy in non-melanoma skin cancers. In a phase II trial, the total response rate of cetuximab is almost 50% in patients with SCC (Wollina, 2012). Gholam et al reported that; combination of cetuximab with paclitaxel established a major reduction in the skin metastases of a heavily pretreated patient with EGFR-positive, TN breast cancer (Gholam, 2007). Therefore, EGFR inhibitor cetuximab could be an option for skin metastases of breast cancer patients with EGFR overexpression.
Molecular Approaches
Breast tumors with early skin metastases expressed E-cadherin infrequently (Sihto et al., 2011). It was also shown that activating ras mutations and allelic loss of the c-met proto-oncogene are never involved in either the initiation or metastasis to skin in human breast cancer (Rochlitz et al., 1989;Ng et al., 1997). Besides, breast cancer patients with skin metastases and cutaneous local recurrences may exhibit different growth patterns due to different degrees of angiogenesis (Colpaert et al., 2003). Vascular endothelial growth factor (VEGF-D) expression was detected only in the inflammatory subtype of breast cancer and in tumors which developed an inflammatory skin metastasis (Kurebayashi et al., 1999). So, there could be a possible relationship between the expression level of VEGF and the development of skin metastasis. The recognition of different degrees of hypoxia-driven angiogenesis in cutaneous breast cancer deposits may have important implications for the utilization of antiangiogenic therapies. Meanwhile, Bevacizumab which is a VEGF-A inhibitor, was found to be harmful in selected breast cancer patients with metastatic cutaneous involvement (Cottu, 2011). This deleterious side effect of bevacizumab in patients with skin metastases could possibly be related with the wound healing impairments of this drug.
Protein expression pattern of the primary breast carcinoma may influence the distant metastatic site. In order to understand the underlying mechanisms of organspecific metastases, several gene and protein expression signatures have been identified in the primary tumor. These expression markers often include various genes coding for adhesion molecules, such as activated leukocyte cell adhesion molecule which demonstrated more significant protein expression in breast cancer skin metastases compared with other metastatic sites (Ihnen et al., 2011). Circulation of the leukocytes share many similarities with tumor cell migration and metastasis, which is critically regulated by chemokines and their receptors. Expression of chemokine receptors in the primary tumor predicts the site of metastatic relapse in patients with axillary lymph node positive breast cancer (Andre et al., 2006). If confirmed, these findings could have important clinical implications regarding the treatment and follow-up of patients with primary breast cancer.
Conclusions
The discomfort of skin metastases increases as the tumor spreads and more tissue destruction occurs. Topical interventions are attractive for its easy self-administration without any major systemic interference. Combination cryosurgery with hyperthermia, radiation therapy and its modalities, topically applied miltefosine or imiquimod and radiobiological approaches like re-irradiation in conjunction with liposomal doxorubicin showed only modest activities in a relatively heavily pretreated patient population, but they are very effective in order to palliate the symptoms (Fritz 2000;Smorenburg, 2000;Kouloulias 2003;Adams, 2012;Hachisuka, 2012). However, all these treatments are not long-term solutions and more efficient medications are necessary. Thus, it is essential to define specific genetic alterations of the tumor cells which increase their metastatic potential. Owing to their therapeutic opportunity, organ specific targets could yield to more effective treatments which may enhance the activity of chemotherapies and improve the prognosis of metastatic breast cancer patients. | 2017-09-27T03:44:18.978Z | 2014-01-01T00:00:00.000 | {
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85131543 | pes2o/s2orc | v3-fos-license | Arbuscular Mycorrhizal Colonization Enhances Biochemical Status in and Mitigates Adverse Salt Effect on Two Legumes
Symbiotic association between arbuscular mycorrhizal (AM) species and host plant roots improves plant growth and protects them from several abiotic stress factors. In the present study, the effect of Glomus mosseae and Glomus fasciculatum as an individual inoculation and in combination was studied on two legumes ( Glycine max and Cyamopsis tetragonoloba ) under soil salinity stress gradient [1.04 (control) to 8.26 dS/m]. Individual and co-inoculation of both the AM fungi alleviated adverse salt effect, with improvement in plant dry weight matter and biochemical parameters. However, these two isolates worked better in combination with respect to higher accumulation of soluble carbohydrate, reducing sugar, protein, proline concentration etc. C. tetragonoloba showed better response as compared to G. max in relation to improvement in nutritional profile under salt stress after AM treatment. As compared to non-mycorrhizal counterparts, co-inoculation with G. mosseae and G. fasciculatum in C. tetragonoloba enhanced total chlorophyll (14.83% at soil salinity of 3.78 dS/m), soluble carbohydrate (17.26% at soil salinity of 5.94 dS/m), proline (8.79% at soil salinity of 3.78 dS/m) while exposed to different soil salinity levels. Also, co-colonization with both the isolates showed more root colonization (%) and may be responsible for the better effect in salt stress alleviation. Electrolyte leakage of mycorrhizal plants was lowered at soil salinity gradient of 2.10 to 8.26 dS/m and hence, maintained membrane stability. These two isolates can be utilized as bio-inoculant in alleviation of adverse salt effect in soil in association with the two test legume plants.
Introduction
Salinization of soil is becoming a severe agricultural constraint in arid and in semi arid regions of India. Presence of excess salt concentrations in soil solution arises mainly due to the heavy use of chemical fertilizers and use of low quality water (during irrigation of agricultural land) which contains soluble salts in large amount (Al-Karaki, 2000;Chartzoulakis and Klapaki, 2000). When salt concentration in soil reaches beyond the normal range, it makes the land unproductive as it results in nutrient imbalance in plants, hampers water availability followed by stunted growth and leads to reduced crop productivity Hopkins and Huner, 1999). Development of salt tolerant crop varieties and physico-chemical methods for removal of excess salts from agricultural soils have been tried (Cuartero and Fernandez-Munoz, 1998;Muralev et al., 1997). These approaches have been successful but are costly and hence, a new alternative attempt has taken up to tackle the deleterious effects of saline soils which involve inoculation of salt tolerant arbuscular mycorrhizal (AM) fungi in agricultural crop.
AM fungi are considered as obligate symbionts and are naturally associated with roots of around 90% vascular plants (Allen, 1991). AM fungi improve plant health by providing essential mineral nutrients from soil. They maintain physiological processes of the host plant, as well as protect it from several biotic and abiotic stresses (Augé, 2000;Ruiz-Lozano, 2003;Sylvia and Williams, 1992). In salt stressed soil, phosphate ions usually precipitate along with Ca 2+ , Mg 2+ , Zn 2+ and are less available to plants (Azcon-Aguiler et al., 1979). But, AM symbiosis in plants enhances the uptake of less mobile phosphorus by extending their external hyphal network beyond nutrient depletion zone. In mitigation of salt stress, AM fungi also improve plant growth, hormonal status, increase nutrient acquisition, maintain osmotic balance, reduce ion toxicity etc. (Juniper and Abbott, 1993;Lindermann, 1994;Ruiz-Lozano, 2003).
With increasing world population, the demand for food, mainly cereal based protein rich diet is increasing, especially in developing countries. Due to scarcity of productive agricultural land and ever increasing population growth, the developing countries are facing the problem of food crisis and hence, protein-rich leguminous food may be one of the substitutes (Sadik, 1991;Weaver, 1994). In such scenario, cultivation of legume plants is promising considering protein content and presence of carbohydrate pool. It is reported that under abiotic stress condition, improvement of biochemical and antioxidant profile of the host legume plant was observed following colonization with AM fungi (Porcel and Ruiz-Lozano, 2004). Several reports suggest that mycorrhizal inoculation with single AM isolate reduces the detrimental effects of salinity, but salt tolerance level of the plant as well as mycorrhizal efficiency to respond the host varies among different plant species and mycorrhizal fungi (Burke et al., 2003;Tian et al., 2004). Hence, in alleviation of salt stress for particular plant species, selection of specific AM fungi as an individual or in combination of two or more is essential. The interaction between legume plant and combined inoculation of two different AM fungi is not well studied before and hence, the present study was aimed at investigating the individual and combined effects of Glomus mosseae and Glomus fasciculatum on two different crop legumes under soil salinity stress. A comparison between two crop legumes Glycine max and Cyamopsis tetragonoloba was undertaken in relation to improvement in plant growth and biochemical status. G. max is considered as inexpensive source of proteins and is also valued for its high oil content. C. tetragonoloba is an annual legume and is an important source of guar gum. Guar gum is directly used in dairy industry and in textile industry after its derivatization. Salt tolerant AM fungi were used in this study as bio-inoculants for cultivation of legumes in salt stressed soil, as both the legumes have nutritional significance.
Chemicals
All the chemicals used in this study were of analytical reagent grade.
Plant Material
Seeds of G. max and C. tetragonoloba used in this study were obtained from Naik Seeds Pvt. Ltd., Pune, Maharashtra (India). Seeds were surface sterilized using sodium hypochlorite solution (0.5% w/v): reverseosmosis water (1:3), followed by rinsing with reverseosmosis water. Surface sterilized seeds of G. max and C. tetragonoloba were directly placed on moist filter paper for germination. At first true leaf stage, seedlings of almost equal length were used for transplantation in pot.
Mycorrhizal treatment and experimental design G. mosseae (Nicol and Gerd) and G. fasciculatum (Thaxt.) Gerd and Trappe used in this study were isolated in the laboratory of University of Pune, earlier from Lonar Crater Lake, Buldhana and Sakri, Dhule, Maharashtra respectively, by wet sieving and decanting method (Datta, 2012;Gerdemann and Nicolson, 1963;Schenck and Perez, 1990). Spores were propagated in Zea mays L. for three months. G. mosseae and G. fasciculatum soil based inocula contained 150 to 160spores/10 g air-dried soil with AM colonized roots (~75% colonization) and 100 to 110spores/10 g airdried soil and roots of ~53% AM colonization respectively. These soil based inocula were used for further study.
The experiment was designed in a complete randomized design, consisted of four mycorrhizal treatments (NM: non-mycorrhizal, Gm: G. mosseae, Gf: G. fasciculatum and Gm+Gf: G. mosseae + G. fasciculatum), five soil salinity levels [1.04 (control), 2.10, 3.78, 5.94 and 8.26 dS/m], two test plants (G. max and C. tetragonoloba), with five replicates (three plants per replicate). In Gm and Gf treatment, 50 g of G. mosseae and 75 g G. fasciculatum soil based inocula were used. However, in Gm+Gf treatment, 25 g of G. mosseae and 40 g of G. fasciculatum were used. Variable amount of soil based inocula were applied to achieve almost similar number of spores per pot (~800spores/pot). Mycorrhizal treatments were provided by placing respective soil based inoculum 3 cm below the seedling prior to seedling transplantation. Plants without mycorrhizal inoculum served as non-mycorrhizal control. Sand-soil volume (3 kg) was kept constant in all mycorrhizae treated and untreated pots. On every alternate day, plants were irrigated with tap water (sieved through 105 μ sieve) and with Hoagland solution (X/10) twice a month (Hoagland and Arnon, 1940). Irrigation was done in such a way to make soil water content of around 40% field capacity. After one month of seedling transplantation, saline stresses were provided and the stress was increased gradually to prevent shock. Saline solution was continuously applied until the target salinity level reached. Plants were grown for four months in greenhouse (at 25-35 °C, relative humidity: 65-70%, photon flux intensity: 300-350 μmol/m 2 /S).
Plant dry weight
After two months of seedling transplantation, plants from each treatment were harvested and washed in distilled deionized water. Fresh plants were kept in oven at 60 °C for 24h for drying and then dry weights were recorded.
Total chlorophyll content
The youngest fully expanded fresh leaves from each treatment were collected for estimation of total chlorophyll content following the method of Strain and Svec (1966). Fresh leaf samples were first extracted in 80% (v/v) acetone and then subjected to centrifugation. Concentrations of chlorophyll a and chlorophyll b of the supernatant were estimated by using spectrophotometer. Total chlorophyll content was determined and the value was expressed in terms of mg/g leaf samples.
Soluble protein content
Fresh plant material (both shoot and root tissues) from each treatment was used for determination of soluble protein content. Sample was extracted in phosphate-buffered saline and then soluble protein content was detected by dye binding assay using Coomassie Brilliant Blue G 250 (Bradford, 1976). BSA was used as standard for the preparation of calibration curve.
Soluble carbohydrate content
Fresh plant material (both shoot and root tissues) from each treatment was subjected to acid hydrolysis and then total soluble carbohydrate content was estimated using anthrone method (Hedge and Hofreiter, 1962). Glucose was used as a standard for preparing the calibration curve.
Reducing sugar content
Fresh plant material (both shoot and root tissues) from individual treatment was extracted in hot ethanol for two successive times and total reducing sugar content was determined using 3,5-dinitrosalicylic acid method (Miller, 1972). Standard glucose solution was used for preparation of calibration curve.
Free proline content
Free proline content was determined by using Ninhydrin acid reagent (Bates et al., 1973). Fresh plant material (both shoot and root tissues) from each treatment was extracted in sulfo-salicylic acid (3% w/v) and filtered through Whatman No. 2 filter paper. Filtrate was then treated with glacial acetic acid and acid ninhydrin reagent and was kept for boiling (1 h). After boiling, toluene was added to the reaction mixture and vortexed. Toluene layer was separated and absorbance of sample was measured at 520 nm using UV/Vis spectrophotometer (Shimadzu UV 1601). Standard proline was used for preparation of calibration curve.
Electrolyte leakage (EL)
Fresh leaf tissue from each treatment was used for determination of electrolyte leakage. Leaf samples were cut into small pieces of almost equal length and used for EL measurement. 0.5 g sample was taken in a test tube containing distilled deionized water and then placed in water bath (at 32 °C) for 2 h. After incubation, tubes were cooled to 25 °C and initial electrical conductivity (EC) was measured using conductivity meter (Hanna,HI 8733). Then, the samples were autoclaved for 20 min and final EC was measured using conductivity meter (Hanna, HI 8733) after cooling the tubes at 25 °C (Dionisio-Sese and Tobita, 1998). EL of fresh leaf samples was calculated using the following formula: EL = (Initial EC/ Final EC) × 100 where EL = Electrolyte leakage; EC = Electrical conductivity.
AM root colonization (%)
Root samples were washed with deionized water, followed by cutting it into 1 cm pieces. The root pieces were thoroughly mixed and a sub-sample (0.5 g) was cleared in hot KOH solution (10% w/v, at 90 °C) for 1 h. Cooled root samples were washed with deionized water and placed in HCl (10% v/v) for 3 min and stained with trypan blue (0.05% w/v) for 15 min at 90 °C (Phillips and Hayman, 1970). Percentage of AM colonization in root samples was estimated by gridline intersects method (Giovannetti and Mosse, 1980).
Statistical analysis
Data were analyzed by ANOVA followed by Duncan's test and values of each treatment followed by different letters indicate statistically significant difference at P<0.05 level. SPSS v. 9.0 was used for the statistical data analysis.
Results
In the present study, soil salinity hampered growth and biochemical status of both the test plants regardless of mycorrhizal treatment. However, it was observed that, mycorrhizal colonization in G. max and C. tetragonoloba, reduced the extent of deleterious salt effect and improved plant growth even under provided soil salinity. Although, both the mycorrhizal isolates showed positive response in enhancement of host plant growth, but combined inoculation of both the isolates was found superior in this respect, followed by individual inoculation with Gm and Gf.
Plant dry weight G. max and C. tetragonoloba plants grown under control treatment (1.04 dS/m) had relatively more dry weight content than the corresponding plants from saline stress treatment (2.10 to 8.26 dS/m). The values were significantly declined as soil salinity was increased from 2.10 to 8.26 dS/m regardless of mycorrhizal inoculations (Tab. 1).
With increasing soil salinity gradient dry weights of test plants were reduced, but under control (1.04 dS/m) and at each level of soil salinity (2.10 to 8.26 dS/m) mycorrhizae inoculated plants had significantly higher dry weight than respective non-mycorrhizal plants. Moreover, inoculation with mixed mycorrhizal fungi was found to be superior over the individual inoculations. Individual effect of soil salinity and mycorrhizal fungi on dry weight was found significant for both the plants. However, their interaction was mainly significant with respect to dry weight content of G. max (Tab. 1).
Total chlorophyll content
Regardless of mycorrhizal inoculations, the test plants grown under control treatment (1.04 dS/m) had significantly higher chlorophyll content and the concentration was reduced with increase in salinity stress (Tab. 2).
However, at each level of soil salinity (2.10 to 8.26 dS/m) and in control treatment (1.04 dS/m), mycorrhizal colonization (individual and combined) in test plants resulted in significantly higher chlorophyll content as compared to respective non-mycorrhizal control plants (Tab. 2).
It was also observed that, regardless of salinity treatments, among the three types of mycorrhizal inoculations, combined mycorrhizae inoculated plants (G. max and C. tetragonoloba) showed higher chlorophyll content, than the corresponding plants inoculated with individual isolates (Tab. 2).
Soil salinity and mycorrhizal inoculations had significant effect on total chlorophyll content in G. max and C. tetragonoloba plants, and their interaction was
Soluble protein content
Regardless of mycorrhizal inoculations, as compared to control (1.04 dS/m), salt stress (at salinity level of 2.10 dS/m) resulted in significant enhancement of soluble protein content of both the test plants, but with further increment in salinity stress level from 3.78 to 8.26 dS/m, a significant reduction was noticed (Tab. 3).
Although, higher levels of soil salinity had negative impact on plant protein content, test plants inoculated with Gm and Gf isolates (individual and in combination) showed a significantly higher protein content as compared to non-mycorrhizal plants grown under control (1.04 dS/m) and at each level of soil salinity (2.10 to 8.26 dS/m). Individual and combined effect of salinity and mycorrhizal inoculations were found significant on soluble protein contents of the test plants (Tab. 3).
Soluble carbohydrate content
In C. tetragonoloba plant, a steady, significant increment in soluble carbohydrate content was noticed, when the soil salinity was raised from control (1.04 dS/m) to 3.78 dS/m and with further increase in saline stress; the magnitude of carbohydrate content got reduced significantly irrespective of mycorrhizal inoculations. However, in G. max plant, the concentration of soluble carbohydrate significantly increased up to salinity stress level of 5.94 dS/m, followed by a decreasing trend regardless of mycorrhizal inoculations (Tab. 4).
With increasing soil salinity either up to third or fourth level (3.78 or 5.94 dS/m), soluble carbohydrate content increased steadily in mycorrhizae treated and untreated test plants; although comparatively higher concentration was found in plants following mycorrhizal inoculations (Gm and Gf). Combined inoculation of both the mycorrhizal isolates worked better in this regard. The individual and interactive effect of soil salinity and mycorrhizal inoculations were found significant on carbohydrate content of test plants (Tab. 4). with increasing soil salinity stress from control (1.04 dS/m) to 3.78 dS/m. However, with further increment in saline stress, a significant reduction was noted (Tab. 5). Even though, due to saline stress gradient (up to 3.78 dS/m), reducing sugar content was increased in mycorrhizae inoculated and non-mycorrhizal test plants, but Gm and Gf colonized (with individual and combined) test plants had significantly higher amount of reducing sugar over corresponding non-mycorrhizal plants under control (1.04 dS/m) and provided salinity stress gradient (2.10 to 8.26 dS/m) (Tab. 5).
Reducing sugar content
Moreover, test plants colonized with both Gm and Gf isolates had reducing sugar contents to a little higher amount as compared to corresponding plants with individual Gm and Gf isolates. Individual effect of soil salinity and mycorrhizal colonization and their interaction were found significant on reducing sugar contents of test plants (Tab. 5).
Free proline content
Proline concentration in test plants was significantly improved under increasing soil salinity up to 5.94 dS/m, regardless of mycorrhizal inoculations. Then it was reduced (P<0.05) in mycorrhizal and non-mycorrhizal plants at the highest level of soil salinity (8.26 dS/m) (Tab. 6).
It is worth noting that, even though, increasing soil salinity gradient (up to 5.94 dS/m) resulted in enhancement of proline concentration, but as compared to non-mycorrhizal plants, mycorrhizal plants had significantly higher proline content at control (1.04 dS/m) and soil salinity gradient (2.10 to 8.26 dS/m) in case of both the plants. Plants with combined mycorrhizal treatment showed more proline content over the corresponding plants colonized with individual Gm and Gf isolates, when exposed to control (1.04 dS/m) and soil salinity stress gradient (2.10 to 8.26 dS/m) (Tab. 6).
Soil salinity and mycorrhizal inoculations when considered individually and in combination showed a significant effect on proline content of G. max and C. tetragonoloba plants (Tab. 6).
Root colonization
At the end of the whole study, non-mycorrhizal plants remained uncolonized whereas, AM colonization was detected in mycorrhizae inoculated test plants exposed to control (1.04 dS/m) and soil salinity gradient up to 8.26 dS/m (Tab. 8).
Due to individual and combined AM inoculations, the magnitude of root colonization was different and relatively higher AM root colonization was noticed in Tab Soil salinity and mycorrhizal inoculation when considered individually a significant effect on AM root colonization was observed in case of both the test plants. However, their interaction was mainly non-significant on this parameter (Tab. 8).
Mycorrhizal response on plant dry weight and various biochemical parameters
The overall effect of AM inoculations with Gm and Gf isolates (individually and in combination) on test plant dry weight and accumulation of various biochemical constituents was determined over respective non-mycorrhizal plants grown under control (1.04 dS/m) and each level of soil salinity (2.10 to 8.26 dS/m) (Fig. 1a to 1f).
In G. max and C. tetragonoloba plants, AM inoculations (Gm and Gf) increased dry weight yield, chlorophyll, protein, proline, carbohydrate and reducing sugar content to a maximum amount at soil salinity either of 3.78 dS/m or of 5.94 dS/m. At control (1.04 dS/m) and various soil salinity levels (2.10 to 8.26 dS/m), the effect of individual and combine mycorrhizal inoculations (with Gm and Gf isolates) was more pronounced in G. max over C. tetragonoloba, in response to higher accumulation of several biochemical constituents (protein and reducing sugar) and dry weight yield (Fig. 1a to 1f).
However, better accumulation of total chlorophyll, proline and carbohydrate content was noticed in C. tetragonoloba as compared to G. max, following individual and combined inoculation with AM isolates under various levels of soil salinity. Moreover, it was observed that, regardless of test plants and soil salinity, the acquisition of biochemical constituents was found to be higher following combined inoculation with Gm and a.
389
Tab. 8. Percent root colonization of mycorrhizal and non-mycorrhizal G. max and C. tetragonoloba grown under control (1.04 dS/m) and at increasing soil salinity levels (2.10 to 8.26 dS/m)
Soil salinity (dS/m) AM inoculation
Chlorophyll content (mg/g) Fig. 1. Mycorrhizal response on enhancement of (a) dry weight yield, (b) total chlorophyll, (c) soluble protein, (d) free proline, (e) soluble carbohydrate, (f) reducing sugar content in individual and combined AM inoculated G. max and C. tetragonoloba over corresponding non-mycorrhizal plants exposed to control (1.04 dS/m) and increasing soil salinity gradient (2.10 to 8.26 dS/m) Gf than their individual colonization.
Discussion
This study investigated the effect of two indigenous AM isolates in promoting growth in terms of dry weight matter and various biochemical parameters at different levels of salt stress.
Dry weight of mycorrhizae treated and untreated G. max and C. tetragonoloba reduced significantly with progressively increasing soil salinity stress, and this may be because of ion toxicity or indirect effect of osmotic imbalance between soil and plant caused by salt ions (Abdel-Latef, 2010). Also, the stunted plant growth under salt stress is normally occurred when high salt contents in soil prevent cell division directly or indirectly (Singh and Chatrath, 2001). However, mycorrhizal inoculation mitigated dry matter reduction in test plants under each salinity level as compared to respective nonmycorrhizal plants. Similar type of results were obtained in previous reports in which AM fungi improved plants dry biomass under salinity stress Gharineh et al., 2009). Present study indicated that individual mycorrhizal inoculation significantly improved dry matter accumulation in test plants as compared to respective non-mycorrhizal plant, but co-inoculation was found to be superior in this respect under salinity stress. It is already known that AM fungi are able to adapt edaphic environmental conditions and isolates from saline soil would have a better salt tolerance capacity (Copeman et al., 1996;Kaya et al., 2009). In our study, Gm was isolated from saline soil as a dominant spore and showed a greater positive response than the other isolate (Gf), but both of these worked better in co-inoculation treatment. Although, mycorrhizal inoculation resulted in enhanced dry weight in both the test plants, but, magnitude of dry weight increment due to mycorrhization was found better in G. max. Better growth of mycorrhizae treated plants has been correlated with more nutrient acquisition in AM mediated plants (Plenchette and Dupponois, 2005).
In the present study, increasing soil salinity gradually reduced chlorophyll content of mycorrhizae inoculated and uninoculated test plants. This result is in agreement with previous report where chlorophyll content in maize plant was declined under saline stress (Sheng et al., 2008). The reduction in chlorophyll content at higher salinity level is mainly because of suppression of chlorophyll synthesizing enzyme activity and less uptake of magnesium ion by plants (El-Desouky and Atawia, 1998;Murkute et al., 2006). But mycorrhizal inoculation can alleviate this problem to some extent and in our study, at each salinity level, significantly higher chlorophyll content was detected in leaves of G. max and C. tetragonoloba plants as compared with respective nonmycorrhizal plants. Thus, Gm and Gf association (individually and in combination) enhanced the photosynthetic ability of G. max and C. tetragonoloba under salinity stress, which is in agreement with earlier report (Sonnazzaro et al., 2006). Data from present study indicated that, regardless of host plant, maximum chlorophyll accumulation occurred by AM fungi in coassociation as compared to their individual association. At each soil salinity level leaves of C. tetragonoloba showed better chlorophyll accumulation as compared to G. max.
When plants grow under saline conditions several types of salt stress proteins accumulate in plant to maintain osmotic balance, act as storage form of nitrogen and furthermore they can be re-utilized later once stress is over (Singh et al., 1987). In the present study, protein content followed an increasing trend at low range of salt stress however, higher soil salinity significantly reduced soluble protein contents in G. max and C. tetragonoloba plants irrespective of mycorrhizal inoculations. However at each salinity level protein content was improved significantly after mycorrhizal colonization as compared to corresponding non-mycorrhizal plants, even though maximum protein accumulation was observed in coinoculated test plants. Reduction in protein concentration with increasing soil salinity was observed, because salt stress interferes with nitrogen acquisition and utilization by interrupting protein synthesis (Aslam et al., 1984;Frechill et al., 2001). In present study, mycorrhizal inoculation mitigated the reduction in protein content in plants grown under saline gradient (2.10 to 8.26 dS/m) and control (1.04 dS/m) treatment and may be because extra radical mycelia of AM fungi easily takes up inorganic nitrogen and transports it to intra radical mycelia. Hence, nitrogen can be easily transferred from fungus to plant in AM association (Govindarajulu et al., 2005). Moreover, percentage accumulation of soluble protein was found maximum in C. tetragonoloba plant, followed by G. max. This finding might be related to the fact that specific nitrogen containing compound accumulate in varying concentration in different plant species under salinity stress (Rabie and Almadini, 2005).
In this study, plants exposed to lower level of salt stress (1.04 to 3.78 dS/m) contained better carbohydrate content than the plants exposed at higher salt stress and may be because of the fact that, in response to soil salinity, soluble carbohydrates have been proved to accumulate in plants to adjust osmotic potential and also serving as a carbon storage compound (Murakeozy et al., 2003;Parvaiz and Satyawati, 2008). Soluble carbohydrate contents of both the test plants were reduced significantly at higher level of salinity stress. However, mycorrhization in all the test plants significantly improved soluble carbohydrate contents compared to respective non-mycorrhizal plants exposed to control (1.04 dS/m) and soil salinity stress (2.10 to 8.26 dS/m). It was also observed that better carbohydrate accumulation was achieved by combined AM inoculation irrespective of plant species. The positive correlation between mycorrhization of host plant and soluble sugar accumulation was also reported earlier (Al-Garni, 2006;Datta and Kulkarni, 2013). When both the test plants are compared, C. tetragonoloba plant accumulated better carbohydrate than G. max plant.
Increasing soil salinity (upto 3.78 dS/m) enhanced the content of reducing sugar in test plants and this may be due to accumulation of several osmolytes (reducing sugar) in plants to remove osmotic disturbance which comes into effect under salt stress and this accumulated reducing sugar also acts as carbon storage (Parida and Das, 2005). Increment in reducing sugar content in various plant species (wheat, grapevine) has already been reported when they were exposed to salt stress (Kerepesi and Galiba, 2000;Khatkar andKuhad, 2000, Singh et al., 2000). But mycorrhizal inoculations increased reducing sugar accumulation in test plants over the nonmycorrhizal plants at various soil salinity levels. Mycorrhizal inoculations with Gm and Gf isolates adjust osmotic balance in a better way and may help in higher osmo-protection.
With increasing soil salinity consistent increase in proline content in both the test plants (mycorrhizal and non-mycorrhizal treated) was observed. Under salinity stress exposure, proline (the protective osmolyte) accumulates for adaptation of salt stress, maintains osmotic balance and also acts as energy and nitrogen reservoir to be used by plant (Ashraf and Foolad 2007;Jindal et al., 1993). It was observed that, mycorrhizal treatment significantly improved proline concentration and their accumulation as compared to non-mycorrhizal plant under increasing soil salinity stress gradient. But a maximum proline accumulation was observed in cocolonized plant. More proline accumulation after AM colonization has already been reported earlier (Jindal et al., 1993). Better proline accumulation in C. tetragonoloba may be correlated with fact that more proline accumulates in less salt tolerant plant as a symptom of stress for maintaining osmotic adjustment (Wang et al., 2004).
Data of present study clearly indicate that electrolyte leakage can be positively correlated with provided salinity stress. In all the test plants, by increasing stress level (2.10 to 8.26 dS/m), a significant reduction in electrolyte leakage was observed in AM inoculated G. max and C. tetragonoloba plants as compared to non-mycorrhizal plants. Less electrolyte leakage in mycorrhizae treated test plants may be correlated with higher electrolyte concentration in AM plants and this helps to maintain membrane stability (Garg and Manchanda, 2008). Mycorrhization in host plants is responsible for lowering electrolyte leakage at 2.10 to 8.26 dS/m soil salinity stress and this finding is in agreement with the other report (He et al., 2007).
Root colonization percentage was reduced significantly in mycorrhizae treated test plants, when salinity level was increased gradually. At each stress level, Gf inoculated plants had the least colonization percentage, however, Gm and co-inoculation with both AM fungi colonized the test plants roots in higher amount. The reduction in magnitude of percentage colonization under increasing salt stress might be due to inhibition of AM spore germination, interruption in hyphal growth in soil or reduction in arbuscule formation etc. (Hirrel, 1981;McMillen et al., 1998;Pfeiffer and Bloss, 1988). Between the two test plants, percentage of mycorrhizal colonization (by individual isolate and in combination) was found to be higher in C. tetragonoloba than G. max under various levels of salt stress (2.10 to 8.26 dS/m) and control treatment (1.04 dS/m). More percentage of AM root colonization (individually and in combination) in C. tetragonoloba plant may be responsible for better accumulation of total chlorophyll, proline, carbohydrate contents etc. under salt stress and protected C. tetragonoloba plant from detrimental effect of salt stress in a better way.
Conclusion
G. mosseae and G. fasciculatum symbiotic interaction (as individually and in combination) with G. max and C. tetragonoloba plants, alleviated the deleterious effect of salt stress by improving various biochemical parameters. Even though, individual AM fungi showed positive effect in mitigation of salt effect, but co-inoculation with both Gm and Gf isolates worked better in this respect. Out of the two test plants, C. tetragonoloba showed better growth profile as compared to G. max in response to coinoculation by both the AM isolates under salt stress. Mycorrhization reduced electrolyte leakage profoundly and could maintain osmotic balance by stabilizing membrane stability. Hence, these two AM fungi can be further utilized as bio-inoculant in salt affected soils. | 2019-03-22T16:17:41.927Z | 2014-09-22T00:00:00.000 | {
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237534990 | pes2o/s2orc | v3-fos-license | Diagnostic Accuracy of FEC-PET/CT, FDG-PET/CT, and Diffusion-Weighted MRI in Detection of Nodal Metastases in Surgically Treated Endometrial and Cervical Carcinoma
Abstract Purpose: Preoperative nodal staging is important for planning treatment in cervical cancer and endometrial cancer, but remains challenging. We compare nodal staging accuracy of 18F-ethyl-choline-(FEC)-PET/CT, 18F-fluoro-deoxy-glucose-(FDG)-PET/CT, and diffusion-weighted-MRI (DW-MRI) with conventional morphologic MRI. Experimental Design: A prospective, multicenter observational study of diagnostic accuracy for nodal metastases was undertaken in 5 gyne-oncology centers. FEC-PET/CT, FDG-PET/CT, and DW-MRI were compared with nodal size and morphology on MRI. Reference standard was strictly correlated nodal histology. Eligibility included operable cervical cancer stage ≥ 1B1 or endometrial cancer (grade 3 any stage with myometrial invasion or grade 1–2 stage ≥ II). Results: Among 162 consenting participants, 136 underwent study DW-MRI and FDG-PET/CT and 60 underwent FEC-PET/CT. In 118 patients, 267 nodal regions were strictly correlated at histology (nodal positivity rate, 25%). Sensitivity per patient (n = 118) for nodal size, morphology, DW-MRI, FDG- and FEC-PET/CT was 40%*, 53%, 53%, 63%*, and 67% for all cases (*, P = 0.016); 10%, 10%, 20%, 30%, and 25% in cervical cancer (n = 40); 65%, 75%, 70%, 80% and 88% in endometrial cancer (n = 78). FDG-PET/CT outperformed nodal size (P = 0.006) and size ratio (P = 0.04) for per-region sensitivity. False positive rates were all <10%. Conclusions: All imaging techniques had low sensitivity for detection of nodal metastases and cannot replace surgical nodal staging. The performance of FEC-PET/CT was not statistically different from other techniques that are more widely available. FDG-PET/CT had higher sensitivity than size in detecting nodal metastases. False positive rates were low across all methods. The low false positive rate demonstrated by FDG-PET/CT may be helpful in arbitration of challenging surgical planning decisions.
Introduction
In patients with endometrial or cervical cancer, nodal metastatic disease adversely affects prognosis (1,2). In cervical cancer, the 5-year relative survival rates for patients with disease localised to the cervix is 92% compared with 56% for those with positive pelvic lymph nodes (LN; ref. 3). In endometrial cancer, the 5-year diseasefree survival is 90% in patients without LN metastasis, but 60% to 70% in those with pelvic LN metastasis and 30% to 40% in those with para-aortic LN metastasis (1). Accurate LN staging is required for prognostic stratification and treatment planning, as well as tailoring the surgical approach and delineating the extent of radiotherapy in both cervical and endometrial cancer. Knowledge of LN status is of paramount importance to stratify patients to radical hysterectomy versus primary chemoradiotherapy, particularly in cervical cancer.
In endometrial cancer, most patients will undergo at least a hysterectomy and bilateral salpingo-oopherectomy as primary treatment. The International Federation of Gynecology and Obstetrics (FIGO) include the LN status in the tumor staging of the patients, and so national and international guidelines recommend surgical LN staging in high-risk subtypes (4,5). However, there has not been any prospective evidence so far to demonstrate therapeutic value of systematic lymphadenectomy (LND) in endometrial cancer (6)(7)(8)(9). Even though sentinel LN techniques are now gradually replacing systematic LND in multiple guidelines (10)(11)(12), its implementation is not yet homogenous around the world due to infrastructural, financial, and governance challenges. For that reason many patients still undergo LND with all the associated sequelae such as lymphocyst formation, lymphorrhea and lymphedema in addition to higher surgical morbidity. The accurate and reliable preoperative identification of LN-positive patients, therefore, still represents an unmet need to adequately tailor therapeutic management.
In cervical cancer, radical hysterectomy is not recommended in LNpositive patients, and so the accurate preoperative identification of even microscopically involved LN would spare those patients unnecessary surgical morbidity and direct them to chemoradiotherapy with exact tailoring of the extended field.
Although the preoperative detection of nodal metastases would be practice-changing, conventional imaging techniques, including CT and MRI, that rely on LN size criteria and morphology (shape, contour, signal intensity) are largely unreliable (10). Few prospective multicenter trials with a histological reference standard to guide management exist (11).
Several functional imaging techniques have been evaluated in an attempt to improve preoperative nodal staging. DW-MRI, which assesses tissue diffusion properties and cellularity, is now widely established for routine use in pelvic MRI in cervical and endometrial cancer staging although published results of DW-MRI in nodal diagnosis are variable (13).
FDG-PET/CT is established in staging locally advanced cervical cancer and incorporated into guidelines due to its diagnostic superiority (12,14). The role of FDG-PET/CT in endometrial cancer has not been clearly established with a lack of evidence to date (15). In general, FDG-PET/CT has high specificity for nodal involvement in advanced cervical and endometrial cancer but low to moderate sensitivity remains problematic, particularly in earlystage disease (15)(16)(17).
Choline-PET imaging is a surrogate marker of accelerated cell membrane metabolism in cancer and has an established role in imaging of prostate cancer, including nodal assessment for high-risk staging (18). In cell line studies of endometrial cancer, the expression and activity of choline kinase alpha is increased with a several-fold increase in the uptake of 3 H choline in endometrial cancer cells compared with normal endometrial stromal cells (19). Early pilot studies of 11 C choline in gynecologic cancer show promise (20,21); however, 18 F-labeled fluoro-methyl and fluoro-ethyl choline have a longer radioactive half-life (110 minutes) and are therefore more practical to use. The use of Fluoro-ethyl-choline (FEC)-PET/CT in detecting nodal metastases in endometrial and cervical cancer has not been previously explored.
Our hypothesis was that any of DW-MRI, FEC-PET/CT or FDG-PET/CT could preoperatively identify LN metastases, with sufficiently high accuracy, to replace the need for surgical LN staging but also identify those patients who should not undergo surgery, in eligible patients with seemingly operable cervical and high-risk endometrial cancer.
Study design and participants
MAPPING is a multicenter, prospective observational study evaluating the diagnostic accuracy for detecting nodal metastases using different imaging methods. Ethics approval, ARSAC licence, and MHRA approvals were obtained (Research Ethics Committee reference number 11/LO/1465). The study was sponsored by Barts Health NHS Trust. The Centre for Experimental Cancer Medicine (CECM), Barts Cancer Institute, Queen Mary University of London had overall responsibility for trial management. All participants gave written informed consent.
Eligible patients were aged ≥18 years, with newly diagnosed histologically confirmed cervical or endometrial cancer and were eligible and fit for surgical lymphadenectomy, as per the decision of a multidisciplinary tumor board and assessment by a specialist surgical and anaesthetic team. Pretreatment FIGO stage (2009) was established on the basis of clinical examination and standard-ofcare imaging (CT and MRI). Patients with cervical cancer were eligible if the pretreatment FIGO stage was considered stage IB and local disease appeared operable. Patients with endometrial cancer were eligible if high-risk features for LN metastases, that is, histologic grade was high-grade (including grade 3 endometrioid adenocarcinoma, serous, clear cell, or carcinosarcoma) with myometrial invasion on MRI, or MRI-based FIGO stage II or above. Patients were ineligible if unable to provide written informed consent, were pregnant or had contra-indications to MRI or PET/CT (Supplementary Table S1).
Translational Relevance
Nodal status is a highly important prognostic factor in endometrial and cervical cancers and has a significant impact on patient risk stratification and management.This is the first multicenter prospective study to directly compare the diagnostic performance of FEC-PET/CT, FDG-PET/CT, and DW-MRI for the detection of nodal involvement in surgically staged endometrial and cervical cancers, with a strict histologic reference standard. No technique had sufficient sensitivity to obviate the need for surgical nodal staging in radiologically node-negative patients, in cases where nodal staging is considered appropriate. However, the low false positive rate may contribute to patient risk stratification in cases with difficult surgical decision making, for arbitration in borderline surgical cases: In cervical cancer, an FDG-positive case could support a decision to redirect patients from radical surgery to chemoradiotherapy options. In endometrial cancer, an FDGpositive case could allow planning of a more tailored therapeutic approach. By identifying the site and extent of avid nodes, a targeted surgical dissection of the area of interest could be planned, avoiding morbidity from unnecessary radicality. Moreover, a preoperative discussion with the patient about their systemic and radiotherapeutic options and how those would complement any surgical approach, would be facilitated.
Procedures
All imaging tests were performed before surgery and within the national standard time frame of preoperative care. Standard-of-care MRI scan was performed first, and as per local practice. FDG-and FEC-PET/CT scans had to be performed on separate days, and the DW-MRI scan could be performed alongside either of these, and at the same time as the conventional MRI ( Supplementary Fig. S1).
MRI
Standard-of-care MRI scan for staging was performed as per local protocol. The MRI field of view included the pelvis and para-aortic regions up to the level of the left renal vein. For nodal evaluation, readers had a minimal dataset that included axial T1, axial T2, and study-specific axial DW-MRI (b values 0, 300, 600, 900, and 1,200) with associated calculated ADC maps, following optimization with ice-water phantom (Supplementary Table S2). The details of standard-of-care MRI and DW-MRI are provided in Supplementary Table S2A-S2C.
FDG-PET/CT and FEC-PET/CT
FDG-and FEC-PET/CT scans were performed using a standard protocol (Supplementary Table S2D) based on UK NCRI PET Research Network guidance. Each center had been accredited by the NCRI PET Research Network for multicenter trials. All scans were acquired from base of skull to upper thighs as 3D acquisitions with TOF if available. Low-dose CT acquisitions were made for attenuation correction and image fusion. FDG-PET/CT scans were acquired at a median of 60 minutes postinjection (range 57-80) and FEC-PET/CT scans at a median 60 minutes (range 55-74) and the two scans were acquired on different days.
Participants fasted for ≥4 hours prior to FDG injection (median 370 MBq, range 217-436 MBq) and scanning only performed if blood glucose was <10 mmol/L. Fasting was not required prior to FEC injection (median 293 MBq, range 209-369 MBq). Patients were asked to void their bladder before both scans. Both tracers were classified as investigational medical products. Patients were contacted 24 hours following scans to record any adverse events.
Reader evaluations
All imaging scans were read by one local and two central radiologists. The central reviews were coordinated through the UK NCRI PET core lab (PET) and the trials unit (MRI), and discordant reviews were resolved by consensus. All radiologists were accredited core members of the gyne-oncology multi-disciplinary team and/or PET/CT experts. All readers were aware of the clinical diagnosis (endometrial or cervical cancer) but when assessing each scan they were blinded to all other imaging scans, surgical findings and final histology. The local radiologic assessment was used for the main analyses to be consistent with patient management and treatment being guided by the local MRI evaluation.
Evaluation of nodes using the standard MRI, DW-MRI, and PET scans was based on a 6-point confidence score: 1, definitely benign; 2, probably benign; 3, low confidence benign; 4, low confidence malignant; 5, probably malignant; 6, definitely malignant. A score of 5 or 6 was classified as test-positive.
Conventional MRI nodal diagnosis based on MRI size and morphology
The anatomic location of nodes that were ≥5 mm and their short and long axis diameter were recorded. If there were multiple nodes in one anatomic region, the largest or most suspicious node was used for the nodal descriptors (fatty hilum, homogeneous appearance, necrosis, or irregular margin). Nodal diagnosis was then made using the size (in mm), or nodal morphology (based on the 6-point confidence score); both without reference to the DW-MRI.
The diagnosis based on nodal size criteria was considered using three cutoffs, analyzed separately: (i) short axis diameter >9 mm; (ii) short axis >10 mm or (iii) "size ratio" criteria, whereby a node <8 mm short axis is considered benign, node >10 mm short axis is considered metastatic (and therefore positive), but a node with a short axis between 8 and 10 mm is only considered positive if the short axis to long axis ratio is over 0.8 (i.e., a round node; ref. 22).
Nodal diagnosis based on DW-MRI
Nodes were identified on the high b value DW-MRI as noncontinuous high signal intensity (SI) round or ovoid structures that corresponded with a node on the anatomic images. Nodes that retained very high SI on the high b value images, without T2 shinethrough, were considered to be positive based on the 6-point confidence score.
Nodal diagnosis based on PET/CT
The anatomic location of any focally increased tracer uptake that was higher than background adjacent tissue corresponding to a node of any size on the CT was recorded and assessed according to a 5-point scale (A, normal FDG/FEC uptake; B, mild increased FDG/FEC uptake likely not to represent tumor involvement, C, equivocal FDG/FEC uptake; D, moderately increased FDG/FEC uptake likely to represent tumor involvement; E, intensely increased FDG/FEC uptake representing tumor involvement). The 6-point confidence level was used for nodal diagnosis.
Primary reference standard
The primary reference standard was confirmed nodal histology obtained by surgical lymphadenectomy. The surgeon was made aware of the position of any suspicious node prior to the lymphadenectomy to ensure the highest likelihood of correlating all suspected positive nodes based on imaging tests. The surgeon recorded the site of resected nodes that were labeled for histologic correlation according to the predefined protocol.
All retrieved nodes were analysed by expert gyne-oncology histopathologists at each site and the anatomic location of all nodes, nodal size, and presence of metastatic disease were recorded. The histopathologist was blind to all image findings, although they were aware of the original histopathology results from preoperative biopsy of the primary tumor, as per standard practice.
In cases where imaging suggested an involved node but histopathology was negative, further reviews were undertaken to avoid an uncertain reference standard due to the possibility that the node was not retrieved at surgery. First, any postoperative imaging was reviewed by an expert consensus to be certain that all suspicious nodes had been retrieved at surgery. If the suspicious node remained in situ, then the case was not eligible for analysis by the primary reference standard, as the correlation with histology was uncertain. Second, the most suspicious node underwent ultrasectioning and immunostains to ensure very high sensitivity for micrometastases. Where histopathology was positive but imaging negative, then the imaging was deemed falsenegative.
The final histologic subtype and pathologic stage of the primary tumor were recorded. If no nodal tissue was obtained in the surgical specimen, the case was not eligible for analysis with the histologic primary reference standard. Secondary reference standard A secondary reference standard was specified to allow for inclusion of patients in whom definitive histopathology was unavailable (either no surgery, no nodal tissue retrieved or suspicious node not resected), and therefore could not be included in analyses using the primary reference standard. This secondary reference standard was comprised by a consensus panel that reviewed all available imaging and clinical information for 9 months from when the patient was recruited to impute the presence and position of any positive nodal sites, for example by identifying progression in a node or clear regression following treatment. If no follow-up was available, the patient was excluded from analysis.
Outcome measures
The primary outcome measures were sensitivity and false positive rate [FPR]) for histologically confirmed nodal metastatic disease. Sensitivity is the proportion of cases who are test-positive among all who have confirmed metastatic disease. FPR (specificity) is the proportion of cases who are test-positive among all those who do not have metastatic disease. These were estimated for each of the DW-MRI, FEC-, and FDG-PET/CT scans, and also (using standard MRI) nodal size criteria and morphology. Secondary outcome measures were positive and negative predictive values. Analyses were performed according to cervical or endometrial cancer, and on a per-patient level (all surgically resected nodal regions considered together) and per-nodal-region level (right pelvis, left pelvis, para-aortic).
Statistical considerations
Sensitivities of DW-MRI, FDG-and FEC-PET/CT were compared with that of nodal size, using a McNemar paired test. The primary analysis was based on patients who had both DW-MRI and FDG-PET/ CT, as these were mandatory for study inclusion and FEC-PET/CT was optional.
Sample size assumptions were based on results for conventional MRI nodal size, DW-MRI (original calculation based on Thoeny HC et al, ESUR 2009 abstract MS8 p45) and FDG-PET/CT (23).
For comparing sensitivity of DW-MRI (93%) with nodal size (47%), at a fixed FPR of 25% (the published estimate for DW-MRI), using a discordant paired analysis, 19 subjects with confirmed histologic nodal disease were required (80% power, and one-sided statistical significance of 0.017 to allow for three main comparisons). Assuming 15% of patients have nodal disease, 127 patients were needed in total. For comparing the sensitivity of FDG-PET/CT (73%) with nodal size (27%), at a fixed FPR of 3% (the published estimate for FDG-PET/ CT), required 23 patients with confirmed nodal disease and therefore about 150 in total.
The target study size was taken as the greater of the two main comparisons: n ¼ 150 patients. No preliminary data were available for FEC-PET/CT, but we expected that FEC-PET/CT would have a diagnostic performance similar to either DW-MRI or FDG-PET/CT, and thus not require a larger sample size. A statistically significant difference was taken to be <0.034 (two-sided, as per the design and power calculation).
Interrater agreement between local and the central consensus reviews was assessed using Cohen kappa statistic.
Results
Participants were included from 5 gyne-oncology tertiary referral centers in National Health Service (NHS) hospitals in England (Supplementary Table S2A). Consent was obtained from 162 patients between October 2012 and July 2017 (Fig. 1). Among the 145 patients who had any study imaging and surgery, surgery was performed laparoscopically in 64, open in 42, robotically assisted procedure in 32 and 7 did not undergo surgery [advanced disease on PET (n ¼ 1), investigator decision (n ¼ 2), patient decision (n ¼ 1), unable to comply with study schedule (n ¼ 2)]. The diagnosis was endometrial cancer in 98 and cervical cancer in 47 patients ( Table 1).
Our primary analyses were based on the 118 who had both DW-MRI and FDG-PET/CT, and definitive histopathology according to the primary reference standard; baseline patient and tumor characteristics are in Table 1 and Supplementary Table S3
McNemar tests found FDG-PET/CT to have significantly better sensitivity than short axis >10 mm (P ¼ 0.016) while all other comparisons were not statistically significant.
On a per-region basis (n ¼ 267 regions among the 118 patients, mean 2.3 regions per patient), diagnostic performances differed only slightly (Supplementary Table S4A-S4C) and McNemar tests found FDG-PET/CT outperformed both short axis >10 mm (P ¼ 0.006) and size ratio criteria (P ¼ 0.04) with regards to sensitivity. Distant metastases were described on PET/CT in 8 of 162 patients, 6 included in the primary reference standard and 2 excluded due to final histology being ovarian cancer (n ¼ 1) and no histology or follow-up imaging therefore no nodal reference standard available (n ¼ 1; Fig. 1).
Among 40 patients with cervical cancer, 10 (25%) had confirmed histologic nodal metastases, only 1 of which had a LN short axis > 10 mm. The sensitivities were lower, ranging from 10% (size ratio criteria, morphology and short axis >9 mm) to 30% (FDG-PET/ CT; Table 3). There was no significant difference in modalities among these patients in terms of sensitivity or FPR. Only 4 patients had an MRI FIGO stage greater than 1B1 (as expected due to patient selection for operability) and in these cases the FPR was 0% for all tests (Supplementary Table S5).
The above results were based on patients who had both the DW-MRI and FDG-PET/CT scans. Quantitative values for FDG-and FEC-SUV and ADC in positive and negative nodes are provided in Supplementary Table S6.
Secondary reference standard
Eighteen patients were not included in the main analyses because they did not have definitive histopathology correlation using the primary reference standard. This included 4 patients who did not undergo surgery, 3 patients in whom a suspected positive node remained visible on postoperative imaging therefore not considered resected (Fig. 2, all included in secondary reference standard), 3 in whom lymphadenectomy was attempted but no nodal tissue retrieved and 8 who underwent surgery but lymphadenectomy was abandoned during surgery for clinical reasons. Of these 18 patients, 6 in total had postoperative imaging and follow-up to 9 months. Combining these with the 118 who had the primary reference standard, gave a total of 124 patients for analyses based on the secondary reference standard (Fig. 1). 25.8% (32/124) had nodal metastasis. Results using the secondary reference standard did not differ substantially from results based on the primary reference standard and are available in Supplementary Table S7A-S7C. Twelve patients had no histology or imaging follow-up and therefore could not be included (Fig. 1).
Central review
Diagnostic performance based on the independent central review did not differ substantially from the local radiologist assessment (Supplementary Table S8). Interrater agreement between local and central consensus reviews was high for each imaging modality, with kappa statistics of 0.70, 0.86, and 0.79 for DW-MRI, FDG-, and FEC-PET/CT, respectively.
Adverse events
The MAPPING study was conducted as a study of investigational medicinal products, according to MHRA guidance. Adverse events were collected for all patients who underwent at least one of the trial PET scans (n ¼ 147 participants, Supplementary Table S9). One patient had mood alteration and another had vomiting, both thought to be related to FEC-PET/CT. One patient had hyperglycaemia and another developed a rash, these were considered related to FDG-PET/ CT. There were no serious adverse events.
Discussion
In this multicenter prospective cohort study, with a strict histologic reference standard, we failed to demonstrate a significantly higher sensitivity, on a per patient basis (n ¼ 118), using FEC-PET/CT, FDG-PET/CT, or DW-MRI compared with standard MRI in the radiologic detection of nodal metastases in seemingly operable cervical (sensitivities of 20-30% compared with 10%) and in endometrial cancer (sensitivities of 70%-87.5% compared with 75%). FEC-PET/CT, studied as a possible alternative to FDG, was not found to improve diagnostic performance compared with FDG-PET/CT, with sensitivity of 66.7% in the entire cohort (25% in cervical cancer and 87.5% in endometrial cancer). As such, we could not establish any preoperative imaging that had sufficient sensitivity to obviate the need for surgical LN staging in seemingly operable early-stage cervical and operable endometrial cancer. On a per-region basis (n ¼ 267), FDG-PET/CT outperformed both short axis >10 mm (P ¼ 0.006) and size ratio criteria (P ¼ 0.04) with regards to sensitivity. Importantly, we demonstrated very low false positive rates for LN metastases in both cervical and endometrial cancers (ranging from 2.5%-6.8%) across all imaging modalities. This high specificity suggests that positive preoperative imaging can be relied upon to indicate nodal involvement, even in the case of non bulky LN, so that indication for surgery and extent of surgical radicality can be appropriately modified.
The performance of functional imaging techniques was better in the cohort of endometrial, compared to cervical cancer patients. This may be attributed to the fact that only patients with early-stage cervical cancer with non-bulky LN on conventional imaging were included, because only those were eligible for surgery, as opposed to patients with endometrial cancer, where those with bulky LN were considered operable and hence included in our study.
In the cohort of patients with endometrial cancer, radiologic detection of nodal metases was demonstrated to be reasonably sensitive (FDG-PET/CT 80%, FEC-PET/CT 87.5%) and highly specific (false positive rate: FDG-PET/CT 5%, FEC-PET/CT 4%). Of all the modalities, PET/CT had higher performance than MRI with or without DW-MRI, although this was not statistically significant. This first study using FEC-PET/CT in endometrial cancer demonstrated good diagnostic performance but not statistically better than FDG-PET/CT. In endometrial cancer, FDG-PET/CT is not currently used routinely but the high negative predictive value (NPV: FDG-PET/CT 93.2%, FEC-PET/CT 95.8%) could be used to re-inforce standard-ofcare imaging to arbitrate difficult or borderline surgical decisions concerning surgical lymphadenectomy. Surgical LN staging is recommended in apparent uterine-confined high-risk endometrial cancer (5) but there is no prospective evidence so far to demonstrate a therapeutic value of systematic LND (9). A corroboration of a borderline or suspicious morphologic imaging test with a functional imaging test could support the decision to proceed with nodal dissection and allow planning of surgical approach and extent to minimize morbidity related to the surgery and subsequent increased risk of lymphoedema. Our study provides prospective multicenter evidence that would support the use of FDG-PET/CT in endometrial cancer cases with difficult surgical decision-making, in order to direct surgical resection to the positive LN avoiding more extensive lymphadenectomy. In cases with a positive PET/CT, the surgical plan could include limited targeted resection of individual accessible positive nodes at the time of hysterectomy, followed by adjuvant therapy or a decision to avoid nodal dissection altogether to treat with targeted radiotherapy depending on the overall tumor dissemination pattern and patient's profile. In cases that are PET/CT-negative, but intermediate to high risk disease, there is increasing evidence for the use of sentinel lymph node technique as a viable alternative for systematic LND (24). This is the first study to report the use of FEC-PET/CT as an alternative to FDG-PET/CT in the nodal staging of endometrial cancer. The rationale to study this was choline-PET/CT has shown promise in the nodal staging of high risk prostate cancer (25) and supportive feasibility data of 11 C-choline-PET in gynecologic cancers (20,21). In our study, FEC-PET/CT performed well, but with no statistical difference to FDG-PET/CT. In daily practice, FDG is more widely available and therefore more practical.
There are no similar multicenter prospective studies in the last five years in gynecologic malignancies. Eight prospective studies have evaluated endometrial cancer with FDG-PET/CT and surgicalpathologic reference standard between 2009 and 2019 (with inclusion of more than 30 patients, range 37-220; refs. [26][27][28][29][30][31][32][33]. The range of sensitivities was between 45.8% and 93.3% and FPRs between 3.0% and 8.8% apart from one study with a higher FPR of 17.9% (31). The largest multicenter prospective study of FDG-PET/CT in endometrial cancer (27), reported per patient sensitivity of 59% (23/39) for nodal metastases in those patients that had histologic confirmation, a little lower than 80.0% in our study. Criteria for nodal involvement and details of surgical reference standard were not described. A more recent single-center prospective study in patients with high-risk endometrial cancer found a lower sensitivity of 45.8% (11/24), specificity of 91.1% (72/79) for nodal metastatic disease in 103 patients (33). In this study, there was a similar prevalence of nodal involvement in the cases that underwent nodal dissection (23.3% vs. 26% in our study) and more FIGO stage 1A cases, although they did not mention the size of the nodes. Their sensitivity is lower than other studies included in a meta analysis that reported the pooled sensitivity of 68% (34). In addition, the reference standard for false positive PET cases was not clear: in our study, we reviewed postoperative imaging to ensure the suspicious node was removed, and excluded cases where the suspicious node was still present on postoperative imaging, to ensure an accurate reference standard. There are only three prospective studies assessing diagnostic accuracy of MRI for LN metastases in endometrial cancer with more than 30 evaluable patients with surgical-pathologic reference standard (including 46, 181, and 220 patients) between 2009 and 2015 (27,31,35). The sensitivities using size criteria (short axis >10 mm) for LN metastases were from 75%, 69.2%, and 40% with FPRs of 19%, 6.5%, and 3.5% respectively. More recently a prospective study developing and testing radiomic features to predict nodal involvement has been published and this is an exciting area of future research (36).
In early-stage presumed operable cervical cancer, we found that no imaging method, including DW-MRI, FDG-, or FEC-PET/CT was able to reliably identify nodal metastases in normal sized/nonbulky LNs with sufficient sensitivity. Our results confirm the findings of a single-center prospective cohort (32.1% sensitivity FDG-PET/CT compared with 30.0% in our study), that the role of FDG-PET/CT in staging of early cervical cancer is limited as the prevalence of nodal involvement is low, and if present, nodal metastases are often small volume and below the sensitivity of PET (37). Although in our cohort 25% (10/40) had at least one positive node, the majority of these nodes were subcentimetre. Importantly, we had no false positive case on FDG-PET/CT in patients with cervix cancer. This information is highly relevant in cases where the choice of surgery may be difficult: a negative FDG-PET/CT could allow a decision for surgery to go ahead, at least for surgical nodal staging, whereas the finding of a positive node could safely and reliably exclude these patients from radical hysterectomy and direct them to chemoradiotherapy. Early data that suggest individual nodal sensitivity of FDG PET/CT in endometrial and cervical cancer could be enhanced by image fusion with DW-MRI with potential complementarity between the modalities but was beyond the scope of our study (38).
There are limitations to our study. Our results are suggestive (but not conclusive) of a difference in diagnostic performance between cervical and endometrial cancer. However, there are insufficient cases to reliably analyse these separately because when the study was designed there were few publications on this, limiting the power calculation. Study recruitment was challenging as patients needed to undertake repeat study MRI for the study diffusion sequence, as well as For all imaging, a positive test result is defined as a confidence score of 5 or 6 (see Materials and Methods); for short axis, a positive result is defined in the table, and for size ratio criteria, see Materials and Methods.
Figure 2.
A 55-year-old with endometrial carcinoma. Cor/axial T2-weighted MRI (i), axial ADC (ii), axial FDG fused (iii), axial FEC fused (iv) shows focal tracer uptake in an 8-mm rounded left external iliac node (row A, arrows, Ã tumor,^ureter), and a 9-mm rounded right common iliac node (row B, arrows). Histopathology was negative. Follow-up axial FDG fused (v) and axial contrast-enhanced CT (CECT) (vi) confirmed the nodes were not removed at surgery. study-specific FDG-PET/CT and optional FEC-PET/CT prior to the planned surgery. In addition, many recruited participants did not complete all the imaging as per study protocol, in particular FEC-PET/ CT due to logistic challenges of coordinating manufacture timings and radiotracer distribution in a multicenter study with short time-frames for imaging prior to surgery. Several cases could not be evaluated with the primary reference standard as we could not be fully confident that the suspicious node on imaging had been correlated at histology. By having a stringent primary reference standard, we ensured a high likelihood of correlated imaging and nodal histology. However, this led to several patients excluded from the primary analysis, where the suspicious node was not successfully surgically resected. Nevertheless, analyses based on the secondary (follow-up) reference standard were similar to the primary analyses. Although our main analyses were based on 118 patients (less than the target of 150), the sample size was based on the number of histologically confirmed cases with nodal metastasis, and we observed 30 cases, above the target of 19-23.
In conclusion, in this prospective multicenter study with strict histologic reference standard, no imaging technique provided sufficient sensitivity to obviate the need for lymph node dissection in cases where this is considered appropriate, in patients with early-stage operable cervical cancer and in patients with intermediate-to highrisk endometrial cancer. However, the very low false positive rates, in all modalities, could be helpful in treatment planning. In patients with cervical cancer, with borderline appropriateness for radical surgery, MRI findings could be supported by FDG-PET/CT, as false positive scans are highly unlikely and therefore a positive scan would allow avoidance of inappropriate surgery, or could direct surgical resection for confirmation. In patients with endometrial cancer, in cases where there is uncertainty regarding the risk/benefits of lymphadenectomy, confirmatory FDG-PET/CT may add support to MRI to decide on proceeding to surgical nodal dissection, as well as directing the surgical approach. | 2021-09-17T06:17:23.836Z | 2021-09-15T00:00:00.000 | {
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255850261 | pes2o/s2orc | v3-fos-license | Genetic mapping reveals new loci and alleles for flowering time and plant height using the double round-robin population of barley
Abstract Flowering time and plant height are two critical determinants of yield potential in barley (Hordeum vulgare). Despite their role in plant physiological regulation, a complete overview of the genetic complexity of flowering time and plant height regulation in barley is still lacking. Using a double round-robin population originated from the crossings of 23 diverse parental inbred lines, we aimed to determine the variance components in the regulation of flowering time and plant height in barley as well as to identify new genetic variants by single and multi-population QTL analyses and allele mining. Despite similar genotypic variance, we observed higher environmental variance components for plant height than flowering time. Furthermore, we detected new QTLs for flowering time and plant height. Finally, we identified a new functional allelic variant of the main regulatory gene Ppd-H1. Our results show that the genetic architecture of flowering time and plant height might be more complex than reported earlier and that a number of undetected, small effect, or low-frequency genetic variants underlie the control of these two traits.
Introduction
The increase in world population, the reduction of available arable land, and climate change represent some of the greatest challenges that humanity faces now and in the near future (Vyas et al., 2022).One answer to these challenges is to reduce the influence of biotic and abiotic stress factors and that way increase crop productivity (Khush, 2013).Of particular importance are yield increases of cereals (Araus et al., 2008), crops that are essential for human nutrition as they alone contribute about 44.5% of the calorie uptake of the world population (FAO, 2019).In addition, they are important for animal feed and beverage production (FAO, 2020).
Flowering time is a critical determinant of yield potential in cereals (Hill and Li, 2016).Indeed, after flowering, grain filling starts (Cockram et al., 2007), and this process has maximal efficiency if it coincides with optimal environmental conditions (Wiegmann et al., 2019).Therefore, plants and farmers have adopted several strategies to synchronize the phenological stages to environmental conditions (Anderson and Song, 2020).
Barley (Hordeum vulgare L.) is ranked fourth among the most cultivated cereals worldwide (FAO, 2020).This species is characterized by great environmental plasticity that allows it to be cultivated at different latitudes, with extremely dissimilar temperature and photoperiod conditions (Dawson et al., 2015).The adaptive success of barley is also due to the selection of favorable allelic variants at the main genes determining the transition from the vegetative to the reproductive phase (Turner et al., 2005;Comadran et al., 2012;Göransson et al., 2019).Three types of genes have been identified as being responsible for the modulation of flowering time in barley: genes that act under the influence of photoperiod, genes that act under the influence of temperature, and genes, called earliness per se that act independently of environmental variables (Fernández-Calleja et al., 2021).
The main genes whose expression is influenced by the photoperiod are Ppd-H1 (Turner et al., 2005) and Ppd-H2 (Kikuchi and Handa, 2009).Ppd-H1, which is located on chromosome 2H, is the major determinant of the response to long-day conditions in barley, acting jointly with HvCO1 and HvCO2 (Campoli et al., 2012).At the same time, Ppd-H1 indirectly influences the response to vernalization by promoting the expression of Vrn-H3 (Mulki and von Korff, 2016).Ppd-H2 is the second main driver of the photoperiod response in barley, but unlike Ppd-H1 it acts in short-day conditions.The non-functional allelic variant of Ppd-H2 allowed the expansion of the cultivation area of barley at higher latitudes (Casao et al., 2011).
The major determinants of the response to temperature are genes involved in the vernalization process.Vrn-H1, located on chromosome 5H, promotes flowering after the plant has satisfied its vernalization requirement (Yan et al., 2003).Furthermore, Vrn-H1 inhibits the expression of Vrn-H2, which is located on chromosome 4H.Vrn-H2 delays flowering, allowing the plant to fulfill its cold needs (Yan et al., 2004;Deng et al., 2015).Therefore, the interaction between Vrn-H1 and Vrn-H2 is one of the main mechanisms that allow the control of flowering time in winter or facultative barley varieties (Yan et al., 2004).The third vernalization gene is Vrn-H3, on chromosome 7H (Yan et al., 2006).Vrn-H3, when not repressed by Vrn-H2, promotes flowering by allowing the transition from the vegetative to the reproductive phase in longday conditions (Hemming et al., 2008).
Within the earliness per se genes group, the major determinant is HvCEN, located on chromosome 2H.Because its expression is not directly influenced by environmental variables, the allelic variants of HvCEN allowed the adaptation of barley to new areas through the regulation of flowering time (Comadran et al., 2012).In addition, three other genes have been described as circadian clock-related earliness per se genes which, although not directly influencing flowering, alter the expression of Ppd-H1: HvELF3 (Faure et al., 2012), on chromosome 1H, HvLUX1 (Campoli et al., 2013), on chromosome 3H, and HvPHYC (eam5) (Nishida et al., 2013), on chromosome 5H.Furthermore, mutations in HvELF3 can affect the expression of HvGI (Dunford et al., 2005), causing earlier flowering (Zakhrabekova et al., 2012).Finally, other genes initially reported to control other quantitative traits have also been described as having an influence on flowering time or flower development: HvAP2 (Shoesmith et al., 2021), on chromosome 2H, and Hv20ox2 (sdw1/denso) (Bezant et al., 1996;Jia et al., 2009), on chromosome 3H.
Another key trait responsible for determining production performance in cereals is plant height (Mikołajczak et al., 2017).An adequate height allows the plant to obtain a lower exposure to lodging and a higher harvest index, but on the other side, it is essential to keep the spikes far from the soil to reduce the risk of yield losses caused by infectious diseases (Vidal et al., 2018).Plant height and flowering time are two inter-related traits.This is because flowering is possible when the meristem has switched from the vegetative to the reproductive phase.For this reason, many of the genes controlling flowering time, such as Ppd-H1 (Turner et al., 2005), Vrn-H1 (Wiegmann et al., 2019), Vrn-H2 (Rollins et al., 2013), Vrn-H3 (Arifuzzaman et al., 2016), Hv20ox2 (Jia et al., 2009), HvCEN (Bi et al., 2019), and HvAP2 (Patil et al., 2019), have a pleiotropic effect on plant height.In addition to these genes, other genes involved in the biosynthesis of brassinosteroids, such as HvBRD on chromosome 2H, HvBRI1 (uzu) on chromosome 3H, HvDWF4 on chromosome 4H, HvCPD and HvDEP1 on chromosome 5H, and HvDIM on chromosome 7H (Dockter et al., 2014;Wendt et al., 2016), have been described as being involved in plant height regulation of barley.Some of the above-mentioned genes, such as HvAP2 and the genes regulating brassinosteroid biosynthesis, have been identified based on mutant approaches (Dockter et al., 2014;Shoesmith et al., 2021).Natural variation was also exploited through screening of germplasm collections (Comadran et al., 2012) and bi-parental (Von Korff et al., 2006;Schmalenbach et al., 2009;Rollins et al., 2013;Arifuzzaman et al., 2014) or nested association mapping populations (Maurer et al., 2015;Nice et al., 2017).When multi-parental populations were examined instead, the experiments included a restricted number of inbred lines (Cuesta-Marcos et al., 2008) and/or the selected parental inbreds were from a restricted geographical range (Cuesta-Marcos et al., 2008;Afsharyan et al., 2020).All these factors reduce the likelihood of identifying genes and allelic variants with low population frequency (Yu et al., 2006).Therefore, the utilization of segregating populations derived from genetic resources with high genotypic and phenotypic diversity could allow the identification of further genes that are involved in flowering time and plant height regulation.This has the potential to facilitate and speed up breeding and provide new targets for genetic modification through, for example, CRISPR platforms.In turn, this could help to extend the cultivation area of barley by allowing its adaptation to new environmental conditions.Furthermore, the knowledge gained in barley has a high potential for transfer to other cereal species that are genetically close but have a polyploid chromosomal structure, such as tetraploid (Triticum turgidum var.durum) and hexaploid (Triticum aestivum) wheat (Langridge, 2018).
In this study, a multi-parent population was used to explore the genetic landscape of flowering time and plant height in barley with the objectives of: (i) determining the genetic variance components in the regulation of flowering time and plant height, (ii) obtaining a comprehensive understanding of the genetic complexity of flowering time and plant height in barley by single and multi-population QTL analyses, and (iii) identifying candidate genes for the detected QTLs regulating flowering time and plant height and detecting new allelic variants of genes responsible for the control of these two traits.
Plant material and genotypic evaluation
The plant material used in this study consisted of a population designated as HvDRR (Hordeum vulgare Double Round-Robin).
The population originated from the crossings of 23 parental inbred lines, including 11 cultivars and 12 landraces (Shrestha et al., 2022), in a double round-robin scheme (Stich, 2009) (Supplementary Table S1).The parental inbred lines were chosen from a diversity panel of 224 spring barley accessions selected from the Barley Core Collection (BCC) (Pasam et al., 2012) to maximize the combined genotypic and phenotypic richness index (Weisweiler et al., 2019).
Starting from the 45 F 1 s, a single seed descent strategy was applied to develop between 39 and 145 recombinant inbred lines (RILs) for each of the 45 sub-populations, totaling 4065 RILs (Casale et al., 2022).For flowering time (FT), 3972 RILs were phenotyped, while for plant height (PH) 4025 RILs have been characterized.The plants were phenotyped at generations F 5 to F 7 .The RILs were genotyped as individual plants at the F 4 generation using a 50K single nucleotide polymorphism (SNP) genotyping array (Bayer et al., 2017).
Phenotyping
FT evaluation was carried out in Cologne (50.960188 N,6.860009 E) from 2017 to 2019, Mechernich (50.601335 N, 6.645622 E) from 2018 to 2019, and Quedlinburg (51.787661 N, 11.205353 E) from 2018 to 2019.PH was evaluated in the same environments except for Quedlinburg, totaling five environments.All the experimental fields were located in Germany.At the Cologne and Mechernich environments, 33 seeds were sown in single rows of 1.6 m length.In Quedlinburg, double rows of the same length were sown.The inter-row distance was 20 cm.Fertilization and plant protection followed local practices.In each environment, an augmented design was used.RILs of the HvDRR population and the inbreds of the diversity panel were planted in single rows with one replicate and only the parental inbreds of the HvDRR population were replicated 15-20 times per environment.The percentage of parental inbreds sown in the field, in relation to the total number of plants of the HvDRR population (parental inbreds included), ranged from 8.73% (Cologne, 2019) to 9.97% (Cologne, 2017 andQuedlinburg, 2018).
FT was recorded as days after sowing when 50% of the plants within the (double) row were flowering.PH was measured as the mean across all emergent plants within a row as height in cm from the ground to the top of the spike (without measuring the awns) when the plant was fully developed.
Statistical analyses
The collected phenotypic data were subject to statistical analysis using the following linear mixed model: where Y ijk indicated the observed phenotypic value for the ith genotype in the jth environment within the kth replication, μ the general mean of the trait, G i the effect of the ith genotype, E j the effect of the jth environment, and e ijk the random error.For the calculation of adjusted entry means, the genotypic effect was considered fixed, while the environmental effect was considered random.
The broad sense heritability (h 2 ) was calculated as: (2) where V g represented the genotypic variance and c the mean of the standard errors of the contrasts among all pairs of genotypes (Piepho and Möhring, 2007).For the calculation of the genotypic variance (V g ), model 1 was used, but all effects were considered random.In addition, we calculated h 2 , when applying for each environment a correction based on the augmented design considering different grid sizes, and then estimating V g and c across the environments.
In order to quantify the interaction between genotype and environment, we used a second linear mixed model: where (G:E) ij represented the interaction between the ith genotype in the jth environment, which was fitted to the data of the parental inbreds.
QTL analyses
Two different QTL analyses were performed in this study on the HvDRR population: multi-parent population (MPP) and single population (SP) analyses.
The estimation of genetic maps necessary for the SP analysis, as well as that of the consensus map used in the MPP analysis, have been described by Casale et al. (2022).
For each sub-population and each trait, an SP QTL analysis was performed, based on the adjusted entry means for each RIL calculated with model 1, using the following scheme.First, standard interval mapping using the Haley-Knott regression algorithm (Knott and Haley, 1992) was applied, followed by forward selection in order to determine the number of QTLs to include in the model.Then a forward and backward selection algorithm was applied to perform multiple QTL mapping.Model selection was based on the highest penalized logarithm of odds (LOD) score with penalties determined through 4000 permutations.A two-dimensional genome-wide scan was performed to detect epistatic interactions between all pairs of loci in the genome.The SP analyses were carried out with the R package 'qtl' (Broman et al., 2003).
Confidence intervals for the QTLs detected via SP were calculated using a 1.5 LOD drop method (Manichaikul et al., 2006).
The MPP analyses were performed by jointly analysing all sub-populations using an ancestral model that took into account the degree of relatedness among the parental inbreds (Garin et al., 2017).The degree of relatedness was calculated by clustering the haplotypes.The haplotype window size was chosen as the consensus genetic map distance for which the linkage disequilibrium, measured as r², was 0.2 (Giraud et al., 2014) (Supplementary Table S2).The MPP analysis was performed using the R package 'mppR' (Garin et al., 2015).
To exclude potential effects created by using the consensus map, we performed an association mapping analysis across the entire HvDRR population.A mixed model approach was used, where the known pedigree of the individual RILs was used to calculate a pedigree-based kinship matrix that modeled the covariance among RILs.
Genomic prediction
Genomic predictions of FT and PH in the HvDRR population were performed by genomic best linear unbiased prediction (GBLUP) using the following model (VanRaden, 2008): where y was the vector of the adjusted entry means of the considered trait (FT or PH), 1 was a unit vector, μ the general mean, Z the design matrix that assigned the random effects to the genotypes, and u the vector of genotypic effects that were assumed to be normally distributed with , in which K denotes the realized kinship matrix between inbreds and σ 2 u the genetic variance of the GBLUP model.In addition, ε is the vector of residuals following a normal distribution N (0, Iσ 2 e ).The prediction ability of the GBLUP model was evaluated by Pearson's correlation coefficient (r) between observed and predicted phenotypes.
To assess the model performance, 5-fold cross-validation with 20 replications was performed.In that case, the prediction ability was defined as the median of the prediction abilities across the 20 runs of each 5-fold cross-validation.
Candidate gene analysis and allele mining
The candidate gene analysis was performed for those QTLs from the SP analysis that did not carry inside their confidence intervals previously reported genes controlling the corresponding trait, explained ≥15% of the phenotypic variance, and had a confidence interval ≤30 cM.For the QTLs that fulfilled these criteria, all the genes within the confidence interval were extracted using the Morex v3 reference sequence (Mascher et al., 2021).Next, variant calling data of SNPs, causing tolerated and deleterious mutations, insertion and deletions (INDELs), and predicted structural variants, obtained as described by Weisweiler et al. (2022), were used to identify genes that were polymorphic between the two parental inbreds of the sub-population in which the QTL was detected.For each gene, we took into account all the polymorphisms inside the coding, non-coding, and, for structural variants, potential regulatory regions of the gene within 5 kb up-and downstream of the gene.Subsequently, we performed a weighted gene co-expression network analysis (WGCNA) to identify modules of co-expressed genes that were associated with the phenotypic variability of the traits.The mRNA sequencing experiment of leaf samples of 21 parental inbred lines, described by Weisweiler et al. (2019), was the basis for this analysis.The selected soft thresholding power was 2, based on the scale-free topology criterion (Zhang and Horvath, 2005).We predicted the gene networks for the three modules with the highest and the three lowest correlations for both traits.In order to obtain a comprehensive understanding of the networks, we selected genes with a gene-module membership P<0.01 and, within them, the top 30% of gene-gene interactions based on the weight of the interactions.Because of the high number of gene-gene interactions in the module 'turquoise', we selected the top 5% of interactions with the highest weight.For the 'lightyellow' and 'tan' modules, we did not filter the interactions based on weight.Furthermore, for the 'black' module we selected the genes with a gene-module membership P<0.05.In the next step, the results of the WGCNA and SP QTL analyses were combined: we further filtered the polymorphic genes within the confidence intervals based on their membership of a module (Wei et al., 2022).The genes within the three modules with the highest and the three with the lowest correlation with the trait under consideration were evaluated for their functional annotation.We selected as candidate genes those with an annotation similar to that of genes previously reported to control the trait under consideration in barley and those for which functional annotation has been described to be involved in plant vegetative or reproductive development.All the analyses for the calculation of the weighted gene co-expression networks were performed with the R package 'WGCNA' (Langfelder and Horvath, 2008).
To confirm the accuracy of the whole genome sequencing data, we performed Sanger sequencing of the 23 parental inbreds for Ppd-H1.To predict if amino acid substitution appearing at a conserved position had a potential effect on the protein function, the SIFT algorithm was used (Vaser et al., 2016).In addition, we performed PCR, as described in Karsai et al. (2005), to check the presence/absence of the three Vrn-H2 genes.
Fine mapping of QTLs by association genetics
We used association genetics in the diversity panel of Pasam et al. (2012) to fine-map the QTLs that did not carry within their confidence intervals genes reported to control the corresponding trait, explained ≥15% of the phenotipic variance, and had a confidence interval ≤30 cM.We used the phenotypic data of the 224 inbreds collected in our field trials and the genotypic information available from Milner et al. (2019).To construct the kinship matrix among the 224 inbreds, we used all the SNPs in the SNP matrix.Association analysis was performed using only polymorphisms from QTLs fulfilling the above-mentioned criteria.For association analysis, we used a mixed model approach, implemented for the variance component (Kang et al., 2010), with the R package 'statgen-GWAS' (van Rossum et al., 2022).
Phenotypic variation and covariation
FT and PH were evaluated for each RIL across seven and five environments, respectively.For both traits, the environmental variance (E) was about two to three times higher than the genotypic variance (G) (Table 1).Furthermore, for FT, the variance of the interaction between genotype and environment (G:E) was, in the parental inbreds, about half of G, while, for PH, G:E was about 87% of G.The values of broad-sense heritability, for the whole HvDRR population, on an entry means basis were high to very high, ranging from 0.76 for PH to 0.86 for FT (Table 1).
To take into account possible intra-environmental variation, the phenotypic values were adjusted using moving grids of three different sizes, exploiting the possibilities of an augmented design.Because of the lack of considerable field effects, the resulting heritability values across all environments and for all three examined grid sizes were reduced compared with the analysis without adjustment.Therefore, we decided to discuss in the following only results from analyses where intraenvironmental variation was not corrected for.
Across all environments, the first sub-population to flower was HvDRR35, where RILs flowered on average 58 d after sowing.In contrast, the latest sub-population to flower was HvDRR46 for which, on average, RILs flowered 79 d after sowing (Fig. 1; Supplementary Table S3).HvDRR46 was also the sub-population with the smallest plants, with a mean height of 48 cm.In contrast, HvDRR12 was, with a mean of 87 cm, the sub-population with the tallest plants (Fig. 1; Supplementary Table S3).HvDRR09 was the sub-population with the lowest coefficient of variation (CoV) value for FT (2.73 d), while the highest CoV was observed for HvDRR43 (23.31 d) (Supplementary Table S3).Regarding PH, the sub-population with the smallest variability was HvDRR15 (CoV=3.83cm), while the highest CoV, 30.53 cm, was observed for HvDRR46 (Supplementary Table S3).The CoV was, for the diversity panel across the same environments, 7.35 d for FT and 14.01 cm for PH (Supplementary Table S3).
The differences between the mean of the parental inbreds and the mean of the sub-populations were also examined as these were an indicator for the presence of epistasis.For FT, the differences between the means of the parental inbred lines and the respective sub-populations were statistically significant (P<0.05) in 22 cases.Among them, the highest differences were observed for sub-populations HvDRR43, with 7.2 d, and HvDRR46, with 10.5 d.For PH the differences of the means of the sub-populations and the parental inbreds were significant (P<0.05) in 14 cases.The strongest differences between the parental inbreds and the progeny mean were observed for sub-populations HvDRR10, with 9.62 cm, HvDRR12, with 9.88 cm, and HvDRR11, with 10.60 cm.All these sub-populations had Sanalta as common parental inbred (Fig. 1; Supplementary Table S1).
Across all sub-populations, the correlation coefficient of FT and PH was −0.012 (Supplementary Fig. S1).However, considerable differences were observed for the single sub-populations (Fig. 2).HvDRR28 was the sub-population with the highest correlation coefficient (0.44), while the sub-population where the two traits were most negatively correlated was HvDRR43 (−0.77) (Supplementary Fig. S2).
Multi-parent population analysis
The MPP identified 21 QTLs each for FT and PH, distributed across all seven chromosomes (Fig. 3).The analysis was performed using the genetic haplotype window sizes estimated from the extent of linkage disequilibrium (Supplementary Table S2).The percentage of phenotypic variance explained by all the QTLs detected in the MPP analysis was 39.1% and 24.9% for FT and PH, respectively.For FT, the confidence interval of 17 QTLs overlapped with the interval of at least one QTL identified in the SP analysis (Supplementary Tables S4, S5).Out of 21 QTLs identified for PH, 16 overlapped with one or more QTLs detected in SP analysis (Supplementary Tables S4-S6).Among the QTLs detected for both traits, the intervals of two pairs of QTLs overlapped: FT-MP-Q3 with PH-MP-Q3 and FT-MP-Q19 with PH-MP-Q20.
The additive effect of the 23 parental inbreds for the 21 QTLs for FT ranged from −2.42 d, observed for Ancap2 at FT-MP-Q5, to 5.14 d, for Kombyne at FT-MP-Q13 (Fig. 4).However, in about 92% of cases, the additive effect for FT was between −1 and 1 d (Supplementary Fig. S3).For PH, the effect ranged from −3.88 cm for Kombyne at PH-MP-Q15, to a maximum of 1.99 cm at PH-MP-Q5, for seven parental inbreds (Fig. 4).Also in this case more than 90% of the additive effects had a value between −1 and 1 cm (Supplementary Fig. S3).Among the allele effects detected for FT, 20 were conferred exclusively by landraces (nine negative and 11 positive additive effects) and 27 exclusively by cultivars (10 negative and 17 positive additive effects).For PH, the allele effects derived from landraces were 18 (12 negative and six positive additive effects), while the effects inherited exclusively from cultivars were 16 (10 negative and six positive additive effects).The crossing design underlying our population allows estimation of the number of alleles at each QTL.The QTLs with the highest number of significantly different allele effects and thereby with presumably alleles were for FT, FT-MP-Q4 and FT-MP-Q19, with six significantly different effects each, and for PH, PH-MP-Q2 and PH-MP-Q4, with seven different effects each (Fig. 4).To verify the consistency of the consensus genetic map, we performed association mapping with a GWAS-type mixed model approach.Overall, four QTLs for FT and seven for PH, from the MPP, contained in their interval G represents the genetic variance, E the environmental variance, G:E the variance explained by the interaction between G and E for the parental inbreds, e the residual error, and h² the heritability of the trait, which was calculated for the whole HvDRR population.
Genomic prediction ability
The prediction abilities of the GBLUP model across the HvDRR population were high with values of 0.89 and 0.87 for FT and PH, respectively (Supplementary Table S7).To compare the prediction performance of the GBLUP model with those of the detected QTLs, we used the squared prediction abilities.The coefficient of determination (r²) obtained by genomic prediction without cross-validation was 0.79 for FT and 0.76 for PH.The cross-validated prediction abilities were 0.77 for both FT and PH.
For FT, HvDRR27 was the sub-population with the highest values of explained variance (77.75%), while for PH the subpopulation with the highest percentage of explained variance was HvDRR22 (78.54%).The lowest percentages of explained variance by the detected QTLs were observed for FT in HvDRR23 (16.91%) and for PH in HvDRR04 (10.27%) (Supplementary Tables S5, S6).
Out of 89 QTLs identified in the SP analysis for FT, 43 mapped to chromosome 2H (Fig. 5).A cluster comprising 21 QTLs was located at the beginning of chromosome 2H.The region covered by the confidence interval of these QTLs included Ppd-H1.Also for other major effect genes, QTL clusters were identified: six QTLs at the end of chromosome 4H, whose confidence intervals included Vrn-H2, 10 QTLs on the long arm of chromosome 5H, a region in which Vrn-H1 and HvPHYC were positioned, and 11 QTLs at the beginning of chromosome 7H, in which Vrn-H3 was located.Other QTLs included additional genes within their confidence intervals such as HvELF3, HvCEN, Hv20ox2 (sdw1/denso), HvFT4 (Pieper et al., 2021), and HvAP2 (Supplementary Table S5).
Single population analysis for PH identified 80 QTLs, where these QTLs were characterized by wider confidence intervals compared with those detected for FT (Fig. 6; Supplementary Table S6).As for FT, the chromosome with the highest number of QTLs was 2H.A cluster, including 14 QTLs, included within its confidence interval Ppd-H1.Other clusters of QTLs included in their confidence intervals HvAP2, Hv20ox2 (sdw1/ denso), Vrn-H1, and Vrn-H3 (Supplementary Table S6).
However, we identified 16 QTLs for FT and 31 QTLs for PH where no genes previously described for the control of the trait were present within their confidence interval (Supplementary Tables S5, S6), although the confidence intervals of most of these QTLs overlapped with previously reported QTLs.Among the 16 QTLs detected for FT, the QTLs with the lowest number of genes in the confidence interval were qHvDRR02-FT-5.1 and qHvDRR31-FT-5.2.The QTLs included 52 and 71 genes, respectively, which reduced to 35 and 45 when neglecting the low-confidence genes.qHvDRR31-FT-5.2 was, with 3.4 cM, the QTL with the shortest genetic confidence interval.For PH, qHvDRR48-PH-4.1 was the QTL with the lowest number of genes in its confidence interval (115 low-and high-confidence or 79 high-confidence genes).The QTL with the shortest confidence interval was qHvDRR22-PH-7.1 with 3.9 cM.
Eight sub-populations showed significant epistatic interactions between loci on a genome-wide scale.In total, 10 significant epistatic interactions were detected, nine for PH and one for FT.Two epistatic interactions each were observed for sub-populations HvDRR34 and HvDRR44 (Supplementary Table S8).
Allele mining
For FT, 21 sub-populations showed a QTL co-localizing with Ppd-H1.For 14 of these, a QTL that included Ppd-H1 was also identified for PH.Sixteen of the 21 sub-populations were polymorphic for the causal SNP 22 of Ppd-H1 (Turner et al., 2005).However, five sub-populations (HvDRR02, HvDRR04, HvDRR20, HvDRR23, and HvDRR48), for which the QTL confidence intervals included Ppd-H1, did not segregate for this polymorphism.All non-polymorphic sub-populations for SNP 22 had HOR1842 or IG128104 as parental inbred lines (Supplementary Table S1).Through Sanger sequencing, we identified the presence of a unique SNP in HOR1842 and IG128104 in the CCT domain of Ppd-H1 (Fig. 7).The primers used to amplify Ppd-H1 are listed in Supplementary Table S9.Based on the SNP position on the Ppd-H1 coding sequence of Morex, we refer to it as SNP 1945.SNP 1945 determines the synthesis of a threonine instead of an alanine (Supplementary Fig. S5).The SIFT algorithm predicted that the amino acid substitution caused by SNP 1945 affected a conserved position and, thus, might negatively affect the protein function.All the parental inbreds of the above-mentioned sub-populations carried a guanine (SNP 22 early allele), in correspondence with SNP 22 but the RILs that inherited SNP1945 from HOR1842 or IG128104 (adenine), tended to have later flowering compared with RILs that carried a thymine in correspondence of SNP 1945.
At the Vrn-H2 locus, an FT QTL was detected in six subpopulations.We evaluated by PCR, as described in Karsai et al. (2005) (Supplementary Table S9), the presence/absence of the three causal Vrn-H2 genes.Out of six sub-populations, five were polymorphic for the genes regulating the Vrn-H2 locus.In HvDRR29, both parental inbred lines, HOR8160 and IG128216, had the complete set of genes (Supplementary Fig. S6).
We did not detect new functional polymorphisms for Vrn-H1 and HvPHYC.However, we identified a significant number of RILs that were recombinant for these two genes which might be a valid resource for future functional studies (Supplementary Table S10).
Candidate gene analysis
The candidate gene analysis was performed for the QTLs detected in the SP analysis that did not carry in their confidence interval previously reported genes controlling the trait under consideration, explained ≥15% of the phenotypic variance, and had a confidence interval ≤30 cM.For these QTLs, we combined the results of QTL mapping with variant calling data and results from WGCNA.Through WGCNA, 27 different gene modules were detected across all the expressed genes in the barley genome (Supplementary Fig. S7).The correlation of the gene expression of modules and the adjusted entry means ranged from −0.52 to 0.49 for FT and from −0.54 to 0.47 for PH.Interestingly, the module with the highest correlation was the same for both traits.After selecting genes within the QTL range and that were included in one of the three modules with the highest or the lowest correlation (Supplementary Fig. S8), we searched for candidate genes.The most represented class of genes for the two traits was that of receptor-like kinase, followed by genes involved in the ethylene pathways, and genes coding for F-box proteins (Supplementary Table S11).
In addition to the function-based candidate gene analysis, we used association genetics in the diversity panel to fine-map the selected QTLs using the genome-wide genotyping-by-sequencing data of Milner et al. (2019).For FT, none of the polymorphisms in the QTL confidence intervals was significantly associated with the phenotype.For PH, we identified four significant SNPs associated with the phenotype (Supplementary Fig. S9).
Discussion
With this study, we aimed to obtain a comprehensive overview of the environmental and genotypic contributions to the regulation of FT and PH in barley.We performed MPP and SP analysis to elucidate the genetic complexity underlying the control of FT and PH.Finally, we identified candidate genes and new allelic variants using additional approaches such as WGCNA and association genetics.
The double round-robin population shows high variability of flowering time and plant height
We observed, with a range of adjusted entry means of 51.2-105.7 d as well as 12.6-101.2cm for FT and PH, respectively, a higher phenotypic diversity among the RILs of the HvDRR population (Fig. 1) compared with previous studies (Cuesta-Marcos et al., 2008;Maurer et al., 2015;Arifuzzaman et al., 2016;Nice et al., 2017;Afsharyan et al., 2020).Also, the standard deviation of the adjusted entry means of the RILs was higher for both traits than that described in previous studies (Pauli et al., 2014;Wang et al., 2014) (Supplementary Table S3).These observations might be due to the higher number of RILs and the selection of the 23 parental inbreds with maximal genotypic and phenotypic richness.
In addition, the variation observed for FT and PH in the diversity panel of 224 spring barley inbreds (Pasam et al., 2012) was similar to that observed in individual sub-populations.However, it was considerably smaller (FT) and more influenced by a few outliers (PH) compared with the diversity observed in the entire HvDRR population (Fig. 1).We observed that relative to the genotypic variance the variance components of environment and genotype-environment interaction were higher for PH than FT (Table 1).This finding was in discordance with a previous study where the variance component of the genotype-environment interaction was higher for FT than for PH (Rodriguez et al., 2008).This result might be explained by the higher variability of edaphic and meteorological variables (e.g.precipitation and temperature) of our study, which influenced PH more strongly than FT (Li et al., 2003), but also the lower difference in latitude among the environmental locations in our study compared with that of Rodriguez et al. (2008).
The high phenotypic variability and heritability values, combined with high-quality genotypic data, suggest that the HvDRR population is a valuable tool for detecting new genetic variants controlling agronomic traits in barley.
QTL analyses uncovered the role of genetic background in determining the correlation between FT and PH
The correlation between FT and PH differed across the HvDRR sub-populations (Fig. 2; Supplementary Fig. S2).
These results were in agreement with those of previous studies that have detected positive and negative correlations between FT and PH (Von Korff et al., 2006;Schmalenbach et al., 2009;Maurer et al., 2016;Nice et al., 2017).The high variability of the correlation coefficients could be due to the great genotypic diversity of the parental inbreds used in our study.To understand this aspect better, we considered in detail the co-located QTLs for FT and PH in the SP analysis.
For the sub-populations with the most negative correlation between FT and PH (HvDRR10, HvDRR11, and HvDRR43), all QTLs detected for FT were also detected for PH, although additional QTLs were observed for PH (Supplementary Tables S5, S6).For four of the five FT/PH QTL pairs, the parental inbred line conferring a positive additive effect for PH revealed a negative additive effect for FT and vice versa (Supplementary Tables S5, S6; Supplementary Fig. S2).In addition, the three sub-populations with the highest correlation coefficient (HvDRR19, HvDRR28, and HvDRR29) also had QTLs falling within the same interval for the two traits (Figs 5,6).In this case, for the three overlapping QTL pairs, the positive additive effect was given by the same Fig. 6.Genetic position of the QTL detected in single population analysis for plant height projected to the consensus map.The position of the QTL confidence intervals is shown as a vertical bar to the right of the chromosome.The color of the bar indicates if the sub-population was obtained by crossing two landraces (yellow), two cultivars (blue), or a landrace and a cultivar (green).The position of the QTL confidence intervals detected through multi-parent population analysis is represented by purple bars.The known regulatory genes previously described as being responsible for plant height regulation and their genetic position are reported in red.The positions of the markers at the borders of each QTL are also reported.
To increase the resolution of the dissection of the genetic origin of the correlation between FT and PH, we exploited MPP analysis.For each of the two traits, we identified 21 QTLs (Fig. 3).The QTL profiles obtained through MPP analysis for both traits had peaks falling within neighboring regions for the main genes previously reported to control FT and PH, such as Ppd-H1 and the vernalization genes.The diversity of the parental inbreds and the large number of sub-populations as well as the total RILs resulted in a high mapping resolution that led to narrow confidence intervals.We observed a pleiotropic effect only for two QTL pairs (FT-MP-Q3/PH-MP-Q3 and FT-MP-Q19/PH-MP-Q20). FT-MP-Q3/PH-MP-Q3 included in their interval HORVU.MOREX.r3.1HG0075860, which was functionally annotated as a transcription factor, while FT-MP-Q19/PH-MP-Q20 included in their intervals Vrn-H3.Therewith, our results suggested that FT and PH variations, with the exception of two QTLs, may be caused by independent genetic factors (Figs 3,5,6; Supplementary Tables S4-S6).
Multi-parent and single population analyses revealed new genome regions as well as genomic variants involved in the control of flowering time and plant height
The number of QTLs identified through MPP analysis (Fig. 3; Supplementary Table S4) was, with 21 each, higher than the number of QTLs detected in earlier studies using bi-and multi-parental populations of barley (Von Korff et al., 2006;Cuesta-Marcos et al., 2008;Schmalenbach et al., 2009;Rollins et al., 2013;Arifuzzaman et al., 2014;Pauli et al., 2014;Maurer et al., 2015;Nice et al., 2017).In the study of Hemshrot et al. (2019), a total of 23 QTLs were identified for FT.However, QTLs with the same genetic position were detected, which reduced the number of non-overlapping QTLs to 13.The reasons for the higher number of QTLs detected in our study compared with earlier studies were most probably the greater number of RILs and environments as well as the selection of very diverse parental inbreds (Stich, 2009;Weisweiler et al., 2019).We compared the physical positions of the QTLs with previous studies (Laurie et al., 1994;Druka et al., 2011;Pauli et al., 2014;Maurer et al., 2015;Nice et al., 2017;Hemshrot et al., 2019;Afsharyan et al., 2020) and did not find any previously reported QTLs co-localizing with FT-MP-Q1, FT-MP-Q2, FT-MP-Q3, FT-MP-Q10, FT-MP-Q11, FT-MP-Q17, FT-MP-Q18, PH-MP-Q6, PH-MP-Q10, and PH-MP-Q19.
The use of a consensus map for our analyses could have led to these observations.This is because structural variants have the potential to lead to inconsistencies in such maps.However, the peaks detected by a GWAS approach, in which the physical order of the markers was used, were in very good accordance with the results of the MPP analysis and, thus, we consider the discrepancies introduced by the consensus map to be of minor importance (Supplementary Fig. S4).
Our observations suggested that the genetic complexity of FT and PH is higher than initially reported.Furthermore, for both traits, the percentage of explained variance by a genomic prediction model was about twice the percentage of variance explained by the QTLs detected in the MPP analysis (Supplementary Table S7).This result suggested that many small effect QTLs remain undetected.
The difference between the percentage of variance explained by a genomic prediction model and that explained by the QTLs detected in the MPP analysis was greater for PH compared with FT (Supplementary Table S7).This observation suggests that PH is more influenced by small effect (and undetected) QTLs than FT.In addition, the total proportion of variance explained by the detected QTLs was lower for PH (25.7%) than for FT (37.4%).This trend was in agreement with the observation that epistatic interactions played a bigger role for PH than for FT (Supplementary Table S8).
The SP QTL analyses detected 89 QTLs for FT and 80 for PH (Figs 5,6; Supplementary Tables S5, S6).We compared the physical position of QTLs reported in earlier studies (Laurie et al., 1994;Druka et al., 2011;Pauli et al., 2014;Maurer et al., 2015;Nice et al., 2017;Hemshrot et al., 2019;Afsharyan et al., 2020), wherever possible, with those of the QTLs detected in our study.We observed for 166 QTLs a co-localization with previously reported QTLs.However, three QTLs, one for FT and two for PH, did not overlap with other previously reported QTLs.The novel QTLs detected by SP were qHvDRR30- 1.The percentage of variance explained by these QTLs was relatively low for qHvDRR30-FT-3.1 (4.3%) but higher for qHvDRR24-PH-3.1 (26.2%) and qHvDRR48-PH-4.1 (19.5%).Therefore, qHvDRR24-PH-3.1 was selected for finemapping, as well as qHvDRR28- For 21 sub-populations, an FT QTL co-localized with the Ppd-H1 locus.Five of these sub-populations (HvDRR02, HvDRR04, HvDRR20, HvDRR23, and HvDRR48) were not polymorphic for SNP 22 (Fig. 7; Supplementary Table S1).SNP 22 is located within the CCT domain of Ppd-H1 (Turner et al., 2005) and was described as the only functional polymorphism of Ppd-H1 (Turner et al., 2005).Another polymorphism of Ppd-H1, SNP 48 (Jones et al., 2008), had previously been associated with FT variation.However, the study of Sharma et al. (2020, Preprint) did not observe hints that SNP 48 was the causal SNP of the Ppd-H1 mutation.In addition, in none of the above-mentioned five sub-populations was SNP 48 segregating.All five sub-populations had HOR1842 or IG128104 as parental inbreds (Supplementary Table S1).From the whole genome sequencing data of the parental inbreds (Weisweiler et al., 2022), followed by Sanger sequencing, we identified a not previously reported polymorphism, SNP 1945, that is unique to HOR1842 and IG128104 (Fig. 7).SNP 1945 is located within the CCT domain of Ppd-H1 and it causes the synthesis of threonine instead of alanine (Supplementary Fig. S5).In the sub-population HvDRR24, whose parental inbreds were HOR1842 and IG128104, we did not detect a QTL for either FT or PH in the genome region of Ppd-H1.In addition, HOR1842 and IG128104 originated from the same geographical region (south-central Asia).Furthermore, we observed that the additive effect for the FT QTL co-localizing with Ppd-H1 was, with about 3.5 d, higher in sub-populations segregating for SNP 22 compared with about 2.3 d for the five subpopulations that did not segregate for SNP 22 (Supplementary Table S5).For the latter sub-populations, the additive effect assumed a positive value for the RILs that inherited the Ppd-H1 allele from HOR1842 or IG128104.These observations support the hypothesis that HOR1842 and IG128104 carry the same causal polymorphism and that SNP 1945 is the causal polymorphism for the QTL in those sub-populations that were monomorphic for SNP 22 as well as a new functional allelic variant of Ppd-H1.
A similar observation was made for the QTL co-localizing with Vrn-H2.The Vrn-H2 locus has been described as one of the main loci responsible for the difference between winter and spring barley varieties (Distelfeld et al., 2009).This difference is caused by the total deletion of a complex of three genes (ZCCT-Ha, ZCCT-Hb, and ZCCT-Hc) in spring barley or, in facultative barley, of a partial deletion (Karsai et al., 2005;Fernández-Calleja et al., 2021).Surprisingly, we observed for four of the HvDRR parental inbreds the complete set of Vrn-H2 causal genes in spring barley varieties (Supplementary Fig. S6).This observation suggested that the role of Vrn-H2 in the vernalization requirement may be more complex than previously assumed and not merely based on the presence/absence of the Vrn-H2 genes.In addition, for HvDRR29, both parental inbreds, HOR8160 and IG128126, carried the three Vrn-H2 causal genes (Supplementary Fig. S6).Similarly to Ppd-H1, it could be hypothesized that HOR8160 or IG128216 carried a new functional allelic variant of Vrn-H2 or that an additional gene, acting on the phenotype in a similar way to ZCCT-Ha:c, was present within the same QTL confidence interval.However, although HOR8160 and IG128126 have been classified as spring barley varieties (Pasam et al., 2012), it cannot be excluded that they originated from winter or intermediate genotypes.
These two examples suggest that the genetic complexity of the studied traits might be higher than anticipated from the simple comparison of co-localizing QTLs and can now be resolved using multiple segregating populations together with next-generation sequencing of the parental inbreds.Finally, cloning of the underlying genes will complement our understanding of the regulatory mechanisms of FT and PH.
Candidate gene analysis for a subset of the QTLs
We first extracted the polymorphic genes among the parental inbred lines within the confidence interval of the QTLs that explained ≥15% of the phenotypic variance, had a confidence interval ≤30 cM, and did not carry in their confidence interval any previously reported gene controlling the trait under consideration.Then, we combined this screening with the results of the WGCNA, selecting the three modules that showed each the lowest and highest correlation with FT and PH (Supplementary Fig. S7).
Among the FT QTLs fulfilling the above-mentioned criteria, qHvDRR28-FT-2.2 had the highest percentage of explained variance and the shortest genetic confidence interval.Two candidate genes identified for this QTL encoded the pseudo-response regulator 3 (PRR3) HORVU.MOREX.r3.2HG0170150 and the ethylene-responsive transcription factor HORVU.MOREX.r3.2HG0170460 (Supplementary Table S11).
Pseudo-response regulator is the same class of genes as Ppd-H1.The role of these genes is critical for the regulation of the plant circadian clock (Eriksson and Millar, 2003;Mizuno and Nakamichi, 2005) which is involved in the control of flowering time (Hayama and Coupland, 2004).Five different subgroups belonging to this class of genes have been reported: PRR1, PRR3, PRR5, PRR7 (to which Ppd-H1 belongs), and PRR9 (Matsushika et al., 2000).Phylogenetic analyses grouped the five sub-groups into three main clusters: PRR1, PRR5-PRR9, and PRR3-PRR7 (Nakamichi et al., 2020).Although genes belonging to all three clusters have been described as controlling FT or being influenced by the photoperiod, the only cluster containing genes from grass species described as being dependent on the photoperiod and at the same time controlling FT was PRR3-PRR7 (Nakamichi et al., 2020).Therewith this gene is an interesting target for further functional studies.
Genes responsible for ethylene biosynthesis are involved in a multitude of developmental processes throughout the plant life cycle (Bleecker and Kende, 2000).The concentration of ethylene also influences gene networks that regulate flowering to optimize the timing of the transition from the vegetative to the reproductive stage in relation to endogenous and external stimuli (Iqbal et al., 2017).Although further studies are needed to identify the ethylene pathways in barley, in rice, overexpression of an ethylene receptor (ETR2) was associated with delayed flowering (Hada et al., 2009).The delay was linked with an up-regulation of a homologous gene of GIGANTEA and TERMINAL FLOWER 1/CENTRORADIALIS (Hada et al., 2009); both of these classes of genes are involved in barley in the control of flowering since HvGI (Dunford et al., 2005) and HvCEN (Comadran et al., 2012) belong to them.Ethylene is also involved in plant growth (Dubois et al., 2018), and its role in vegetative development has been described in barley (Patil et al., 2019).In addition to the one found in qHvDRR28-FT-2.2, we identified two ethylene-responsive transcription factors (HORVU.MOREX.r3.7HG0685230 and HORVU.MOREX.r3.2HG0182430) in qHvDRR22-PH-7.1 and qHvDRR29-PH-2.1 (Supplementary Table S11).Besides being an ethylene-responsive transcription factor, HORVU.MOREX.r3.2HG0182430 also belongs to the same class of genes as HvAP2.
In addition to functional data, we used association genetics to fine-map the detected QTLs using the diversity panel that was evaluated in the same set of environments as the HvDRR population.For FT, none of the polymorphisms from Milner et al. (2019) that were located in the QTL confidence intervals were significantly associated (P<0.05) with FT variation.The reason for this discrepancy was most probably that association mapping panels have a low power to detect marker-trait associations in the case of low-frequency alleles (Myles et al., 2009), which is overcome by using segregating populations as in the HvDRR population.For PH, low-significance marker-trait associations have been detected.However, one of the polymorphisms was in proximity (<150 kbp) to HORVU.MOREX.r3.3HG0222500, a candidate gene detected for qHvDRR24-PH-3.1 through the WGCNA approach (Supplementary Table S11; Supplementary Fig. S9).
These results suggest that the integration of QTL analyses with other omics datasets supports the detection of candidate genes regulating traits of agronomic interest.
Conclusions
The great phenotypic variability observed for FT and PH in the HvDRR population suggests that this population will be a powerful genetic resource to detect new regulatory mechanisms that could allow the extension of the barley cultivation area or its adaptation in changing environmental conditions.Furthermore, it was observed that environmental variables affected these traits and that the environmental component had a greater influence on PH compared with FT.In addition, our study provides a comprehensive summary of the genetic architecture of FT and PH and forms the basis for future QTL cloning studies.Finally, the detection of novel QTLs, but also the observation that additional alleles or genes segregate at known loci like Ppd-H1 and Vrn-H2, suggests that the studied traits are genetically more complex than previously reported.
Supplementary data
Supplementary data are available at JXB online.'darkgreen','purple','tan','lightyellow','green','blue',and 'turquoise'. Fig. S9.Negative decadic logarithm of the P-value for association tests of sequence variants in QTL without previously reported genes for the control of the trait within their interval, explaining ≥15% variance, and with interval ≤30 cM for flowering time and plant height.
Table S1.Crossing scheme of the 45 HvDRR subpopulations and number of RILs for each sub-population.
Table S2.Genetic and physical distances for which the linkage disequilibrium measured r² reached a value of 0.2.
Table S3.Average of the adjusted entry means, standard deviations, and coefficients of variation across all 45 sub-populations and the diversity panel for flowering time and plant height.
Table S4.Summary of the results of the multi-parent population analysis for flowering time and plant height.
Table S5.Summary of the results of the single population analysis for flowering time.
Table S6.Summary of the results of the single population analysis for plant height.
Table S7.Prediction ability of the genomic SNP marker data for flowering time and plant height without cross-validation and with 5-fold cross-validation across all sub-populations.
Table S8.Genome-wide epistatic loci detected in the HvDRR population.
Table S10.Recombinant RILs between Vrn-H1 and HvPHYC for sub-populations having a QTL in the Vrn-H1 and eam5 genomic region.Table S11.List of candidate genes in the confidence interval of selected QTL that carried a polymorphism among the parental lines.
Fig. 1 .
Fig. 1.Violin plots for adjusted entry means for flowering time and plant height of each HvDRR sub-population and for the 224 inbreds of the diversity panel.DAS, days after sowing.The green circles represent the adjusted entry means of the parental inbreds of the sub-population.The orange lines represent the mean of the adjusted entry means of the recombinant inbred lines of the respective sub-population.
Fig. 2 .
Fig. 2. Distribution of correlation coefficients between flowering time and plant height calculated for the HvDRR sub-populations.
Fig. 3 .
Fig. 3. Negative decadic logarithm of the P-value of the multi-parent population analysis for flowering time (top) and plant height (bottom) using an ancestral model.On the x-axis, the position on the consensus genetic map is reported.Each dashed line indicates the peak position of the corresponding QTL.
Fig. 4 .
Fig. 4. Heat map of the effects of the parental inbreds at the QTLs detected through multi-parent population analysis for flowering time (top, in days after sowing) and for plant height (bottom, in cm).Indexed letters indicate the significance of the difference (P<0.05) between the effects of the same QTL.
Fig. 5 .
Fig. 5. Genetic position of the QTL detected in single population analyses for flowering time projected to the consensus map.The position of the QTL confidence intervals is represented as a vertical bar parallel to the right of the chromosome.The color of the bar indicates if the sub-population was obtained by crossing two landraces (yellow), two cultivars (blue), or a landrace and a cultivar (green).The position of the QTL confidence intervals detected through multi-parent population analysis is represented by purple bars.The genetic positions of the known genes regulating flowering time in barley are shown in red.The positions of the that flank each QTL are also reported.
Fig. 7 .
Fig. 7. Genomic sequence of the last exon of Ppd-H1 of Morex, Igri, Optic, Golden Promise, Triumph, and the 23 parental inbreds of the HvDRR population.SNP 22 is highlighted in yellow, SNP 1945 in orange.At the top, the gene structure of Ppd-H1 is given.Lines indicate the positions of SNP 21, SNP 22 (Turner et al, 2005), and SNP 1945 within the last exon.
Fig. S1 .
Histogram and correlation plot between flowering time and plant height across all 45 HvDRR sub-populations.Fig. S2.Histograms and correlation plots between flowering time and plant height, for each of the 45 HvDRR sub-populations.Fig. S3.Effect size of the QTL detected through multiparent population analysis for flowering time and plant height for each of the parental lines.Fig. S4.GWAS-type mixed model approach for flowering time (FT) and plant height (PH) of the HvDRR population.
Fig. S5 .
Fig. S5.Amino acid sequence of the terminal region of Ppd-H1 of Morex, Igri, Optic, Golden Promise, Triumph, and the 23 parental inbreds of the HvDRR population.Fig. S6.Gel pictures of PCRs performed to detect the presence/absence of ZCCT-Ha:b and ZCCT-Hc as described in Karsai et al. (2005).Fig. S7.Heat map of the module-trait relationships for plant height and flowering time.Fig.S8.Network predictions for modules 'orange', 'black','darkgreen', 'purple', 'tan', 'lightyellow', 'green', 'blue', and 'turquoise'.Fig. S9.Negative decadic logarithm of the P-value for association tests of sequence variants in QTL without previously reported genes for the control of the trait within their interval, explaining ≥15% variance, and with interval ≤30 cM for flowering time and plant height.TableS1.Crossing scheme of the 45 HvDRR subpopulations and number of RILs for each sub-population.TableS2.Genetic and physical distances for which the linkage disequilibrium measured r² reached a value of 0.2.TableS3.Average of the adjusted entry means, standard deviations, and coefficients of variation across all 45 sub-populations and the diversity panel for flowering time and plant height.TableS4.Summary of the results of the multi-parent population analysis for flowering time and plant height.TableS5.Summary of the results of the single population analysis for flowering time.TableS6.Summary of the results of the single population analysis for plant height.TableS7.Prediction ability of the genomic SNP marker data for flowering time and plant height without cross-validation and with 5-fold cross-validation across all sub-populations.TableS8.Genome-wide epistatic loci detected in the HvDRR population.TableS9: Lists of primers used to amplify Ppd-H1 and Vrn-H2.TableS10.Recombinant RILs between Vrn-H1 and HvPHYC for sub-populations having a QTL in the Vrn-H1 and eam5 genomic region.TableS11.List of candidate genes in the confidence interval of selected QTL that carried a polymorphism among the parental lines.
Fig. S8 .
Fig. S5.Amino acid sequence of the terminal region of Ppd-H1 of Morex, Igri, Optic, Golden Promise, Triumph, and the 23 parental inbreds of the HvDRR population.Fig. S6.Gel pictures of PCRs performed to detect the presence/absence of ZCCT-Ha:b and ZCCT-Hc as described in Karsai et al. (2005).Fig. S7.Heat map of the module-trait relationships for plant height and flowering time.Fig.S8.Network predictions for modules 'orange', 'black','darkgreen', 'purple', 'tan', 'lightyellow', 'green', 'blue', and 'turquoise'.Fig. S9.Negative decadic logarithm of the P-value for association tests of sequence variants in QTL without previously reported genes for the control of the trait within their interval, explaining ≥15% variance, and with interval ≤30 cM for flowering time and plant height.TableS1.Crossing scheme of the 45 HvDRR subpopulations and number of RILs for each sub-population.TableS2.Genetic and physical distances for which the linkage disequilibrium measured r² reached a value of 0.2.TableS3.Average of the adjusted entry means, standard deviations, and coefficients of variation across all 45 sub-populations and the diversity panel for flowering time and plant height.TableS4.Summary of the results of the multi-parent population analysis for flowering time and plant height.TableS5.Summary of the results of the single population analysis for flowering time.TableS6.Summary of the results of the single population analysis for plant height.TableS7.Prediction ability of the genomic SNP marker data for flowering time and plant height without cross-validation and with 5-fold cross-validation across all sub-populations.TableS8.Genome-wide epistatic loci detected in the HvDRR population.TableS9: Lists of primers used to amplify Ppd-H1 and Vrn-H2.TableS10.Recombinant RILs between Vrn-H1 and HvPHYC for sub-populations having a QTL in the Vrn-H1 and eam5 genomic region.TableS11.List of candidate genes in the confidence interval of selected QTL that carried a polymorphism among the parental lines.
Table 1 .
Variance components of the multi-environment linear mixed model and heritability values for flowering time and plant height | 2023-01-17T14:11:22.321Z | 2023-01-13T00:00:00.000 | {
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219134109 | pes2o/s2orc | v3-fos-license | A REVIEW ON ENERGY EFFICIENCY OF STEEL PLANTS IN INDIA ON ENERGY EFFICIENCY OF STEEL PLANTS IN INDIA.”
: The steel industry being highly energy intensive in nature is one the major consumers of energy. The iron and steel industry is the largest energy consuming manufacturing sector in the world. It is therefore that the question of fuel or energy has been of the highest importance in steel making, and one can boldly claim that all other conditions remaining constant, saving or wasting of fuel can make the difference between a profit or a loss of a steel plant. Energy conservation in steel plants is very crucial to ensure the competitiveness of the steel producing industries and to minimise environmental impacts. India's leading iron and steel companies, scored averages at best in Centre for Science and environment green rating test. The Indian iron and steel sector's energy consumption of 6.6 GCal per tonne, is 50 per cent higher than the global best practice. The integrated steel plants in India have the opportunities to strengthen their operations and minimise energy losses and wastages to reduce specific energy consumption by 5-6%. To reduce the gaps between India and developed countries we have to follow the technological advancement and implementation of innovative strategies at every stage of the operation of steel plants. The specific energy consumption in the Indian steel industry is high compared to that in advanced countries. Data for four integrated steel plants in India have been analysed. World crude steel production reached 1.621 million tones (Mt) in 2015. To meet the needs of our growing population, steel use is projected to increase by 1.5 times that of present level by 2050 .
Introduction
The Indian Iron and Steel industry is vital to the nation's development efforts and to support the required rapid economic growth. Steel finds its application in a wide range of sectors such as automobile, power, machine goods, and infrastructure. Energy efficiency and low carbon growth have emerged as key pathways to reduce the nation's energy intensity and emissions intensity. The industry has taken several initiatives to conserve energy at each sub process by adopting best technologies and innovative process operations or the usage of alternate materials. The efficient use of energy has always been one of the steel industry's key priorities. World steel study estimates Electricity Electrical energy is one of the major components, which largely supports the Electric Furnace, machinery operations, drives, fans, lighting and other process supported systems. Among the three major process routes in steel making, 24% and 31% utilise electrical furnaces such as Arc furnace and Induction furnace routes respectively. The power required by the steel industry is estimated to increase to 16,000 MW in 2025-26 from around 8200MW in 2016-17 . Electrical furnaces melt the charged materials to produce specialised steel using electrical energy. A variety of steels are manufactured through electrical furnaces. Reducing the burning losses of iron and readily oxidising the alloying elements are other advantages of electrical furnaces. Use of electricity remains relatively stable. Electricity generally used for the operation of rolling mill motor systems, in electric arc furnaces for melting of scrap and in rolling mills.
Petroleum fuels
Purchased petroleum fuels like fuel oil, furnace oil, Low Sulphur Heavy Stock (LSHS) to meet any short fall in the availability of fuels in high temperature furnaces.
Gaseous fuels
By-product coke oven gas, blast furnace gas and also coal for fuels are used to meet the fuel shortage.
All these forms of energy are used in various forms of Kilns and Furnaces which comprise the bulk of a steel plant, e.g., All these furnaces consume huge quantities of all types of fuels e.g., solid, liquid, gaseous and electric as mentioned above.
The quantity of energy required sub process-wise for production of one ton of steel throughout the world are given in the following diagram: The specific energy consumption figure for production of one ton of steel has been calculated for the last half a century (1940 -1990) and it has been observed that though very little progress has been made in this regard, in fact the figure has gone higher upon some steel plants in recent years, it is obvious that in the developed countries the specific energy consumption has decreased from a level of 9-12 G Cal / t to 5-7 G cal / t in the last 40-50 years. On the other hand the present specific energy consumption level in the Indian Steel Plants is rather high ranging between 9-15 G Cal / t and download there is hardly any perceptible download trend in the S.E.C. data.
Barriers and Challenges
The Indian iron and steel industry has been working consistently and contributing to the infrastructure development and economic growth of the country in the face of several barriers and challenges. Some of these can be summarized as follows: • Availability of raw materials such as ores and coal resources • Supply chain and associated infrastructure (roadways and railways) • Variation in the international prices • Land acquisition and grant of environment clearance [12] • Techno-economics, and production efficiency benchmark in compliance with international standards • Sustaining in a competitive environment due to global trade agreements • Reckonable restrictions and high tariff barriers To overcome these challenges, suitable economic policy framework needs to be formulated to facilitate continuous technology advancements and adoption of energy saving measures with specific national policy mechanisms such as PAT.
The Indian Steel industry will need to adopt suitable economic policy and investment framework to facilitate continuous upgradation to best available technology which will result in reduction in the energy consumption.
On a study of the energy consumption in steel plants it will be revealed that -70% of the energy is consumed in iron making stage in the Blast Furnace. Therefore the energy conservation efforts in the iron making areas have to be accorded a very high priority.
The major consuming centres of the petroleum fuels in the steel plants are also to be given high priority as it contributes to about 14% of the total fuel bill of the steel plants while it contributes only about 3% of the total energy input.
The amount of Coke Oven Gas and Blast Furnace gas generated and consumed in Steel Plant also requires attention because these two gases produced in-situ plays an important role in steel making.
Sintering Plants
Sintering process can contribute to energy conservation in our steel plants in a big way though in an indirect manner, it is known that the percentage of a sinter in the blast furnace reduces the coke rate. Therefore the following steps should be taken-• To increase the bed thickness to a level of 400-450 mm in order to fully utilise the fuel consumed. • To increase the capacity of the blowers.
• To recover sensible heat of the sinter which is discharged at a temperature of 700-8000C.
A process similar to coke drying quenching process for recovery of the sensible heat of the hot sinter has been developed in Japan.
Coke Ovens
The coke ovens in Indian Steel Plants suffer from the three following defects e.g., Low Coke Oven Gas yield -The yield of coke oven gas varies in India from 23-290 nrWper tonne of coal with 18-21% ash and 22-C6% volatile matter as against 320 NmJ per tonne of coal charge using coal having 8% ash and volatile matter of 27%. [13] High C.O. gas usage for under firing -Specific Heat Consumption in Indian Coke Ovens varies from 0.6 to 0.7 G Cal / ton of coal charged , it has been observed that not only specific heat consumption is high but also the uses of coke oven gas for under firing is high.
High Wastage Energy -Heat balance of Coke Ovens reveals that the various heat losses e.g., sensible heat of the coke and the gas and the stack losses are 1.3x106 G Cal per year.
For energy conservation in Coke Ovens in Steel Plants the following methods have been proposed: To reduce the use of coke oven gas for coke oven under firing it is essential that the health of the Coke Ovens be properly maintained.
In addition improved combustion control, maintaining proper thermal regime in the ovens it is necessary to introduce automatic control of air and fuel ratio in the ovens.
To increase the yield of Coke Oven it is essential to minimise leakage and avoid incidence of undercharging of ovens.
Increased yield of Coke Oven gas contributes to improvement in the gas balance of the steel plant.
To introduce waste heat recovery technologies like preheating of coal charge, coke dry quenching technology, it is necessary to introduce waste heat recovery system which will bring down the requirement for fuel input from a level of 0.56 G Cal per tonne of coal charge to 0.5 G Cal per tonne of coal charge.
The energy value accounts for the quality of energy, higher temperature being suited for energy generation. The sensible heat accounts for low temperature steam as the main target for heat recovery. There is a significant energy recovery potential for sinter plants.
The total sensible heat recovery possible from ISP is 5.53 GJ/trs out of which 4.88 GJ/trs is commercially practiced. As the steam pressure increases, the amount of steam generated decreases due to increase in boiling point and steam enthalpy. The boiler efficiency assumed for the calculations is 90%. The steam can be utilised in the process wherever required.
WHR Boilers (WHRB) can be installed at coke oven and hot stove exit points. Waste heat can be utilised in the following ways:
1) Power -Steam Rankine Cycle 2) Power -Organic Rankine Cycle 3) Integration with Captive Power Plant
The exhaust gases emanating from different sections of an ISP are at a high temperature. The enthalpy of the exhaust gases can be utilised to supplement the heat requirements of the steel plant.
The exhaust gases from different processes are made to pass through heat exchangers to generate steam. The steam generated from each section is collected and used to run a turbine to generate electricity.
The following are the technologies available for producing power from exhaust gases of an iron and steel plant.
The potential for WHR from different sections together is in the range of 12 -56 MW depending on the capacity of the plant.
Scrap Utilization
Scrap is an important raw material for secondary steel makers which reduce the energy demand per ton of crude steel production. According to the Bureau of International -what? recycling scrap purchases by steel works worldwide has increased by 17.2% to 340million tonnes in2010
Use of Alternative Fuels in Blast Furnace
Blast furnace is the most energy intensive sub process in an ISP. The main energy source for the process is coke which is expensive. India has low reserves of coking coals and mostly depends on import of coking coals. It is necessary to search for alternative fuels injection in blast furnace to reduce energy consumption and minimize the import dependency. The possible alternative energy sources that can be used in the blast furnace operation are biomass, biochar, waste plastics, natural gas, fuel oil, etc.
Availability and Quality of Coal
Coal is another major source and according to present situation the availability of coking coal is a matter of serious consideration for steel industry for different reasons. Direct usage of Indian coking coals is a constraint due to high ash content. It should either be washed or blended with imported coal before usage. The production of quality coking coal is low in India. As per 2011 findings, the total coking coal reserves in India was 33.474 billion tonnes with 17.67 billion tonnes of "substantiated" category and total non-coking coal resources of 252.39 billion tonnes with 96.33 billion tonnes of "substantiated" category. The total coal requirement including coking coal is 613.62 million tonnes to meet the 2016-17 targets.
Environmental Pollution Norms
Iron and Steel sector has high emissions due to the energy intensive processes utilised. Indian steel industry emissions are higher than the global steel producers and emission levels and this could be due to technology or raw material variations. The emission standards for Iron and Steel industry are described below
Coke Ovens
The effluent standards for coke ovens are suspended solids 100 mg/l, oil and grease 10 mg/l, Ammonical nitrogen as N 50 mg/l and phenol 1 mg/l Stack emission standards are SO2 800 mg/Nm3, NOx 500 mg/Nm3, PM 50 mg/Nm3 and sulphur in coke oven used for heating is 800 mg/Nm 3
Sinter Plant
The effluent standards for suspended solids, oil and grease are 100 mg/l and 10 mg/l respectively from sinter plant Stack emission standards are PM is 150 mg/Nm 3
Blast Furnace
The suspended solids 50 mg/l, oil and grease 10 mg/l, ammonical nitrogen as N 50 mg/l are the effluent standards for blast furnace. Stack emission standards are SO2 800 mg/Nm 3 NOx 500 mg/Nm 3 , PM 50 mg/Nm 3 and sulphur in coke oven used for heating is 800 mg/Nm 3 .
Summary and Conclusions
The Indian Iron & Steel sector has plants which are energy efficient and are comparable to the best in the world after adjusting for the availability and quality of the key inputs such as iron ore and coal. Some of the major plants in India recover most of the useful by-product gases and reuse their inherent calorific value in other processes. However, there are plants which operate under conditions of smaller scale, older technology, lower grade iron ore and coal and without adequate access to knowledge of best practices and finance options for deployment of EE measures.
The key levers for improving EE in a typical Iron and Steel plant include sub-process Energy Efficient technologies such as Pulverised Coal Injection, Coke Dry Quenching and Top Recovery Turbine can be deployed in order to contribute to energy savings. Recycling Blast furnace slag, Waste Heat Recovery options, effective Alternate Fuel Resources are other such options that can be implemented in iron and steel plants.
Energy efficiency in India is a national priority given that large sections of rural households do not have access to energy. The Indian iron and steel industry and energy policy implementing agencies such as the BEE have embarked on a cooperative journey which is vital to the country's roadmap for low-carbon inclusive growth. | 2020-05-07T09:16:17.640Z | 2020-02-24T00:00:00.000 | {
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242392373 | pes2o/s2orc | v3-fos-license | Analysis on Data Characteristic and Mechanism of Wind Vibration Disaster of Power Transmission Line
In this paper, characteristic identification and mechanism analysis on wind vibration disaster of power transmission line are carried out by using the data driving method. To provide technical support for prevention and control of wind vibration disaster to power transmission line, statistics are made in this from three dimensions i.e meteorology & geology, line structure and disaster characteristics to the wind vibration data of power grid conductors. Then frequency distribution characteristics of wind vibration disasters for conductors under different parameter conditions has been analyzed, and the correlation between various factors and wind vibration disasters according to analysis results has also been discussed.
Introduction
A certain recording data of wind vibration disasters of conductors in China has been accumulated [1][2] during the running of power transmission line over years, including structural and non-structural data of multiple types, such as character, table and picture, and the types of wind vibration disaster involve aeolian vibration [3], sub-span oscillation [4] and windage yaw [5]. In recent years, under the background of power grid intelligence and energy network development, it is urgently necessary to implement deep exploration and machine learning to data of conductor wind vibration disasters, so as to realize the characteristic identification and mechanism analysis on wind vibration disasters of conductors of power transmission line from the perspective of data driving [6], thus providing technical support for prevention and control of wind vibration disasters of power transmission line. This paper will sort the recording data of several typical types of wind vibration disasters of conductors of power transmission line such as aeolian vibration, sub-span oscillation and windage yaw, make statistics of data distribution characteristics from meteorological and geological environment parameters and line structure parameters, and talk about the relationship between wind vibration disasters of conductor and the environmental and structural parameters.
Statistical Rule of Disaster Data Information
The fault recording information about wind vibration disasters of conductors collected in this paper includes three types of data, i.e. aeolian vibration, sub-span oscillation and windage yaw, and the quantity of collected data is 191 pieces in total. The data time distribution is relatively wide, and the data contents mainly include basic information of line, meteorological and geological information, conductor structural parameters and description of three kinds of wind vibration disasters.
The basic information of line includes line name, voltage class, terrain (plain, mountainous region and hill), altitude (grading as per height), line tendency and years of operation; the meteorological information includes wind speed, wind direction, included angle between wind direction and line, temperature, humidity, precipitation type, conductor ice coating thickness; and structural parameters of conductor include quantity of loops of pole tower (single loop and double loops), conductor model, conductor arrangement mode, and quantity of conductor ] bundles (single-bundle, two-bundle, fourbundle, and six-bundle); the description of wind vibration disaster information includes disaster occurrence duration (insulation windage yaw duration, aeolian vibration duration, sub-span oscillation duration), section No., pole tower No., span, and anti-vibration device installation situation, spacer installation situation, windage yaw angle, insulator type, ground wire model, anti-vibration device type, damage type (conductor or ground wire breaking, strand breaking, and anti-vibration device), spacer distance, quantity of spacers, tripping or not, description of other faults (machinery, electrical apparatus, etc., including fault distance measurement, damage detail description, etc.), and description of adopted measures.
Targeting artificial recording data, conduct quantitative processing to the descriptive language in the Table, and the table corresponding to the processing is as shown in Table 1.
Statistical Result of Disaster Data
According to data contents, the statistical analysis of fault data is conducted according to three dimensions, i.e. meteorology and geology, line structure and disaster characteristics, and the histogram is adopted to represent disaster distribution characteristics. Figure 1, the wind speed statistics adopts grade for division, and see Table 1 for the scope of wind speed which the grade corresponds to; the quantity of samples with wind speed grade of 6 is the most, while a certain proportion of samples are distributed within the wind speed scope of grades 9-12 in a centralized way.
Meteorology and Geology Dimension
In Figure 2, the scopes of peak values of included angle between wind direction and line are 40°-50°, 60°-70°, and 80°-90°; in which, the quantity of data samples within the 80°-90° is the most; therefore, it is preliminarily judged that the wind vibration disaster of conductor may occur more easily when the included angle between wind direction and line is almost vertical. Figure 3, the temperature is distributed within the scope of -10 to 10℃ in a centralized way, so the line disaster will occur more easily at relatively low temperature, and there is the data of -30/30 temperature data recording; this data refers to the scope of temperature of recording data of a time, and it is preliminarily inferred that the line in the environment where the temperature difference is relatively large may have line disaster easily. Explaining from the perspective of dynamics, at low temperature, the tension and internal force levels of conductor are high, and there will be fatigue and wear under aeolian vibration easily; when the temperature is relatively high, the conductor sag will be increased, and the windage yaw amplitude will also be increased obviously, and then the windage yaw discharging fault will occur easily. Figure 4 is the statistical histogram of wind vibration terrain, includes three terrains, i.e. plain, mountainous region and hill. In a word, the proportion of wind disasters of lines in plain terrain is relatively large. Figure 5, it can be known that the altitudes of lines suffering from wind vibration disaster are mainly centralized within the scope of 1,400m, and another part of lines are in the regions with altitudes of 0m -800m; in which, the altitudes are centralized within the scope of 400m. This paper believes that the above mentioned distribution pattern is mainly determined by natural distribution characteristic of lines, and the influence of altitude on wind vibration of conductor is not taken into consideration temporarily.
Line Structure Dimension
In Figure 6, the years of operation of line is within 10 years generally, and that of a small number of lines is 15-20 years, and some lines have operated for 35 years, which is relatively long. It is preliminarily judged that the wind vibration disaster of conductor has relatively poor correlation with years of operation of line, and the wind vibration damages of current most lines are mainly caused by severe environment and structural defect factors, the wind vibration faults of only a small number of lines are caused by material aging and degradation of materials for they have been put into operation for a long time. Figure 7, it can be known that among the data this time, the quantity of lines of pole tower with single loop and that with double loops are 80 and 110 respectively, and the data distribution of samples is relatively balanced. It is preliminarily inferred that the influence of quantity of loops of pole tower on different types of wind vibration is relatively low.
From Figure 8, it can be known that the proportion of data amounts of single conductor, twobundle and six-bundle in the wind vibration sample data is relatively large, and the proportion of fourbundle and eight-bundle sample data is relatively low. Figure 9, it can be known that the windage yaw angle of insulator is mainly centralized within 10-30°, and the quantity of data samples with angle greater than 30° is relatively small.
Line Structure Dimension
From Figure 10, it can be known that the duration of samples which undergo aeolian vibration among the data is 8 hours mainly, and the duration of a small number of samples which undergo aeolian vibration is 1 hour. Figure 11 and Figure 12, it can be known that the duration of sub-span oscillation fault is mainly centralized within two time periods, i.e. 1-2.5 hours and 7-8 hours; the sub-span sections where the sub-span oscillation may occur most easily are 35m-40m and 60m-70m. | 2019-12-19T09:15:56.540Z | 2019-12-13T00:00:00.000 | {
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9154909 | pes2o/s2orc | v3-fos-license | The first megatheropod tracks from the Lower Jurassic upper Elliot Formation, Karoo Basin, Lesotho
A palaeosurface with one megatheropod trackway and several theropod tracks and trackways from the Lower Jurassic upper Elliot Formation (Stormberg Group, Karoo Supergroup) in western Lesotho is described. The majority of the theropod tracks are referable to either Eubrontes or Kayentapus based on their morphological characteristics. The larger megatheropod tracks are 57 cm long and have no Southern Hemisphere equivalent. Morphologically, they are more similar to the Early Jurassic Kayentapus, as well as the much younger Upper Cretaceous ichnogenus Irenesauripus, than to other contemporaneous ichnogenera in southern Africa. Herein they have been placed within the ichnogenus Kayentapus and described as a new ichnospecies (Kayentapus ambrokholohali). The tracks are preserved on ripple marked, very fine-grained sandstone of the Lower Jurassic upper Elliot Formation, and thus were made after the end-Triassic mass extinction event (ETE). This new megatheropod trackway site marks the first occurrence of very large carnivorous dinosaurs (estimated body length >8–9 meters) in the Early Jurassic of southern Gondwana, an evolutionary strategy that was repeatedly pursued and amplified in the following ~135 million years, until the next major biotic crisis at the end-Cretaceous.
Introduction
During the first 30 million years of their evolution, dinosaurs constituted a relatively morphologically non-diverse group of land vertebrates compared to contemporaneous crurotarsans [1,2] with which they shared many Late Triassic ecosystems. The outset of the Jurassic witnessed the global evolutionary radiation of Dinosauria, with events associated with the end-Triassic mass extinction (ETE) and the Triassic-Jurassic boundary (TJB) often hypothesised to have played an important role [3]. The earliest Jurassic is thus a period of particular interest as it spans a post-extinction recovery period during which dinosaurs continued to thrive and diversify globally [3,4]. This observation has been instrumental in recent interpretations that favour abiotic contingency ("opportunism") [5,6] over competitive superiority in explaining the ultimate success of the dinosaurs [3,7]. PLOS During the Late Triassic and Early Jurassic, the largest carnivorous dinosaurs seldom surpassed 5 m in body length, as evidenced by both the skeletal and ichnofossil record [5]. Olsen et al. [5] suggested that "ecological release" associated with the disappearance of incumbent non-dinosaurian archosaurian predators across the TJB possibly explains the sudden leap in carnivorous dinosaur size thresholds in the Early Jurassic, as evidenced by the ichnospecies Eubrontes giganteus.
Thus, body size constraints have been linked to shifts in global ecosystem composition, with medium to larger sized dinosaurs argued to be able to command a greater range of morphospace following the ETE [2,3]. This hypothesis, which potentially explains the appearance of relatively large-bodied theropod dinosaurs within the earliest Jurassic, was based mainly on the ichnology of the Newark Supergroup (USA) and has been received with some scepticism e.g., [8,9].
Amongst dinosaurs, Theropoda is remarkable for demonstrating continuous evolutionary novelty throughout the history of the clade, a phenomenon which is particularly noticeable at nodes proximate to the theropod-bird transition [10]. In the last decade, there have been many studies interested in dinosaurian, and specifically theropod, macroevolutionary patterns, with the dynamics of changing body size playing a central role within this literature [2,3,10]. However, reconstructing early theropod evolutionary patterns is much more problematic, with studies focused on discrete (morphological) characters depicting gradualistic rates of change for the group across the TJB [2]. In contrast, investigations utilising mass-estimates suggest a significant increase in body size for Theropoda within the Late Triassic [10]. A source of this confusion is the lack of theropod body fossils from this period. Theropod body fossils from the Early Jurassic are relatively rare, with Coelophysis bearing the largest number of well-preserved specimens globally [11,12].
In contrast, the theropod ichnite record is comparatively rich and represents a good source of additional information independent of skeletal material. In general, Upper Triassic strata are dominated by an abundance of theropod ichnites between 20 cm to 25 cm in length (grallatorid-sized) [8]. However, there does appear to be a preponderance for an increase in their size in the Early Jurassic based on the increased number of larger theropod ichnites, with the oldest known example of Eubrontes (35 cm) in North America (in a unit that is 10 ky younger than TJB-defined by palynomorphs [5]) and 34 cm in South Africa [13]. Despite the abundance of these Lower Jurassic theropod tracks and trackways, they are currently of relatively low generic diversity due to similarities in track morphology. This limits descriptions to within the one plexus of three ichnogenera (i.e. Grallator, Anchisauripus and Eubrontes) which are primarily distinguished by differences in size [8,14].
In southern Africa, rare theropod body fossils and isolated teeth are present in Lower Jurassic rocks [15][16][17][18]; whereas theropod tracks are common from the Late Triassic (e.g. [19]) and into the Early Jurassic (e.g. [20,21]). These tridactyl tracks and trackways, despite their conservative foot morphology, allow for a more holistic treatment of theropod evolution, over the TJB. Here, we report, for the first time, a tridactyl dinosaur trackway site from the Elliot Formation in the Roma Valley (Maseru District, Lesotho; Fig 1) that preserves the largest Early Jurassic theropod trackway to date.
This new ichnofossil data from western Lesotho considerably expands the range of bodysize displayed by carnivorous dinosaurs in the Early Jurassic in Gondwana, providing insight into the rate and tempo of body-size increase experienced by Theropoda across the ETE and the TJB (i.e., a punctuated, "release-type" versus a more step-wise body-size increase mirroring that of large-bodied dinosaurian herbivores, represented primarily by Sauropodomorpha). In addition to the morphological description of the tracks, we also document the associated taphono-sedimentary context of the trackway site and, using photogrammetry, provide a three-dimensional view to illustrate the morphology of the tracks. Large tracks, herein, refer to any ichnite greater than 40 cm in length and megatheropod tracks refer to the tridactyl theropod tracks >50 cm.
Geological background and stratigraphy of the megatheropod trackway site
The Matobo megatheropod trackway site is located within the uppermost Elliot Formation, 1.8 km west of the National University of Lesotho main entrance in the Roma Valley (Maseru District, Lesotho; Fig 1). It lies on an informal road between the villages of Ha Mokhosi and Ha Matobo. Although the megatheropod trackways were discovered by the authors, the site is immediately adjacent to the Matobo trackway site that was briefly documented by Ambrose [22].
The Roma Valley itself is carved into the Lower Jurassic successions of the upper Stromberg and lower Drakensberg Groups of the Karoo Supergroup ( Fig 1B). The valley floor and sides expose the sedimentary rocks of the upper Elliot and Clarens formations, whereas the hilltops are often capped by Karoo continental flood basalts that were dated at 183±1.0 Ma [23]. Outcrops of the older, Triassic-age rocks (e.g., Molteno and lower Elliot formations) are scarce and limited to the westernmost part of the valley, while mafic dolerite intrusions (also part of the Drakensberg Group) are relatively common.
The Roma Valley has yielded only a modest assemblage of vertebrate fossils [22]; however, the area has a rich ichnological record. For example, Ambrose [22] described, albeit very briefly, 14 fossil vertebrate track sites located in or close to the Roma Valley and incorporated some drawings by the members of the 1998 British Schools Exploring Society Expedition. Despite intensive searches, our team could only relocate some of the sites listed in Ambrose [22]. This is most probably because the sites were destroyed by local building-stone quarrying activities, which are currently under way informally.
Material and methods
Field work was conducted under a field permit (permit number: NR/M/E/10) issued by the Lesotho Government Department of Mines and Geology. Field evidence was collected in the form of macroscopic observations of the ichnofossil bearing sedimentary rocks and their vertical and lateral distribution at the study locality, Matobo site (29˚27 0 08.57@S, 27˚42 0 08.51@E; Roma, Maseru District). The outcrop was photographed and described with enough detail to produce an in-depth characterization of the sedimentary facies, which entailed the documenting of lithological, geometric, and sedimentary structures etc. The dinosaur tracks were measured in situ and recorded in detail via photographs, photomosaics, and sketches using ImageJ software. Matobo trackways and tracks have been labelled alphabetically 'A'-'D'. Where applicable, track length (TL), track width (TW), length of digits (te: II, III, IV), interdigital angles (II^III, III^IV, II^IV), metatarsophalangeal length (FL-te), pace length (PL), stride length (SL), and pace angulation (PANG) were measured for each trackway (Table 1). Track length (TL) is considered, here, as the length from the tip (but not including claw mark where preserved) of digit III to the base of the heel margin. Track measurements were taken as described in [13].
The Surface areas of all ichnites have been measured using ImageJ software on scale-calibrated photographs and figures. Using the freehand drawing tool, the perimeter of the track was outlined as accurately as possible. ImageJ then generated an area measurement for the outlined, irregular surface. This was done for the metatarsophalangeal area as well as for each individual digit (exclusive of claw mark impressions, which are differentially preserved). Individual digits' surface areas were measured by using a straight line linking the hypex between digits II^III and III^IV as a base line. If the medial and lateral hypices were not on the same level, a straight line was drawn across from the higher hypex. The lateral hypex shows the most variability in theropod tracks [41] and therefore this method aims to avoid any pitfalls associated with this variability. The area below this line was treated as the metatarsophalangeal 'heel' pad region. This method, while not taking into account many morphological details, is a rough guide to estimate the proportions of 'heel' to digit area.
A digital model, using 2-D cartography and photogrammetry, was made for the large trackway surface from the site. Photogrammetric models were undertaken using a Canon PowerShot EOS D1200 (Focal length 28 mm, 5184 x 3456 resolution) following the methods provided in Mallison and Wings [42]. AgisoftPhotoscan (standard version 1.1.4) software was used to process point clouds. Three-dimensional models were converted to colour maps in the open source CloudCompare software (v.2.6.1, http://www.danielgm.net/cc/). Orthophotos were used for individual track D footprint images. Rubber silicon replicas were made of trackway D and are housed in the Ichnology Collection of the Evolutionary Studies Institute (ESI) at the University of Witwatersrand, South Africa (accession number: BP/6/735). All 3D surface models and their raw data are deposited at Figshare.
Sedimentology of the megatheropod trackway site
The Elliot Formation in the vicinity of the megatheropod trackway site within the Roma Valley is a~185 m thick succession of clastic sedimentary rocks, of which~25 m belongs to the lower Elliot and~160 m to the upper Elliot formations (Fig 1C). The former is exclusively exposed in patchy outcrops that are to the west of the trackway site and below the level of diagnostic carbonate nodule conglomerates (facies Gcm) of the uEF (Figs 1B and 2).
The uEF at Matobo can be subdivided into two major facies associations based on their shared characteristics of sedimentary features, geometries, lithology and grain size. The finegrained facies association (Figs 1C and 2) is dominated by deep red, maroon to deep pink laminated and massive mudstones (facies Fl, Fm) that contain desiccation cracks, rootlets, and large, in situ carbonate nodules (Fig 2A, 2G and 2F). The laminated mudstones (facies Fl; Fig 2G) show rhythmical bedding and comprise silty mudstones and clay drapes with rare conchostracans ( Fig 2F).
The mudstones are interbedded with fine-grained sandstone beds, which are either single storey (< 50 cm thick; Fig 2A, 2G and 2F) or multi-storey and form upward-fining successions that are up to 10 m thick (Figs 1C and 2A). The latter, forming the coarse-grained facies association at Matobo, is typically based by a~25 cm thick, massive, poorly sorted, bone-bearing, reworked carbonate nodule conglomerate (facies Gcm- Fig 2C and 2D) that is laterally traceable in excess of 100 m. In this regionally recurring and unique conglomerate in the uEF [36,37,48], the nodules range from rounded to sub-angular, are poorly to moderately sorted, and are grey, white-to-red in colour. The clasts commonly form a clast-supported fabric ( Fig 2D). The rest of the multi-storey sandstone package is dominated by very fine-and fine-grained sandstones (with subordinate medium-grained sandstones) that are either massive, with or without clasts (facies Sc, Sm; Fig 2B and 2C), or ripple cross-laminated (facies Sr) towards the top of the succession, where ripple marked surfaces are vertebrate track bearing ( Fig 2E). The clast-rich sandstones (facies Sc; Fig 2B), another regionally recurring and unique rock type in the uEF [36,37], is light pink or deep red, maroon and contains poorly sorted, 1-4 cm angular, The overall geometry of the coarse-grained facies association is channel-like, whereas the interbedded mudstones and sheet-like, fine-grained sandstones are tabular and laterally persistent (Fig 2A). Based on local and regional sedimentological and palaeontological evidence (also see [36,48]), the former is interpreted as ephemeral fluvial channel fills, whereas the latter as palaeo-pedogenically altered sediments of floodplains with sheet-flood deposits and shallow playa lakes. The former were pedogenically altered (rootlets), whereas the latter were temporarily inhabited by conchostracans and received seasonal sediment supply (rhythmical bedding) characteristic of fluvio-lacustrine settings under seasonally wet, semi-arid climatic conditions.
Description of tracks and trackways
Approximately twenty tridactyl tracks, in varying degrees of preservation (preservation grade between 1 and 2; [49]), are present on the Matobo palaeosurface. The tracks appear to be both true tracks (with natural casts) and undertracks at the top of a sandstone bed. Of these tracks, eleven form four trackways (hereafter referred to as Matobo A, B, C, and D) and the remaining are discrete, scattered footprint impressions. These remaining nine tridactyl tracks are discussed in Ambrose (2003) (Fig 3). In contrast, at the northern extremity of the palaeosurface, Matobo A and,~4 m to the west, Matobo D are shallowly impressed. The palaeosurface substrate consistency was highly variable over short distances and the size of the track and, by extension the animal, did not have bearing on the competency of the substrate. Unfortunately, the integrity of the Matobo palaeosurface is affected by traffic in the form of carts, livestock, and occasional minibuses, which use most parts of the surface as an informal road. This damage is manifested in the form of NE-SW running grooves that can be observed in photogrammetry models (see supplementary material and Fig 4).
Tracks of Matobo A, B and C are moderately large in size (TL: 30-40 cm), tridactyl, digitigrade and elongate (TL/TW = 1.2-1.5) with weak to moderate mesaxony (av. 0.5; based on the anterior triangle ratio l/w of Lockley [49]; Table 1; Fig 3). The digits are transversely thick relative to their anteroposterior length, tapering to V-shaped tips, and are fairly straight but show slight divergent curvature along the tips of digits II and IV. They lack digital pad impressions and rarely preserve pointed claw marks (e.g. tracks A2, B1; Fig 3). High divarication of digits II^IV (56˚-58˚) is notable and the interdigital divarication angle II^III (27˚-30˚) is marginally larger than III^IV (28˚-29˚; Table 1). V-shaped hypices between digits are noted ( Fig 3B, 3C and 3D). The heel margin, which is the metatarsophalangeal pad impression of the foot, is rounded and U-shaped in Matobo B and to a lesser extent in Matobo A (more V-shaped). Matobo C's posterior margin is more V-shaped in appearance.
Matobo A is a 4 m long trackway comprised of 5 consecutive tracks ( Fig 3B). The preservation of the tracks along the extent of the trackway is relatively consistent. The tracks have an average length (TL) and width (TW) of 32 and 23 cm, respectively, giving an average TL/TW ratio of 1.4. Tracks appear more gracile and digitigrade than Matobo B and C on the same surface, but this appears to be largely a function of substrate firmness. The trackway is narrow with the pace angulation (PANG) ranging between 139˚and 155˚. The pace length varies Table 1 between 0.4 m and 1.0 m with a general decrease in pace length from track A1 to A5 (west to east; Fig 3B). Accordingly, the stride length decreases between track A1 and track A5 from 1.8 m to 1.2 m ( Table 2). The speed of the animal, based on the calculated morphometric hip height, shows a corresponding deceleration from 1.2 ms -1 at the start of the trackway to 0.7 ms -1 at the end of the trackway ( Table 2).
Matobo B consists of two tracks still containing some of their natural sandstone casts (Fig 3; Table 1). Tracks B1 and B2 ( Fig 3C) have an average length and width of 40 cm and 32 cm, respectively, and a TL/TW ratio of 1.2 (Table 1). These represent the second largest tridactyl theropod tracks, to date, in the Elliot Formation at~40 cm long ( Fig 3C; Table 1). The digits appear relatively straight with pointed tips; however, the natural casts obscure the latter and prevent more accurate morphological observations. This may have also contributed to the exaggerated length and width of these tracks relative to Matobo A and C. Morphological detail (and hence preservation) of the right pes impression B2 (Fig 3C') is better, as noted by the defined and relatively undistorted nature of the digits, than that of the right pes (B1; Fig 3C"). This appears as a function of the competency of the substrate.
The two tracks of Matobo C have an average TL and TW of 31 and 21 cm, respectively, giving a TL/TW ratio of 1.5. Morphologically, the tracks fit into the abovementioned general description of the Matobo site tracks and have no additional/unique features besides the more V-shaped appearance of the posterior margin. As previously noted, the right pes impression (Fig 3D') shows is better preserved with no distortion of the digits.
Very large, tridactyl, digitigrade, right (D1) and left (D2) pes tracks are situated in the easternmost sector of the Matobo palaeosurface (Matobo D; Figs 3 and 4). The average track length and width is 57 cm and 50 cm, respectively, and the TL/TW ratio is 1.1 ( Table 1). The tracks show weak to moderate mesaxony with a calculated mesaxonic index of 0.5. Digit impressions are shallowly depressed (Fig 4B and 4C). Digits are straight with rounded ends and lacking claw marks, although the photogrammetry images (Fig 4A", 4B" and 4C") show more pointed digits tips than can be observed in the field (or from the line drawing). The digits do not taper but maintain a fairly uniform width along their length. There is no hallux impression. The length of digit II is 41.5 cm in D1 and 44.5 cm in D2, thus digit II is shorter than digits IV (49 cm long), and both are shorter than digit III (57 cm long). The free length of digit IV is greater than digit II because the hypex of digit IV is lower than the medial hypex. The ratio of the posterior margin of the heel to the base of the hypex of digit II and digit IV averages 2.3 and 3.2 for D1 and D2, respectively. Interdigit angles have low variability between tracks with an average of 33˚and 29˚for II^III and III^IV, respectively (Table 1). Total divarication (II^IV) is 66˚in D1 and 60.5˚in D2. There are no digital (phalangeal) pads impressions preserved and the metatarsophalangeal 'heel' pad forms a distinct, small, semi-circular impression (Fig 4B and 4C). The 'heel' margin is V-shaped. In both tracks, this depression is 17 mm deep proximally and shallows to 14 mm towards the distal digit margin and tip of digit III (Fig 4B" and 4C"). The depth of the 'heel' and digits is slightly irregular (Fig 4B" and 4C"), and the depth of digits III and IV appear deeper than digit II.
Intra-track variation at Matobo. Bivariate morphological analyses, as presented in Fig 5A, show moderate morphological intra-track variability in Matobo A, B and C (Table 1; Figs 3 and 5). This is a phenomenon commonly related to the rheology of the substrate, which is controlled by moisture content and sediment properties (e.g., grain size, sorting). Intra-track variability is low for pes pairs from Matobo B, C, and D despite the perceived changes in the substrate consistency (Figs 3C, 3D and 4A). Conversely, the variability observed in Matobo A (Fig 5A) is more likely related to the slowing gait of the animal (from 1.2 to 0.7 m s -1 over a distance less than 4 m; Fig 3; Table 2) than to the substrate, which shows no marked changes in consistency along the length of the trackway. Fig 5B illustrates the morphological ratios of the Matobo tracks and the bounding ranges of well-known Lower Jurassic tracks [50]. The imprecise placement of Matobo A-D within the comparable North American ichnotaxa fields (Fig 5B) is likely related to differences in the Gondwana forms versus these Laurasian ichnotaxa.
The averaged values for Matobo C plot outside of the ranges of Eubrontes, Kayentapus hopii, and Kayentapus damarensis [51], and closer to the field of Kayentapus minor (Fig 5B). [50] using the (TL-te)/TW versus te/TW ratios. Demarcated fields are for Eubrontes isp., Kayentapus minor, Kayentapus hopii and the Southern hemisphere Kayentapus damarensis [51]. The lines labelled '3:1', '2:1' and '1:1' denote the ratio of digit III extension of either one-third, one half or equal to metatarsophalangeal length [50]. Abbreviations: TL-foot length, TW-foot width, te-toe extension: digit III projection length past digit II and IV. https://doi.org/10.1371/journal.pone.0185941.g005 Conversely, averaged values for the Matobo A and B plot within the range given for Eubrontes isp. but as outliers on the ratio line '2:1' (digit III extends half the metatarsophalangeal length) (Fig 5B). The U-shaped posterior metatarsophalangeal margin (hereafter referred to as the 'heel') typical of Eubrontes is seen in Matobo B and this lends support for their placement within the Eubrontes-range, but less so with Matobo A. Additional shared features of Matobo A and B with this ichnotaxon are their moderately large size (av. 34 cm), average TL/TW of 1.2-1.4, weak to moderate mesaxony (av. 0.5; Table 1), broad digits (digit II and IV being approximately subequal in length), and claw marks preferentially preserved. Conversely, Matobo C, while sharing several similar gross morphological features (e.g., broad, thick digits,) with Matobo A and B (Fig 5A), is morphologically closer to Kayentapus-like tracks (Fig 5B). It displays a wide divarication and a more V-shaped heel typical of Kayentapus-like tracks [52,53]. Matobo A also displays some Kayentapus-like traits with respect to its narrow pace angulation (139˚-155˚) and large stride length (1.8 m-1.2 m; Table 2), which are similar to the type material of K. hopii (pace angulation: 174˚, stride: 1.8-1.9 m; [53]). In general, the wider II^IV divarication angles (av. 57˚) of Matobo A, B, and C are greater than the range and uppermost limit of Eubrontes (40˚; [54]). Wider divarication is common to Kayentapus-like tracks [53] and a wide II^IV divarication angle (62˚) has been reported for other Lesotho tridactyl tracks, for instance Neotrisauropus deambulator Ellenberger [54] (UEF, Moyeni, Quthing District), which is an ichnite that has been designated to Kayentapus by Piubelli et al. [52].
In contrast to Matobo A-C, Matobo D is significantly longer (by 17-25 cm), with narrower digits, a slightly wider total (II^IV) divarication angle (63˚), lower TL/TW (1.1 versus 1.4-1.5 range for Eubrontes; Table 2) and a V-shaped posterior margin. Despite the low TL/TW ratio (1.1), which falls below the reported threshold values of >1.25 for theropods [55], this trackway is considered of theropod origin due to a suite of other morphological characteristics.
Comparative ichnology
The V-shaped posterior margin of Kayentapus is considered to be one of its more distinguishing attributes [53], and is noted to occur in the Early Jurassic and then again in the Early Cretaceous as represented by the theropod ichnite Irenesauripus Sternberg [56]. In addition to the Northern Hemisphere occurrences, Kayentapus has been described from Madagascar [57] and Namibia [51,58], but has not been formally recognised in Lesotho. Piubelli Table 1; N refers to the number of tracks measured. Distance measurements in cm; angles in degrees. This data was obtained from measurements of scaled photos taken from publications cited. https://doi.org/10.1371/journal.pone.0185941.t003 Matsieng, Qalo, and Matelile localities [20] and have not been viewed by the current authors (due to inaccurate locality information), but are considered Early Jurassic in age (zone B/5 in Ellenberger, [24]). In fact, Ellenberger [24] suggested that Kleitotrisauropus (Kainotrisauropus) is comparable with Eubrontes of Lodève in France. In contrast, the ichnospecies Kainotrisauropus morijiensis was noted by Ellenberger [20] as reminiscent of K. minor of the Hettangian in France. A formal revision of these ichnotaxa is needed to formalise their potential assignment to Eubrontes, Grallator, or Kayentapus Similarities between Eubrontes and Kayentapus isp. have been noted in the southern African/ Gondwana tracks described by Wagensommer et al. [51]. The Jurassic Namibian tracks reported in their study are medium to large (range of FL = 25-35 cm, FW =~13-25 cm), have robust digits, moderate total divarication (~40˚; in line with expected range of Eubrontes), and interdigital angles between II^III = 10-15˚and III^IV = 25-30˚. Wagensommer et al. [51] designated tracks ONP VII_1 to Eubrontes giganteus and ONP I_1 to Kayentapus damarensis. The latter tracks (ONP I_1) were assigned to Kayentapus because, in comparison to Eubrontes, the greater toe extension and shorter TL/TW ratio were considered diagnostic of that ichnogenus with respect to Weems's [50] foot measurement ratios. However, as noted by Wagensommer et al. [51], this does not take into account other features such as the divarication angle, robustness of the digits, or stride. Weems [50] argued that these characteristics are difficult to consistently measure because of variation relating to either pace or substrate. The underlying issue, however, relates to the homogeneity of theropod dinosaur foot morphology globally during this time. Thus, the lack of marked differentiation between ichnotaxa is potentially a reflection of gross similarities in foot morphology and the generalised manner of bipedal locomotion typical of Early Jurassic theropods [59].
In the current study, the Matobo tracks also present evidence for and against their assignment within both Eubrontes and Kayentapus. Again, this seems to trend with other southern African tridactyl tracks which do not completely conform to Northern Hemisphere standards. Despite the suggested affinities to Eubrontes for Matobo A and B within Weems' [50] scheme, their higher II^IV divarication angle, moderate mesaxony, and V-shaped posterior margin also suggests a possible referral to Kayentapus. The latter possibility is further supported by comparable morphological rations (te/TW and {TL-te)/TW}). However, the evidence against placement within Kayentapus rests on the divarication between digits III and IV equal to or less than between digits II and III, and the significantly larger FL and FW.
Track B is considered as being more Eubrontes-like due to its significantly U-shaped heel margin in combination with its large track size and robust digits (Figs 3C and 5). In contrast, the dimensions of Matobo trackway A are broadly consistent with those of K. minor, while the V-shaped heel and wide divarication of both track A and C (features not taken into account in the plot of Fig 5B) also suggest a possible referral to Kayentapus (Fig 5A). As previously discussed, there are several globally recognised Late Triassic-Early Jurassic theropod ichnotaxa: Eubrontes, Gigandipus, Anchisauripus (synonymised by most with Grallator), Grallator, and Kayentapus. While the size of the track is by no means a distinguishing factor, none of these ichnotaxa are wholly comparable to Matobo D, although the closest morphological affinities are seen in the ichnogenus Kayentapus. Thus, for the ostensible purpose of classification, Matobo D is considered here as Kayentapus-like, although its relationships to other large, globally occurring ichnites is given further exploration in the comparative ichnology discussion below. In summation, despite the wide II^IV divarication angle for Matobo A-D and the relative symmetrical distribution of the interdigital angles II^III and III^IV, the morphological evidence presented above (digit width, TL, TW etc.; Table 1 and Fig 5) places at least Matobo A and B into the ichnogenus Eubrontes ( Fig 5B) and Matobo C and D as belonging to a Kayentapus-like animal.
Comparative ichnology with large tracks
Matobo A-D are broadly assessed against other large, valid ichnotaxa, irrespective of age differences of up to 100 million years or geographical occurrence (Tables 3 and 4). Matobo D represents a unique megatheropod trackway in the Early Jurassic. There are no Early Jurassic theropod tracksites of comparable size excepting several large footprints reported from the uppermost part of the Sołtyków outcrop of Poland (presumed Hettangian in age; Tables 3 and 4; Fig 6 [60, 61]). One of the first described gigantic Polish tracks (Muz. PIG 1661.II) was studied by Gierliński et al. [60], and is reported as 54 cm long, with robust digits, claw impressions and, uniquely, a large metatarsophalangeal area constituting 33% of foot length. Gierliński et al. [62] suggested this track was most similar to Upper Jurassic theropod footprints (i.e. Megalosauripus; [63,64]) based on the large metatarsophalangeal area. Three other large tridactyl forms are reported from the Sołtyków site (39 cm TL Kayentapus minor; [61]; 35 cm TL Eubrontes isp; [61]; 50-65 cm TL large theropod footprints e.g. MPT.P/146 [61]) and are presented here (Table 3).
There are several commonly occurring tridactyl taxa in the Early Jurassic, with Eubrontes giganteus representing one of the larger forms (! 25cm; [54]). Other large (> 40 cm) Eubrontes-like tracks are reported from the Middle Jurassic (cf. Eubrontes, from the Rosewood-Wallon coalfield, Australia; Fig 6 [65]) and Lower Cretaceous (Eubrontes (?) glenrosensis, Glen Rose Formation, Trinity Group, USA; Fig 6 [66]). Eubrontes and large Eubrontes-like Hip height and body length measurements are in metres; track length (TL) is provided in cm; N refers to the number of tracks measured; if more than one track was measured from the same trackway, TL is an average value. Allometric body length is calculated from allometric hip height, and morphometric body length is calculated from morphometric hip height. https://doi.org/10.1371/journal.pone.0185941.t004 Lower Jurassic megatheropod tracks from the Elliot Formation tracks (e.g. [54,61]) typically have a TL/TW ratio of 1.4-1.5, digit impressions that are thick and sturdy, a short digit III extension and digits II and IV which project equally far along the axis of digit III [8,54]. The total divarication angles between 25˚-40˚ [54]. Table 3, Fig 6) is a gracile tridactyl track with a comparable age distribution to the Matobo tracks. This ichnogenus has been variously considered an independent ichnogenus (wider digit divarication and overall pace; [67]), 'lumped' within the Grallator-Eubrontes spectrum [54,68], or considered synonymous within Eubrontes [8,69]. Milner et al. [68] considered Kayentapus valid with respect to the divarication of digit IV and the length of projection of digit III. Interestingly, Kayentapus shows considerable variation both between tracks and within a single trackway, making an explicit morphological diagnosis for this ichnogenus difficult [64,68]. Lockley et al. [70] have further recognised the 'Kayentapus-Magnoavipes' morphotype, which identifies large theropod tracks (TL 35 cm) with very high divarication angles of 90˚to 100˚. Matobo D does not show this morphological characteristic, Table 3 for details. https://doi.org/10.1371/journal.pone.0185941.g006 Lower Jurassic megatheropod tracks from the Elliot Formation nor is there marked variability between tracks-although the latter may be accounted for by small sample size (N = 2). However, Matobo D, Kayentapus hopii, and Kayentapus minor ( Table 2) share the V-shaped posterior margin, relatively slender/gracile digits, and comparable TL/TW ratio, although the latter (K. minor) is smaller in size with respect to both the TL and TW. The interdigital angles are very similar between these tracks (Table 3), with divarication angles of Matobo D (II^III = 33˚, III^IV = 29˚, II^IV = 63˚) being similar to the Polish specimens of Kayentapus [61] and the North American Kayentapus hopii [67]. Dissimilarities are present with K. hopii when looking at the morphological (TL-te)/ TW and te/ TW ratios (Fig 5; Table 3). However, Matobo D's (FL-te)/TW ratio of 0.4 is more comparable with K. minor (0.5) than K. hopii (0.2) [70,71].
The ichnogenus Megalosauripus is a common Late Jurassic-Cretaceous form hypothesised to represent the megalosaurid (Tetanurae) dinosaurs dominant in this time period [63,72]. This ichnogenus is not considered to occur prior to the Late Jurassic [60]. In comparing Matobo D to the Lower Jurassic (~Hettangian) Polish representative, tentatively cf. Megalosauripus isp. (and 'giant polish theropod'; Tables 3 and 4; [60,62,73]), similarities are mainly noted in the TL (cf. Megalosauripus isp. of 49 cm and 54 cm with claw impression), track measurement ratio of (FL-te)/TW (metatarsophalangeal region/track width), phalangeal length/ total TL ratios and total divarication angle (Table 3). However, despite some morphological parallels, the considerably less robust and distinct lack of a hallux impression set Matobo D apart from this Polish ichnotaxon. That said, a hallux impression is more likely a reflection of the rheology of the substrate than a function of the particular walking gait or style, especially given the generalised foot anatomy of basal theropods.
In summation, although Matobo D can be shown to be morphologically distinct from Eubrontes and Eubrontes-like tracks, distinguishing the former from either Kayentapus or Irenesauripus is much more problematic. However, despite the noted morphological similarities between Matobo D and the Cretaceous Irenesauripus, their great age disparity suggests that these similarities are primarily due to convergence. Matobo D is therefore interpreted as representing either an especially large variant within the Kayentapus morpho-spectrum, or a novel ichnotaxon. We consider it here, based on the morphological description provided, to be a much larger variant of the North American ichnogenus Kayentapus and as such we assign it to the ichnogenus Kayentapus. However, given it unique size and morphological traits (i.e. greater free length of digit IV vs. II) we hereby establish a new ichnospecies.
Kayentapus ambrokholohali ichnosp. nov., Fig 4. Diagnosis: two very large, 57 cm long and 50 cm wide, gracile tridactyl pes impressions with wide total divarication (II^IV) is 63˚. The metatarsophalangeal pad of digit IV is well-defined and the margin is V-shaped. The free length of digit IV is greater than that of digit II. Depth of track depression is 17 mm proximal to the metatarsophalangeal pad and shallows to 14 mm towards the distal digits.
Etymology: ambro-Ambrose (derived from the Latin name Ambrosius) meaning "immortal"; This is in honour of Emeritus Professor David Ambrose for his detailed recording of the localities of several of the Roma trackways. It was during an attempt at the relocation of these sites that the newly exposed trackway was discovered. And, kholohali, from the Sesotho 'kholo', meaning 'big/large/great'; and 'hali', meaning "much/very" after their unexpectedly large size.
Role of the metatarsophalangeal pad ratios in diagnoses
Because larger animals are more likely to have better developed metatarsophalangeal pads [76], we examine here the importance of the utility of measurements drawn from the metatarsophalangeal pad length (MPL) and metatarsophalangeal surface area (MSA) ( Table 3; Fig 7). However, it is important to note that the heel and digit surface areas may be variable between tracks and sites, and that several ways of measuring dimensions can be made from line Table 3. https://doi.org/10.1371/journal.pone.0185941.g007 Lower Jurassic megatheropod tracks from the Elliot Formation drawings. This is because these ratios, much like the track impression as a whole, are dependent on several external factors, i.e. animal weight and speed, sediment properties, sediment collapse around digits and secondary factors relating to their degree of preservation. Lallensack et al. [41] point out that the size of the heel surface area may vary due to the additional impression of the metatarsus or partial impression of the foot.
Generally, the greatest variability between ichnotaxa is shown in the ratio MSA to total digital surface areas (Fig 7), whereas the ratio of MPL:TL shows more consistency despite the differences reflected in surface areas. With respect to the ratio of the MPL:TL (Fig 7), and not taking age in consideration, Kayentapus hopii has the smallest metatarsophalangeal pad/ total foot length, whereas Eubrontes (?) glenrosensis and the Polish theropod track of Niedźwiedzki [61] have the largest. The MP to total digital surface area ratio for Matobo D falls within a ± 5% range with the following ichnotaxa: Irenesauripus, cf. Megalosauripus isp., I. mclearni, and I. acutus, confirming their moderately small metatarsophalangeal pad area/ total digit surface area. Similarities in these morphometrics are more marked between Matobo D and Irenesauripus isp., cf. Megalosauripus isp. and Kayentapus isp.
Large metatarsophalangeal pad area to total digit surface area suggests a bulky, robust 'heel' pad, and when contrasted against a low length ratio can imply a 'heel' pad dimension which is short but wide (i.e. as in Eubrontes isp., Eubrontes (?) glenrosensis and the large Polish theropod track; Fig 7). Interestingly, the taxa most dissimilar to Matobo D are those which exhibit broad, thick digits of equal surface area to the metatarsophalangeal pad, or ones for which the MPL makes up a significant proportion of TL (Figs 7 and 8); e.g., Eubrontes cf., Eubrontes (?) glenrosensis and the large theropod taxa described by Niedźwiedzki [61] from Poland.
Trackmaker identity
The Matobo tracksite represents some of the larger Early Jurassic theropod tracks in southern Africa. Certainly, Matobo D represents the largest Early Jurassic track, globally. Other globally occurring contemporaneous megatheropod tracks (cf. Megalosauripus isp. and large Polish theropod; Table 4) are solely represented by the Sołtyków site tracks, Holy Cross Mountains (Poland), which are 54 cm in size [60,78]. These tracks were found in the Zagaje Formation which is considered to be Hettangian in age based on sequence stratigraphic analysis [79] and its early 'Liassic' (Early Hettangian) flora [80], which has been critiqued [81]. Other similar sized ichnites are represented by the much younger Lower Cretaceous material (e.g. [72,82]). In addition to the Matobo tracks, the hip heights and body lengths of the theropod track makers have been estimated from several other globally occurring large theropod ichnites, and these had estimated hip heights (h) of 1.5-2 m and body lengths that ranged from~6 to 9 m (Table 4; Fig 8A and 8B). When compared to hip heights and body lengths reconstructed from fossil material [45], the calculated heights and lengths obtained from track lengths show a linear trend (Fig 8B).
Reconstructed body dimensions for Matobo A and C give estimated hip heights similar to those estimated for the ichnogenera Eubrontes isp. (see [54]), Kayentapus hopii (from [67]) and Gigandipus ( [68]; Tables 3 and 4; Fig 8A). Estimated hip heights for Matobo B are Tables 3 and 4). Comparable vertebrate fossil hip and body length for the track maker of Matobo B is the allosaurid Allosaurus (as is the case for K. minor, Eubrontes, and Gigandipus; Table 4). Matobo A and C are more likely to be associated with an animal of dimensions resembling Dilophosaurus (see discussion below), Ceratosaurus (Upper Jurassic; Morrison Formation, USA; Lourinhã and Alcobaça formations, Portugal) [83]; or Elaphrosaurus (Tendaguru Formation, Tanzania, [84]; Fig 8B).
Matobo D, being the largest of the Matobo tracks, yields a very large track maker with a hip height of up to 2.7 m and a body length of~9 m (Fig 8A; Table 4). Other comparable theropod ichnites for which hip height and body length range could be extrapolated are the Polish large theropod, Irenesauripus, I. mclearni, I. acutus, Eubrontes cf. (left), Eubrontes (?) glenrosensis ( Fig 8A; Table 4).
Remarkably, estimated body length and hip heights for Matobo D are comparable to (age dissociated) tyrannosaurid and allosaurid vertebrate fossil material, and represents the upper limit of the Upper Jurassic Allosaurus (occurring in North America, Portugal, and Tanzania; [85]). The unprecedentedly large dimensions of Matobo D are all the more noteworthy when contrasted against known theropod body fossils from the latest Triassic and earliest Jurassic. Well-preserved theropod material from this time period is rare, and there is a distinct lack of comparable vertebrate body fossil material from the Lower Jurassic. Gierliński et al. [62] have proposed that some large Lower Jurassic tracks may also be considered to be made by (basal) allosaurids, but there are little to no body fossil evidence to support this claim despite the occurrence of these large tracks. Globally, the large theropod dinosaur trackmakers appear more consistently in the Middle to Upper Jurassic, and are mainly thought to be allosaurids and megalosaurids with robust metatarsus and phalanges and inferred well-developed metatarsophalangeal pads [64,76].
In general, the limited vertebrate fossil evidence for larger prints (!30 cm TL) has meant that many have been, in part, attributed to the relatively large (6 m body length,~400 kg; [86]) Early Jurassic theropod Dilophosaurus. On the basis of their foot anatomy, the tracks of these animals are considered likely to be robust, with a large metatarsophalangeal pad area/total digit surface area. It is not considered to produce gracile tracks, but rather larger, robust tracks such as Matobo B (Figs 7 and 8).
Allosauroid-like material from the Elliot Formation of South Africa has been tentatively reported by Ray and Chinsamy [16] based on SAM-PK-K10013, which is a large, recurved and finely serrated tooth. Ray and Chinsamy [16] suggest that the tooth possibly belongs to a basal theropod but its stratigraphic position within the Elliot Formation is unknown. Other large, serrated teeth have been found within the Elliot Formation (e.g. SAM 383; [87]), although their stratigraphic and biological affinities are unstudied/unknown, and it remains plausible that some of this material is crurotarsan (i.e., "rauisuchid") in nature. In general, skeletal remains of carnivorous dinosaur fossils are rare in the Upper Triassic-Lower Jurassic of southern Africa, whereas ichnites of these animals are much more common. To date, the oldest tridactyl theropod ichnite reported from South Africa is from the Upper Triassic uppermost Molteno Formation (Maclear trackway site, TL between 16-19.5 cm; [19]), whereas the youngest theropod tracks are in the lower Clarens Formation of Lesotho and South Africa (e.g., [20,24,88]). None of these southern African tridactyl theropod ichnites are associated with theropod skeletal remains.
The first theropod body fossils in South Africa are found in the Lower Jurassic upper Elliot Formation (UEF) and are represented by only two theropod genera, Dracovenator regenti [18] and Coelophysis (Syntarsus) rhodesiensis [89]. Coelophysis is suspected as the maker of the abundant Grallator isp. [30] traces in the UEF, although this ichnite, due to the range of sizes, probably represents a variety of small and medium-sized theropods [20,30,89]. In contrast, D. regenti is known only from fragmentary skull material with an estimated total skull length of 500 mm [18]. Yates [18] considers the general dimensions of Dracovenator similar to the theropod Dilophosaurus wetherilli (5-6 m in length) and, indeed, Dracovenator has been recovered from within the putatively monophyletic "dilophosaurid" clade [86]. The track lengths estimated for Dracovenator, in the size range of~25-34 cm, would be similar to the TL reported for Matobo A, B (Eubrontes-like) and C (Kayentapus-like) here. The foot morphology of Dilophosaurus has also been suggested to share several morphological analogies with the ichnite Kayentapus [90]. Taken together, the morphological characteristics of Matobo D (in particular the size, narrowness and length of the digits), suggest that the tracks were made by a relatively gracile, carnivorous dinosaur with an allosaurid-like bauplan. Nonetheless, this suggestion awaits substantiation via the discovery of additional fossil theropod material from the poorly sampled basal rocks of the Jurassic.
Conclusions
Our discovery of a new megatheropod trackway and several large tracks suggests that, in comparison to the Upper Triassic, the size range of the theropod trackways and, by extension that of their body size, rapidly expanded in the Early Jurassic. In southern Africa, where theropod body fossils are extremely rare, it also suggests an unappreciated degree of diversity of theropods active during this time. Currently, it is unclear whether the appearance of megatheropods is a consequence of a) "ecological release" following the extinction of non-crocodylomorph crurotarsans during the end-Triassic biocrisis event [5]; or b) a similar set of ecological stimuli that led to the progressive size increase of Sauropodomorpha beginning in the mid-Norian. In any case, our results suggest that very large theropods appeared in the fossil record of southern Africa prior to the Pliensbachian, mostly likely in a relatively short window of evolutionary time following the TJB, and not during the Middle Jurassic as currently suggested by the body fossil record. The appearance of mega-carnivores in the Early Jurassic is of great interest, and augurs an evolutionary phenomenon that was repeated on multiple occasions throughout the remainder of the Mesozoic, producing such iconic taxa as Allosaurus in the Upper Jurassic as well as Spinosaurus and Tyrannosaurus in the Upper Cretaceous. Furthermore, this study corroborates recent assessments of Elliot Formation biostratigraphy, with the semi-arid environment of the upper Elliot Formation able to support both small and very large sized theropods, as well as a diverse array of early sauropodomorphs (i.e. McPhee et al. [40]). Ecological incentives to increase body size may thus be related to rapid changes in both climate and predatorprey dynamics at the outset of the Jurassic, with the expansion of dinosaurian niche space accommodating a range of novel strategies not possible prior to the TJB [91]. This also has bearing on the manner in which sauropodomorph size thresholds represent a good predictor of theropod size increases and vice versa, with this relationship warranting in-depth future investigation. | 2018-04-03T00:00:38.064Z | 2017-10-25T00:00:00.000 | {
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57964358 | pes2o/s2orc | v3-fos-license | SYSTOLIC BLOOD PRESSURE IS A VALID MARKER OF IN-HOSPITAL SURVIVAL IN ACUTE CARDIOGENIC PULMONARY EDEMA - ANALYSIS OF 1.397 PATIENTS
Bearing in mind the prevalence and dramatic clinical picture of acute cardiogenic pulmonary edema (ACPE), as well as impermissibly great early and late mortality, the goal of this paper was to examine if blood pressure (BP) is a predictor of in-hospital outcomes in ACPE. The analysis includes 1.397 patients with ACPE, treated at the Department for Cardiovascular Diseases, Clinical center, Niš, from 1993-2005. According to systolic blood pressure (SBP) on admission, patients were divided into three groups: low SBP (L; <100mmHg), normal SBP (N; 100-139 mmHg) and high BP (H; ≥140mmHg). In-hospital mortality was significantly higher in the subgroup of L vs N (62.75% vs 15.79%, p<0.001), as well as in the subgroup N vs H (15.79% compared to 7.26%; p<0.0001). Serum creatinine concentration was significantly higher in the subgroup of L vs N; the level of Na+, the thickness of the interventricular septum in diastole and the prevalence of preserved LV contractile function were higher in the subgroup H compared to N; the concentration of K+ in serum was higher in subgroup L vs H and AF was more represented in the L subset as compared with the N subset. Therefore, the systolic BP is an important predictor of in-hospital survival, not only in AMI, but in acute cardiogenic pulmonary edema, too. In the study including the largest homogeneous group of patients with acute cardiogenic pulmonary edema without AMI (1.397 persons), we found that patients with hypotension (systolic BP<100 mmHg) died in hospital about 9 times more often (62.75% vs 7.26%) than patients with elevated systolic BP on admission. Acta Medica Medianae 2015;54(3):45-50.
Introduction
Heart failure (HF) is a very important medical, and therefore a social problem for many reasons: high and growing prevalence, high mortali-ty, compromised quality of life, numerous comorbidities, high treatment cost (1-4). General prevalence of HF in Europe is 1-2% and significantly higher among those older than 60 years: over 10% for men and 8% for women. Acute HF has high annual mortality: 20-30% (5, 6).
Acute cardiogenic pulmonary edema (ACPE) is the most difficult type of retrograde failure of the left heart and represents 16.2% of acute HF; the direct triggers are: hypertensive crisis, arrhythmia (including atrial fibrillation (AF), acute myocardial infarction (AMI) myocardial ischemia without necrosis of cardiomyocytes, valvulopathy, infection (particularly of the lungs), anemia, inadequate medication, etc. (5,(7)(8)(9)(10)(11)(12)(13)(14). Depending on concrete etiopathogenetic factors, therapy should be tailored to each individual patient (6, 9). As the heart impact is an important component of blood pressure (BP), especially of the systolic BP, insufficient left ventricle (LV), in general, is not able to raise BP. Therefore, BP is low in complicated forms of chronic and even acute HF.
Aims
The aim of this work was to examine if the BP is an indicator of in-hospital mortality in ACPE.
Patients and methods
The analysis covers 1397 patients having ACPE, with rates >50% at lung fields at auscultation, as well as with oxygen saturation <95%, who were hospitalized at the Department for Cardiovascular Diseases, Clinical Center Niš, from 1993-2005. There were 50.90% men with the average age of 69.98±9.45 years (23-94 years). AMI was the exclusion criterion, but shock was not. According to the systolic BP (SBP) at the time of hospitalization, patients were divided in three subgroups: with low SBP (L;<100 mmHg), normal SBP (N;100-139 mmHg), and high SBP (H;>140 mmHg) (1, 15).
Global LV contractile function is considered preserved if the LV ejection fraction (EF) was ≥45 %, according to numerous publications, but without definitive evidence (5, 16). Statistical analysis were performed using commercial software (SPSS, Chicago, Illinois, version 19). A p value <0.05 was considered statistically significant.
Results
More females were in the subgroup H compared with the subgroup N: 54.8% vs 44.6%; p< 0.01. In-hospital mortality was significantly higher in the subgroup L vs N (62.75% vs 15.79%, p< 0.001), as well as in the subgroup N vs H (15.79% Interventricular septum thickness in the diastole (measured by echocardiography) was higher in the subgroup H than N (12.4±2.4 vs 11.4±2.8 mm, p<0.05). AF was more prevalent in the subgroup L vs N (54.5% compared to 28.0%; p<0.001) (Figure 3).
There were no statistically significant differences among subgroups in other tested parameters, such as age, prevalence of diabetes mellitus, serum glucose levels and LV diastolic diameter.
Discussion
Our finding that more females were in the subgroup H compared to subgroup N was due to the fact that arterial hypertension is a very common cause of HF in older women and is often characterized by preserved LV global systolic function (3, 17).
The probability of surviving hospitalization was directly proportional to the value of SBP on admission, with large, nine-fold gradient between those with high and low BP (Figure 1). It is known that SBP is essential for the prognosis of acute HF, as well as AMI (1, 18). Pulmonary edema is a form of acute HF and the most severe type of retrograde LV failure. In this study we have confirmed that systolic BP was a valid prognostic parameter in ACEP. The confirmation is reliable, given the large number of examined patients. Therefore, in ACEP patients without AMI, SBP as an extremely simple and universally available marker was able to stratify patients according to the risk of death. The explanation is obvious: high BP in ACEP is both the cause and a consequence of the severity of the disease and consequent stress and involves mainly good inotropic and chronotropic response to excessive myocardial sympathetic and catecholamine stimulation. In a large registry, almost half of the hospitalized patients with HF had BP >140/ 90 mmHg (3).
In addition to prognostication, this recognition of the importance of higher SBP as a marker of in-hospital survival has therapeutic implications (19)(20)(21). It is necessary to take a lot of care not to reduce the BP excessively. Noninvasive ventilation with positive pressure may be useful (22,23). It helps to preserve lung function and provides condition for better BP control, by liberalization infusion of fluids in part. SBP was a significantly better marker of in-hospital survival compared with DBP in our patients with ACEP. It is unusual that we have shown just the opposite for long-term survival (24). A meaningful explanation is that there is prognostic information both in SBP (which is largely related to the LV global contractility and more important in the acute phase of pulmonary edema) and in DBP (which is in relation to peripheral vascular resistance, and thus the organ perfusion pressure, including vital ones, which is important for the longterm prognosis of ACEP patients).
The finding that the serum creatinine and K+ levels were significantly higher in the L vs N subgroup is probably due to the acute prerenal kidney damage. All the more so because the vast majority of patients with chronic kidney disease (CKD) have arterial hypertension, and in the absence of acute prerenal kidney damage one would expect a higher not lower BP in those patients with ACEP, who have CKD (25).
The results of this work (that a subgroup of patients with higher SBP on admission has more pronounced thickening of the interventicular septum and that this H subgroup has more patients with preserved LV systolic function) can be easily explained by the higher prevalence of arterial hypertension (3). High BP can trigger ACEP, but once when a patient is in ACEP, deterioriation of clinical course toward shock is less likely.
AF is almost twice more prevalent in the subgroup with low BP. There are two rational explanations of this situation: 1. AF reduces cardiac output, and, consequently, the BP.
2. AF and low BP are both the consequences of the reduced LV function.
AF is common in acute heart failure and a marker of bad prognosis during hospital stay and after discharge, despite continuous improving the prognostic trend in acute heart failure (26).
Due to great importance of AF in HF, the new European Society of Cardiology (ESC) Guidelines advice is to search for other possible causes of AF (like hyperthyreosis), triggers (like alcohol abuse), and to evaluate a need for anticoagulant therapy (3). | 2019-01-23T18:49:32.126Z | 2015-09-15T00:00:00.000 | {
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219779663 | pes2o/s2orc | v3-fos-license | Empirical Modeling of Liquefied Nitrogen Cooling Impact during Machining Inconel 718
Featured Application: Implementation of a machine learning procedure to model the temperature drop in the cryogenic cooling of Inconel 718. Machine learning is performed by an adaptive neuro-fuzzy inference system (ANFIS). Based on a representative set of cryogenic cooling experiments, the formed response surfaces (of temperature drop in Inconel 718) are represented as a set of fuzzy logic rules. Since we are dealing with a small set of experiments (input–output datapoints), the leave one subject out (LOSO) version of k-fold cross-validation is adopted. LOSO o ff ers to exploit the small datasets and by them assess and tune the overall ANFIS modeling performance. A machine learning-based model is designed to minimize the RMSE prediction error by finding the optimal set of hyper-parameters (external ANFIS input parameters). The optimization is performed with the particle swarm optimization (PSO) algorithm. Abstract: This paper explains liquefied nitrogen’s cooling ability on a nickel super alloy called Inconel 718. A set of experiments was performed where the Inconel 718 plate was cooled by a moving liquefied nitrogen nozzle with changing the input parameters. Based on the experimental data, the empirical model was designed by an adaptive neuro-fuzzy inference system (ANFIS) and optimized with the particle swarm optimization algorithm (PSO), with the aim to predict the cooling rate (temperature) of the used media. The research has shown that the velocity of the nozzle has a significant impact on its cooling ability, among other factors such as depth and distance. Conducted experimental results were used as a learning set for the ANFIS model’s construction and validated via k-fold cross-validation. Optimization of the ANFIS’s external input parameters was also performed with the particle swarm optimization algorithm. The best results achieved by the optimized ANFIS structure had test root mean squared error ( test RMSE ) = 0.2620, and test R 2 = 0.8585, proving the high modeling ability of the proposed method. The completed research contributes to knowledge of the field of defining liquefied nitrogen’s cooling ability, which has an impact on the surface characteristics of the machined parts.
Introduction
Materials such as high temperature alloys (nickel, titanium, etc.) are usually employed in the manufacturing processes of high added value products, especially in the space, aircraft and military Appl. Sci. 2020, 10 industries [1]. These sorts of materials have excellent mechanical and thermal properties which are maintained even when the materials are exposed to high temperatures. Additionally, such materials have low thermal conductivity and get plastically hardened at high temperatures, hence their extremely poor machinability [2]. The nickel-based alloy Inconel 718, which is the subject of the research in this paper, is a high temperature alloy. It is a corrosion (oxidation) resistant material that can be used at high temperatures while maintaining its hardness characteristics. This is due to its good tensile strength, fatigue life, creep properties and rupture strength. However, due to these specific material characteristics, this alloy is generally difficult to machine [3]. According to these mentioned facts, it could be possible to machine those materials by the usage of cooling lubrication fluids, such as a high-pressure jet and liquefied nitrogen, etc. The development of the industry in the field of machining processes has been striving to achieve a higher level of productivity and environmental awareness. This includes minimizing the use of cooling lubrication fluids based on artificial ingredients and increasing cutting speeds. The use of ecologically impeccable cooling lubrication fluids that do not harm people's health and are environmentally friendly has been promoted. Thus, it is necessary to use an environmentally acceptable coolant in the manufacturing industry. For this purpose, liquefied nitrogen as a cryogenic coolant has been explored since the 1950s in the metal cutting industry [4].
The main characteristic of this technology is the use of a cryogenic fluid (liquefied nitrogen) as a cooling fluid during the cutting process, at very low temperatures. The main advantage of liquefied nitrogen is the cooling of the cutting zone, where it is the highest temperature. A deficiency of using liquefied nitrogen as cooling fluid during the machining process is a lack of knowledge in terms of the physical properties of the used media and consequently there are very few publications about them in scientific journals. In past publications on the field of cryogenic machining, artificial intelligence methods were only used to predict machinability aspects: tool wear, cutting forces and surface roughness. In the field of measuring temperatures during the machining process and designing empirical models, the few works are the following. Mozammel and Dhar [5] conducted experiments on measuring temperatures in the hard turning of AISI 1060 steel with coated carbide inserts and developed the predictive empirical model of cutting temperatures with neural networks. Chinchanikar and Choudhury [6] performed experiments on the machining of hardened steel and reached the modeling of the correlations between cutting parameters and different machine aspects including tool wear, chip morphology and surface integrity. Ezugwu et al. [7] reached a correlation between cutting parameters and output process parameters such as tool wear and cutting force in the high speed machining of Inconel 718 with neural networks. Elbaz et al. used a genetic algorithm [8] and particle swarm optimization (PSO) [9] as a learning method of the adaptive neuro-fuzzy inference system (ANFIS) in order to optimize the positions of membership functions (MFs) (making the search space significantly bigger since more independent variables were included in optimization) and increase the ANFIS's accuracy based on leave out cross-validation.
The aim of the paper is to explain in detail the influence of the characteristic aspects of the placement of the nitrogen inlet on the treatment process. Designing an empirical model can help us to understand the influence of the cooling medium used in the machining process. Within the research, a set of experiments was conducted where the material Inconel 718 was cooled by a moving liquefied nitrogen jet, with the aim of measuring the temperature in the plate. The empirical model was designed with ANFIS, which has the ability to predict and explain the cooling impact of liquefied nitrogen in Inconel 718 (temperatures). The results were processed with neural networks combined with fuzzy logic and evaluated with performed experiments. At the end, the external ANFIS hyper-parameters were fine-tuned with the PSO algorithm to improve the modeling degree. Since we were dealing with a small set of experiments (input-output datapoints), the adopted leave one subject out (LOSO) version of k-fold cross-validation offered to exploit the small datasets and by them assess and tune the overall ANFIS modeling performance.
Experimental Setup Description
A set of experiments was conducted where the temperature was measured in a nickel super alloy material (Inconel 718). To obtain the most efficient results in terms of temperature field in the specimen, the input parameters of the process were defined in correlation with the measured temperature of the material. A sufficiently large sample of experimental data was presented as the basis for designing the machine learning-based model.
Liquefied nitrogen was applied on the Inconel 718 plate with the following dimensions: 250 × 146 × 2.5 mm. The plate was polished at R a < 0.3 um on the upper side where the flow of the media was applied. On the bottom side of the plate, 3 sets of 5 holes within 5 mm distance were manufactured by EDM or electrical discharge machining ( Figure 1). Each set of holes was at the same depth. The nozzle (internal diameter d n = 1 mm) was clamped on the magnetic holder on the spindle of the Computer numerical controlled (CNC) machine and moved across the middle of the plate, right above the middle hole set on the polished surface of the plate.
Experimental Setup Description
A set of experiments was conducted where the temperature was measured in a nickel super alloy material (Inconel 718). To obtain the most efficient results in terms of temperature field in the specimen, the input parameters of the process were defined in correlation with the measured temperature of the material. A sufficiently large sample of experimental data was presented as the basis for designing the machine learning-based model.
Liquefied nitrogen was applied on the Inconel 718 plate with the following dimensions: 250 × 146 × 2.5 mm. The plate was polished at Ra ˂ 0.3 um on the upper side where the flow of the media was applied. On the bottom side of the plate, 3 sets of 5 holes within 5 mm distance were manufactured by EDM or electrical discharge machining ( Figure 1). Each set of holes was at the same depth. The nozzle (internal diameter dn = 1 mm) was clamped on the magnetic holder on the spindle of the Computer numerical controlled (CNC) machine and moved across the middle of the plate, right above the middle hole set on the polished surface of the plate. The nozzle was moved across the middle of the plate, right above the middle hole on the grinded surface of the plate. In the experiments, five commercial thermocouples Ni Cr-Ni (K-type, diameter 1 mm, insulated, Tt = -200 to 205 °C) were used. The thermocouples were fixed to the holes with a power tape. To assure contact of the thermocouple with the workpiece, a thermo paste based on silicon oil with metal oxides was used. The nozzle diameter was 1 mm and moved 15 mm above the plate. Liquefied nitrogen was piped through the nozzle under pressure measured on the liquefied nitrogen dewar p = 1.8·10 5 Pa.
For data acquisition, National instruments (NI) acquisition card was used. The input parameters were determined and with them the design of the experiments. The temperature of the material T The nozzle was moved across the middle of the plate, right above the middle hole on the grinded surface of the plate. In the experiments, five commercial thermocouples Ni Cr-Ni (K-type, diameter 1 mm, insulated, T t = -200 to 205 • C) were used. The thermocouples were fixed to the holes with a power tape. To assure contact of the thermocouple with the workpiece, a thermo paste based on silicon oil with metal oxides was used. The nozzle diameter was 1 mm and moved 15 mm above the plate. Liquefied nitrogen was piped through the nozzle under pressure measured on the liquefied nitrogen dewar p = 1.8 × 10 5 Pa.
For data acquisition, National instruments (NI) acquisition card was used. The input parameters were determined and with them the design of the experiments. The temperature of the material T [ • C] was measured, depending on the changing of the input parameters: velocity of the nozzle v, depth of the measuring temperature d and distance from the moving nozzle x. The value range for the moving nozzle speed v was defined based on the comparable cutting speed recommended for machining Inconel 718. By the determination of the values, the aim was to determine the temperature drop in the material after the nozzle had moved across the plate. This was compared to one workpiece revolution in the turning process or milling process. The design of the experiments consisted of 35 experiments at different nozzle moving speeds v = (5, 15, 25, 35 m/min), at different depths at which the temperature in the material was measured d = (0.1, 0.5, 1 mm), and at different plate-nozzle distances x = (0, 5, 10 mm). Figure 2 shows the experimental setup (the clamping of the nozzle onto the machine spindle and the detail that shows the diameter of the jet dispersion on the surface) [10].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 16 [°C] was measured, depending on the changing of the input parameters: velocity of the nozzle v, depth of the measuring temperature d and distance from the moving nozzle x. The value range for the moving nozzle speed v was defined based on the comparable cutting speed recommended for machining Inconel 718. By the determination of the values, the aim was to determine the temperature drop in the material after the nozzle had moved across the plate. This was compared to one workpiece revolution in the turning process or milling process. The design of the experiments consisted of 35 experiments at different nozzle moving speeds v = (5, 15, 25, 35 m/min), at different depths at which the temperature in the material was measured d = (0.1, 0.5, 1 mm), and at different plate-nozzle distances x = (0, 5, 10 mm). Figure 2 shows the experimental setup (the clamping of the nozzle onto the machine spindle and the detail that shows the diameter of the jet dispersion on the surface) [10]. Table 1 shows the results obtained by measuring the temperatures in the Inconel 718 cooled by the liquefied nitrogen. It can be deduced that the temperature drop in the material significantly decreased with the increasing nozzle speed and this had a greater impact on its cooling ability. Table 1 shows the results obtained by measuring the temperatures in the Inconel 718 cooled by the liquefied nitrogen. It can be deduced that the temperature drop in the material significantly decreased with the increasing nozzle speed and this had a greater impact on its cooling ability. Figure 3 shows an experiment on the measured temperature-time distribution for a moving speed of v = 5 m/min. Five temperature-time curves for each thermocouple were positioned at the same depth (d = 0.5 mm) to provide enough information for describing the temperature field in the plate with the aim of obtaining the most efficient empirical model with neural networks. The process Appl. Sci. 2020, 10, 3603 6 of 16 lasted 1.8 s, and the difference to 2.4 s was predicted for balancing the heat flux in the system. The highest temperature difference (blue ∆T = 5.5 K) appeared in the middle thermocouple, which was in accordance with the expectations since the nozzle moved across the middle of the plate.
The minimum temperature was reached after 1.1 s; consequently, a time delay in the process appeared. The reason for this phenomenon was in the accumulated gaseous nitrogen film on the specimen which prevented the greater cooling impact of applying a liquid nitrogen jet on the plate [10].
The Empirical Model-Adaptive Neuro-Fuzzy Inference System (ANFIS)
Adaptive neuro-fuzzy inference systems (ANFIS) belong to a family of hybrid systems. They use a supervised learning on the learning algorithm which has a function similar to the model of the Takagi-Sugeno fuzzy inference system [11]. An ANFIS combines the advantages of fuzzy logic (FL) and artificial neural networks (ANNs) for modeling purposes. ANNs operate in a black box manner and they have no capability of explaining a particular decision; on the other hand, ANNs have the ability to predict the output of a complex system through a learning process based on the examples provided [12,13]. FL itself possesses no learning ability but is able to provide mathematical descriptions in the form of linguistic terms and the structure of IF-THEN rules [14], offering an insight into decisions. An ANFIS can be used in a wide range of applications since it possesses the ability to handle large amounts of data in nonlinear and dynamic systems, especially when relationships between data are not fully understood. Figure 4 shows the ANFIS scheme. The ANFIS structure consists of five connected network layers, to . The first and fourth layers contain adaptive nodes while the other layers contain fixed nodes represented in circular shapes. The adaptive nodes contain parameters that are modified to achieve the desired input-output mapping, while the fixed nodes contain none. The architecture of the ANFIS is presented based on two fuzzy IF-THEN rules and the first order Sugeno model as follows:
The Empirical Model-Adaptive Neuro-Fuzzy Inference System (ANFIS)
Adaptive neuro-fuzzy inference systems (ANFIS) belong to a family of hybrid systems. They use a supervised learning on the learning algorithm which has a function similar to the model of the Takagi-Sugeno fuzzy inference system [11]. An ANFIS combines the advantages of fuzzy logic (FL) and artificial neural networks (ANNs) for modeling purposes. ANNs operate in a black box manner and they have no capability of explaining a particular decision; on the other hand, ANNs have the ability to predict the output of a complex system through a learning process based on the examples provided [12,13]. FL itself possesses no learning ability but is able to provide mathematical descriptions in the form of linguistic terms and the structure of IF-THEN rules [14], offering an insight into decisions. An ANFIS can be used in a wide range of applications since it possesses the ability to handle large amounts of data in nonlinear and dynamic systems, especially when relationships between data are not fully understood. Figure 4 shows the ANFIS scheme. The ANFIS structure consists of five connected network layers, l 1 to l 5 . The first and fourth layers contain adaptive nodes while the other layers contain fixed nodes represented in circular shapes. The adaptive nodes contain parameters that are modified to achieve the desired input-output mapping, while the fixed nodes contain none. The architecture of the ANFIS is presented based on two fuzzy IF-THEN rules and the first order Sugeno model as follows: where: • x and y are the inputs, • A i and B i are the membership functions (MF), • f i is the output, and • p i , q i and r i are the linear design parameters (consequent parameters), determined during the training process [16].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 16 where: • and are the inputs, • and are the membership functions (MF), • is the output, and • , and are the linear design parameters (consequent parameters), determined during the training process [16].
In all nodes are adaptive nodes. Inputs are presented as and . The output of a node is the membership value , , that specifies the degree of input (or ) belonging to fuzzy set (or ): we adopt the Gaussian MF by: where and are the shape parameters (premise parameters) of an MF. Layer is the rule layer with fixed or nonadaptive nodes labeled as . As an output, it computes the firing strength (activation level) of each rule with the AND logical operator (or with other types of T-norm operators with general performance): In is the normalization layer with fixed nodes, labeled as . The outputs of this layer are normalized representations of each rule: Layer contains adaptive nodes. The output of each node is the product of normalized firing strength and a first order polynomial (for a first order Sugeno model): In l 1 all nodes are adaptive nodes. Inputs are presented as x and y. The output of a node is the membership value O 1,i , that specifies the degree of input x (or y) belonging to fuzzy set A i (or B i ): we adopt the Gaussian MF by: where a i and c i are the shape parameters (premise parameters) of an MF. Layer l 2 is the rule layer with fixed or nonadaptive nodes labeled as π. As an output, it computes the firing strength w i (activation level) of each rule with the AND logical operator (or with other types of T-norm operators with general performance): In l 3 is the normalization layer with fixed nodes, labeled as N. The outputs of this layer are normalized representations of each rule: Layer l 4 contains adaptive nodes. The output of each node is the product of normalized firing strength and a first order polynomial (for a first order Sugeno model): Appl. Sci. 2020, 10, 3603 8 of 16 Layer l 5 contains a single fixed node, labeled as . It computes the summation of all the input signals coming from the nodes in layer l 4 : Consequent and premise parameters are computed during the training process, based on the back-propagation gradient descent algorithm or with hybrid learning [15].
Particle Swarm Optimization
The particle swarm optimization algorithm (PSO) stochastic metaheuristic computational method was developed by Eberhart and Kennedy in 1995 [17]. PSO and other metaheuristic algorithms do not guarantee a global optimal solution, yet they have proven to be essential in near-optimal solution searches in vast nonlinear multidimensional solution spaces [18]. The algorithm mimics the intelligence of a flock of birds (swarm), based on the socio-psychological correlations [19] expressed by mathematical formulae over the bird's (particle's) position and velocity. The position of a particle represents a possible solution, while velocity can be described as the pulling tendency of a particle in the direction of the next solution search. PSO enables an iterative solution search by moving particles through the search space, trying to improve the candidate's solution regarding the given fitness function representing a measure of quality. Fitness function represents how good the multidimensional position of an i-th particle is. Particles move according to their personal and global best positions. Conceptually, personal best (pbest id ) imitates a particle's autobiographical memory: it tends to push each particle to the place at which the current particle was most satisfied. Global best (gbest) is conceptually similar to a collective knowledge: it tends to pull all particles in the direction represented by the best position (achieved until now) of any particle in a swarm.
In the first iteration, particles' positions are generated randomly as multidimensional constrained vectors, with initial velocities set to zero. Through the iteration process of the solution search, the particles' velocities v t id are altered with: and positions x t id with: where: • id subscript represents the current particle, • t is the current iteration, • w is the inertia weight coefficient, • C 1 and C 2 are the acceleration coefficients, and • rand 1 () and rand 2 () represent random number generators.
To control the system's convergence tendencies, a set of restriction coefficients w, C 1 and C 2 was adopted, according to Kennedy and Clerc's recommendations [20], that has proven to work sufficiently well on a variety of different optimization problems. The positions x t id were represented as external ANFIS input parameters. The PSO algorithm was set to stop after a certain number of iterations.
Workflow and Validation
In this study, we presented two cases: case 1 consisted of the ANFIS modeling and prediction of the temperature difference output. It started by dividing the dataset (input-output data points) into training and testing sets. The training set was used for the ANFIS model construction and the testing set for evaluating the ANFIS model on the data yet unseen by the model. In k-fold cross-validation, the original dataset was randomly partitioned into k equal sized sub partitions. The k − 1 sub partitions were used as training data and a single partition was used for the testing of the ANFIS structure. During the cross-validation procedure, all external ANFIS input parameters, such as number of membership functions (MF), type of MF, number of epochs, learning step, increase in learning step and optimization method, were kept fixed. The whole cross-validation procedure was then repeated k times, with each of the k sub partitions used exactly once as testing data. In the majority of cases presented in this paper, the leave one subject out (LOSO) validation scheme (k = 35) was adopted, partitioning the whole dataset into 35 equal parts and performing 35 iterations of learning with 34 folds and testing on the remaining fold (yet every iteration a different fold). The LOSO procedure promised better overall generalization of the model's performance and enabled the use of the whole dataset for both the model's learning and its testing. The root mean squared error (RMSE) was used as a fitness function and represented the quantitative evaluation of the ANFIS's performance.
Case 2's procedure was structurally the same as that of case 1, except for an additional part represented by the PSO algorithm, which was set to optimize the external ANFIS input parameters. As the PSO fitness function, the RMSE testing error was adopted. The PSO algorithm was set to minimize the RMSE testing error and consequently find optimal ANFIS input parameter settings ( Figure 5).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 16 35) was adopted, partitioning the whole dataset into 35 equal parts and performing 35 iterations of learning with 34 folds and testing on the remaining fold (yet every iteration a different fold). The LOSO procedure promised better overall generalization of the model's performance and enabled the use of the whole dataset for both the model's learning and its testing. The root mean squared error (RMSE) was used as a fitness function and represented the quantitative evaluation of the ANFIS's performance. Case 2's procedure was structurally the same as that of case 1, except for an additional part represented by the PSO algorithm, which was set to optimize the external ANFIS input parameters. As the PSO fitness function, the RMSE testing error was adopted. The PSO algorithm was set to minimize the RMSE testing error and consequently find optimal ANFIS input parameter settings ( Figure 5).
Case 1
During the ANFIS model's formulation, the number of MFs for each input, type of MF, number of learning epochs, initial step size and step size increase rates were varied. In case 1, variations of different types of MF were used, such as generalized bell-shaped, triangular, trapezoidal, pi-shaped, Gaussian's and Gaussian combination MF. The type of MF has not altered the results in a significant manner. Modeling with a different number of MFs for each input (dependent variable) was also performed. Increasing the number of MF number, computational time and number of IF-THEN rules formed by the ANFIS also increase. Input MFs were generated using the grid partition clustering method that uniformly partitioned the input variable ranges and created a single-output Sugeno fuzzy system. Other clustering methods, such as subtractive clustering, have achieved lower modeling precision rates. A Gaussian MF was adopted and the ANFIS structure ( Figure 6) was trained for 100 epochs using back-propagation and a hybrid algorithm to determine the premise and consequent parameters, with an initial step size equal to 0.1 and a step size increase rate of 1.10. Results are stated in Table 1 for the LOSO cross-validation. The ANFIS structure trained with three MF and the hybrid learning algorithm computed the lowest training RMSE ( = 8.37 e-7) but a relatively high testing RMSE (= 0.8594). Lowering the number of epochs did not result in improving
Case 1
During the ANFIS model's formulation, the number of MFs for each input, type of MF, number of learning epochs, initial step size and step size increase rates were varied. In case 1, variations of different types of MF were used, such as generalized bell-shaped, triangular, trapezoidal, pi-shaped, Gaussian's and Gaussian combination MF. The type of MF has not altered the results in a significant manner. Modeling with a different number of MFs for each input (dependent variable) was also performed. Increasing the number of MF number, computational time and number of IF-THEN rules formed by the ANFIS also increase. Input MFs were generated using the grid partition clustering method that uniformly partitioned the input variable ranges and created a single-output Sugeno fuzzy system. Other clustering methods, such as subtractive clustering, have achieved lower modeling precision rates. A Gaussian MF was adopted and the ANFIS structure ( Figure 6) was trained for 100 epochs using back-propagation and a hybrid algorithm to determine the premise and consequent parameters, with an initial step size equal to 0.1. and a step size increase rate of 1.10. Results are stated in Table 1 for the LOSO cross-validation. The ANFIS structure trained with three MF and the hybrid learning algorithm computed the lowest training RMSE (= 8.37 × 10 −7 ) but a relatively high testing RMSE (= 0.8594). Lowering the number of epochs did not result in improving the generalization ability of the discussed ANFIS that tended to overfit the training set. The computational time of the ANFIS with back-propagation learning and two MFs achieved the best results with the input parameters stated in the above paragraph. The discussed ANFIS was about 5.6 times faster compared to the ANFIS structure with the hybrid learning algorithm and three MFs, 4.8 times faster than the ANFIS with back-propagation learning and three MFs and had a similar computation time to the ANFIS structure with hybrid learning and two MFs.
Based on the set of results in Table 2, further experiments ( Figure 7) were performed on the ANFIS structure with the same ANFIS input parameters and back-propagation learning algorithm and two Gaussian MFs. To produce the results in Figure 7, the 10-fold cross-validation was used. Two Gaussian MFs were used due to their simplicity and high performance (low computation). In further experiments, six different initial step sizes (0.001, 0.005, 0.01, 0.05, 0.1 and 0.5) and six different step size increase values (1.00001, 1.001, 1.1, 1.2, 1.3 and 1.4) were used for the ANFIS model construction. The ANFIS structure with the 0.1 initial step size and 1.001 step size increase achieved the best test RMSE (= 0.4195) as can be seen in Figure 7a. The train RMSE of the mentioned structure was 0.1254 as depicted in Figure 7b. The computational time of the ANFIS with back-propagation learning and two MFs achieved the best results with the input parameters stated in the above paragraph. The discussed ANFIS was about 5.6 times faster compared to the ANFIS structure with the hybrid learning algorithm and three MFs, 4.8. times faster than the ANFIS with back-propagation learning and three MFs and had a similar computation time to the ANFIS structure with hybrid learning and two MFs.
Based on the set of results in Table 2, further experiments ( Figure 7) were performed on the ANFIS structure with the same ANFIS input parameters and back-propagation learning algorithm and two Gaussian MFs. To produce the results in Figure 7, the 10-fold cross-validation was used. Two Gaussian MFs were used due to their simplicity and high performance (low computation). In further experiments, six different initial step sizes (0.001, 0.005, 0.01, 0.05, 0.1 and 0.5) and six different step size increase values (1.00001, 1.001, 1.1, 1.2, 1.3 and 1.4) were used for the ANFIS model construction. The ANFIS structure with the 0.1 initial step size and 1.001 step size increase achieved the best test RMSE (= 0.4195) as can be seen in Figure 7a. The train RMSE of the mentioned structure was 0.1254 as depicted in Figure 7b.
Case 2
The particle swarm optimization algorithm (PSO) was used for the optimal external ANFIS input parameters search in the so called PSO-ANFIS procedure. Optimization was set to find the minimum testing RMSE based on the LOSO cross-validation procedure by altering the initial step size (ISS) and step size increase (SSI) via PSO. ANFIS input parameters such as number of MFs, type of MF and number of epochs were kept fixed (two Gaussian MFs, trained for 100 epochs with the backpropagation learning algorithm). The ANFIS structure was trained based on the back-propagation learning algorithm to determine the premise and consequent parameters.
One hundred PSO particles were used in the optimum search. PSO was set to work for 100 iterations, with a set of restriction coefficients = 0.729, = 1.496 and = 1.496. Each PSO particle's fitness function value was calculated with LOSO: in our case, this was 35-fold crossvalidation. The convergence of the PSO algorithm is shown in Figure 8. The initial positions of the PSO particles, represented by ISS and SSI, were created with a random number generator at intervals of (0,10) and (1,10), respectively. Case 2, consisting of the ANFIS model and the PSO module, proved to achieve similar optimum values in multiple runs with different initial positions for the ISS and SSI. Additional external parameters, such as iteration number, number of MFs or step size decrease rate could be introduced in a similar manner to ISS and SSI.
Case 2
The particle swarm optimization algorithm (PSO) was used for the optimal external ANFIS input parameters search in the so called PSO-ANFIS procedure. Optimization was set to find the minimum testing RMSE based on the LOSO cross-validation procedure by altering the initial step size (ISS) and step size increase (SSI) via PSO. ANFIS input parameters such as number of MFs, type of MF and number of epochs were kept fixed (two Gaussian MFs, trained for 100 epochs with the back-propagation learning algorithm). The ANFIS structure was trained based on the back-propagation learning algorithm to determine the premise and consequent parameters.
One hundred PSO particles were used in the optimum search. PSO was set to work for 100 iterations, with a set of restriction coefficients w = 0.729, C 1 = 1.496 and C 2 = 1.496. Each PSO particle's fitness function value was calculated with LOSO: in our case, this was 35-fold cross-validation. The convergence of the PSO algorithm is shown in Figure 8. The initial positions of the PSO particles, represented by ISS and SSI, were created with a random number generator at intervals of (0,10) and (1,10), respectively. Case 2, consisting of the ANFIS model and the PSO module, proved to achieve similar optimum values in multiple runs with different initial positions for the ISS and SSI. Additional external parameters, such as iteration number, number of MFs or step size decrease rate could be introduced in a similar manner to ISS and SSI.
This section presents an analysis of the results of the experimental data and the ANFIS model output on the nitrogen cooling impact during the machining of Inconel 718. Table 3 and Figure 9 represent the predicted temperature differences of the ANFIS structures compared to the real experiment values. The first three columns in Table 3 present the experimental values (v, d and x), the fourth column the experimental value (∆T) and the fifth column the temperature difference (∆T case1 ) of the testing set predicted in case 1 based on the LOSO cross-validation. The sixth column represents the temperature differences (∆T case2 best ) of the testing set predicted in case 2. The optimized ANFIS structure in case 2 with ISS = 1.6764 and SSI = 7.7766 achieved the testing RMSE equal to 0.2620 and a training RMSE of 0.3372. The respective testing and training R 2 values were equal to 0.8585 and 0.8912. This section presents an analysis of the results of the experimental data and the ANFIS model output on the nitrogen cooling impact during the machining of Inconel 718. Table 3 and Figure 9 represent the predicted temperature differences of the ANFIS structures compared to the real experiment values. The first three columns in Table 3 was equal to 0.3450°C, while the optimized ANFIS structure in case 2 achieved an average temperature difference between the experimentally measured temperature drop ( ΔT ) and the temperature drop predicted by ANFIS based on the LOSO testing set (|ΔT − ΔT |) equal to 0.2619°C. Neural networks combined with fuzzy logic in the ANFIS system offer an insight into their decisions. Figure 10 presents the Fuzzy interference system (FIS) properties of the discussed ANFIS. Output ( represented by a first order Sugeno model) was calculated based on eight IF-THEN rules that represent all combinations of input MFs (= 2 ). The premise parameters are stated as and , where represents the mean and the width of the -th Gaussian MF. The first rule states that if the input 1 belongs to MF1, input 2 to MF3 and input 3 to MF5, then output is determined by = Predicted and mesured temp. difference Input-output datapoint index Figure 9. Column bar chart representing the temperature differences between experimental values ∆T (blue bars), predicted PSO-ANFIS ∆T case2 best (orange bars) and ANFIS ∆T case1 (gray bars).
The average difference between the experimentally measured temperature drop (∆T) and the temperature drop predicted by the ANFIS based on the LOSO testing set in case 1 (|∆T − ∆T case1 |) was equal to 0.3450 • C, while the optimized ANFIS structure in case 2 achieved an average temperature difference between the experimentally measured temperature drop (∆T) and the temperature drop predicted by ANFIS based on the LOSO testing set (|∆T − ∆T case2 best |) equal to 0.2619 • C.
Neural networks combined with fuzzy logic in the ANFIS system offer an insight into their decisions. Figure 10 presents the Fuzzy interference system (FIS) properties of the discussed ANFIS. Output (∆T represented by a first order Sugeno model) was calculated based on eight IF-THEN rules that represent all combinations of input MFs (= 2 3 ). The premise parameters are stated as a i and c i , where a represents the mean and c the width of the i-th Gaussian MF. The first rule states that if the input 1 belongs to MF1, input 2 to MF3 and input 3 to MF5, then output is determined by ∆T = m 1 * input1 + p 1 * input2 + q 1 * input3 + r 1 . The consequent parameters of the 1-st rule, denoted as m 1 , p 1 , q 1 and r 1 , are equal to −0.5706, −0.1155, −0.0434 and 8.896, respectively.
Conclusions
In the past decade, sustainable machining has become increasingly important. The use of an alternative cooling lubrication fluid which is not harmful to the environment and human health is encouraged. Nowadays, cryogenic machining sets sustainable trends in machining technologies, which represents guidelines for the implementation of sustainable technologies in production, where the use of harmful cooling lubrication is reduced. The modern guidelines of machining emphasize the consumption of cooling lubrication fluids which do not affect human health and the environment. As part of these trends, the idea of sustainable development at all levels of life has emerged. This paper explains the aspects of the use of liquefied nitrogen as a cooling medium in cryogenic machining and thus contributes to basic knowledge in the field of cooling effects, promising greater involvement and promotion of sustainable engineering in the industry. The objective of this research was to analyze the thermal influence of liquefied nitrogen on workpieces depending on variation of the input parameters to obtain correlation with the measured temperature.
The effect of the conductance of liquefied nitrogen on workpieces was reduced due to the formation of a gaseous film which had very low thermal conductivity cca. 0.03 W/mK and a low heat transfer coefficient in comparison to the liquid nitrogen phase. The nozzle must be placed as close as possible to the cutting zone to ensure the most efficient cooling capacity of the medium and thus the smallest possible dissipation of the jet.
To predict temperature drops in the material, various versions of ANFIS models were applied to predict the temperature difference of Inconel 718 cooling with liquefied nitrogen. Various ANFIS model configurations were proposed and discussed. The ANFIS model (case 1) and the ANFIS structure combined with PSO (case 2 ) were proven to achieve low modeling errors of the temperature drops in Inconel 718. The best values of RMSE achieved were low, stating the ANFIS's modeling ability of liquefied nitrogen cooling. Since the study was conducted with a small number of experiments (input-output datapoints), the LOSO k-fold cross-validation procedure was adopted, enabling the more representative accuracy of the presented ANFIS models, which were not suited to fixed and/or small sets of datapoints. LOSO offered to exploit the small datasets and by them assess the overall ANFIS modeling performance. Depending on the nature of the problem, additional external ANFIS input parameters (not only ISS and SSI) can be included. Including additional external input parameters prolongs the computation time, which is still shorter compared to other hybrid approaches since fewer independent variables are included.
Conclusions
In the past decade, sustainable machining has become increasingly important. The use of an alternative cooling lubrication fluid which is not harmful to the environment and human health is encouraged. Nowadays, cryogenic machining sets sustainable trends in machining technologies, which represents guidelines for the implementation of sustainable technologies in production, where the use of harmful cooling lubrication is reduced. The modern guidelines of machining emphasize the consumption of cooling lubrication fluids which do not affect human health and the environment. As part of these trends, the idea of sustainable development at all levels of life has emerged. This paper explains the aspects of the use of liquefied nitrogen as a cooling medium in cryogenic machining and thus contributes to basic knowledge in the field of cooling effects, promising greater involvement and promotion of sustainable engineering in the industry. The objective of this research was to analyze the thermal influence of liquefied nitrogen on workpieces depending on variation of the input parameters to obtain correlation with the measured temperature.
The effect of the conductance of liquefied nitrogen on workpieces was reduced due to the formation of a gaseous film which had very low thermal conductivity cca. 0.03 W/mK and a low heat transfer coefficient in comparison to the liquid nitrogen phase. The nozzle must be placed as close as possible to the cutting zone to ensure the most efficient cooling capacity of the medium and thus the smallest possible dissipation of the jet.
To predict temperature drops in the material, various versions of ANFIS models were applied to predict the temperature difference of Inconel 718 cooling with liquefied nitrogen. Various ANFIS model configurations were proposed and discussed. The ANFIS model (case 1) and the ANFIS structure combined with PSO (case 2) were proven to achieve low modeling errors of the temperature drops in Inconel 718. The best values of RMSE achieved were low, stating the ANFIS's modeling ability of liquefied nitrogen cooling. Since the study was conducted with a small number of experiments (input-output datapoints), the LOSO k-fold cross-validation procedure was adopted, enabling the more representative accuracy of the presented ANFIS models, which were not suited to fixed and/or small sets of datapoints. LOSO offered to exploit the small datasets and by them assess the overall ANFIS modeling performance. Depending on the nature of the problem, additional external ANFIS input parameters (not only ISS and SSI) can be included. Including additional external input parameters prolongs the computation time, which is still shorter compared to other hybrid approaches since fewer independent variables are included. | 2020-05-28T09:17:01.434Z | 2020-05-22T00:00:00.000 | {
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263701863 | pes2o/s2orc | v3-fos-license | Prevalence of HIV in Patients with Malignancy and of Malignancy in HIV Patients in a Tertiary Care Center from North India
Background and Objectives: People living with HIV/AIDS are at an increased risk of de-veloping cancer. The goals of this study were to obtain data on the prevalence of HIV in the cancer population and vice versa at a major tertiary cancer and HIV center in North India. Methods: This cross-sectional study was conducted over a 3-year period from July 2013 to June 2016, wherein successive HIV positive patients from an anti-retroviral therapy (ART) center were screened for malignancy. Simultaneously, successive cancer patients at the cancer center were screened for HIV. Baseline demographic details, risk factors, and laboratory investigations were ob-tained for all the patients. Results: Among the 999 HIV-positive patients at the ART center, the prevalence of malignancy was 2% (n=20; 95% confidence interval (CI) 1.13, 2.87). Among the 998 patients with a malignancy, the prevalence of HIV infection was 0.9% (n=9; 95% CI 0.31, 1.49). Weight loss, loss of appetite, and fever were the most common symptoms in patients with HIV and cancer. Among 29 patients with HIV and cancer, AIDS-defining cancer was found in 19 patients; non-Hodgkin’s lymphoma was the most common malignancy reported (n=13). Interpretation and Conclusion: There is a low prevalence of HIV in cancer patients as well as a low prevalence of cancer in HIV patients. AIDS-defining cancers remain much more common than non-AIDS-defining cancers. With the increased coverage of ART, it is expected that non-AIDS-defining cancers will increase, as is evident from data from more developed countries.
Human immunodeficiency virus (HIV) infection is a risk factor for cancer [1,2].HIV-associated cancers can be divided into acquired immune deficiency syndrome (AIDS)defining cancers (ADC) and non-AIDS-defining cancers (NADC) [2].The availability of effective antiretroviral therapy (ART) has lowered the incidence of cancer but there has been a simultaneous increase in the life expectancy of people living with HIV [3].Consequently, the overall incidence of cancer and AIDS-defining cancer has stabilized over the last two decades (except invasive cervical cancer), while there has been an absolute increase in NADC [2][3][4][5][6].HIVassociated cancer is one of the leading causes of death in HIV patients [7][8][9].ADC include non-Hodgkin's lymphoma (NHL), invasive cervical carcinoma, and Kaposi's sarcoma (KS) [10].NADC include lung cancer, anal cancer, Hodgkin's lymphoma, etc.The standardized incidence ratio (which compares the cancer rate in patients with HIV to the expected rate in the general population) [11] varies from 2 in lung cancer to 498 in Kaposi's sarcoma.NHL remains the most common cancer in patients with HIV worldwide [2,12].
Risk factors for cancer in HIV include immunosuppression, oncogenic potential of HIV [13], co-infection with other oncogenic virus such as hepatitis B and C [13], human herpes virus-8 (HHV-8) infection [14,15], human papilloma virus (HPV) [16], Epstein-Barr virus (EBV) [17,18], etc. HIV can also sensitize the cells to the oncogenic potential of tobacco, as suggested by an increase in the risk of lung cancer in people living with HIV (three times the general population) after adjustment for smoking status [13].
In 2016, there were 36.7 million people living with HIV worldwide; 2.1 million of these lived in India [19,20].Based on regional records, NHL is the most common cancer overall [21][22][23][24] and Kaposi's sarcoma is rare [23].Increasing access to ART [25] is expected to decrease the overall incidence of cancer in this population, but the spectrum of cancer in people living with HIV in India needs to be better understood for effective screening.
We aim to look at the prevalence and spectrum of cancer in HIV patients at a tertiary care referral center in North India.
METHODS
From July 2013 to June 2016, we used a structured history and physical examination to screen successive adult (≥18 years) HIV-positive patients, irrespective of whether or not on any anti-retroviral therapy (ART), who presented at the ART center at the hospital for malignancy.The institute's ethics committee approved the protocol.Written informed consent was obtained from all the participants prior to their participation in the study.
Baseline demographic data were recorded regarding education, occupation, marital status, status of spouse and children, and likely mode of transmission.CD4 count was assessed for all the patients with HIV and cancer at the ART center.CD4 cell counts were measured by flow cytometry using BD FACS CALIBUR and fluorescein monoclonal antibodies (Becton Dickenson Biosciences, San Jose, CA, USA).
We assessed for known risk factors for malignancies such as current or former smoking, tobacco chewing, and alcohol intake.Those suspicious for malignancy were further evaluated with radiological investigations such as X-ray, ultrasound, computed tomography (CT) scan, magnetic resonance imaging (MRI), and positron-emission tomography (PET)-CT scan of the region of interest.All the malignancies were confirmed by histopathological examination.The time since the diagnosis of HIV and malignancy was recorded for all the patients.The overall prevalence of malignancy in HIV was calculated.
All the consecutive patients who presented at the cancer center with a confirmed diagnosis of a malignancy were screened for HIV antibodies using ELISA.All patients, irrespective of type and stage of malignancy were included in the study after due consent.Biopsy reports of all the patients diagnosed elsewhere were reviewed at this hospital for confirmation of diagnosis.The patients who tested positive for HIV were referred to the ART clinic at the All India Insti-tutes of Medical Sciences (AIIMS) for HIV treatment and follow-up.
The study was continued till 1000 patients each were recruited at the ART center and the cancer center respectively.
All the patients with HIV and cancer were followed-up for treatment response and outcome for one year postenrollment.
The data were recorded in a predesigned Excel spreadsheet.All the participants were given a unique code number.We calculated the prevalence of cancer in the HIV patients and the prevalence of HIV infection in patients with cancer over the three-year period.The descriptive statistics were calculated, including means and standard deviations for the continuous variables and percentages for the categorical variables.One-way ANOVA with Bonferroni's correction was used to find significant differences in the data following parametric distribution, while the Wilcoxon rank-sum test was used to find differences in CD4 counts between the groups.
RESULTS
There were 999 HIV-positive patients enrolled from the ART clinic and 998 patients from the cancer clinic.Most of the patients at the ART center were males, whereas most patients at the cancer center were females.
Of the 999 HIV-positive patients, 20 patients were diagnosed with a malignancy.
Most of the patients in the three groups did not smoke or consume significant amounts of alcohol.The patients with HIV and cancer were older than those with HIV only (44.8±10.5 vs 36.7±9.2 years, p-value <0.05).
Almost half of the patients with HIV and cancer had fever, involuntary weight loss, and loss of appetite in the preceding six months, while only one-third of the patients in the HIV only group had these symptoms.There was no significant difference in the CD4 counts in groups 1 and 3 at the time of diagnosis of cancer (median CD4 count 246 vs 188, p-value >0.05).
Among the patients with HIV and cancer (n=29), 22 were male and 7 were female.Overall, 19 patients had AIDSdefining cancers while 10 had non-AIDS-defining cancers.The most common malignancy was non-Hodgkin's lymphoma (13/29) followed by invasive cervical carcinoma (6/29).No case of KS was observed.
There was only one case of hepatitis B co-infection in patients with HIV and cancer.At one-year follow up, 19 patients were alive, 8 patients had died, and 2 patients could not be traced.
DISCUSSION
Our study suggests that approximately 2% of HIV patients had cancer, while 1% of cancer patients had HIV infection.AIDS-defining cancers remain more common than non-AIDS-defining cancers.In more developed countries with a higher life expectancy, non-AIDS-defining cancers predominate.It is expected that better coverage of ART, control of communicable diseases, and increased longevity would lead to an increase in non-AIDS-defining cancers and a decrease in AIDS-defining cancers [2,26,27].
The prevalence of HIV infection in cancer patients is very low, but it is approximately four times the national population (the national prevalence of HIV infection in India is 0.26%) [28].In a similar report from Maharashtra, India, 3,832 patients were studied and the prevalence of HIV infection was only 1.2% [29].
AIDS-defining cancer such as NHL was around four times more common in patients with HIV compared to HIVnegative cancer patients.At the same center as the present study (AIIMS), a retrospective study analyzing malignancy in HIV patients during the initial years of ART (2005-2013) suggested a similar infrequent occurrence of malignancy in HIV, with NHL being the most common [30].Most regional studies from India concur that NHL is the most common malignancy in HIV and KS is very rare [21][22][23][29][30][31][32][33].One of the reasons postulated is the low seroprevalence of HHV-8 in the Indian population [34].However, a recent study by Munawwar et al. reported that 26% of 165 HIV-positive ART-naïve Indian men (90% heterosexual) had antibodies against HHV-8 [35].The role of factors additional to HHV-8 in the pathogenesis of KS needs to be explored.
Diffuse large B-cell lymphoma is the most common subtype of NHL overall as well in the HIV population [2,23,24].While EBV is commonly associated with immunodeficiency-related Hodgkin's lymphoma, its role in the pathogenesis of NHL in HIV varies, with most cases of primary CNS lymphoma expressing EBV proteins while rarely being associated with Burkitt lymphoma [36][37][38].Outcomes of lymphoma have been worse in HIV-positive patients [39][40][41].With effective chemotherapy and ART, there has been a better outcome reported with some studies showing similar outcomes as in HIV-negative patients [39,[42][43][44].
Patients with HIV are susceptible to chronic infection by oncogenic strains of HPV.In addition to causing cervical cancer, HPV also causes anal cancers and head and neck cancers [2,45].There is a high prevalence of HPV infection in HIV-infected women; almost one in four such patients have HPV co-infection with a high rate of intraepithelial neoplasia [46].
In this study, we observed a low prevalence of malignancies at the time of HIV diagnosis.We screened many successive patients at the ART and cancer center.It is yet to be observed whether HIV positive patients with malignancies have similar outcome as seronegative patients of the same stage.Drug interactions among chemotherapeutic agents and ART are also largely unknown.Though we followed HIVcancer patients for one-year post-enrollment, stage of the diagnosed malignancy, treatment given and response is unknown.With effective ART and improvement in immunity it is expected that incidence of malignancy will be reduced, follow-up studies will help understand natural history of this disease.
HIV-related cancers are preventable through vaccination and early and highly active ART [2,47,48].Screening of all HIV patients with clinical and radiological examinations should be conducted for early diagnosis and treatment of cancer.With this study, we have created a national HIVcancer registry that will help to understand the spectrum of HIV-related malignancy in the hospital and communitybased setting.
CONCLUSION
There is a low prevalence of cancer at the time of HIV diagnosis in North India.Similarly, HIV infection is rare in cancer patients.AIDS defining cancers remain most preva-lent, while Kaposi's sarcoma is rare.Follow-up of these patients is required to see whether these patients have similar outcomes as compared to seronegative patients of the same stage.
ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
The protocol was approved by ethics committee of all India Institute of Medical Sciences, New Delhi, India.
HUMAN AND ANIMAL RIGHTS
No animals were used in this research.All humans research procedures followed were in accordance with the standards set forth in the Declaration of Helsinki principles of 1975, as revised in 2008 (http://www.wma.net/en/20activities/10ethics/10helsinki/).
CONSENT FOR PUBLICATION
Written informed consent was taken from all subjects before participation in this study. | 2018-11-09T20:33:54.334Z | 2018-07-01T00:00:00.000 | {
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236177396 | pes2o/s2orc | v3-fos-license | Cosmological evolution with a logarithmic correction in the dark energy entropy
In a thermodynamical model of cosmological FLRW event horizons for the dark energy (DE), we consider a logarithmic corrective term in the entropy to which corresponds a new term in the DE density. This model of $\Lambda (t)$ in an interacting two-component cosmology with cold dark matter (DM) as second component leads to a system of coupled equations, yielding, after numerical resolution, the evolutions of $\Lambda (t)$, the Hubble $H(t)$, vacuum density $\Omega \_{\Lambda}(t)$, deceleration $q(t)$ and statefinder $R(t)$ and $S(t)$ parameters. Its results, compatible with an initial inflation and the current observations of the so-called"concordance model", predict a graceful exit of early inflation and the present acceleration and solve in the same time the age and coincidence problems. Moreover they account for the low-$l$ CMBR power spectrum suppression.
Introduction
The observations of SNe of type Ia [1] [2], showing that the universe has probably recently entered on a phase of acceleration, raised up a renewal of interest in the cosmological constant (CC) problem [3] [4].
Since then an increasing number of observational evidences for such an acceleration has been settled.This acceleration together with other cosmological indicators such as the flatness (k = 0) from the CMB anisotropies observed by Boomerang, Proxima and WMAP [5], the present values H 0 by the HST key project [6], Ω b0 and Ω m0 by observations of the light elements, SBBN and cluster baryon fraction from X-ray emission [7]), led to the so-called "concordance model" [8] based on the ΛCDM model.This model is the simplest improvement of the old standard cosmological model (including inflation).More precisely, the simplest theoretical candidate explaining this accelerated expansion is a cosmological constant Λ, or some component, called dark energy (DE): any term in the energy-momentum tensor which can produce the same negative pressure, such as a scalar field [9], called quintessence [10], or a bulk viscosity [11].
However, the re-introduction of the CC Λ leads to new intriguing puzzles.Particularly, we have to face the old CC problem: the discrepancy between the predictions of the effective Quantum fields theory concerning the large vacuum energy density and the tiny upper limits observed in astrophysics for the CC.Besides, it gives rise to a new issue, the so-called "coincidence problem", where one has to explain why the CC starts recently to dominate the matter.Assuming Λ = constant, it can only be solved by an unsatisfying fine-tuning.Finally, the introduction of Λ needs also to be conciliated with the inflation in the early universe.
In these conditions, we have to improve the ΛCDM model in order to solve these puzzling issues.
Therefore, we propose a Λ(t)CDM model, namely the simplest modification of the ΛCDM model which allows the best account of the theoretical expectations and the observed data.Some "ansatze" were already proposed to solve the CC problem before the discovery of the present universe acceleration (for a review, see [12]).By contrast, the present model, namely the event horizon Thermodynamics (ehT) model, is derived from a thermodynamical approach and not based on an ad hoc hypothesis for the law Λ(t).Such a phenomenological model is an indispensable preliminary to outline the main laws that any further treatment at the statistical or/and quantum level will have to reproduce, as for example the Stefan's law has guided the Planck law's advent for the black body radiation.We thus proposed a model with a variable vacuum energy density [13] [14], based on the generalization of the Thermodynamics of the black hole event horizons (BH e.h.) [15] to the Friedmann-Lemaître-Robertson-Walker event horizons (FLRW e.h.) [16].This model allows to reconstitute the entire evolution from the early inflation to late acceleration (for another approach, see also [17]).
In this article, we extend this phenomenological model by introducing a logarithmic correction in the Λ-entropy.Such a corrective term in the entropy was recently computed for the entropy of any thermodynamical system and applied to Black Hole by S. Das et al. [18].This correction due to statistical fluctuations around an equilibrium state has an universal character.As the cosmological FLRW e.h.presents notable thermodynamical analogies with the BH e.h.(albeit some differences [19]), we examine the effects of the introduction of this new term in the Λ(t)CDM model.It appears that the derived expression for Λ(r), with r the radius of the event horizon (e.h.), can then be naturally interpreted as a renormalisation relation and allows to describe the main observed features of the evolution of the universe.In this scheme, the obtained universe is initially submitted to a local phase of de Sitter inflation (Ω Λ (0) = 1).The vacuum density parameter begins to decrease and there is a graceful exit of inflation.The parameter Ω Λ passes through a small minimum, then grows again, which allows to solve the "coincidence problem".It tends asymptotically to a de Sitter universe.At any instant t, H(t) and Λ(t) are decreasing functions.
The paper is organized as follows.In Section 2, we set the basis of the general relativistic framework in which our model for Λ(t) will be developed.In Section 3, we present the ehT model for the vacuum (or DE) energy density.
Then in Section 4, we examine the influence of the entropy's correction term in the vacuum energy density and its relevance in the renormalisation approach.In Section 5, we deduce the equations of evolution for H(t) and r(t), and present in section 6, their numerical resolution, in particular, the graphic of Ω Λ (t).Section 7 is devoted to a brief conclusive summary.
An interacting two-component model
Let us briefly recall the minimal assumptions underlying our model.In order to set our notations, let us first write the Einstein equations: with χ = 8πG c 4 and Λ 0 a positive constant (the GR bare cosmological constant).Only for this choice of coefficients in Eq. ( 1), the Bianchi identity ∇ β T αβ = 0 is satisfied [20] [21].
Assuming a perfect fluid of energy density ρ T and pressure P T , the energymomentum tensor is given by T αβ = ρ T u α u β − P T ∆ αβ with ∆ αβ := g αβ − u α u β , and the index "T " means "Total".
In accordance with the WMAP observational results of the CMB's anisotropies [5], the metrics is taken to be the spatially flat (k = 0) FLRW metrics.With these assumptions, Equation (1) reduces to with As Λ vac := χρ 1 with ρ 1 variable, Λ varies.In the following, we refer to Λ as the " variable cosmological constant".It can be considered as an "effective" CC.The corresponding dark energy (DE)-component has the energy density : ρ Λ = Λ χ .We do not make any further assumption on a separated energy conservation for each component.In the old standard cosmological model, this assumption is made only for each era of "domination" of one component (e.g."era dominated by the radiation", "era dominated by the matter", today "era dominated by the DE", or in the very early universe "era dominated by the inflation (or the inflaton)").If such an assumption can seem reasonable for these domination eras (and so appears more as an approximation), it is not the case for the transient periods, as for instance the very recent epoch of transition from dark matter (DM) to DE domination.
Anyway, the complete energy conservation due to the Bianchi identity is more general than the separated conservations, which necessitate some supplementary assumption.For example, in the Λ(t) and quintessence models, a separated conservation law for each component implies a "variable" equation of state.By contrast, the present model assumes the equations of state in a thermodynamical sense, i.e. given once for all, with an interaction between the two components.This kind of approach with components interaction has already been considered (see e.g.[22][23]).
Let us sketch the rough scenario of the universe evolution.Initially, setting ρ 1 = ρ Max and ρ 2 = ρ 2 min ≃ 0, the universe undergoes a de Sitter inflation, with Λ M = Λ vacM − Λ 0 > 0. Then ρ 1 decreases or equivalently, Λ decreases with the universe expansion.In the same time, ρ 2 is increasing.Finally, ρ 1 tends to Λ 0 or equivalently Λ tends to Λ min ≃ 0 ,which is again a de Sitter evolution.Today, we observe a very small value for Λ = χρ 1 − Λ 0 with a very large vacuum energy density ρ 1 given by quantum field theory (today ρ 1 is of the order of Λ 0 ).For a reasonable (Planck) largest cut-off, we obtain Assuming a variable cosmological "constant" solution (as considered here), this scheme appears as an hardly intelligible fine-tuning.It is the so-called "old problem" of the cosmological constant (CC).Besides, the recent domination of the vacuum energy by dust constitutes the "new puzzle" of the CC (or "coincidence problem").At the beginning, ρ 2 represents the energy density of the radiation and the ultrarelativistic matter.For sake of simplicity, we shall only consider in the following (section 6) the non relativistic matter (γ = 1).
We propose a phenomenological model of this DE or, equivalently, this variable cosmological "constant" (or this vacuum energy density) able to produce the main properties of this scenario.More particularly, the two problems of the CC are solved in the framework of our model where Λ runs from a large initial value Λ M (a huge inflation in the very early universe) to a weak final constant Λ m which yet starts again to dominate, leading to "the coincidence problem".In the two cases, the same DE, with a density Λ is used to explain the universe acceleration.This DE is negligible or dominates, depending on the considered period, and its density decreases all along the universe expansion.
The variable DE density Λ = χρ Λ is the first component of the two perfect fluids constitutive of the universe.In the de Sitter space-time, this cosmological "constant", or vacuum energy density, obeys the equations of state [15][24]: where P Λ and T Λ represent its (negative) pressure and its temperature respectively, h is the Planck constant, c the light velocity and k the Botlzmann constant.Each thermodynamical variable is linked to the proper radius r of the event horizon at a given instant by the relations We can also introduce the horizons number density n Λ defined as usual in Thermodynamics by the inverse of the specific volume of the horizon [13] Introducing the expression (6) of the temperature in the preceding relation, we obtain a third equation of state: Equation ( 9) together with Eq. ( 4) and ( 5) define a local equilibrium state for the DE component in the de Sitter spacetime.Let us now consider the same DE component in the spatially flat FLRW space-time.The three thermodynamical equations of state (4), ( 5) and ( 9) have to remain valid in FLRW spacetime because the thermodynamical equations of state of any actual component (e.g.P = ρ 3 , or the Stefan law ρ = σT 4 , for the equilibrium radiation) are always supposed to be independent of the chosen spacetime.Besides, the definition (8) of n Λ remains also valid in the (spatially flat) FLRW spacetime.Consequently, from ( 8) and ( 9), we find that this DE component is associated to the FLRW e.h.via the relation (7).Using (7), ( 8) and ( 9), we obtain the expression (6) for T Λ and (5) for Λ in the FLRW spacetime too.
Relation (7) has also been independently derived from holographic principle [25].Albeit the choice of r is not necessarily a priori given in the holographic approach (e.g. the Hubble horizon H −1 can be chosen instead [26][27], or the particle horizon r p as well [28]), it is the unique choice in this framework that leads to an admissible equation of state [29] and which is strongly supported by the SnIa observational results [30] [14].
Differentiating the definition of r with respect to the time (e.g.see eqs.(3.4) and (3.6) in [13] or ( 9) and ( 10) in [14])) yields the relation where H is the Hubble parameter and the dot means the time derivative.
In order to set a complete local thermodynamics, we need to determine the entropy density ns Λ where s Λ is the specific entropy.To reach this goal, we use the Gibbs equation which at the specific level is given by: where is the specific energy, v Λ ≡ 1 nΛ the specific volume.With Eq. ( 4), we deduce
Fluctuations and renormalization
In a recent article, a logarithmic corrective term in the expression of the entropy of any thermodynamical system was derived by S.Das et al. [18].This correction due to small thermal fluctuations around an equilibrium reads for BH entropy: where α ′ is a constant.Introducing the same correction in the cosmological eh model, the expression of the specific entropy becomes where L P is the Planck length (defined by L 2 P := χhc = 4l 2 P , with l 2 P := Gh c 3 in the I.S. units), α is a constant of dimension [r 2 ] and C an arbitrary constant.As usual, the sign of the entropy is negative for the cosmological horizon (e.g.[16] [13]).As noticed by S.Das et al. [18] and Major and Setter [31], when there is not conservation of the number of particles in an open sytem, the sign "-" before the α-term has to be replaced by a sign "+".This is precisely the case here for our Λ-component, which is not separately conserved.
Let us split the constant C into where r 2 is associated to the corrective term and r 3 to the expression of s Λ without correction.The expression of s Λ becomes ). ( 16) The constant factor α := r 2 1 has the dimensions of a surface.Let us remark the omission of the Bekenstein factor k , ∀r ≥ r(0) where r(0) is the initial event horizon (see Section 6).The simplest choice for r 2 and r 3 is given by r 2 = r 3 = r M , where r M is the maximum radius (possibly infinite).Hence, s Λ is always positive with s Λ = 0 when r = r M .For r = r M − ǫ with ǫ ≪ r M , we deduce r 2 3 − r 2 ≃ 2ǫr M and 2r 2 1 ln( rM .A corrective term is only obtained if r 1 ≪ r M as will be verified in Section 6.Finally, for r = r m , we have (r 1 ln( rM rm ).For r 1 ≪ r M , the ln-term is a corrective term and tends to s → s M = 8πk It follows that r 1 has not necessarily to be very small with respect to r but only with respect to r M .
Let us examine the effects of such a new term in s Λ (14) on the expression of Λ .
Differentiating Eq.( 14) for s Λ with respect to r and introducing this expression into Equation ( 12) with ( 6) and ( 8), we obtain a differential equation for Λ and r, which yields after integration Λ = 3 r 2 (1 + ( where the boundary conditions are set such that Λ tends to 0 when r → ∞.This relation is an extension of the expression (7) of Λ when thermodynamical fluctuations are taken into account.
It is worth noting that this relation is strongly akin to a renormalization relation (e.g.see first Eq.(3.3) in [32], or Eq. ( 33) in [33], see also [34]).In the same way, Padmanabhan ([35], Eq.( 19)) and Myung ([36], Eq. ( 14)) write a renormalized vacuum energy density by stressing a hierarchical structure of the form: where the first term is interpreted as the energy density necessitating to be renormalized, the second term as the vacuum fluctuations and the third one as the de Sitter thermal energy density (the dimensional constants and the factor 3 were restored for convenience).Padmanabhan and Myung have introduced this form in the case L 1 = L P , here we use an intermediate scale L 1 greater than L P .A value of L 1 accurate to observational results will be determined in Section 6. Setting Λ 1 := 3 , and multiplying the relation ( 19) by χ, we obtain: Assuming Λ 1 to be the bare CC Λ 0 of the G.R. (eqs.( 1) and ( 3)), this expression is of the same form as the expression ( 18) derived in our model.Let us discuss an intriguing point.The second term in the r.h.s. of (18) has by construction to be interpreted as a term of fluctuations, whereas it is the first one in the r.h.s. of (20) which is interpreted as fluctuations by Padmanabhan and Myung.Let us consider the solution to this apparent discrepancy.Relation ( 18) is derived from considerations concerning fluctuations by use of a Taylor expansion.It can be considered in the framework of renormalization as the truncature of a convergent series up to its first terms (see [33] after their Eq.( 35)).In particular, Eq. ( 18) is valid for the two following limits: For r ≫ r 1 , Eq (18) represents the first terms of a Taylor expansion and Λ tends to (7) which itself tends to its minimal constant limit Λ m = 3 r 2 M when r tends to its maximum limit r M (IR cutoff).For these large values of r, we are concerned with the inflation undergone today by the universe.Therefore the fluctuations we considered in ( 14) and ( 18) are these recent fluctuations.In this limit, the model ( 7) was studied in [14].
For r ≪ r 1 , Eq (18) can be rewritten as a Taylor expansion for little values of r and Λ tends towards another de Sitter state when r reaches its minimum limit r m (UV cutoff).For these values of r, what is concerned is the maximum value of Λ, during the inflation formerly undergone by the very early universe: . Padmanabhan and Myung consider these early fluctuations in (19).Considered as a renormalisation relation, Eq. ( 18) is also valid for any intermediate value of r and especially when r ≡ r 1 .Assuming the given expression (18) for Λ, we derive now the evolution of the universe.
Evolution equations for H and r
In a spatially flat FLRW cosmology (k = 0, metrics signature + ---), the field equations (2) for a two-component perfect fluid with the variable CC as first component are given by • From now on, the index 2 for the second component of the fluid is understood.Equations ( 22) and ( 23) lead to By defining (24) and ( 10) become where the expression (18) of Λ has been introduced.Eqs ( 26) and ( 27) constitute a system of two coupled first order differential equations satisfied by {Y (t), H(t)}.We introduce the dimensionless variables where Y 1 and H 1 are two, a priori independent, arbitrary constant scales for Y and H respectively.We also define the dimensionless time τ Hence, ( 26) and ( 27) become with the dimensionless ratio of the scales Y 1 and H 1 and The apostrophe denotes the derivative with respect to τ .Finally, we set where Λ 1 is an arbitrary constant scale for Λ.With ( 18), ( 34) yields where is the dimensionless ratio of the scales Λ 1 and Y 1 .We deduce which furnishes a scale for the dimensionless CC density parameter Ω Λ defined as or, with ( 34)-( 37) and (28), Finally, setting τ = 0 Ω Λ (0) = 1, with (39) gives Now it only remains to fix y(0), besides the parameters X 1 , α 1 , n , l 1 and γ.
Results
We numerically solve the equations ( 30) and ( 31) and obtain the solutions y(τ ) and h(τ ), for the initial conditions (40) with By choosing r 2 = r 3 = r M ≃ 10 26 m (see Section 4, Eq. ( 17)), the condition ( 17) is satisfied for any r ≥ r(0) ≃ 10 −11 m (this last value will be derived in the following).
As the current values of the observational data (Ω Λ (τ 0 ) = 0.7 , H(τ 0 ) = 72 kms −1 M pc −1 ) of the ΛCDM model [37][38] are model-dependent, we use the values derived in the framework of our Λ(t)CDM model from the studies of SnIa observational constraints [14] where τ 0 represents the present instant.Let us emphasize that the use of the values derived from the ΛCDM concordance model would only marginally modify the following results.
The different scales H 1 ,Y 1 , and Λ 1 can be deduced from these two values (42).We deduce the instant τ 0 from the plot of the function Ω(τ ) Setting this value in y(τ ) and h(τ ) and using (35), we deduce 30) and ( 31) with the initial conditions ( 40) and ( 41).
It reaches its present day value 0.63 at τ 0 (43) or t 0 = 4.88084 × 10 17 s = 15.4664Gyr.This value is in good agreement with the observational results derived from the "concordance model" ( [38] Section 2.5.p.3) and allows to avoid the age crisis (see e.g.[39]).With a linear extrapolation of H(τ ) during τ 3 − τ 0 , we roughly evaluate the redshift, namely z(τ 3 ) ≃ 1.01826, where the acceleration begins again.This value is in good agreement up to the previous approximation with the exact value of the transition redshift z T = 0.965 found in [14].
In the same time, the "coincidence problem" is resolved.The date of the coincidence can be defined by different ways : for instance by the instant t m (see above), i.e. the instant from which Ω Λ grows again, or by the instant t 3 (see above) when the universe inflates again, or even, more recently, by the instant t 4 = 3.61505 × 10 17 s = 11.4554Gyr when Ω Λ = 1 2 = Ω m .In the following, we use this last definition.
The long period about from 6.39 × 10 −40 s to t 4 ≃ 2.8 × 10 17 s when Λ does not dominate, i.e. when Ω Λ < 0.5, allows to maintain the standard model and its afferent results all along this period.
Another interesting result to be noted is the shape of the curve Ω Λ (τ ) ( fig. 1) which presents a symmetry with respect to τ = 0 , suggesting a period for Ω Λ (τ ).Each curve ( Ω Λ , h, y, λ, H, Λ) is (quasi-) symmetrical with respect to t = 0.The functions Λ(τ ) and H(t) decrease (increase resp.) when τ ≥ 0 (τ ≤ 0 resp.).There is a "big bounce" on an inflationary space at t = 0, with a period that is stretched over more than twice t 0 and another one at a very large time on a quasi-de Sitter space.We studied above the results given for τ ≥ 0 only.The first inflation is "strong" (at t = 0) when Λ is maximum, the second one is "weak"(at t → ∞) when Λ is minimum (possibly→ 0).Today, Λ is on the way to its minimum (maybe zero), and the universe is dominated by the small cosmological constant (37).
We can also obtain the R(t) ( or "jerk" j(t) ) and S(t) "statefinder" parameters [40] and q(t) deceleration parameter for the model with for their present day values The positive sign of R 0 indicates the present overinflation, (either increasing of inflation, or trend towards inflation).One can also evaluate R in the early period of deflation (i.e.desinflation).For instance at τ = 0.509275 when inflation was present, though decreasing, because Ω Λ (τ = 0.509275) = 0.4 > 1 3 , we obtain R(τ = 0.509275) = −1.414< 0.We can yet obtain R < 0 for Ω Λ < 1 3 during some time after the end of the initial inflation.For example at τ = 0.51151 when Ω Λ = 0.2 < 1 3 , we have yet R(τ = 0.51151) = −0.20005.The instant when R changes of sign is given by τ 9 = 0.51204075 or t 9 = 3.07026 × 10 −39 s.Since then, R is positive.
The comparison of the "kinematical" values (53) to observations (SNAP supernova data ) could also be a test for our model (see the remark of [40], Section 4) and would allow the measurement of the production source of CDM [41] which is constrained by the statefinder parameters and the "snap" parameter Finally, this model also supports the low-l CMBR power spectrum suppression suspected in the WMAP data.Indeed, the event horizon plays naturally the role of a dynamical IR cut-off translatable to a wave number cutoff [42] [43] as for the holographic model [25] [30].Applying the same procedure as [42] [43] for the Dirichlet boundary condition λ c = 2r 0 , we roughly evaluate the influence of this cutoff.In the integrated Sachs-Wolfe (ISW) effect, the IR cutoff appears as the lower limit of the comoving momentum which truncates the summation giving the coefficient C l of the power spectrum.For a power law spectrum with a spectral index n = 1 (Harrisson-Zeldovich), we have for the low l [42]: where j l are the spherical Bessel functions.In flat spacetime, the multipole number l is given by l = k l (η 0 − η * ) (55) where (η 0 − η * ) is the comoving distance to the last scattering surface.The definition of the conformal time in flat space η = − r a with the Dirichlet limits condition leads to l c = π((1 Taking the current value z * = 1089 [5,44] and using (18), we obtain r * r0 can be evaluated with Λ * and Λ 0 from Table 1 and we find : l c = 5.15.For the value Λ ′ * of the end of the recombinaison, we get : l ′ c = 6.76.These values are in the expected range.
Conclusion
From the FLRW e.h.entropy with thermal fluctuations (Eq.( 14)), we obtained the corresponding law for the cosmological constant Λ(r) as a function of the event horizon radius r (Eq.( 18)).We used the striking similarity of this last expression (18) with a renormalization relation (Eq.( 20)) to reinterpret (18) in terms of such a (truncated) renormalization relation (20).
Using this Λ(r) law in an interacting two-component Λ(t)CDM cosmological model in a FLRW spacetime, we derived a system of coupled equations for H(t) and r(t) (Eqs.(30) and ( 31)).Through a numerical resolution (Section 6), we obtained the curves of evolution of Ω Λ (figs. 1 to 3), the table 1 of the values of the main cosmological parameters (H, Ω Λ ) at different times in the evolution of the universe, and the evolution of the deceleration and statefinder parameters.
The event horizon thermodynamical model presented here allows us to account for most of essential features of the cosmology such as the value of the age of the universe,∼ 15.5Gyr (safe from the age problem [45]), the ratio Λ[0] Λ[τ0] ∼ 5 × 10 112 , as well as the exit of inflation after about t 2 ≃ 23000 Planck times, the existence of a minimum ∼ 5 ×10 −20 for Ω Λ attained after about 3 ×10 −11 s, the coming back of the inflation (after about 7.24Gyr from the beginning), followed by the recent coincidence, at 11.4Gyr, with the matter density, but also allows us to address the entropy problem, and the creation of (dark) matter from the vacuum energy.Besides, the model does not perturb the results of the old standard model for the period going from 6.39×10 −40 s≃ 10000 Planck times to 2.87 × 10 17 s = 11.4Gyrall along which the DE is subdominant ( Ω Λ < 0.5 ).Finally, it can render an account of the low-l CMBR power spectrum suppression, for an angular scale corresponding to multipole numbers lower than l c ∼ 7.
l 2 P
in most papers by choice of the Planck units (h = c = k = G = 1).The expressions of r 2 and r 3 are constrained by setting r | 2014-10-01T00:00:00.000Z | 2006-03-13T00:00:00.000 | {
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5321093 | pes2o/s2orc | v3-fos-license | The hidden symmetry algebras of a class of quasi-exactly solvable multi dimensional operators
Let $P(N,V)$ denote the vector space of polynomials of maximal degree less than or equal to $N$ in $V$ independent variables. This space is preserved by the enveloping algebra generated by a set of linear, differential operators representing the Lie algebra $gl(V+1)$. We establish the counterpart of this property for the vector space $P(M,V) \oplus P(N,V)$ for any values of the integers $M,N,V$. We show that the operators preserving $P(M,V) \oplus P(N,V)$ generate an abstract superalgebra (non linear if $\Delta=\mid M-N\mid\geq 2$). A family of algebras is also constructed, extending this particular algebra by $\Delta -1$ arbitrary complex parameters.
In the case of operators of one real variable acting on a scalar function, the possibilities of finite dimensional invariant vector spaces are rather limited [4].
Up to a change of the variable and a redefinition of the function, the vector space can only be the set P (N) of polynomials with degree less than or equal to N.
The relevant operators are the elements of the enveloping algebra of sl(2) whose generators are suitably represented by three differential operators [1,4]. The QES equations they define therefore possess an sl(2) hidden symmetry.
When the number of variables or (and) the number of components of the function is (are) larger than one, the number of possible invariant vector spaces and hidden symmetry algebras increases considerably. The scalar QES operators in two variables were classified in [5,6]. Seven inequivalent spaces of functions appear to be possible. Correspondingly, the hidden symmetry algebras can be of several types, e.g. sl (2), sl(2) ⊗ sl(2), sl (3). A few cases of this classification generalise easily to operators involving an arbitrary number of variables. In particular, the case labelled 2.3 in ref. [6] can be extended to the space P (N, V ) of polynomials of maximal degree less than or equal to N in their V independent variables. The related algebra is sl(V + 1).
The construction of the matrix operators in one variable preserving the direct sum P (N 1 ) ⊕ . . . ⊕ P (N k ) has also been considered [7,8]. These operators are closely related to graded algebras. As an example, the case P (N) ⊕ P (N + 1) is related to the graded Lie algebra osp(2, 2) [9].
The purpose of this paper is to classify the operators preserving the vector The dimension of P (N, V ) is given by The set of linear differential operators preserving P (N, V ) can be perceived as the enveloping algebra generated by the following operators These (V + 1) 2 independent operators fulfil the commutation rules of the Lie algebra gl(V + 1). Acting on the finite dimensional space P (N, V ), they lead to an irreducible representation of this algebra. The commutation relations are Within the representation (4), the Casimir operators of gl(V + 1) have the values where C is any operator which commutes with all J's, satisfy also the relations (5). For instance, this is the case for the (V + 1) 2 − 1 independent operators (sinceC 1 ≡ 0) which form an irreducible representation of sl(V + 1) when acting on P (N, V ). The usual form [1] of the of sl(2) generators is recovered for V = 1. These operators play a major role in the topic of quasiexactly solvable equations.
More generally, an element, say A, of the enveloping algebra constructed over the J b a (or theJ b a ) is a quasi-exactly solvable operator preserving P (N, V ). That is to say that the spectral equation admits C V +N V solutions. Recently, the Calogero and Sutherland quantum hamiltonians were shown to be expressible in terms of the operators J b a [10], this result reveals the hidden symmetries of these models.
Operators preserving P(M,V) ⊕ P(N,V)
We now put the emphasis on the 2×2 matrix operators which preserve the vector Without loss of generality, we assume the integer ∆ to be non negative. In order to classify the operators preserving (11) we define a list of generators. First the "diagonal" generators that we choose as for 0 ≤ a, b ≤ V . They are built as a direct sum of two operators of the type (4)-(7), translated by (7) in such a way that that J 0 0 (N, ∆) is proportional to the unit matrix. The interest for this translation will appear later.
The "non diagonal" generators naturally split into "Q operators", proportional to the matrix σ − (as usual σ ± = (σ 1 ± iσ 2 )/2). and "Q operators" proportional to the matrix σ + . It is convenient to write them by using a multi index [A] ≡ a 1 , a 2 , . . . , a ∆ . For later convenience we also define [Â i ] as the set [A] where the index a i has been removed. We choose the non diagonal generators respectively as where δ represents the overal degree in x 1 , . . . , x V of the monomial x a 1 . . . x a ∆ and where the scalar operators q [B] , fully symmetric in their ∆ indices b k , are defined by The operators Q [A] (and similarly the Q
This result (whose demonstration follows the same lines as in the scalar case [4]) allows to write formally all the operators preserving (11). However, in order to classify these operators, it is useful to set up normal ordering rules between the generators. In particular, these rules allow to write any product of operators (the enveloping algebra) in a canonical form, e.g. as a sum of terms where, in each term, the Q operators (if any) are written on the left, the Q operators (if any) on the right and the J operators in between. As we show next, such rules exist for the operators (12),(13),(14).
Normal ordering rules
The operators (12) obey the commutation rules (5) and assemble into a reducible representation of gl(V + 1) when acting on the vector space (11). The dimension of it is By construction, the operators under the adjoint action of the generators J b a (N, ∆). More precisely, we have The explicit form of the generators leads to the value k = ∆. The first Casimir constructed with (12), i.e.
plays the role of a grading operator : The product of any two operators Q (and separately of two Q's) vanishes, hence also their anticommutator The evaluation of the anti-commutator {Q, Q} is more involved. Its form can be guessed from the covariance under gl(V + 1), from the symmetries of Q and Q in their indices and from the fact that the anti-commutator involves at most derivatives of the order ∆. It is therefore likely that the anticommutator {Q, Q} should be expressed as a combination of the tensors where the operator S[.] denotes the sum over all permutations of all indices entering in the argument [.]. After calculation, we found the following relations between (12), (13), (14), and the parameters α k are numbers which are uniquely determined by the polynomial equation As a consequence of (24), the right hand side of (23) is an even (resp. odd) polynomial in the operators J if ∆ is even (resp. odd). We would like to stress that this particularly simple expression is due to the labelling of the generators and to the translation used in (12). A priori, the undetermined coefficients α k could be 2 × 2 diagonal matrices.
The non vanishing parameters α k appear only for k = ∆, ∆ − 2, ∆ − 4, . . . and read as follows for the first few values of ∆ :
Abstract algebras
We now investigate the possibility that the operators (12),(13),(14) represent the generators of an abstract associative algebra. We will see that there are two types of such algebras that we note generically A(V, ∆) and B(V, ∆). A few cases are known to coincide with Lie algebras [9,7] A(1, 0) ≃ sl(2) ⊗ sl(2) (26) For ∆ > 1, A(1, ∆) corresponds to a non linear superalgebra [7]. The algebra A(1, 2) was treated in great detail in [11]. Here we want to move away from the case V = 1 in order to access the hidden symmetries of the operators preserving (11) in general.
With the aim to promote the normal ordering rules of the previous section into a set of relations defining an abstract associative algebra, we first note that the operators J b a (resp. Q, Q) should naturally be interpreted as the bosonic (resp. fermionic) generators of the algebra (this refers of course to the most natural choice of the commutator or of the anti-commutator used to exchange the order between these generators). Therefore, we expect some graded algebras to come out. However, it is well known that the knowledge of a particular representation (here (12), (13), (14)) is not sufficient in general to infer the whole algebraic structure : the Jacobi identities are not automatically fulfilled. In the present case, the identities which are not obeyed are those involving a {Q, Q} (or a {Q, Q}) anticommutator (remember that they vanish). Although we can try to modify the whole set of (anti) commutation relations, we limit our research of the underlying abstract algebras in relaxing only the relation (21). In order to present the way to modify it, a few notations are worth introducing.
Due to its symmetry in the indices [A], the representation defined by the Q [A]
(and similarly by the Q [A] ) corresponds to a Young diagram with one line of ∆ boxes. The products assemble into a representation of gl(V + 1) under the adjoint action of the operators J. This representation can be decomposed into irreducible pieces. The symmetry of Q is such that the irreducible representations appearing in the decomposition of (28) correspond to the Young diagrams consisting of two lines with total number of 2∆ boxes. When applied to the anticommutators The Young element S Y corresponding to this Young tableau reads with With these notations, we are ready to describe the conditions we choose for the anticommutations of two Q operators . We restrict them by imposing In the case ∆ = 1, the relation (33) just implies that all anticommutators of two operators Q vanish (and similarly for two Q). The algebra A(V, 1) is linear, it coincides with the Lie superalgebra denoted spl(V + 1, 1) in the classification [12]. If V = 1 one recovers the algebra osp(2, 2) (remember the equivalence of osp(2, 2) with spl(2, 1)).
for any permutation σ of the 2∆ indices. The total number of independent constraints is not as easy to find as in the case 1; we obtained it in two particular cases and
Exceptional solutions
It should be stressed that associative algebra could also exist with the same structure as above, i.e. with (5), (17), (18), (33) and (23) but where some of the parameters α k are 2 × 2 diagonal matrices. That is to say they depend on the Casimir operators constructed with the gl(V + 1) subalgebra generated by the operators (12). We could not solve this problem for general values of ∆ but we studied completely the cases ∆ = 1, 2, 3. We obtained one new solution in the case ∆ = 2, V = 1, p = 1. The most general relation for {Q, Q} which is compatible with associativity depends on four parameters. It is of the form where β is the additional parameter while C 1 , C 2 represent the Casimir operators (6) computed in the representation (12).
Proof of proposition 2
Let us come to the proof of proposition 2. The relevant Jacobi identities are The application of S Y (see (31)) to this equation and the use of (33) lead to the necessary and sufficient conditions Let us first suppose that the anticommutation relations of Q and Q take the form rather than the more general one (23). Using (42) together with (17), and separating the terms, say X ′ , which come out without k (through (17)) from the terms, say kY ′ , which come out linear in k, the expression (41) becomes In (44) and (45) Since X ′ + kY ′ has to be zero identically, every coefficient of every (independent) operator entering in it has to be zero. This allows a great simplification in the necessary and sufficient conditions.
• The terms labeled . . . in (44) and (45) can be forgotten altogether. Indeed, the terms where the Q's are in the first position are independent of the terms where they are not.
• The symmetry on the [B] can also be eliminated. Every term, for every value of the indices b k , has to vanish on its own.
• Let us introduce the notations for some arbitrary fixed value of b 1 and where c k is absent as a lower index and b 2 , . . . , b ∆ have also fixed values..
With these simplifications, the condition X ′ + kY ′ = 0 reduces to the necessary and sufficient condition X + kY = 0 with and The operator X + kY is composed of exactly two types of independent operators.
They can be written canonically as Indeed : • The indices of the Q operator have to be completely symmetrical. Hence they must belong to the first line of the Young tableau and by symmetry of S Y can be chosen as the [A] set.
• If the index in W is taken in the first line, it can be chosen to be c ∆ . This is due to the fact that any of the indices (except the those belonging to the set [A] which already pertain to the Q) in the first line is equivalent by symmetry to any other in the first line. The remaining indices for the V can the be chosen in any order and for example in the natural order.
• If the index in W is taken in the second line, it can be chosen to be c 1 .
Indeed any of the indices in the second line is equivalent by symmetry to any other in the second line. The remaining indices for the V can again be chosen in any order and for example in the natural order.
The remaining task is to extract in X and in Y the number of times the operators O 1 and O 2 occur. This is a rather delicate operation in terms of the symmetries involved. Let us call X i (resp. Y i ) with i = 1, 2 the coefficient of the operator O i in X (resp. Y ) With these notations the condition X + kY = 0 becomes equivalent to To now compute these four coefficients, we will make use of the fundamental theorem of group theory which states that, if P is any permutation of the elements
S[A] = P S[A] = S[A]P (53)
Computation of X 1 Let us rewrite X as Hence, from the 2 2p terms in E x we can restrict ourselves to the transposition (a i , c i ) which comes with a minus sign. At the same time i can be restricted to the range 1, 2p. The sommation on j then has one term with j = i. For the terms with j = i, the c j in Q has to belong to the set j = 2p + 1, . . . , ∆ in order to be able to replace it by an a by the first symmetry operator S in (54). Hence the restricted part of X, sayX, is composed of two pieces, sayX α andX β , Using (53), we easily conclude that in X α the following coefficient appears The following coefficient then appears The extra factor as compared to the coefficient coming out of X α is due to the extra sommation over j.
Summing up the results (59,57), we find Computation of Y 1 The same technique applied to Y 1 is much simpler as the relevant term in E x is simply the identity. Hencê Collecting again the factors, we find Computation of X 2 The relevant term in E x is again the identity and the reduced part of X which can lead to a term of the form O 2 (51) iŝ The sommation on j has to be restricted to those values in the first line of the Young diagram. A transposition (a i , c j ) can then be factored out of S[A, c 2p+1 , . . . , c ∆ ] as well as a transposition (a i , c ∆ ), i.e. in total a cyclic permutation (a i , c ∆ , c j ) . We find Collecting the factors as usual, we find The extra factor (∆) comes from the sommation over i.
Computation of Y 2
In this last case the relevant term in E x is again the identity and the reduced part of Y which can lead to a term of the form O 2 (51) iŝ Collecting the terms, we find We can now summarize the conditions coming from (52) : 1. The conditions coming from the Jacobi identities are thus two in number if p = 0 (the condition for the operator O 1 to be defined) and if ∆ = 2p (the condition for 0 2 to be defined). These conditions are incompatible.
2. More generally, the anticommutators of two Q's cannot vanish for more than one representation. The conditions coming from the Jacobi identities involving two Q and one Q also lead to exactly the same conditions. Hence the anticommutators which are chosen to be zero for the anticommutations of two Q's on one side or of two Q's on the other side must be identical.
Summary and Conclusions
The operators preserving globally a system of two polynomials in V variables (V ≥ 1) and of degrees N and N − ∆ (∆ ≥ 0) respectively can be constructed as the elements of the enveloping algebra of certain superalgebras.
In this paper, we have constructed a family of such associative, non linear superalgebras. Any of these algebras is specified by V , by ∆ and by a set of ∆ + 1 complex numbers noted α k with k = 0, 1, . . . , ∆. They are generated by (V + 1) 2 (bosonic) operators All the supplementary conditions on the products of the operators Q (and of the operators Q) necessary to guarantee associativity (equivalent to the generalised Jacobi identities) are given by our proposition 2. | 2014-10-01T00:00:00.000Z | 1997-01-13T00:00:00.000 | {
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56230822 | pes2o/s2orc | v3-fos-license | Development of e-learning for English class
In the twenty first century, teachers are required to have a digital literacy skill. They must be able to integrate technology in learning process. It was already conducted by a teacher in one of public junior high schools in Jakarta. She searched the materials from the internet but she had a problem to adjust the learning materials to her students’ needs and characteristics. Therefore, this study was undertaken to explore deeply how to develop e-learning in English class based on her students’ needs and characteristics. This study employed research and development methodology. The participants were an English teacher and the first graders of junior high school. The result showed that this e-learning used PHP, Framework Bookstrap and MySQL. The e-learning also used Adobe Flash and Adobe Illustrator to make some animated videos. It consisted of listening, reading, and grammar which the topic focused on descriptive texts comprising some themes referring to 2013 curriculum. Those themes were people, animal, thing, place, and occupation. The development of e-learning deserved to be implemented after it was validated by the expert, evaluated by the teacher and tested to the students. It is expected to contribute as supplemental learning media in English learning process.
Introduction
The proliferation of information communication and technology grows rapidly nowadays.That phenomenon can be seen from the high use of cell phone and the internet around the world.3.15 billion people use the cell phone and 3 billion people use the internet [1,2].Internet is highly demand in our life [3].The use of the internet in Indonesia increases 100 percent each year [4].It offers all information needed around the world [5].It affects the education world [3].It influences the teaching and learning process in the class [6,7,8].When the previous observation was conducted, one of teachers at public junior high school in West Jakarta, Indonesia used the laptop and projector as well the speaker when teaching English in the class.She taught her students using video downloaded from the internet.Sometimes, she used the online learning such as Edmodo to teach her students.She was a portray of a teacher in the twenty first century that she had the digital literacy.The teacher needs the ability to use the information and communication technology in their classroom such as the internet use [9].The Ministry of Education and Culture of Republic of Indonesia also determine the national literacy movements.One of them is the digital literacy.It means that Indonesian government has been supporting schools to implement the digital literacy.Nowadays, they applied computer-assisted test for the national test in some schools for some areas around Indonesia.
Based on the previous interview, even though the teacher integrated the technology in teaching English in the classroom, she had problem to adjust the material she found from the internet to meet her students' needs and characteristics.She also had difficulty to find resources such as listening.Although, information and technology centre of the Ministry of Education and Culture provides the e-learning in website (http://edukasi.kemdikbud.go.id/), it cannot be accessed because it always comes up "file or directory not found' command.The e-learning also has not been much more developed in Indonesia [10,11].To address those problems, this study is important to undertaken by developing English e-learning.There are some various terminologies used addressing the e-learning: web-based learning, online learning, computer-based learning or computerbased instruction.Morrison and Aparicio et al. call it online learning [12,13].Even though it has different names, it is used in teaching and learning process involving the technology such as computer network (internet/ intranet) or multimedia [13,14,15].It has some functions: supplementary and substitution functions.Supplementary function means that it can be combined with the conventional learning which emphasizes on face-to-face learning in the classroom.It is used to enrich the learning process.On the other hand, substitution function means using e-learning fully in learning process [11].A study showed that the e-learning and traditional education are still used together.They complete each other in order to reach the educational result [16].
Some studies found that the e-learning was more effective than traditional instruction [17].It has some advantages [14].It can support the learning process [3,18,19,20].It gains the academic achievement, motivation, performance and attitudes [14, 15 ,17].It facilitates the teachers to provide learning materials and they ask their students to accomplish and submit the assignments or exercises.The learning process is also fun, interactive and effective by using it [19].It provides the flexibility of time, place, learning speed, instructional standardization and effective instructional.The slow students can learn the material or tutorial provided in the e-learning.It can help them [4].The students also can learn autonomously [14].
The previous studies were undertaken by some researchers in developing the elearning.Yaniawati and Muis et al. conducted the e-learning for mathematic class.Munir dan Prabowo developed the e-learning using SMS Joomla for vocational high school students in Bantul, Indonesia which its content was about computer sciences.Turino et al. developed e-learning for teaching listening skill for senior high school (SMA 4) in Semarang, Indonesia.He designed it using Adobe Flash Professional CS3 in swf format.Setyowati focused on reading skill for senior high school students, thus she designed the e-learning for reading.Sutisna developed the e-learning for teaching grammar class at English Class Department, Pakuan University, Indonesia.The result showed that students' understanding on grammar and their motivation increased when they used e-learning and they could access it easily.It's a useful tool for the lecturer too when teaching grammar [21].Siswono and Liona also developed e-learning which emphasized on phonology, syntax, semantic, and pragmatic.They provided stories with proper grammar and the result showed that the teacher was easier to teach grammar to the students and it helped students to understand the grammar well.Solak & Cakir developed the course of elearning consisted of basic English skills which contain animation and interactive presentation for higher education level.The students also were able to discuss with their teachers in a synchronized way.Rymanova et al. developed e-course using Moodle as the Learning Management System which its contents were lexical, grammar, and speaking skill.From the studies, it could be concluded that the e-learning for English class concerning on some English skills.It is developed for senior high school level and mostly for higher education level [22].In USA, almost 90 percent of universities use it [4] and in Korea 70 percent [23].Thus, this study develops listening, reading, and grammar for English learning at junior high school level especially for first grade.The study aims to explore and describe deeply the development of the English the e-learning by applying a need analysis.It is required to design the instructional material for the e-learning [24].
Method
This study employed research and development methodology.Its aim was to yield a product [25].It had many kinds of model used.Those models had different steps and processes [6].This study applied model developed by Gall, Gall and Borg but did not fully complete all processes due to the time constraint.The process consisted of conducting need assessment, planning (identifying skills that will be learnt, generating the objective and learning activities, and having expert's validation), revising the products referring to the feedback given by the experts, conducting the field testing of the product to three students and teacher, and revising the product based on students and teachers' feedback.
This study was conducted from January to June 2017.The participants of this study were first graders and an English teacher of 89 Junior High School in West Jakarta, Indonesia.Interviewing the teacher was undertaken to obtain the data what she and her students needed.The document analysis (the syllabi) was also required to write the objective so it could be used a supporting data.The instruments used to validate media and content of e-learning were given to the experts.The questionnaire for teacher and students are also provided to get their feedback.
Result and Discussion
Based on the need analysis, students required to learn listening and reading skills which focused on descriptive texts.Those descriptive texts had some themes such as people, animal, place, thing, and occupation.The materials referred to the 2013 curriculum.Not only listening and reading skills, they were also necessary to learn grammar such as simple present tense, adjective, adverb, and preposition.After analysing the needs, the next steps were making the e-learning content, determining the name of e-learning website, making the logo, making the login for the students and teacher (administrator), developing English Gate by using PHP, Framework Bootstrap, and MySQL, making Entity Relationship Diagram (ERD), inputting the content into database system, hosting all database systems created in mini Raspberry Pi 3, sending English Gate as English e-learning to be validated, and testing English Gate to three students.
The content of e-learning consisted of listening, reading, and grammar.The listening contained the audio by recording an American native speaker's voice.It provided the audio with the animation, vocabulary (English-Indonesia), and multiple-choice questions.When students clicked the words in vocabulary, they had sound.The reading provided some texts with the sound, pictures, vocabulary, and short-answer questions.The students had to type the answer in the box provided.The grammar had the animated video which aimed to help the students to learn adjective, adverb, preposition and simple present tense.After they watched the animated video, they could play the game by accomplishing some exercises related to the grammar they learnt before.The exercises were in fill-in-the blank format.The students also could see their result and the answer key after they finished answering the exercises.The animations were created by using some software such as Adobe Flash and Adobe Illustrator.
English Gate was chosen as the name of the English e-learning.The logo was created using Adobe Illustrator.It provided login for the students and teacher (administrator).Students' login could be used to learn the material, do the exercises and see their scores.Administrator's login could be used to see the students' score, assess her/his students' score, register students' data, edit and delete the questions and answers.It used PHP as a back-end, Framework Bootstrap as front-end and MySQL as a database system.Entity Relationship Diagram (ERD) was used to describe the logical structure of database.
Fig. 1. Example of ERD
The contents were inputted into the database system.They were the homepage of English Gate consisting of home, login, about us, listening, reading, and grammar; username and password of administrator for login in the administrator dashboard; the questions by clicking 'manage question' in the administrator dashboard.All those data base systems created were hosted in mini Raspberry Pi 3 that functioned as web server.Thus, the IP address of the web server was 192.168.1.200.
Fig. 3. Administrator dashboard
English Gate was sent to two experts and teacher to validate its media and content.The result showed that the content was good and the content expert and the teacher did not give any suggestion about the content.On the other hand, the media experts gave some feedback.The feedback was the homepage display should be added and provided the manual how to use English Gate.Referring to those suggestion, it was revised and sent back to be validated again.The second validation showed the e-learning deserved to be applied and tested to some students.
These results of this study were not fully similar to the previous study.The development of the e-learning (English gate) consisted of login, homepage, interactive learning material (listening, reading and grammar) consisting of animated video, and quizzes with the feedback.The previous study found that the components of the e-learning contents were varied.This result study showed that Morrison developed it consisting of register, login, personal profile, material consisting of pre-assessments, texts, pictures, simulations, interactive exercises, downloaded materials, quizzes, bookmarks, post-assessments, and feedback [19].It's a little bit different from Morrison, Munir and Prabowo developed their e-learning content comprising curriculum, syllabus, materials and tests.Turino, et al. also provided login, homepage, syllabi, evaluation, and online consultation and chatting forum.Login was for the administrator, students, and teachers.The homepage consisted of main menu, register, and learning menu which offered downloaded material for students and teachers.The materials were in audio, Microsoft Word, Microsoft PowerPoint, PDF, and animation format.The listening materials developed used Adobe Flash Professional CS3.The syllabi were also provided, if the teachers wanted to delete or add them, they could do it.The evaluation comprised the questions and answers with various format such as multiple choice and true-false.The online consultation and chatting forum were designed to facilitate the students' interaction and participation.Almost similar to the previous researchers, Siswono dan Liona developed their e-learning consisting of login, learning plan, lesson, story, exercise, and dictionary.The learning plan provided a year learning planning.The lessons were the materials given to the students.The story feature consisted of animations with different themes.The dictionary was useful for the students to find the meaning of words [27].Rymanova with their instructors or teachers.The tests of units had some variations such as multiple choice, matching, short answer and other forms [14].
English Gate was tested to three students.They were observed by recording them when they used it.The result showed that they could use it well.Then they were given a questionnaire sheet to know their response in using it.All of them urged that they were happy to use it and more understand to learn English.The vocabulary also helped them to understand the meaning of words.Some of them gave suggestion to improve the assessment of reading because they thought their answers were correct but they had unsatisfying scores.Therefore, the teacher had to rechecked their reading answers and scores.Some studies showed that the content of the e-learning was not absolutely same one another.The important thing was it gave a significant impact for the students.It could be accessed autonomously by the students, flexible use, effective and efficient.Siragusa (2000) revealed that the main components in online learning were content, interaction, feedback, and interface design.The content had to align to the purpose and students' ability.The interaction referred to the communication between the students and teachers or instructors.The feedback referred to the teachers' comment or suggestions given to the students both individually or in group.The interface referred to easy navigation that could help and respond the students.The use of image, colour, sound, animation could entice the students [17]
Conclusion
English Gate is a web-based learning that can be used to learn English especially listening, reading, and grammar which it is developed for first graders of junior high school.Its contents are login, homepage, interactive learning material (listening, reading and grammar) consisting of animated video, and quizzes with the feedback.It is also equipped with the vocabulary (English-Indonesia) as aid tool for the students.It is expected to give a contribution as a learning source for students to enhance their English ability.This study needs some further improvement in reading assessment because students' possibility answers are varied.Further research is also required to investigate the effectiveness of English Gate as e-learning if it is applied in some classes.This study is supported by grant of Ministry of Research, Technology and Higher Education of the Republic of Indonesia.
et al. developed the e-learning, namely e-course, which consisted of the general data (teachers' personal data), unit feature (instructional unit, materials such as vocabulary and grammar, glossary, activities and supplementary resources from internet, and assessment feature.The materials were video, animation, text or images.They also provided Forum, Chat, and Wiki which facilitated students to interact SHS Web of Conferences 42, 00077 (2018) https://doi.org/10.1051/shsconf/20184200077GC-TALE 2017 | 2018-12-17T21:39:17.624Z | 2018-01-31T00:00:00.000 | {
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81925112 | pes2o/s2orc | v3-fos-license | Acute Renal Failure Related to Recto Sigmoid Diverticular Perforation
Diverticula in the colon are very common, but only 10-20% of the diverticulum carriers develop a diverticulitis. The inflammation can lead in 15% of the patients to a free perforation. In this case the patient has a recto sigmoid perforation due to a diverticulum. The infraperitoneal free air leads to a compression of the ureters with consensual acute renal failure.
Introduction
Diverticula in the colon are very common, but only 10-20% of the diverticulum carriers develop a diverticulitis [1]. The inflammation can lead in 15% of the patients to a free perforation [2]. In this case report, an unusual localization of the diverticulum appears which leads to an acute renal insufficiency.
Case Report
A 75-year-old female patient was transferred to the central hospital in Bern after a CT scan was made in the peripheral hospital which showed free infraperitoneal air of unclear aetiology.
At the hospitalization the patient was cardiopulmonary stable and afebril (pulse 78, 98% oxygen saturation, blood pressure 111/61, temperature 37.2°C). The clinical exam reported pain in the lower abdomen, without signs of peritonism. The digital rectal examination showed a slight anal prolapse and an emphysema was palpated pararectal. The blood control showed elevated leukocytes (21.8 G/L (neutrophils of 20.31 G/L)), CRP (261 mg/L) and creatinine (234 micromol/L).
The patient reported a general weakness for approximatively 2 weeks with reduced appetite and a single episode of vomiting. In the same time she noticed constipation. The last bowel movement was two days ago, since then she had pain in the abdomen, in the back and the renal flanks on both sides. The patient history showed an appendectomy in childhood and a hysterectomy due to menorrhagia at the age of 41 (1982) (Figures 1-3). An empirical antibiotic therapy was started with rocephin and metronidazole on suspicion of a rectosigmoid perforation. On the same day a rigid rectoscopy and a gynecological examination have been performed which showed no perforation.
Due to the compression induced postrenal renal insufficiency the decision was made to aspirate the free air pararectal to reduce the infraperitoneal pressure. After previous endosonographic presentation of the rectum, 500 ml air and 1 ml bloody secretion was drained, which was used for microbiological examination with no bacterial growth after a few days. For follow-up control a CT scan was made after the decompression (Figure 4).
Figure 4
After decompression with expansion of the rectum.
The day after, blood samples showed decreasing infection values (CRP 220 mg/L, leukocytes 14 G/L) and creatinine (172 micromol/L). On the same day a flexible rectosigmoidoscopy was performed, which showed a small ostium in the rectum at about 6-7 cm from ano, compatible with the perforation site. This ostium was supplied with an OTS (over the scope)-clip ( Figure 5). Due to a rising CRP value of 293 mg/L two days later a CT scan was made. A new fluid collection was found and drained on the same day CT scan controlled. The drainage extracts turbid brown secretion, so the rectosigmoid perforation was confirmed (Figure 6).
Figure 6
Insertion of a pigtail-drainage into the fluid collection.
In the liquid taken from the drainage, intestinal flora was detected in the microbial culture.
Following blood samples showed decreasing the infection values and the kidneys recovered completely. In the follow up CT scan five days after hospitalization, the fluid collection was decreasing with pigtail catheter positioned correctly. The patient was discharged home on the same day with antibiotics and drainage daily flushed by the home care.
Ambulant follow up showed decreasing infection values under antibiotical treatment, with a CRP value of 7 mg/L directly after antibiotics stop. Unfortunately the drainage produced continuously purulent fluid. In the next control the patient had fever and the infection values increased again. After one month a diagnostic laparoscopy was made which showed several inflammatory visceral adhesions. Due to this situs, with active inflammation, a recto-sigmoid resection with descendostoma (Hartmann situation) was performed by a lower median laparotomy. The histopathology showed a purulent-abscessing diverticulitis with granulation tissue without malignancy.
Discussion
Diverticulosis is very common, up to 50% of all individuals older than 60 years have colonic diverticula. About 10-20% develop a diverticulitis [1]. Herniation of the colonic mucosa through the circular muscle layers of the colonic wall leads to diverticula. Mostly the herniation is at the site of penetrating blood vessels through the colon wall [3]. The prevalence increases with age. Risk factors for diverticulitis are obesity and smoking [4].
Diverticulitis can lead to a free perforation in to the peritoneal cavity or generate an abscess. Differential diagnoses of a free colon perforation include tumor, IBD, treatment with NSAIDS and stercoral perforation. Approximately 16% of the patients with acute diverticulitis develop an abscess [5]. Which leads to an operative therapy or a percutaneous drainage is then required. A systematic review has shown that 50% of the abscesses (Hinchey stages IB and II) were successfully drained by radiology [6]. The drainage could eliminate the two-step procedure with a temporary colostomy and lead to an operation with a primary anastomosis [7].
In this case, the perforation was below the peritoneal fold and formed a valve mechanism that resulted in a massively increased infraperitoneal pressure. According to the literature research, no similar case has been published in which a tension pneumopelvis has led to a displacement of the urinary tract with an acute postrenal renal insufficiency and an ascending urinary tract infection.
The pararectal puncture with the release of free air rapidly improved kidney function and minimized the risk of urosepsis. No perforation was detected in the rectoscopy, so a flexible sigmoidoscopy was made. There, the perforation site was located and sealed with an OTSC. However, in the follow up rectosigmoid perforation occurred in the infraperitoneal space.
Retrospectively, it has to be discussed whether the flexible sigmoidoscopy and the forced search for the perforation site was necessary. Although an OTSC could be placed on the possible perforation site, it is questionable whether the infraperitoneal space was contaminated by sigmoidoscopy or not. A percutaneous drainage in the infraperitoneal space, after exclusion of a large perforation by the rectoscopy, would have been debatable. Thus, the pressure in the pelvis would have been minimized and the perforation site might have healed under antibiotic therapy.
Conclusion
Although diverticula in the rectosigmoidal transition are rare, it must be considered when infraperitoneal air is visible. Pressure decrease is recommended to prevent secondary urinary retention. | 2019-03-18T14:06:43.445Z | 2018-01-01T00:00:00.000 | {
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248541684 | pes2o/s2orc | v3-fos-license | Effectiveness of a helmet promotion campaign, China
Abstract Objective To evaluate the effectiveness of a 2020 nationwide helmet promotion campaign, in terms of helmet wearing and correct helmet wearing, aimed at electric bike riders and motorcyclists in China. Methods We obtained 192 hours of film of traffic before (2019) and after (2021) implementation of the campaign at eight road intersections in Changsha, recording cyclist (traditional and electric) and motorcyclist helmet-wearing behaviour during both weekdays and the weekend, and peak and off-peak traffic. We extracted data on rider characteristics and helmet-wearing behaviour. We applied a logistic regression to obtain estimates of helmet wearing and correct helmet wearing, and calculated odds ratios adjusted for rider variables. Findings We filmed 11 525 cyclists and motorcyclists, 5256 (45.6%) before and 6269 (54.4%) after the campaign. We estimated a substantial increase in the overall percentage of helmet wearing from 8.8% (95% confidence interval, CI: 8.0–9.6) to 62.0% (95% CI: 60.8–63.2). After controlling for covariates, we noted that helmet wearing increased in all groups. However, we observed a decrease in the overall percentage of correct helmet wearing from 91.9% (95% CI: 89.4–94.3) to 83.5% (95% CI: 82.3–84.7). Post-campaign, we estimated the highest percentage of helmet wearing for delivery riders (88.8%) and lowest for traditional cyclists (3.8%); we estimated the lowest percentage of correct helmet wearing for three-wheeled motorcyclists (58.8%). Conclusion To increase helmet wearing and correct helmet wearing, we recommend amending the campaign to include traditional cyclists as well as education and legislation on the correct fastening of helmet chinstraps.
Introduction
Cyclists and motorcyclists are vulnerable road users; of the 1 198 289 global road traffic deaths in 2019, cyclists and motorcyclists accounted for 64 693 (5.4%) and 222 357 (18.6%), respectively. 1 The corresponding figures in China, the world's most populous country, are 22 602 cyclist and 40 735 motorcyclist fatalities, contributing to 34.9% and 18.3% of global fatalities, respectively. 1 These statistics are particularly remarkable given the recent achievements in other global health indicators such as life expectancy and maternal and child mortality. 2 Helmet use reduces injuries and deaths among cyclists and motorcyclists, 3,4 and has therefore become a recommended intervention in the World Health Organization (WHO) and United Nations (UN) Global plan: decade of action for road safety [2021][2022][2023][2024][2025][2026][2027][2028][2029][2030]. 5 This report, as well as previous research, also recommends the enactment of strong law enforcement on helmet requirements. [6][7][8][9] Unfortunately, in 2017 just 49 of 167 countries that legally mandate motorcyclist helmets met five best-practice recommendations for helmet-use policies, and only 61 countries were rated as "good" in their helmetwearing law enforcement. 6 The estimated overall percentage of motorcyclists wearing a helmet was only 61.3% in low-and middle-income countries, much lower than the percentage of 94.4% in high-income countries. 6 China, a middle-income country, implemented a national motorcyclist helmet law in 2003, 10 but the percentage of motorcyclists wearing a helmet was only 20% in 2010. 6 The 2003 law does not apply to cyclists (which includes riders of both traditional cycles and electric bikes, i.e. e-bikes) and is not strictly enforced for motorcyclists. According to a WHO/UN report, China only scored six points out of 10 for motorcyclist helmet law enforcement, with a score of 10 indicating regular and consistent enforcement. 6 To address several road safety issues, including the low numbers of both e-bike riders and motorcyclists wearing a helmet, the Chinese government initiated the nationwide road safety campaign One helmet, one belt 11 on 1 June 2020. The helmet promotion part of the campaign incorporated several different approaches, aiming to: increase helmet ownership, accessibility and utilization; strengthen the enforcement of existing laws and regulations; enhance national education; and implement on-site motorist counselling programmes to encourage increased helmet use (Box 1). 13 The comprehensive campaign integrated many elements of empirically supported interventions.
We investigate whether the 2020 helmet-use promotion campaign was effective in increasing both helmet use and correct helmet use. Leveraging data collected for other purposes in 2019 in Changsha, China 14 about one year before the initiation of the campaign, we compared pre-campaign observations with post-campaign data collected during the same season in 2021, about one year after the implementation of the campaign. Although the campaign was aimed at e-bike riders and motorcyclists, we also collected and analysed data concerning traditional cyclists. We converted these observed data from Changsha to estimated percentages of both helmet wearing and correct helmet wearing using a logistic regression model.
Objective To evaluate the effectiveness of a 2020 nationwide helmet promotion campaign, in terms of helmet wearing and correct helmet wearing, aimed at electric bike riders and motorcyclists in China. Methods We obtained 192 hours of film of traffic before (2019) and after (2021) implementation of the campaign at eight road intersections in Changsha, recording cyclist (traditional and electric) and motorcyclist helmet-wearing behaviour during both weekdays and the weekend, and peak and off-peak traffic. We extracted data on rider characteristics and helmet-wearing behaviour. We applied a logistic regression to obtain estimates of helmet wearing and correct helmet wearing, and calculated odds ratios adjusted for rider variables. Findings We filmed 11 525 cyclists and motorcyclists, 5256 (45.6%) before and 6269 (54.4%) after the campaign. We estimated a substantial increase in the overall percentage of helmet wearing from 8.8% (95% confidence interval, CI: 8.0-9.6) to 62.0% (95% CI: 60.8-63.2). After controlling for covariates, we noted that helmet wearing increased in all groups. However, we observed a decrease in the overall percentage of correct helmet wearing from 91.9% (95% CI: 89.4-94.3) to 83.5% (95% CI: 82.3-84.7). Post-campaign, we estimated the highest percentage of helmet wearing for delivery riders (88.8%) and lowest for traditional cyclists (3.8%); we estimated the lowest percentage of correct helmet wearing for three-wheeled motorcyclists (58.8%). Conclusion To increase helmet wearing and correct helmet wearing, we recommend amending the campaign to include traditional cyclists as well as education and legislation on the correct fastening of helmet chinstraps.
Research
Effectiveness of helmet promotion campaign, China Peishan Ning et al.
Study design
We designed a film-based before-andafter study to evaluate the effectiveness of the national helmet promotion campaign. The pre-campaign observations were conducted from 29 June to 21 July 2019, and the post-campaign observations during 2-14 July 2021. We adopted multistage random sampling to determine observation sites (intersections) in Changsha, China. 15 We recorded field observations using smartphone-based high-definition cameras (1080p resolution, Redmi Note 3, Xiaomi, Beijing, China) placed at each intersection to record all passing cyclists and motorcyclists. 15 Because cyclist and motorcyclist behaviour (i.e. adherence to road safety regulations) varies between weekdays and weekends, 16 and because law enforcement is inconsistent between weekdays and weekends, we recorded traffic at each selected intersection over two different days: one during the week and one during the weekend. On each re-cording day we filmed traffic for 6 hours: 3 hours of peak traffic and 3 hours of offpeak traffic. We determined the times of peak traffic through pilot observation. 15 In total, we recorded traffic for 192 hours: two observations (pre-and post-campaign) at eight intersections/ observation for 2 days/intersection (weekday and weekend) for 6 hours/day.
Rider variables
Following techniques described in previous research, 17,18 we transcribed the 192 hours of film to extract the following variables for each rider: (i) sex (male or female); (ii) estimated age group (< 20, 20-49 or ≥ 50 years); (iii) time of week (weekday or weekend); (iv) time of day (peak or off-peak traffic); (v) road user (traditional cyclist, e-bike rider, two-wheeled motorcyclist or threewheeled motorcyclist); (vi) ownership type (public-access or privately owned vehicle), identified by viewer observation of the vehicle; and (vii) occupation (delivery rider or other), determined by viewer observation of the vehicle and/or uniform or apparel of rider.
Sample size
Because the 2019 pre-campaign observations to address other study hypotheses had already been conducted, we calculated the minimum sample size for this study based on study-specific aims and assumptions to assess whether the current sample size met those requirements. Because of potential clustering of riding behaviours at road intersections, we took intracluster correlation into account. Based on data from the 2019 study and relevant literature, 19 assuming α: 0.05 and β: 0.80 (i.e. type I and II error values), helmet-wearing percentages pre-and post-campaign of 8.8% and 13.2%, respectively (i.e. a hypothesized 50% increase in helmet wearing), a cluster size of 650 and an intracluster correlation of 0.005, we determined the minimum sample size to be 3347 for each round of observations.
Outcome measures
The primary outcome was the percentage of riders wearing a helmet, that is, the number of both cyclists and motorcyclists wearing a helmet as a percentage of the total number of filmed cyclists and motorcyclists. The secondary outcome was percentage of helmet-wearing riders correctly wearing a helmet. Following previous research, helmet wearing was recorded as correct if the helmet chinstrap was observed to be firmly fastened under the rider's chin. 3,5,17,18
Statistical analysis
We used χ 2 tests to examine sample characteristic differences between preand post-campaign observations. We estimated the percentages of helmet wearing and correct helmet wearing and constructed 95% confidence intervals (CIs) assuming binomial distributions. Because intracluster correlations for percentages of helmet wearing (0.0786) and correct helmet wearing (0.0442) based on road intersection were both insignificant (P-values of 0.07 and 0.11, respectively), we tested the effectiveness of the national campaign using a logistic regression. We quantified the effect of the campaign on helmet wearing by estimating crude odds ratios (cORs) as well as adjusted odds ratios (aORs), adjusted for the seven rider variables described above. We performed all statistical analyses using Statistical Analysis System version 9.4 (SAS Institute, Cary, United States of America). Statistical Box 1. Multifaceted approach of the 2020 national helmet promotion campaign targeted at e-bike riders and motorcyclists, China
Increasing helmet ownership, accessibility and utilization
Three major processes took place: (i) 1.1 million helmets were donated by companies nationwide through more than 15 000 public events; 11 (ii) over 75 000 public-access helmets were provided at traffic safety counselling points around schools and in rural areas before March 2021; 11 and (iii) over 50 000 public-access e-bikes equipped with smart helmets that must be worn before the e-bike is unlocked were put into use in May 2021 in Changsha, China. 12
Strengthening law enforcement
The current national law only mandates motorcyclists to wear a helmet, but the national helmet promotion campaign targeted both e-bike riders and motorcyclists (but not cyclists). The law enforcement component was led by the Traffic Management Bureau, who organized more than 3000 live streaming events and podcasts in which helmet law was enforced, and over 1000 coordinated and publicized (with reporters invited to participate) field enforcements of helmetuse laws and regulations. The bureau educated 12.2 million e-bike riders and motorcyclists violating helmet laws, and exposed typical cases of non-compliance. 11
Enhancing national education with regards to helmet use
The education component included both offline (e.g. safety posters) and online (e.g. videos, audio, pictures and articles) methods to inform the public of helmet use, correct helmet use, helmet use as legislative or policy requirements, the enforcement plan for helmet use and fines for non-compliance. According to official statistics, over 3000 posters and 1000 educational videos had been produced to promote helmet use by March 2021. Further, over 300 000 educational messages and 20 000 videos had been disseminated through social media (e.g. WeChat and Microblog) and news and short film platforms. These efforts yielded 970 million Microblog readings and 10 billion short film plays. 11
Implementing on-site motorist counselling programmes
On-site motorist counselling programmes, particularly focused on delivery riders, were conducted to persuade e-bike riders and motorcyclists to wear helmets. From May to November 2020, over 6100 on-site motorist counselling programmes were jointly implemented by 1.9 million traffic police departments and catering and express delivery industries across the country. These efforts resulted in over 630 000 delivery riders changing their helmet-wearing behaviour, and over 2300 police warnings were issued to catering and express delivery companies whose employees were not wearing helmets. 11 Effectiveness of helmet promotion campaign, China Peishan Ning et al.
tests were two-sided (i.e. the outcome variable differed across both the preand post-campaign observations) and conducted at P < 0.05.
Ethical statement
The Ethics Committee of Xiangya School of Public Health, Central South University, China approved our study (approval no. XYGW-2021-72), exempting our research from informed consent. All our recorded data were de-identified. This report follows the Strengthening the Reporting of Observational Studies in Epidemiology statement. 20
Results
We recorded the helmet-wearing behaviour of 11 525 cyclists and motorcyclists in total. Both the 5256 (45.6%) recorded before and 6269 (54.4%) recorded after implementation of the national helmet promotion campaign exceeded our calculated minimum sample size required to estimate the percentage of riders wearing a helmet and conduct the desired statistical comparison (Table 1).
We estimated an increase in the overall percentage of all cyclists and motorcyclists wearing a helmet from 8.8% (95% CI: 8.0-9.6) to 62.0% (95% CI: 60.8-63.2) as a result of the national road safety campaign (aOR: 27.7; 95% CI: 24.5-31.3; Table 2). After controlling for covariates, we estimated that the helmet-wearing percentage of all groups increased substantially following the implementation of the campaign, with aORs ranging from 4.4 (95% CI: 1.2-16.4) for riders aged < 20 years to 54.3 (95% CI: 13.3-222.0) for riders of public-access vehicles.
Of cyclists and motorcyclists wearing helmets, we estimated correct helmet wearing in > 70% of all groups at both observation periods, with the exception of three-wheeled motorcyclists after implementation of the campaign (58.8%; 95% CI: 42.3-75.4; Table 3). However, we estimated a decrease in the overall percentage of correct helmet wearing from 91.9% (95% CI: 89.4-94.3) to 83.5% (95% CI: 82.3-84.7) between the two observation rounds, with an aOR of 0.5 (95% CI: 0.3-0.7). We also estimated a significant reduction in correct helmet wearing in most groups, with aORs as low as 0.2 (weekend riders or three-wheeled motorcyclists) to 0.5 (male, riders of privately owned vehicles or non-delivery riders). The decrease in the percentage of other helmet-wearing groups who were wearing helmets correctly was not significant. Of all groups, we estimated that the percentage of helmet wearers wearing helmets correctly was highest among delivery riders after the implementation of the campaign (90.3%; 95% CI: 88.3-92.4; Table 3).
Discussion
The sixfold increase in helmet wearing after implementation of the national road safety campaign suggests that it was a highly successful intervention. Although the effectiveness of the campaign may be diminished in rural areas and smaller cities, where relevant resources are reduced compared with larger cities, 3 we hypothesize that the overall success of respondents had some knowledge of the national road safety campaign. 15 The variation in percentages of riders wearing helmets between groups reflects the priorities of the national helmet promotion campaign: the intervention was targeted at all e-bike riders and motorcyclists, but not traditional cyclists. 13 This inconsistency likely caused confusion among both traditional cyclists and traffic police officers. Governmental efforts to increase use of helmets by riders of public-access vehicles were also an important contributor to the inconsistent success of the campaign. The first set of 50 000 public-access e-bikes equipped with smart helmets were installed in Changsha in May 2021; 12 these e-bikes have built-in fourth-generation (4G) technological communication components that create a wireless internet link between the smart helmet and e-bike, and the e-bike will not unlock for use if the smart helmet is not worn. 12,25 Similar programmes were not introduced for public-access bicycles or motorcycles. Finally, the on-site motorist counselling programmes focused primarily on helmet use among delivery riders, 11,13,26 contributing to the high post-campaign percentage of helmet wearers among this population.
In contrast, we estimated that the overall post-campaign percentage of helmet wearers who were wearing a helmet correctly (83.5%) decreased significantly from the estimated pre-campaign percentage. This overall percentage is also far lower than the WHO target of 100% by 2030. 2,4 Contrary to best practice, 3,6 the enforcement component of the campaign does not require helmets to be properly fastened and therefore does not adequately incorporate public education concerning correct helmet use. 10,11,13 A phenomenon similar to our observation Effectiveness of helmet promotion campaign, China Peishan Ning et al.
was reported in Viet Nam, where an increase in correct helmet wearing was not observed despite substantial increases in helmet wearing following the 2007 implementation of a new policy. Viet Nam revised its national policy in 2008 to address this challenge, after which incorrect helmet use was observed to diminish. 27,28 The decreasing trend in correct helmet use may also reflect the fact that new users of helmets may have had no knowledge of or interest in the correct wearing of their helmet. Helmets may have been worn simply to meet legal requirements and not for personal safety, a possibility supported by the evidence that the presence of visible police enforcement was associated with increased helmet use among motorcyclists. 29 The variations in estimated percentages of correct helmet wearing between groups may be associated with differences in both personal risk perception 30 and external interventions such as on-site motorist counselling programmes. 26,31 Our study had several limitations. First, our observations were limited to one city so generalizability to other locations, particularly rural areas, should be assessed through future research. Second, our study only assessed the short-term effectiveness of the national helmet promotion campaign, about one year after its implementation; the longer-term impact might decrease over time and should be evaluated. Third, viewer judgement during manual transcription of data from film, in terms of riding behaviour and rider characteristics, may have resulted in bias. The viewers were unaware of the study hypotheses and did not participate in statistical analyses, but they did know which round of observations (i.e. pre-or post-campaign) they were coding. Correct helmet wearing was coded based on an imperfect proxy technique from previous research, 17,18 that is, whether the chinstrap was fastened tightly or else unfastened or fastened loosely. Both sex and age were judged inexactly based on physical appearance. Despite these challenges, our pilot research demonstrated a high accuracy in recording sex (99.7%; 304/305) and age group (93.1%; 284/305), and the reproducibility coefficients of over 10 hours of the 192 hours of film exceeded 92.0% for the studied variables. 15 These results suggest that bias from manual transcription was unlikely to substantially change our results. Fourth, because components of the campaign were conducted in a mixed and comingled manner, we could not assess the effectiveness of each component separately.
Our findings have two implications for policy. First, we determined that the multiapproach national helmet promotion campaign was successful and should be maintained, but some aspects of the campaign could be improved to yield a stronger effect. We recommend extending the campaign to cover all riders, including traditional cyclists, rather than focusing on e-bike riders and motorcyclists. According to best-practice recommendations, 6 a requirement not just to wear a helmet but for the helmet to be properly fastened should be considered in legislation and enforcement. We also recommend incorporating cor-rect helmet use into both the goals of the campaign and public education, as well as expanding the administration of onsite motorist counselling programmes to all cyclists and motorcyclists not wearing helmets. One challenge to the implementation of such campaigns is balancing the behavioural components with enforcement of policy. Research suggests that the enforcement of legal requirements is likely to yield more benefit in terms of improved road safety than simply behavioural and/or educational interventions on their own, 6,8,9 but a combination of legal enforcement and education is likely to be most effective. For maximum benefit, carefully developed, theory-driven behavioural and educational interventions should CI: confidence interval; E-bike: electric bike; NA: not applicable; OR: odds ratio. a Odds ratio after adjusting for seven covariates (sex, age group, time of week, time of day, type of road user, public-access versus privately owned vehicle and occupation) for overall percentage of correct helmet wearing, and after adjusting for the remaining six covariates for subgroup percentage of correct helmet wearing. b Crude and adjusted odds ratios were not calculated when one percentage for comparison is 0% or 100%.
Research
Effectiveness of helmet promotion campaign, China Peishan Ning et al.
be implemented along with the creation and then enforcement of legal policies. A second implication of our results is that long-term mechanisms should be established by the government to ensure required resources are available to implement the campaign nationwide in a sustainable and continuous way. Government support or philanthropic donations will continue to be required to implement the intervention broadly, including the distribution of helmets to those who need them in underdeveloped and rural locations. Sporadic and scattered campaign implementation is unlikely to yield lasting benefit.
Eficacia de una campaña de promoción del uso del casco en China
Objetivo Evaluar la eficacia de una campaña de promoción del uso del casco a nivel nacional en 2020, en lo que respecta al uso del casco y a su correcta utilización, cuyo grupo objetivo son los conductores de bicicletas eléctricas y motociclistas en China. Métodos Se obtuvieron 192 horas de filmación del tráfico antes (2019) y después (2021) de la aplicación de la campaña en ocho intersecciones viales de Changsha, en las que se registró el comportamiento de los ciclistas (con bicicletas tradicionales y eléctricas) y de los motociclistas en relación con el uso del casco, tanto en días laborables como en fin de semana, y en horas pico y no pico de tráfico. Se extrajeron datos sobre las características de los ciclistas y el uso del casco. Se aplicó una regresión logística para obtener estimaciones del uso del casco y de su correcto uso, y se calcularon las razones de posibilidades ajustadas a las variables de los conductores. Conclusión Para aumentar el uso del casco y su correcta utilización, se sugiere modificar la campaña para incluir a los ciclistas con bicicletas tradicionales, así como la educación y la legislación sobre la correcta sujeción de las correas de la barbilla del casco. | 2022-05-07T06:23:14.142Z | 2022-03-23T00:00:00.000 | {
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18844439 | pes2o/s2orc | v3-fos-license | Evidence for hard chiral logarithms in quenched lattice QCD
We present the first direct evidence that quenched QCD differs from full QCD in the chiral ($m_q \rightarrow 0$) limit, as predicted by chiral perturbation theory, from our quenched lattice QCD simulations at $\beta = 6/g^2 = 6.0$. We measured the spectrum of light hadrons on $16^3 \times 64$, $24^3 \times 64$ and $32^3 \times 64$, using staggered quarks of masses $m_q=0.01$, $m_q=0.005$ and $m_q=0.0025$. The pion masses showed clear evidence for logarithmic violations of the PCAC relation $m_{\pi}^2 \propto m_q$, as predicted by quenched chiral perturbation theory. The dependence on spatial lattice volume precludes this being a finite size effect. No evidence was seen for such chiral logarithms in the behaviour of the chiral condensate $\langle\bar{\psi}\psi\rangle$.
I. INTRODUCTION
Simulation of full lattice QCD with light quarks, turns out to be very time consuming. Most of the computer time involved in such simulations is spent in calculating (changes in) the determinant of the Dirac operator. For this reason, calculations on the largest lattices are usually performed using the quenched (valence-quark) approximation where this determinant is replaced by unity in the generation of gauge eld con gurations. The questions, how reliable is this approximation and where does it break down, naturally arise. Clearly the quenched approximation does not give the correct running of the coupling constant. On the other hand, hadron spectra and related quantities, calculated with u and d quark masses which are unphysically large, show good agreement between quenched and full QCD.
However, quenched QCD clearly fails in its treatment of the avour singlet pseudoscalar meson { the 0 . In full QCD, the 0 propagator can be represented by the sum The rst term is just the pion propagator and has a simple pole at m 2 . Summing this geometric series shifts this pole to m 2 0 . In the quenched case, only the rst two terms survive. This is due to the absence of closed fermion loops in the quenched approximation. Thus the propagator has a simple pole from the rst term and a double pole from the second, both at m 2 , an unphysical behaviour. In fact, since the second term includes a factor of N f (the number of avours), taking the limit N f ! 0 as is appropriate for the quenched approximation removes this second term in equation 1. Unfortunately, this contribution, with its double pole, reappears in 0 loop contributions. To study how this feeds back into the other propagators and observables of the quenched theory, Sharpe 1,2], and Bernard and Golterman 3,4] developed quenched chiral perturbation theory. The presence of the double pole in the 0 propagator, gives a logarithmic infrared divergence to loops involving the 0 propagator in the chiral limit of this e ective eld theory. This means that amplitudes will develop spurious powers of ln(m 2 = 2 ). These chiral logarithms are \hard" in that they control the chiral limit of the theory. This distinguishes them from the chiral logarithms of full QCD, which are \soft", that is they are multiplied by (powers of) m 2 , and so do not contribute in the chiral limit. Thus quenched QCD should produce results which di er from full QCD, at least in the limit m ! 0. It is important to know how large these e ects are at quark masses small enough to give the correct pion mass, or at least small enough to be able to extrapolate to the correct pion mass, since these e ects represent systematic errors introduced by the quenched approximation.
Prior to our work, no evidence for such chiral logarithms had been reported from simulations of quenched QCD. We report on our measurement of pion masses and h i on 16 3 64, 24 3 64 and 32 3 64 lattices at = 6:0 at staggered quark masses m q = 0:01, m q = 0:005 and m q = 0:0025 (in lattice units). A complete report of hadron masses, and nite size e ects from these simulations will be given elsewhere 5]. At these low quark masses chiral logarithms are evident in the pion mass data, but not directly in the h i data. Kuramashi, et al. 6] had noticed earlier that our preliminary data for the pion mass from a subset of our 32 3 64 lattice con gurations 7], appeared to show evidence for chiral logarithms. We have now completed the nite size analysis needed to rule out this being a nite size e ect.
In section 2 we describe our simulations and measurements, and present our results. These results are discussed in section 3.
II. SIMULATIONS AND MEASUREMENTS
Quenched gauge eld con gurations with = 6:0 were generated alternating one 10 hit metropolis sweep with one overrelaxation sweep. On each lattice, con gurations were separated by 1000 sweeps after discarding the rst 20,000 sweeps for equilibration. We have generated and analysed 200 con gurations on a 32 3 64 lattice, 339 con gurations on a 24 3 64 lattice, and 410 con gurations on a 16 3 64 lattice, allowing us to estimate nite size e ects. For meson spectroscopy we used a \corner" wall source on time-slice 1 to generate our quark propagators.
On the 16 3 64 lattice we also used a second \corner" wall source on time-slice 33. A stochastic estimator for h i was calculated for each con guration using a single gaussian-noise source. We refer the reader to our earlier work at = 6:5 for details of our methods 8].
where T is the di erence in times between the timeslice containing the source and that of the sink, N t is the temporal extent of the lattice and 2 is the local non-goldstone pion. Correlated ts are used in each case. The quantity quality confidence level degrees of freedom error ; (4) introduced by the HEMCGC collaboration 9], is used as a guide when choosing among ts of acceptable 2 /con dence level. This is merely an attempt to quantify our prejudice that good ts should not only have a high con dence level, but should also cover a large range of T values, and have small errors. Table I gives values of m , and table II gives values of m 2 from these simulations, as a function of m q and lattice size.
For full QCD m 2 = 2 m q , as m q ! 0 where is a constant with the dimensions of a mass. In Fig. 1 to parameterize departures from the chiral limit 13]. In the case m 0 = m 2 this yields c = 1:5(1), = 0:09(4) and d = 0:6(7) which, as we could have guessed from the good ts obtained to the form in equation 6, indicates that the introduction of the extra parameter d is not justi ed. Finally, we have attempted to t to the form m 2 =m q = C + am 2 (8) to see if the departure from standard PCAC can be described as an O(m 2 ) correction. Here, the 2 per degree of freedom of the t 6 (con dence level < 2%). Thus the chiral logarithm interpretation is favoured.
For completeness, in Fig. 3 we give an e ective mass plot for m 2 =m q for our 32 3 64 and 24 3 64 lattices.
Here we wish to indicate that the e ect we are seeing is not an arti ce of the systematic errors introduced by our (2) and a = 32:7(4), with a con dence level of 74%. This is such a good t that we conclude that there is no evidence for chiral logarithms in h i, other than those in m .
(Because of this we should not expect a good t if we replace m 2 with m q . Indeed, such a t has a con dence level of only 5%.) Finally we have reanalysed the data from our = 6:5 simulations 8]. Here, although m 2 =m q , does show some dependence on m q , it does not appear to be statistically signi cant. We were able to t this ratio to a constant c = 2:55(4), with a con dence level of 23%. Attempts at ts with chiral logarithms or a term linear in m 2 yielded even worse ts.
III. DISCUSSIONS AND CONCLUSIONS
We have observed clear evidence for quenched chiral logarithms in the pion spectrum of quenched lattice QCD at = 6:0. The reason we were able to observe this e ect appears to be that, by using a large (32 3 64) lattice, we were able to accommodate smaller quark masses (as low as m q = 0:0025) than had been considered previously. By measuring these masses on 16 3 64 and 24 3 64 lattices in addition to the 32 3 64 lattice, we have demonstrated that the pion mass has negligible nite size e ects for spatial boxes of length 24, at these quark masses. Our ts were to the leading-logarithmic expressions of Sharpe 2,10], rather than the 1-loop calculations 1, 3,4]. The reason for this choice is simple: because the leading-logarithmic sum comes from a renormalization-group argument, the arbitrariness of the scale is obvious, and the value of the exponent is independent of . This is not true of the 1-loop result.
Our calculated value of ( = 0:056 (5) mask the chiral logarithms down to rather small quark masses. Let us try to estimate the size of the errors in the pion mass incurred by using the quenched approximation, and coming from quenched chiral logarithms. In the spirit of Gupta, let us assume that at m q = 0:01, this error is negligible. From table 2 we see that the error in the pion mass at m q = 0:0025 is 2:5%. Taking the inverse lattice spacing to be 2GeV , assuming an e ective of 0.1 from m q = 0:0025 down to the physical pion mass, and using m 0 = m , we nd a further error of 6% in going to the physical pion mass. If we had used m 0 = m 2 this error would have been even less. Thus we would expect that the quenched chiral logarithms will introduce an error of 5 { 10% in the pion mass. Of course, at = 6:0, this is a considerably smaller source of systematic errors in the pion mass than the avour symmetry breaking between the goldstone and non-goldstone pions.
We have observed no evidence for quenched chiral logarithms in the chiral condensate h i. At least part of the problem here could be the presence of a large O(m 2 ) term, due to the fact that our de nition of h i is ultraviolet divergent for nite m q . This term tends to mask any other dependence on m q . In addition, as we have seen in the case of the pion mass, power series in m 2 can mask chiral logarithms down to quite low quark masses. There is no reason, a priori, why the chiral logarithms in h i should not be suppressed to even lower quark masses than is the case for m , by the power series behaviour of f .
Our measurements at = 6:5, which do not probe as far towards the chiral limit as those at = 6:0, do not show signs of quenched chiral logarithms. This is unfortunate, since at = 6:5, the pion masses show little sign of avour symmetry breaking, so the systematic errors due to lattice breaking of continuum symmetries are small. Thus had we observed any anomalous behaviour, we could have been con dent that it was a property of the continuum theory.
We conclude, therefore, that anomalous chiral logarithms do appear in at least some physical quantities in quenched QCD. Clearly more work is needed. The e ects we have seen are small, and it is hoped that some scheme similar to the one used by Kuramashi et al. 6] for the 0 mass, in which the e ects of the missing loops are estimated, could be applied to more general amplitudes in quenched QCD.
the CCSF sta at Caltech for their support. 1. m 2 =m q as a function of m 2 . This is a log-log plot, so that the predicted power law behaviour is a straight line.
The solid line is the t to equation 6.
2. h i as a function of m 2 | 2018-04-03T06:07:16.384Z | 1995-02-03T00:00:00.000 | {
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250138300 | pes2o/s2orc | v3-fos-license | Evidence of Internal Validity of the Social Skills Inventory for Caregivers (SSI-CE) in Brazil
With population aging, it is important to develop studies on psychological skills that can facilitate the task of caregivers of a dependent older adult. The Social Skills Inventory for Caregivers of the Elderly (SSI-CE) is an assessment tool for this purpose. Even considering that the instrument already has evidence of validity, the aim of this study is to seek additional evidence of validity for the SSI-CE. To this end, 533 family caregivers of older adults with an average age of 49.7 years were investigated by responding to the following instruments: Sociodemographic Questionnaire, Brazil Economic Classification Criteria, and SSI-CE. Initially, two factor models for the SSI-CE were compared (second-order
able to ask for help from others, seeking information about the illness of the care-recip ient, and assertive communication (Pinto et al., 2016). Furthermore, evidence on the importance of these skills for caregivers shows that the burden experienced by such individual is related to a concentration of several care tasks in a single person (Grazziotin & Scortegagna, 2018;Pereira & Soares, 2015). According to the authors, this feeling could be mitigated by sharing these tasks, which could be agreed based on good quality interpersonal relationships.
It is worth noting that a person's social skills repertoire can also be related to sociode mographic variables. Scheufler et al. (2017), for example, found that 1) the presence of depressive symptoms, 2) the years of education, and 3) the socioeconomic level of older adults was related to their social skills repertoire, indicating that a broader repertoire of social skills could be linked to less depressive symptoms, more years of education and a higher socioeconomic level of older adults in their communities. Similar studies could be developed to assess these differences in other populations, such as the growing population of caregivers of older adults. However, specifically for caregivers, Queluz, Barham, et al. (2019), conducted a review of the literature in which they found that, despite the relationship between social skills and 1) a lower feeling of burden and 2) better psychosocial adjustment on the part of caregivers, the research on social skills in caregivers' older adults is still recent, and it is necessary to deepen the existing knowledge on this subject. Thus, an instrument that could measure the social skills of caregivers would contribute to the advancement of studies in this field.
In order to fill this gap, Queluz et al. (2017) developed the Social Skills Inventory for Family Caregivers of the Elderly (SSI-CE), an instrument that aims to assess the social skills of family caregivers of older adults. After semantic and content evaluation of the SSI-CE, the authors sought evidence regarding the internal structure and measurement reliability. A 24-item structure, divided into three factors: Emotional Expressiveness (α = 0.87), Assertive Communication (α = 0.79), and Information Seeking (α = 0.60) was identified as appropriate for the instrument.
Additional evidence of validity based on the relationship between the SSI-CE and measures that assess theoretically related constructs was found by Queluz et al. (2018). As expected, the SSI-CE was positively correlated with quality of life (r = .30; p < .01) and positive interaction with senior (r = .48; p < .01), and negatively correlated with burden (r = −.43; p < .01), presence of depressive symptoms in caregiver (r = −.28; p < .01) and conflict in their relationship with senior (r = −.34; p < .01). Subsequently, analyses were also conducted to evaluate a potential social-desirability bias of the instrument. In this sense, Queluz et al. (2021) found no correlation between the SSI-CE and the Marlowe-Crowne Social Desirability Scale (r = .07; p = .52), indicating evidence of discriminant validity for the measure.
A psychological assessment instrument needs to present good evidence of validity so that its scores can be accurately measured and interpreted, for example, relation ship with variables already known in the literature and good fit indices, when testing the model statistically (American Educational Research Association [AERA], American Psychological Association [APA], & National Council on Measurement in Education [NCME], 2014). As demonstrated, the SSI-CE already presents several types of evidence of validity, indicating its potential for use. However, as it is a cumulative process (AERA et al., 2014), additional evidence is still needed, so that SSI-CE scores can be properly interpreted. For example, when evaluating a particular construct, it can be hypothesized that different related domains comprise a single general factor of interest. In this case, evidence justifying the calculation of a general score for the construct is needed. Confir matory Factor Analysis (CFA) is adequate to show (or not) the existence of a general factor, as it aims to identify latent variables (factors) manifested by directly observable variables (instrument items; Laros, 2012).
Different factor models can be tested in CFA to verify which one best represents the factor structure of a set of items, such as correlated-factor models, second-order hierarch ical models, or bifactor models (Reise et al., 2010). Some models assume the existence of a general factor that directly or indirectly encompasses all the items of the instrument, such as the bifactor model and the second-order model, respectively. In the bifactor model, there are only first-order latent factors (directly linked to the items), including a general factor (that generically represents the construct that the instrument should measure) and specific factors (that represent more restricted conceptual subdomains of the same construct; Reise, 2012). Thus, each item loads on a specific factor and on the general factor (Rios & Wells, 2014), and there is no relationship between the general and the specifics factors.
In the second-order model, in turn, there are two types of latent variables: first and second order. The second-order factor, which broadly represents the construct to be measured, is directly linked to first-order factors, which reflect specific skills or domains of this construct (Wolf et al., 2011). Therefore, an assessment of both models (bifactor and second-order) would bring empirical support to investigate the presence of a gener al factor for the social skills of caregivers of older adults, indicating the relationship between the observable variables and the latent factors of the instrument. In addition, invariance assessment of the internal structure of the SSI-CE in relation to variables already recognized as related to social skills in other populations might be important.
Given the above, the objective of this study is to seek additional evidence of validity to confirm that the SSI-CE is fit for use. Hence, the study aims to check for evidence of validity based on the instrument's internal structure, empirically testing the existence of a general factor. To this end, the plausibility of a second-order and bifactor structure for the SSI-CE was evaluated. In addition to performing an invariance analysis of the most suitable structure for the SSI-CE in relation to the variables gender, age, presence of dementia, and type of data collection (online or in-person), the reliability values for the general score, if feasible, and for each specific factor of the instrument were also verified.
Method Participants
A total of 533 family caregivers of older adults participated in this study, with an average age of 49.7 years (SD = 14.1), ranging from 18 to 87 years. The majority were females (88.5%), children of the elderly care receiver (62.5%), married or in a stable relationship (57.6%), with complete higher education (40.5%), and belonging to the Brazilian B2 soci oeconomic group (27.8%). Considering the relationship with the elderly care-recipient, other could be neighbors or even formal caregivers who continued care, when the elderly could no longer pay for the service. The sample was non-probability and of convenience. In each state, caregivers were recruited from a national Brazilian program called Serviço de Atendimento Domiciliar (SAD; Home Care Service) or referred by other caregivers or researchers' acquaintances. The inclusion criteria were being over 18 years of age and being relative of the senior they cared for. Caregivers who received any payment to care for the senior were removed from the sample. Data collection was performed at caregivers' homes, at the units of the SAD, or somewhere chosen by them, in order to ensure confidentiality and privacy. Data was also collected online, through a Google Forms interview. From the total sample, 76 participants (14.3%) responded remotely (online). Participants' sociodemographic data are shown in Table 1.
Instruments Sociodemographic Questionnaire
Prepared by the authors of this study to raise the sample characteristics, such as gender, age, education, marital status, and relationship with the senior they cared for. In addition, caregivers were asked whether the senior had been diagnosed with some sort of demen tia.
Brazil Economic Classification Criteria
The Brazil Economic Classification Criteria (ABEP, 2016) aim to assess the socioeconom ic level of families based on their purchasing power, number of durable consumer goods, presence of domestic workers, head of household education level, access to tap water, and whether the household is located on a paved street. Classes are divided into seven tiers: A, B, C, D and E.
Social Skills Inventory for Family Caregivers of the Elderly
The Social Skills Inventory for Family Caregivers of the Elderly (SSI-CE; Queluz et al., 2017) aims to assess the interpersonal repertoire related to the social skills of family caregivers of older adults. The instrument consists of 24 items, divided into three fac tors: Affective Expressiveness (10 items, α = 0.87), Assertive Communication (10 items, α = 0.79) and Training/Information Seeking (4 items, α = 0.60), and has a total Cronbach's alpha of 0.89. The first and second factors generate a score ranging from 10 to 40, while the third generates a score ranging from 4 to 12. The sum of the factors results in an overall score ranging from 24 to 96. The higher the score, the greater the repertoire of social skills reported by the caregiver. Caregivers respond to the instrument by indicating whether they never, sometimes, often or always behave in a certain way in a given situation. Item examples are: "When a family member praises the way I care for our elderly relative, I say thank you"; "I show affection to the older person that I care for through words or gestures. "
Procedure
The first step of the research was contacting the elderly healthcare service to obtain authorization to recruit the caregivers they had access to. After approval, the project was submitted and approved for execution by the Ethics Committee of the Universidade São Francisco (No. 1.876.754/2017). Then, a pre-selection of caregivers who fit the desired pro file within the contact institutions was carried out. When caregivers accepted to be part of the research, an in-person data collection interview was scheduled. Initially, a Free and Informed Consent Form was read and potential questions were answered. Subsequently, caregivers responded to the following instruments: Sociodemographic Questionnaire, Brazil Economic Classification Criteria, and SSI-CE. Data collection sessions were con ducted individually, and participants could choose if they preferred to have items read aloud by the researcher (e.g., in case of low education level) or if they would like to read and answer the questions by themselves. Online data collection was conducted via Google Forms, and participants were recruited from caregiver groups on social media (Facebook and WhatsApp). At the beginning of the survey, caregivers would read the Free and Informed Consent Form and indicate if they accepted to participate. If they did not agree with the consent form, the survey was interrupted. In addition, the e-mail address of the first author of this work was made available in case they wanted to get in touch to have their questions answered. The order of application of the instruments was the same: Sociodemographic Questionnaire, Brazil Economic Classification Criteria, and SSI-CE.
Data Analysis
The data were analyzed using the MPlus software. Descriptive statistics were used to characterize the sample according to sociodemographic aspects. The KMO index (Kai ser-Meyer-Olkin) and Bartlett's sphericity test were examined to apply factor analysis to the data matrix (Hair et al., 2006). Factor analysis was performed using the Weighted Least Squares Mean and Variance (WLSMV) estimation method. The adjustment indices considered were: Comparative Fit Index (CFI ≥ 0.90), Standardized Root Mean Square Residual (RMSEA, ≤ 0.08), Tucker Lewis Index (TLI ≥ 0.90), and Chi-square divided by degrees of freedom (χ 2 /df < 5; Hu & Bentler, 1999). Factor weights greater than 0.40 were considered adequate (Hair et al., 2006;Pituch & Stevens, 2016). The second-order model was found to be more suitable. Therefore, Pearson's correlation was used to verify the level of association between the factors of the SSI-CE and its general factor. The magnitude of the correlations was classified as: weak (< 0.30), moderate (0.30 to 0.59), strong (0.60 to 0.99) or perfect (1.0; Barrett et al., 1981).
For the invariance analysis, measurement invariance and sample heterogeneity mod els were compared using the software JASP 0.12, and CFI changes greater than 0.01 in relation to the previous model were considered an indication of invariance violation (Damásio, 2013). The variables evaluated were: age, gender, presence of dementia in the elderly care receiver, and type of data collection. The internal consistency indices used were Cronbach's Alpha and McDonald's Omega, and values above 0.60 were considered acceptable (Dunn et al., 2014;Ventura-León & Caycho-Rodríguez, 2017). The software JASP 0.14.0 was used for invariance analysis and reliability measures (Love et al., 2019).
Comparison of the Proposed Factor Models for the SSI-CE
The first part of data analysis was to verify if the data matrix is possible for factoring. The KMO value was = 0.92 and p = .001, for Bartlett's Test, indicating that the correlation matrix is factorable (Damásio, 2012). Then, the plausibility of calculating a global score for the SSI-CE was tested, by checking CFA results for the second-order and bifactor models. Subsequently, the results of these models were compared to check which would be more suitable for the instrument. The adjustment index values found for both models are shown in Table 2. In addition, both models are shown in Figure 1, including factor loadings for all items and factors.
SSI-CE Factor Structure When Second-Order and Bifactor Models Are Evaluated, Respectively
Based on the data shown in Table 2, all the adjustment indices evaluated for the secondorder and bifactor models were suitable. However, the bifactor model presented factor loadings below the 0.40 cutoff. Thus, the fit index of the results for the second-order model may empirically justify the existence of a general factor for the SSI-CE, indicating that this is the most plausible model for the instrument. The fact that the SSI-CE is represented by a second-order model means that specific factors are related, and the variations found in these factors can be explained by a broader construct (second-order factor), justifying the calculation of a general score for the latent variable (in this case, social skills), which is supported by theory (Del Prette & Del Prette, 2019). In addition to the adjustment indices, the correlations between the factors of the instrument and of these factors with the general score were also evaluated. Results are shown in Table 3. According to the results shown in Table 3, all factors were significantly correlated with each other and with the general score. The correlations found were either moderate or strong.
SSI-CE Invariance Assessment
Tables 4-7 show the adjustment indices for the factor structure of the SSI-CE for six invariance models (configural, metric, scalar, residual, structural, and latent mean). The variables analyzed were: age, gender, presence of dementia in the elderly care receiver, and type of data collection. Considering the values found for TLI and the comparison with the previous hierarchical model, the variable age did not show measurement invariance. In Table 5, measurement invariance was tested in relation to the variable gender. Table 5 shows that the SSI-CE presented structural and latent mean invariance, confirming that there is latent covariance in relation to the sample. That is, in this case, there is sample heterogeneity in relation to gender. In Table 6, measurement invariance was tested in relation to the variable presence of dementia in the elderly care receiver.
Similarly to gender, results in Table 6 show that the SSI-CE presented structural and latent mean invariance for presence of dementia, indicating sample heterogeneity for this variable (or care condition). Table 7 shows measurement invariance in relation to type of data collection (online or in-person).
According to the results in Table 6, the SSI-CE presented structural and latent mean invariance considering the type of data collection (online or in-person), indicating sam ple heterogeneity depending on how data collection was performed.
SSI-CE Reliability Analysis
In regard to SSI-CE reliability, ω = 0.89 and α = 0.89 were found for the general score. The Affective Expressiveness factor presented ω = 0.86 and α = 0.86, the Assertive Communication factor presented ω = 0.77 and α = 0.76, and the Training/Information Seeking factor presented ω = 0.66 and α = 0.65. All of them are considered suitable reliability values, and the value found for the total score is excellent (Ventura-León & Caycho-Rodríguez, 2017).
Discussion
Many older adults require specific assistance and care from others to obtain or maintain an adequate quality of life for their biopsychosocial well-being. For this reason, taking care of older adults is a task that, in order to be effective, often involves seeking knowl edge (about potential illnesses that may affect them, for example), and also developing skills to adequately meet the set of requirements of this task. Assisting a dependent senior involves daily interactions both with the assisted person and with other people included in this context, such as professionals, other family members, or friends. For this reason, it is essential to develop social skills to help caregivers to adequately meet all the interpersonal demands that may arise from these social exchanges, ensure the well-being of the care receiver, and preserve caregivers' mental health (Pinto et al., 2016;Queluz, Barham et al., 2019).
In order to contribute to research on this subject, the SSI-CE specifically evaluates the social skills associated with the care of elderly family members. Previous studies indicate that the instrument shows good evidence of validity (Queluz et al., 2017(Queluz et al., , 2018(Queluz et al., , 2021. However, following the recommendations of AERA et al. (2014), the aim of this study was to expand the evidence of internal validity of the instrument.
Considering the results of previous studies on the factor structure of the SSI-CE (Queluz et al., 2017), first, it was analyzed whether the items responded better to a bifactor or a second-order hierarchical factor structure. Although the adjustment indices of both models were satisfactory, the second-order factor model fits better to the data and is more parsimonious. It was found that the factors showed high and significant correlations both with each other and with the general factor. This result is a indicator that the general factor explains a greater proportion of item variance compared to the specific factors (Dominguez-Lara & Rodriguez, 2017), and is another indication of the relevance of the second-order model. This result is also supported by the theory, as in previous studies correlations were found between the classes of social skills, which are represented by the factors of different instruments in the area of social skills (Del Prette & Del Prette, 2010Del Prette et al., 2021).
Furthermore, comparing both models, it was found that the factor loadings of the second-order factor model were higher than 0.40 for all items. However, this was not true for the bifactor model, once it had items with poor factor loadings, which did not support the viability of the model (Pituch & Stevens, 2016). The factor weight of each item indicates the amount of variance explained by the factor (Pituch & Stevens, 2016). Thus, the greater the weight, the better indicator of the construct's dimension is the item. According to the postulates of several theorists on this analysis, factor weights higher than 0.40 in samples greater than 350 cases indicate an acceptable and significant propor tion of explained variance for each item, which is a valid criterion not only for decision making on the permanence of items (Costello & Osborne, 2005;Hair et al., 2006;Pituch & Stevens, 2016), but also to assess the suitability of different models. For this reason, the results obtained for the second-order factor model show that the relationships between observable and latent variables are sufficiently consistent and replicable.
Another objective of this study was to analyze SSI-CE measurement invariance ac cording to different variables related to specific social skills for the care of older adults. Results showed that the scores provided by the test have the same meaning in the different conditions assessed (Damásio, 2013): age, gender, presence of dementia, and type of data collection. First, there is evidence that the factor configuration is invariant in the groups formed according to these variables, that is, the number of factors and items that make up each factor is the same for all sets (Kline, 2015). Second, the metric invariance of the scale shows that factor loadings are similar in terms of their latent factors (Putnick & Bornstein, 2016), demonstrating that there was no differential item functioning according to the mentioned variables.
Third, the scalar invariance of the SSI-CE shows that, if there are differences between the group means, they are due to differences in the levels of the latent variables, not to differences in the average scores of the items (Marsh et al., 2018). Finally, results also showed that, in the proposed factor model, it can be assumed that the specific variance and the variance due to the measurement error of each item are the same for all groups, according to the proposed variables. That is, in general, social skills are equally measured, regardless of the effect of age, gender, presence of dementia, and type data collection (Putnick & Bornstein, 2016).
In addition to measurement invariance, invariance was also analyzed due to popu lation heterogeneity. Regarding structure, differences were found according to gender, presence of dementia, and type of data collection. This indicates that the relationships between factors are not equivalent across groups (Damásio, 2013). However, it is impor tant to consider that these results do not indicate deficiencies in the measures of the scale, but rather characteristics of these measures that respond to the heterogeneity the latent trait (i.e., social skills and their dimensions) presents in the different groups (Brown, 2006). In this sense, the information provided by this analysis is extremely useful when complemented with the results of invariance analysis of latent means. In this study, it was found that the means of the latent variables (i.e., the three factors and the general factor) were different across the groups gender, presence of dementia in the elderly care receiver, and type of data collection. In view of this, developing different interpretation standards for each variable would be recommended (Hogan, 2018).
Furthermore, in order to understand the nature of the differences found, it would be important to investigate them in more depth, to find out how they manifest in each group. However, previous studies have already shown an association of social skills with gender (Anme et al., 2010;Coelho et al., 2015;Crombie, 1988;Gaspar et al., 2018;Scheufler et al., 2017). Regarding age, although longitudinal studies that explain changes in social skill levels in different age cycles have not been found, there are studies showing that different age-related variables influence people's social competence, including neuroplasticity and preservation of cognitive functions (Taborsky & Oliveira, 2012). With regard to the presence of dementia in the elderly care receiver, other studies have also shown that caring for highly dependent people, such as older adults with dementia, impacts the psychological well-being of caregivers and the use they make of cognitive resources and adaptation to the environment (Keating et al., 2019;Pinto & Barham, 2014;Queluz et al., 2019). Finally, considering the differences in how data collection was performed, and that there are no previous studies comparing both types of data collection methods with regard to social skills, new analyses should be carried out with larger and more equitable samples between online and in-person data collection, in order to be able to more accurately establish the magnitude of these differences and the size of the effect.
A final analysis carried out was an assessment of the internal consistency of each latent measure of the SSI-CE, in which values considered adequate by the literature were found for all factors (Ventura-León & Caycho-Rodríguez, 2017). However, for the factor Training/Information Seeking, the value found was below 0.70, which indicates that caution is needed when using this measure for diagnosis or classification (Hogan, 2018). Nevertheless, when the factors are composed of few observable indicators or items, the internal consistency coefficients tend to be low because the variability between items is lower and there is a greater susceptibility to underestimating its coefficients, which may be a reason why the reliability values found were low (Ventura-León & Caycho-Rodríguez, 2017).
Some limitations of this study are the need for further analysis of the differences found and the use of samples of equivalent sizes, across groups. In addition, a nonprobabilistic sampling method was used, which is a weakness regarding the sample's representativeness, although different age groups, economic classes, and education levels were included to reduce this bias. Future studies should consider these limitations and continue inquiring about the instrument's evidence of external validity, especially about the predictive power of its scores. It would also be interesting if the authors could establish normative tables for the SSI-CE, considering the heterogeneities for the sample found in this study.
Finally, the data obtained here show that the SSI-CE has new evidence of internal val idity, is represented by a second-order factor structure, and has good reliability indices. These results, together with those of previous studies on the instrument (Queluz et al., 2017(Queluz et al., , 2018(Queluz et al., , 2021, allow it to be consolidated as a relevant and extremely useful measure to continue developing and expanding the field of study of social skills in caregivers of elderly family members. In addition, these results also favor the use of SSI-CE in prevention and intervention programs, in search of quality of life and well-being for this population, care receivers, and others involved in the task of caring for a dependent senior. | 2022-06-30T15:04:24.678Z | 2022-06-28T00:00:00.000 | {
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12463247 | pes2o/s2orc | v3-fos-license | Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis
During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in salamander cells suggest an innate immune response to the alga, with potential attenuation of NF-κB, and metabolic alterations indicative of modulation of insulin sensitivity. In stark contrast to its algal endosymbiont, the salamander cells did not exhibit major stress responses, suggesting that the host cell experience is neutral or beneficial. DOI: http://dx.doi.org/10.7554/eLife.22054.001
Introduction
All vertebrates have a 'microbiome' that includes mutualist ecto-symbionts living in close association with, but not within, their cells (Douglas, 2010). The most substantial vertebrate ecto-symbioses occur in the colon and small intestine and are implicated in physiological processes such as nutrient absorption from undigested complex carbohydrates (Ley et al., 2008;Krajmalnik-Brown et al., 2012). Known endosymbioses in vertebrates, where microbial cells live within the vertebrate cells, are almost exclusively parasitic, causing diseases such as malaria, toxoplasmosis, and chytridomycosis (Douglas, 2010;Sibley, 2004;Davidson et al., 2003). Currently, there is only a single exception. The green alga Oophila amblystomatis enters the cells of the salamander Ambystoma maculatum during early development (Kerney et al., 2011), and co-culture experiments show that the algae consistently benefit the salamander embryo hosts (Small et al., 2014;Graham et al., 2013;Pinder and Friet, 1994).
There is a long history of experimentation on the ectosymbiotic association between O. amblystomatis and A. maculatum: where the alga populates salamander egg capsules that contain developing embryos (Small et al., 2014;Gilbert, 1944). In the ectosymbiosis, the alga appears to benefit from nitrogenous waste excreted by the developing embryo while providing periodic oxygen and photosynthate to the microenvironment of the embryo's egg capsule, aiding salamander development (Small et al., 2014;Graham et al., 2013;Gilbert, 1944). However, the intracellular association between these two organisms was only recently recognized (Kerney et al., 2011), 122 years after the first published description of green salamander egg masses (Orr, 1888).
Perhaps the most intensively studied endosymbiosis between photosynthetic microbes and nonphotosynthetic animal hosts is the facultative mutualistic interactions between various invertebrate cnidarians (i.e. corals and sea anemones) and dinoflagellate endosymbionts (Davy et al., 2012). Such interactions provide the host animals the ability to obtain energy through photosynthesis. Cnidarian-dinoflagellate endosymbioses involve a number of physiological changes in both the host and photo-symbiont on a cellular level. The host animal tends to exhibit a tempered immune response to the ingressing cells (modulated by the host, symbiont, or both) (Detournay et al., 2012), and to express genes necessary for transferring nutrients to the symbiont (Lehnert et al., 2014), receiving nutrients from the symbiont (Lehnert et al., 2014), and use of the endosymbiont-derived metabolites (Lehnert et al., 2014). Less is known about adaptations of the endosymbiotic cells, but they can include modified osmoregulation (Mayfield and Gates, 2007), export of nutrients to the host cell (Lin et al., 2015), and physical changes such as loss of flagella (Muller-Parker et al., 2015).
In this study, we used a dual RNA-Seq approach on wild-collected A. maculatum salamander embryos and their endosymbiont alga O. amblystomatis to characterize the transcriptomic changes that occur in both organisms during this unique endosymbiosis. We isolated free-swimming algal cells living within the egg capsule ('intracapsular environment', triplicate sampling), salamander cells that did not contain algae (N = 50 cells per replicate, quadruplicate sampling), and salamander cells containing intracellular algae (N = 50 cells per replicate, quadruplicate sampling) from the same individuals. We identified differentially expressed genes in both organisms attributed to the intracellular association. The algal endosymbiont undergoes drastic changes in metabolism, displaying signs of cellular stress, fermentation, and decreased nutrient transport, while the host salamander cell eLife digest Throughout the natural world, when different species form a close association, it is known as a symbiosis. One species can depend on another for food, defense against predators or even for reproduction. Corals, for example, incorporate single-celled algae into their own cells. The algae photosynthesize, harnessing energy from sunlight to make sugars and other molecules that also feed the coral cells. In return, corals protect the algae from the environment and provide them with the materials they need for photosynthesis. This type of relationship where one organism lives inside another species is called an endosymbiosis.
In animals with a backbone, endosymbioses with a photosynthetic organism are rare. There is only one known example so far, which is between a green alga called Oophila amblystomatis and the spotted salamander, Ambystoma maculatum. The female spotted salamander deposits her eggs in pools of water, and algae enter the eggs, proliferate, and later invade tissues and cells of the developing embryos. However, it is not understood how similar the interaction between the alga and the salamander is to that in coral-algal symbioses, or whether it is rather more similar to a parasitic infection. Burns et al. now address this question by comparing salamander cells harboring algae to those that lacked algae. A technique called RNA-Seq was used to characterize the changes in gene activity that take place in both organisms during the endosymbiosis. The results show that algae inside salamander cells are stressed and they change the way in which they make energy. Instead of carrying out photosynthesis to produce food for the salamander host -as happens in coral-algal interactions -Oophila amblystomatis is fighting to adapt to its new environment and switches to a less efficient energy producing pathway known as fermentation. Burns et al. found that, in striking contrast to the alga, affected salamander cells do not show signs of stress. Instead several genes that are known to suppress immune responses against foreign invaders are expressed to high levels. This may explain how salamander cells are able to tolerate algae inside them.
The next challenge is to understand how the alga enters salamander cells. The current work identified some potential routes of entry, and follow up studies are now needed to explore those possibilities. Figure 1. Three populations of cells from A. maculatum egg capsules containing stage 39 embryos were collected and prepared for mRNA extraction, cDNA sequencing, and differential expression analysis revealing several hundred significantly differentially expressed genes detected for the salamander and alga. (a) Intracapsular algae (Population 1) were removed from intact eggs using a syringe and hypodermic needle (photo credit: Roger Hangarter). Embryos were decapsulated and washed, and the liver diverticulum region (dashed line), containing high concentrations of algae (red dots), was isolated and dissociated into a single cell suspension (illustration adapted from Harrison, 1969). The dissociated cells were screened for A. maculatum endoderm cells without alga (black arrowheads) and endoderm cells with intracellular alga (green arrowhead). Scale bars on microscope images are 20 mm. (b) Isolated endoderm cell, and isolated endoderm cell with intracellular alga. Scale bars on microscope images are 20 mm. (c) Representative cDNA distribution (bioanalyzer trace) from a population of 50 manually isolated A. maculatum endoderm cells. Peaks at 35 bp and 10380 bp are markers. Due to evidence of lysed A. maculatum cells observed in the cell suspension fluid after dissociation of A. maculatum embryos (debris seen in dissociated A. maculatum microscope images in (a) and (b)), that fluid was tested for the presence of contaminating mRNA. mRNA was not detected in the surrounding fluid, In A. maculatum, 46,549 genes were analyzed for differential expression. A total of 300 genes were identified as differentially expressed with an FDR adjusted p-value less than 0.05 ( Figure 1e). There were 134 genes with higher expression in salamander cells containing intracellular algae and 166 genes with lower expression in those cells. Of those, 74 (55%) of over expressed genes and 71 (43%) of the under expressed genes were assigned putative functional annotations. The genes were grouped into twelve broad functional categories ( Table 2) reflecting the response of A. maculatum cells to the intracellular algae. Transposable elements comprise the largest category of annotated differentially expressed genes (18% of over-and 27% of the under-expressed). Other functional responses include an immune response to the intracellular alga, modulation of the host cell's nutrient sensing, and differential expression of genes related to cell survival and interactions with other cells, including cell-cell adhesion and motility. The complete list of annotated, differentially expressed algal genes can be found in the file Table 2-source data 1.
Phosphate and nitrogen transporters in O. amblystomatis are regulated by phosphate and glutamine levels, respectively Cultures of the symbiotic alga in AF6 media, allowed in vitro testing of algal inorganic phosphate and nitrogen transporter regulation, in response to availability of relevant nutrient sources.
The high affinity phosphate transporter PHT1-2, was regulated by extracellular inorganic phosphate concentration in cultured O. amblystomatis (Figure 2a). The average qPCR expression difference between high (100 mM and above) and low (10 mM and below) phosphate concentrations was 32-fold (p=4.4 Â 10 À15 ), which agrees with both the RNA-seq data (25 fold lower expression in the endosymbiotic alga), and estimates of phosphate concentrations in vernal pool water (low micromolar) (Brodman et al., 2003;Carrino-Kyker and Swanson, 2007) compared to inside amphibian cells (low millimolar) (Horowitz et al., 1979;Burt et al., 1976). A second phosphate transporter, a chloroplast localized sodium dependent phosphate transport protein 1 (ANTR1), was not regulated by extracellular phosphate levels ( Figure 2b). Its low expression level in the endosymbiotic alga is therefore not likely to be related directly to an increased phosphate level of the host cytoplasm.
Dual-RNA-Seq of a vertebrate endosymbiont
This study provides the first transcriptomes for A. maculatum and O. amblystomatis and an in-depth look at gene expression changes of both organisms in their unique endosymbiotic state. The dual-RNA-seq approach has previously been used to investigate intracellular parasitism in vertebrates (Westermann et al., 2016(Westermann et al., , 2012Tierney et al., 2012). However, our analysis represents the first investigation of a vertebrate endosymbiosis where the generalized interaction between the two organisms has consistently been characterized as a mutualism (Small et al., 2014;Gilbert, 1944;Bachmann et al., 1986). Our results also extend dual-RNA-seq methodology to low cell number samples from wild collected, non-model organisms. The transcriptional responses to this cellular association reveals how a vertebrate host responds to an intracellular mutualist and offers insights into the physiological condition of both partners in their endosymbiotic state.
In the host salamander, we identified only a small fraction of the analyzed genes (300/46,549; 0.64%) that are differentially expressed between endosymbiont-bearing vs endosymbiont-free salamander cells. This tempered host response is reminiscent of that of the hosts in coral-dinoflagellate endosymbioses; less than 3% of the analyzed genes were shown to be differentially expressed when the host coral was inoculated with and without a symbiosis competent strain of dinoflagellate (Voolstra et al., 2009;Mohamed et al., 2016). By comparison, the algal response to endosymbiosis from ectosymbiosis was observed to be more pronounced; 4.12% (277/6,726) of the algal genes were differentially expressed, proportionally 6.4 times more genes than in the host salamander. This level of change, nevertheless, is much more subtle when compared to the changes observed between the endosymbiont algal transcriptome and the cultured free-living alga grown in nutrient replete conditions where 40% (2,687/6,726) of the algal genes were differentially expressed.
Expression differences in intracellular algae
The over-expression of heat shock proteins, autophagy related proteins, and other stress inducible genes reveal hallmarks of stress in the intracellular algae (Supplementary file 1). These are undergoing multiple metabolic changes compared to their free-swimming intracapsular counterparts. Intracellular algae parallel the response of the closely related green alga Chlamydomonas reinhardtii to low sulfur levels (Supplementary file 2) under hypoxia, including gene expression changes consistent with a switch from oxidative to fermentative metabolism (Supplementary file 3) (Nguyen et al., 2008;Grossman et al., 2011). This response, relative to intracapsular algae, includes under-expression of photosystem II core components (Supplementary file 4) in the chloroplast and complex I of the electron transport chain in the mitochondrion (Piruat and Ló pez-Barneo, 2005) (Supplementary file 5), along with over-expression of fermentative metabolic pathways that would shuttle pyruvate toward acetyl-CoA, organic acids and alcohols [crucially, over-expression of pyruvate-ferredoxin oxidoreductase (PFOR), phosphate acetyltransferase (PAT), and aldehyde-alcohol dehydrogenase (ADHE)], and potentially produce H 2 gas [over-expression of an iron hydrogenase (HYDA1)] (Supplementary file 3) (Volgusheva et al., 2013;Yang et al., 2013;Catalanotti et al., 2013).
To verify the observed expression differences between intracapsular and intracellular algae, we performed a comparison of expression in the intracellular algae to O. amblystomatis gene expression in unialgal culture in nutrient replete media. A complete analysis of differentially expressed genes between O. amblystomatis cultured in nutrient replete media and intracellular algae revealed 1,805 over-expressed transcripts and 882 under-expressed transcripts in the intracellular algae (indicating 40% of transcripts are differentially expressed, Figure 3). A summary of GO terms enriched among the 1,805 genes over-expressed in intracellular algae relative to algae cultured in nutrient replete media confirms an enrichment in fermentation and stress response processes ( and intracellular-cultured algae comparisons. This includes under-expression of 3 components of the mitochondrial electron transport chain, and consistent over-expression of PFOR, PAT, ADHE and HYDA1. Equivalent photosystem II core components are not significantly under-expressed in intracellular algae compared to cultured algae, suggesting that intracapsular algae over-express photosystem II core components, rather than intracellular algae under-expressing them. This may be due to hyperoxic conditions in the intracapsular environment (Pinder and Friet, 1994), which could lead to oxidative damage to and rapid turnover of the photosystem II core (Richter et al., 1990).
Intracellular Algae vs. Cultured Algae p<0.05
Average log 2 CPM Overall, these metabolic changes suggest that photosynthesis by the alga does not keep up with respirational demand during the endosymbiotic state. In algal fermentation, some photosynthesis components are still active, notably photosystem II (even if it is downregulated) (Volgusheva et al., 2013). However, the complement of over-and under-expressed genes in the alga suggests that neither the diffuse oxygen in these tissues nor the oxygen generated by photosynthesis is sufficient to meet the metabolic demands of the algal cell through oxidative phosphorylation. Instead these cells have switched to fermentation. This is potentially attributable in part to the opaque tissues of the embryonic host, which restrict the necessary photons from reaching algal chloroplasts that would allow more oxygen to be generated by the splitting of water. . Differential expression of fermentation genes in intracellular algae compared to cultured algae. Blue dots are genes that were over-expressed in intracellular algae compared to intracapsular algae. Orange dots are genes that were under-expressed in intracellular algae compared to intracapsular algae. Vertical blue lines represent plus and minus two-fold fold change. The horizontal blue line represents FDR adjusted p-value equal to 0.05. Genes above the horizontal blue line are significantly differentially expressed; genes below the blue line are not. Key fermentation genes, PFOR, HYDA1, ADHE, and PAT are significantly over-expressed in intracellular algae compared to cultured algae, in the same manner as they are over-expressed in intracellular algae compared to intracapsular algae. Several components of complex I of the electron transport chain in the mitochondrion are also significantly under-expressed (CYB, ND2, ND4), though ND1 is over-expressed in intracellular algae compared to cultured algae. DOI: 10.7554/eLife.22054.023 The occurrence of intracellular fermentation is also supported by decreased starch granules in the intracellular algae and transcriptional changes to algal sulfate transport and sulfur metabolism associated genes. Previous analysis of algal ultrastructure revealed intracellular algae had a significant reduction of starch reserves compared to intracapsular algae (Kerney et al., 2011). Intracellular algae had approximately 56% of the starch reserves of intracapsular algae by cross sectional area, which corresponds to around 42% of the starch reserves by volume (Kerney et al., 2011). Starch consumption during algal fermentation is well characterized in other Chlamydomonad algae (Zhang et al., 2002). Two recent studies observed a reduction of intracellular starch reserves to approximately 50% of their peak levels during long term sulfur deprivation (Zhang et al., 2002;Chader et al., 2009). The overexpression of a sulfate transporter and taurine catabolic enzymes (González-Ballester et al., 2010), along with other transcripts associated with sulfur metabolism (Figure 3; Supplementary file 2), indicates that fermentation in intracellular Oophila coincides with sulfur deprivation and closely matches the consumption of starch found in other fermenting algae.
One discrete class of algal changes entails modifications to nitrogen and phosphorous transporters (Supplementary file 6), which are likely attributed to relatively high concentrations of intracellular nutrients compared to the egg-capsule microenvironment (Brodman et al., 2003;Carrino-Kyker and Swanson, 2007;Horowitz et al., 1979;Burt et al., 1976;Vastag et al., 2011;Goff and Stein, 1978). Transcriptional responses, closely matching those seen in our DE analysis, were initiated by mimicking intracellular concentrations of phosphate or glutamine in cultured algal stocks. The algal down regulation of inorganic nitrogen transporters in response to glutamine suggests that the algal endosymbiont is using host glutamine as a nitrogen source. This scenario is supported by the use of glutamine as a sole nitrogen source in other related green algal taxa (Neilson and Larsson, 1980). The changes in transporter expression indicate that metabolite concentration differences in an algal cell's microenvironment can account for potentially all of the observed transcriptional differences in the DE analysis. This not only validates our dual-RNA-seq experimental design but suggests mechanisms of niche-dependent transcriptional regulation consistent with other green algae (Fan et al., 2016), and the potential acquisition of host-derived glutamine for intracellular algal metabolism.
Expression differences in salamander cells with algal endosymbionts
There are interesting parallels to both parasitic infections and other known facultative endosymbioses in the salamander transcripts expressed. These include innate immune responses, nutrient sensing, cell motility and apoptosis/survival. The changes in transcript expression reveal a unique cytosolic relationship between these salamander cells and their algal endosymbionts.
Transposable elements
There are a remarkable number of transposable elements that are differentially expressed between salamander cells with and without algal endosymbionts (Supplementary file 7). Their differential expression may be controlled by the transcriptional regulation of nearby co-regulated genes (Batut et al., 2013). We posit that the observed differential regulation of transposable elements in this study is a function of A. maculatum's extraordinarily large genome (at around 31 Gb, which is approximately ten times the size of the human genome) (Licht and Lowcock, 1991). This large size is likely attributable to a large number of mobile elements in the salamander genome (Keinath et al., 2015), which may share regulatory regions with protein coding genes (Batut et al., 2013). With a few exceptions, the genes annotated as transposable elements have few detectable RNA transcripts (median counts per million of 1.54 and 0.88 for genes with increased or decreased expression in salamander plus endosymbiont samples, respectively) compared to other, non-transposon, differentially expressed genes (median counts per million of 10.34 and 14.82, respectively). Of the 69 genes with sequence homology to known transposable elements, 32 (46%) have homologs in A. mexicanum transcriptomes (Voss, 2016Voss, 2016Stewart et al., 2013;Wu et al., 2013). The transposable elements are largely (68%) long interspersed nuclear elements (LINE retrotransposons), which are typically associated with genome expansions in eukaryotes (Kidwell, 2002). Other differentially expressed transposable elements are PLE retrotransposons (6%), LTR retrotransposons (11%), DIRS retrotransposons (6%), and DNA transposons (3%). Intracellular invasion by a foreign microbe can lead to apoptosis in animal cells (e.g. salmonella [Monack et al., 1996;Kim et al., 1998] or malaria [Kakani et al., 2016]). However, the salamander cells with algal endosymbionts did not show any clear transcriptional signs of apoptosis. The one gene whose primary functional role may be in apoptosis is the salamander transcript (Bcl2-like protein 14, BCL2L14) with higher expression in salamander cells with algal endosymbionts, which contains BCL-2 homology (BH) domains BH3 and BH2 ( Figure 5). Nonetheless, BCL2L14 has been explicitly shown to not be involved in pro-apoptotic regulation (Giam et al., 2012). Other genes with higher expression in salamander cells with algal endosymbionts (e.g. olfactomedin-4, OLFM4; TNFAIP3-interacting protein 1, TNIP1; serine/threonine protein kinase 1, SGK1) have demonstrated anti-apoptotoic functions in other animals (Liu and Rodgers, 2016;Ramirez et al., 2012;Lang et al., 2010). In the lab, intra-tissue, and potentially intracellular, algal cells are detected for prolonged periods during development and post hatching in salamander larvae up to Stage-46, and algal DNA was detected in adult tissues (Kerney et al., 2011). Eventually, the number of detectable algal cells within the larvae decreases (Kerney et al., 2011). This may coincide with the development of the salamander's adaptive immune system, or it could be that the alga stops producing chlorophyll, but are nonetheless maintained within the embryo. There are seven transcripts (e.g. SGK1; GDNF receptor alpha-4, GFRA4; thymosin beta 4, TMSB4) (Supplementary file 8) with higher expression in salamander cells with algal symbionts that are linked to cell survival in different physiological contexts including cancer cell survival and proliferation, and neuronal survival during development (Lang et al., 2010;Enokido et al., 1998;Bock-Marquette et al., 2004). Genes from this category may contribute to building a novel network of gene regulation used to maintain these endosymbionts. The salamander immune response Our data reveals a limited immune response of embryonic cells with algal endosymbionts (Supplementary file 9). The salamander immune response can largely be categorized as an innate immune response, but it also includes components of an adaptive response (e.g., interleukin 7, IL7) that precedes the developmental formation of an adaptive immune system (Charlemagne and Tournefier, 1998). Amphibian immunology has, for the most part, been considered a physiological process of larvae and adults . There are no prior transcriptional datasets on embryonic immune responses, although there is a growing interest in amphibian embryo-microbial interactions (Gomez-Mestre et al., 2006). Therefore, our study fills a gap pending the availability of comparative data from embryonic pathogens such as the oomycete Saprolegnia (Petrisko et al., 2008).
The increased expression of pro-inflammatory interleukins and chemokines (e.g. interleukins, IL-8; IL-7; IL-1b; and C-X-C motif chemokine 10, CXCL10) parallels the transcriptional response of adult frog skin to the chytrid fungus Batrachochytridium dendrobatidis (BD) infection and entry . Naïve BD infected frogs mount a much more dramatic immune response than these isolated salamander cells Ellison et al., 2014;Rosenblum et al., 2009). However, this may, in part, be due to the tissue-level resolution of these studies on chytrid infection, as opposed to the cellular-level resolution of our study, or the comparison of embryos to adults.
Multiple transcriptional differences indicate an increased generation of reactive oxygen species (ROS) in salamander cells containing algae. The NADPH-oxidase family member dual oxidase 1 (DUOX1)is involved in the zebrafish intestinal epithelial cell immune response to Salmonella infection (Flores et al., 2010) and is also more highly expressed in salamander cells with endosymbiotic algae. While this protein is implicated in several physiological processes, its ability to catalyze the generation of reactive oxygen species (ROS) reveals a potentially conserved immune response role in these non-phagocytic embryonic cells. Moreover, we observed under-expression of subunit 6b of cytochrome c oxidase (COX6B1). Reduction of cytochrome c oxidase activity is associated with the generation of ROS through signaling to endoplasmic reticulum NADPH oxidases in yeast (Leadsham et al., 2013). Additionally, the downregulation of STEAP4 leads to lower levels of ROS in mouse osteoclasts (Zhou et al., 2013), whereas we see upregulation of STEAP4 here. Importantly, the generation of ROS does not rely on the presence of high levels of oxygen in vertebrate cells as ROS can be generated in hypoxic as well as normoxic conditions through a variety of mechanisms (Kim et al., 2012;Nathan and Cunningham-Bussel, 2013) although there is no indication that the salamander cells are suffering from hypoxia here.
Four genes with high expression during the intracellular symbiosis (OLFM4; TMSB4; TNIP1; and mucin1, MUC1) are associated with negative regulation of the NF-kB response in other systems (Ueno et al., 2008;Verstrepen et al., 2009;Liu et al., 2010;Sosne et al., 2007). NF-kB is a transcription factor complex that is expressed in all vertebrate cell types, and is involved in a variety of immune responses (Tato and Hunter, 2002;Takada et al., 2005). Of the possible genes transcriptionally regulated by NF-kB (Ali and Mann, 2004;Gilmore Lab, 2016), only effectors in the cytokine/chemokine group were observed as over-expressed in salamander cells containing intracellular algae. Our salamander transcriptiomes contain 295 genes that are homologous to downstream targets of NF-kB identified in other systems. Only five are over-expressed here, IL8, IL1b, CXCL10 and TNIP1 (with TNIP1 also being a negative regulator of NF-kB) (Supplementary file 10-sheet 1, 'NFkB_Expr'). A fifth NF-kB response gene, which we have annotated as a trypsin, but which also has strong homology to Granzyme B (associated with apoptosis) was under-expressed in salamander cells hosting endosymbionts (Supplementary file 10-sheet 1, 'NFkB_Expr'). Increased expression of genes associated with attenuating NF-kB signal transduction has precedent in other intracellular infections (Tato and Hunter, 2002) and possibly symbiotic associations (McFall-Ngai, 2014). The over-expressed OLFM4 and MUC1 are also implicated in establishing the vaginal microbiome, potentially through their roles in modulating NF-kB signaling (Doerflinger et al., 2014;Fields et al., 2014). How the algal cell entry may be affecting these genes and whether NF-kB by-pass is involved in algal cell entry or maintenance remains to be determined.
We did not find modified expression of toll-like receptors (TLR's), which detect pathogen-associated molecular patterns. These function in activating an innate immune response to both bacterial and protistan pathogens (Ashour, 2015) as well as establishment of gut commensals (Round et al., 2011). While TLR's do not require differential regulation for their normal function, established schistosomiasis, entamoeba, trypanosome, and filarial nematode infections all result in down-regulation of TLR transcripts (Ashour, 2015), and a resulting NF-kB mediated response. In our data, we found 10 salamander genes with homology to various TLRs. None were differentially expressed between salamander cells without and salamander cells with endosymbionts. Further, we examined expression of 151 additional genes associated with the TLR response, and found only one over-expressed gene downstream of TLRs, TNIP1, which is a negative regulator of NF-kB, as discussed above (Supplementary file 10-sheet 2, 'TLRs_Expr'). While the lack of differential transcriptional regulation of TLR's or their regulators does not preclude their involvement in algal entry response, it is notable in comparison to other parasitic infections where TLR expression is often down regulated (Ashour, 2015).
One potential benefit of having an intracellular alga may be to prime the embryo's immune system, without over-activating it, granting the invaded embryos additional protection against the microbial environment outside of the egg capsule. One mechanism for this immune priming may be revealed by relatively increased hepcidin expression in salamander cells with intracellular algae. Elevated hepcidin levels are protective against multiple infections of malaria parasites in mammalian models (Portugal et al., 2011) and were shown to enhance resistance to bacterial infection when transgenically over-expressed in zebrafish (Hsieh et al., 2010).
Metabolism and nutrient sensing in A. maculatum
In established endosymbioses between invertebrates and algae, the transfer of organic molecules synthesized by the algal partner allows otherwise non-photosynthetic animals to become partial or complete autotrophs. The exchange of photosynthate from symbiont to host is mediated by a range of molecules including sugars, sugar alcohols, and lipids (Burriesci et al., 2012;Kellogg et al., 1983;Colombo-Pallotta et al., 2010). In the endosymbiosis between A. maculatum embryos and O. amblystomatis, the alga does not appear to be using a canonical photosynthetic process of carbon fixation, oxygen evolution, and sugar production, but is rather metabolizing by fermentation. This metabolic state does not, however, preclude the possibility of metabolite transfer from the intracellular alga to the salamander cells. Under fermentation, the alga may still generate ATP from light energy (Godaux et al., 2015), fix carbon (Godaux et al., 2015) or use alternate molecules as a carbon source (Gibbs et al., 1986). Fermenting algae are also capable of using anabolic reactions to produce sugars and lipids (Gibbs et al., 1986). Indeed, the related alga Chlamydomonas moewusii excretes glycerol, acetate, and ethanol under anoxic conditions (Klein and Betz, 1978). Release of fermentation byproducts such as formate, acetate, and glycerol, or of energy storage molecules such as sugars or lipids into the host cytoplasm could trigger differential expression of nutrient sensing mechanisms within A. maculatum cells.
In A. maculatum cells with an intracellular alga, differentially expressed genes involved in nutrient sensing (e.g. STEAP4; neurosecretory protein VGF, VGF; resistin, RETN; pyruvate dehydrogenase phosphatase 1, PDP1; and calcium/calmodulin-dependent protein kinase 1, CAM-KK 1) (Supplementary file 11) (Wellen et al., 2007;Petrocchi-Passeri et al., 2015;Steppan et al., 2001;Jeoung and Harris, 2010;Witczak et al., 2007) are suggestive of altered metabolic flux through catabolic and anabolic pathways, particularly with respect to insulin production and sensitivity. One differentially expressed algal gene that may be implicated in nutrient exchange with salamander cells is an algal gene with homology to Niemann-Pick type C (NPC) proteins, which is more highly expressed in intracellular algae. These proteins are involved in intracellular cholesterol transport, and are potential mediators of lipid transfer in cnidarian-dinoflagellate endosymbioses (Dani et al., 2014). Intriguingly, increased expression of this potential sterol sensing gene is observed in our intracellular algal transcripts, whereas in cnidarian-dinoflagellate interactions it is the host that utilizes NPC proteins (Dani et al., 2014).
Further metabolic changes include lower expression of maltase-glucoamylase (MGAM), an acid phosphatase, and trypsin-like proteins in salamander cells with intracellular algae (Supplementary file 11). These changes indicate a reduction in glycogen metabolism (Barbieri et al., 1967), and a reduction in the degradation and utilization of yolk platelets that has been shown to be mediated in part by acid phosphatases (Lemanski and Aldoroty, 1977). Collectively, these metabolic changes may be induced by detection of metabolites transferred by the alga, or alternatively, expression changes of these genes might be modulated by autocrine signaling as there is overlap between nutrient sensing and inflammatory responses .
Potential mechanisms for algal entry into salamander cells
Lipoprotein-related protein 2 (LRP2), which is expressed 5.5 fold higher in invaded salamander cells, is part of a family of multi-ligand receptors that trigger endocytosis (Supplementary file 11) (Fisher and Howie, 2006). Dual binding of malaria sporozoites to a human LRP receptor and heparin sulfate proteoglycans mediates malaria sporozoite invasion into liver cells (Shakibaei and Frevert, 1996). All salamander samples also displayed expression of heparin sulfate basement protein (mean CPM 9.82), though it was not differentially expressed in invaded cells. The alga may have surface proteins that interact with the salamander LRP2 receptor however it does not have a recognizable homolog of the malaria circumsporozoite (CS) protein, which is implicated in interactions with LRP and heparin sulfate proteoglycans (Shakibaei and Frevert, 1996). The lack of a recognizable symbiosomal membrane around an intracellular alga (Kerney et al., 2011) suggests that if they do indeed enter through endocytosis, they must escape from or degrade the host-derived vesicle. Interestingly, there are two algal lipase/esterase genes and one gene with homology to the bacterial virulence factor streptolysin S that are over-expressed in endosymbiotic algae (55, 11, and 82-fold). Lipase/esterases are known virulence factors in bacterial parasites . These algal lipase/esterases may have a role in algal entry or endosome escape. While the LRP connection to malaria entry is an interesting parallel, the over-expression of LRP2 may be attributable to other processes in the host cell. For instance LRP2 may be involved in nutrient sensing as it has been implicated in retinol binding protein (RBP) import (Salamander RBP2 was also more highly expressed in cells with endosymbiotic algae) and vitamin homeostasis (Christensen et al., 1999).
The observed increased expression of salamander villin 1 (VIL1) in cells with intracellular algae (Supplementary file 12) may also reveal a pre-disposition of infected cells for algal entry. Similar predispositions exist in hepatocytes infected with a malaria sporozoite. These express high levels of EphA2 prior to parasite entry, which eventually allows a by-pass of host apoptosis (Kaushansky et al., 2015). Villin one is an actin modifying protein that has recently been shown to be required for membrane ruffling and closure following Salmonella typhimurium invasion of intestinal epithelial cells (Lhocine et al., 2015). This host protein is required for successful pathogen entry and is regulated by the bacterial SptP protein through phosphorylation. Similar membrane ruffling has been observed in regions of algal contact with host alimentary canal epithelium coincident with algal entry (Kerney et al., 2011).
The nature of the endosymbiosis
Whether the alga benefits from this endosymbiotic interaction remains unclear. Similar questions of net 'mutualism' persist for the symbiosis between the bobtail squid Eprymna scolopes and the bacterium Aliivibrio fischeri (McFall-Ngai, 2014) although in both systems microbial cells exhibit specific taxic responses to a developing host, suggesting an adaptive origin of these behaviors. In a previous study, we found evidence consistent with vertical transmission. Algal 18S rDNA was amplified from adult oviducts and Wolffian ducts, and encysted algal cells were found inside the egg capsules of freshly laid eggs using transmission electron microscopy (TEM) (Kerney et al., 2011). However, to date, we have not found conclusive evidence for vertical transmission of the alga from one generation to the next. As such, any benefit to the alga in this endosymbiotic interaction remains unknown.
We may speculate that intracellular algae are providing some benefit to its host, as many past light/dark rearing experiments have shown a net benefit to the salamander embryo from their algal symbionts, which presumably included endosymbionts as well (Pinder and Friet, 1994;Gilbert, 1944;Bachmann et al., 1986;Gilbert, 1944). Two recent studies have suggested the transfer of photosynthate from intracapsular Oophila to the salamander host (Graham et al., 2013(Graham et al., , 2014. However oxygenic photosynthesis and fixed carbon photosynthate transfer does not appear to be a significant contribution from intracellular algae to their hosts (Graham et al., 2013). Instead these algae appear to be utilizing fermentation, a common response of chalamydomonad algae to sulfur deprivation and hypoxic conditions (Yang et al., 2015).
In an intriguing parallel to the metabolic state of intracellular Oophila, some obligate intracellular parasites, such as the apicomplexan Plasmodium sp., are 'microaeorophiles:' these generate most of their energy through the incomplete oxidation of glucose to lactate, a fermentative process (Olszewski and Llinás, 2011). Plasmodium falciparum exhibits increased infection and growth at low partial pressures of oxygen Scheibel et al., 1979). Moreover, the observed cell stress response of the alga is reminiscent of that experienced by intracellular apicomplexan parasites (Bosch et al., 2015). Mutualist ectosymbionts like the bacteria Aliivibrio fischeri also use anaerobic metabolism, including fermentation, and express genes consistent with a response to oxidative stress during their association with the bobtail squid Eprymna scolopes (Thompson et al., 2017;Wier et al., 2010).
The primary fermentation products of chlamydomonad algae include glycerol, ethanol, formate, and acetate, along with smaller amounts of CO 2 and H 2 (Catalanotti et al., 2013). Glycerol is recognized as a major mediator of energy transfer between dinoflagellate photosymbionts and cnidarian hosts (Davy et al., 2012). In the context of the salamander-alga endosymbiosis, the related alga C. reinhardtii was shown to export glycerol after osmotic shock (Léon and Galván, 1995), a condition that intracellular Oophila likely experience upon invasion of salamander cells. Additionally, C. moewusii excretes glycerol during fermentation (Klein and Betz, 1978). Formate and acetate fermentation by-products in bacterial ectosymbionts are known sources of energy and bases for biosynthesis of more complex molecules in animal intestinal cells (den Besten et al., 2013;Karasov and Douglas, 2013). Moderate ethanol concentrations, below 2 mM, appear to be well tolerated by animal cells (Castilla et al., 2004), while higher concentrations become cytotoxic, inducing apoptosis and necrosis (Castilla et al., 2004). It is unknown whether intracellular algae are indeed releasing significant quantities of ethanol into host cells, however prior TEM observations (Kerney et al., 2011), and our results suggest that ethanol is below cytotoxic levels as we do not see indication of a necrotic or apoptotic host response.
Although the main comparison in this manuscript was between intracapsular algae and intracellular algae, we also considered differentially expressed genes between algae cultured in nutrient replete medium and intracellular algae. The latter comparison supported fermentation in the intracellular algae, but did not indicate over-expression of additional biosynthetic capabilities including enhanced vitamin biosynthesis or the production and export of other metabolites that might be beneficial to the salamander. Whether the intracellular algae are on the positive end of a net host benefit remains uncertain, however it is clear that the algae have an unconventional 'photosymbiont' role.
Conclusion
To the best of our knowledge there are only two models where the acquisition of horizontally acquired endosymbionts has mechanistic resolution: dinoflagellates in corals and rhizobial bacteria in root nodules. Starting from wild collected samples in a non-model system, we compiled novel transcriptomes of two organisms and revealed gene expression changes associated with their intracellular symbiosis from low cell number samples. These data reveal that life in a vertebrate's cytoplasm induces a stress response in the symbiotic alga. While the alga appears to benefit from high concentrations of phosphate and organic nitrogen sources, our data suggests that the alga is limited in oxygen and sulfur, and is osmotically stressed (Figure 6). The salamander appears to recognize the alga as foreign, but does not mount an immune response comparable to what is seen in amphibian-pathogen interactions, and the salamander may be actively repressing important immune regulators such as NF-kB as well as receiving a nutritive benefit from the endosymbiotic alga ( Figure 6).
Components of the salamander expression profile are relevant to vertebrate interactions with commensal symbionts. For example, three of the genes with higher expression in cells with endosymbionts in this association, OLFM4, MUC1, and IL8 are also up-regulated in human irritable bowel disease (Gersemann et al., 2012). Other roles of OLFM4 and MUC1 include negative regulation of NF-kB signal transduction, and interactions with commensal ectosymbionts in humans (Liu et al., 2010;Doerflinger et al., 2014;Fields et al., 2014). The notable absence of other transcripts (e.g. TLR's) are indicative of endosymbiont tolerance in this system, in sharp contrast to an expected response to vertebrate pathogens.
As in other endosymbiotic associations, A. maculatum and O. amblystomatis engage in a unique dialog involving host tolerance of the symbiont and metabolic cross-talk between partners. Distinctive facets of this metabolic cross-talk include fermentation in the endosymbiont as well as phosphate and glutamine acquisition from the host cytoplasm. This study has dramatically expanded our ability to interrogate this endosymbiotic dialogue on a molecular level by co-opting the dual RNAseq approach established for parasitology research to a non-model mutualism.
Materials and methods
Embryo and alga collection, cell isolation, and cDNA library preparation A. maculatum cells in 90% RNAlater were diluted to a 50:50 solution of cells in RNA later and amphibian-phosphate buffered saline (APBS; PBS + 25% H 2 O to match amphibian osmolarity of 225 ± 5 mOsm/L) because the 90% RNAlater solution was too viscous for single cell isolation. The solution was spread on a glass slide and inspected between the bright field and epi-fluorescence illumination with a Chlorophyll filter set. Fifty salamander cells with intracellular algae and fifty salamander cells without intracellular algae per individual embryo were separated by manual single cell isolation (mouth pipetting) from dissociated embryos with a hand-pulled borosilicate pipette connected to a rubber tube. Each cell was collected directly from 45% RNA later into a microcentrifuge tube containing 200 mL lysis buffer (Extraction Buffer (XB), PicoPure RNA extraction kit; ThermoFisher Scientific). Four biological replicates were collected from four different A. maculatum embryos from the same clutch. Three biological replicate samples of intracapsular algae from the intracapsular fluid of three eggs were also collected-by aspiration with a syringe and 23 gauge needle-for RNA extraction. Additional RNA was prepared from three unialgal strains of Oophila including UTEX LB3005 and LB3006, which were established previously (Kim et al., 2014 from an egg clutch sampled in 2012 from Greenbrook Sanctuary (Palisades, NY)-unfortunately, was lost during a laboratory power-outage. Quadruplicate sampling was made for quantitative analyses of the LB3005 strain. RNA was extracted from each sample using the PicoPure RNA extraction kit following the manufacturer's recommended protocol for 'RNA Extraction from Cell Pellets', starting with incubation of the lysate at 42˚C for 30 min with the modification of adding an equal volume of 70% ethanol to the 200 mL of lysis buffer. RNA for the cultured algal strains was prepared by using a combination of TRIzol (Thermo Fisher Scientific) and a Qiagen RNeasy kit.
Whole cDNA libraries were prepared directly from total RNA using the SMARTer Ultra Low cDNA Kit-HV (Clontech, Mountain View, CA) according to the manufacturer's protocol for a starting sample of 50 cells. Sequencing libraries for the Illumina HiSeq 2500 platform were prepared from the whole cDNA libraries using the Nextera-XT kit (Illumina, San Diego, CA)
De novo assembly
Eleven paired-end whole cDNA libraries with greater than ten million paired reads per library were processed for assembly. There were four libraries from A. maculatum cells without intracellular algae, and four paired libraries (from the same individuals) from A. maculatum cells with intracellular algae, and three libraries from motile intracapsular algae. Quality trimming of the reads was performed with Trimmomatic (v 0.32) (Bolger et al., 2014) to remove low quality bases and adapter sequences. All eleven paired end libraries were used to construct a total evidence assembly using the Trinity algorithm (version trinityrnaseq_r20140717) (Grabherr et al., 2011;Haas et al., 2013). Transcriptomes of cultured algal strains were assembled separately in Trinity.
Total evidence assembly filtering
The total evidence assembly returned 1,533,193 unique RNA-seq contigs that were clustered into 1,345,464 potential gene level (isoforms collapsed) transcript groups. The assembly largely consisted of a mixture of A. maculatum and O. amblystomatis transcripts. There were also 7,193 transcript groups (0.5%) corresponding to a predatory mite, Metaseiulus occidentalis, and 2,641 transcript groups corresponding to a dermal fungus, Malassezia globosa. Sequences corresponding to the mite and fungus were removed by BLASTN homology search (all BLAST analysis was completed using BLAST+ algorithms, v 2.2.28+ (Camacho et al., 2009) using BLAST databases comprised of all known transcript sequences from the genera Metaseiulus and Malassezia. Transcripts corresponding to the alga O. amblystomatis and salamander A. maculatum were recovered by BLASTN against a database consisting of transcript sequences from lab grown cultures of O. amblystomatis and sequences from the model salamander Ambystoma mexicanum (contributed by Randall Voss-University of Kentucky, and from [Stewart et al., 2013;Wu et al., 2013]). Transcripts were further filtered by a BLASTX homology search against a database containing the entire protein complement of: Arabidopsis thaliana, Chlamydomonas reinhardtii, Mesostigma viride, Micromonas pusilla, Ostreococcus tauri, Oryza sativa, O. amblystomatis, Chrysemys picta bellii, Xenopus tropicalis, A. mexicanum, Pseudozyma, Saccharomyces cerevisiae, and the genera Melanopsichium and Leptosphaeria. The assortment of species was chosen due to phylogentic proximity to O. amblystomatis or A. maculatum, or due to best hits from those genera/species found when a selection of transcripts was queried against the nr database. Best hits to plant or green algal species were noted as algal sequences and combined with the results of BLASTN against the O. amblystomatis database. Best hits to salamander or other animal species were noted as salamander sequences and combined with the results of BLASTN against the A. mexicanum database. Best hits to fungal sequences were discarded. The remainder with no known homology were retained and included as putative algal or salamander transcripts based on their expression pattern across samples. One expectation of differential expression analysis is that most genes are expressed equally between control and experimental samples. If expression levels are ordered from low to high expression in control samples and binned in a sliding window of 100 genes per bin, the median expression level in each bin will increase as the index increases. Based on the expectation of equal expression, for the same sets of genes, the median expression level of experimental samples should correspondingly increase.
For a subset of genes confirmed to belong to A. maculatum by BLAST homology, genes were sorted by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values in the samples of A. maculatum without intracellular algae. Median FPKM values of bins of 100 genes, in a sliding window from the lowest expressed gene through the 100 genes with the highest FPKM values were calculated for the samples of A. maculatum with and without intracellular algae. The median FPKM values for the bins of A. maculatum only samples were plotted against the data from the samples of A. maculatum with intracellular algae (Figure 1-figure supplement 2a). At moderate to high expression levels, A. maculatum with intracellular alga FPKM values increased with A. maculatum without intracellular algae FPMK values (Figure 1-figure supplement 2a). But at very low FPKM values, the data were essentially uncorrelated. Median A. maculatum without intracellular alga FPKM values increased (since the data was ordered by those values), but median A. maculatum with intracellular alga FPKM values stayed the same (Figure 1-figure supplement 2a).
Lower limit FPKM values were determined by finding the FPKM value above which A. maculatum without intracellular alga samples and A. maculatum with intracellular alga samples exhibited a positive correlation (Figure 1-figure supplement 2a). Genes with FPKM values below the lower limit of both sets of samples being analyzed for differential expression (i.e. intracapsular algae and intracellular algae) were not included in the analysis. The uncorrelated expression pattern of genes with FPKM values below the threshold suggests that there is either insufficient sequencing depth to compare those genes between the two conditions, or those lowly expressed genes are expressed stochastically in these cells and the fluctuations in expression levels of those genes are not indicative of a biological difference between conditions. The same analysis was completed for A. maculatum cells with intracellular algae by ordering the genes based on their expression levels ( Figure 1-figure supplement 2b), and for intracapsular (Figure 1-figure supplement 2c) and intracellular algal samples ( Figure 1-figure supplement 2d). . The values are reflective of the sequencing depth of each sample, and are close to the widely used FPKM >1 lower limit threshold used in many RNAseq studies (Fagerberg et al., 2014;Shin et al., 2014;Graveley et al., 2011), except for the intracellular alga samples which suffer from low sequencing depth, but nonetheless display correlated expression with intracapsular alga samples starting at low FPKM values.
After determining the lower limit thresholds, the algal gene set consisted of genes with at least one read pair mapping to each of the three intracapsular algal samples or each of the four endosymbiotic cell samples that additionally were not found in the salamander, fungal, or mite BLAST data. Additionally, the genes had to have expression values above the lower limits described above in respective algal samples and below the lower limit for A. maculatum without intracellular alga samples in the salamander only cell samples for those genes. This resulted in a set of 8989 potential algal genes. However, due to a low depth of sequencing of the algal component of the endosymbiotic cell samples, additional filtering was necessary.
Genes that were not detected in intracellular algae could have been missing due to the lower depth of sequencing rather than representing an actual biological difference in expression between the algal populations. To determine what level of expression in intracapsular alga would be needed for a complete absence of measured expression in intracellular alga to be meaningful, the 8,989 algal genes were first ordered by intracapsular algal FPKM. Then the proportion of genes with no expression in intracellular samples was plotted against the median expression level in high-sequencing-depth intracapsular algae in bins of 100 genes in the ordered data. At low FPKM values in the intracapsular algae, up to 58% of the genes were absent from intracellular samples (Figure 1-figure supplement 3b). As expression in intracapsular alga samples increased, the proportion of genes with measurable expression levels in intracellular algal samples increased as well (Figure 1figure supplement 3b). The same relationship was not observed for A. maculatum data sets, where the depth of sequencing between samples was approximately equal (Figure 1-figure supplement 4).
The dependence of gene detection in intracellular algal samples on FPKM level in intracapsular algal samples abated at intracapsular alga FPKM values where 95% or more genes could be detected in intracellular algal samples (Figure 1-figure supplement 3b, vertical red-dashed line). That expression level corresponded to 67.9 FPKM in intracapsular algal genes. After removing genes with no detectable expression in intracellular algae with expression levels below 67.9 FPKM in intracapsular algae, the dependence of gene detection on expression level in intracapsular algae was removed, and the anomalous peak of undetected genes was removed from the expression histogram (see: Figure 1-figure supplement 3a and c). This resulted in a set of 6,781 genes. Due to finding some genes with homology to anomalous organisms such as pine and beech trees without homologs in C. reinhardtii in the set of 6,781 genes (perhaps due to pollen in the low cell number samples), only genes with homologs in the lab strain Oophila transcriptomes were considered. The final set of algal genes used in differential expression analysis consisted of 6,726 genes.
Differential expression analysis
For transcripts derived from wild-collected samples, read mapping and transcript count quantification was accomplished using Bowtie2 (Langmead and Salzberg, 2012) and RSEM (Li and Dewey, 2011) using default parameters. For transcripts derived from cultured alga, read mapping and transcript quantification was accomplished using BBmap (Bushnell, 2016) and Salmon (Patro et al., 2015) (respectively). The read mapping and quantification algorithms used for reads from the cultured alga were implemented due to divergence of the two algal strains. The transcripts common between the two strains were on average 95% similar. BBmap paired with Salmon allowed for relaxed mapping parameters that were able to map the reads to the transcriptome despite differences in sequence composition with increased sensitivity compared to Bowtie2 plus RSEM. Prior to differential expression analysis, data driven abundance and homology filtering was implemented to derive the final gene lists used in differential expression analysis. A detailed account of filtering procedures can be found in Supplementary Materials and methods under the heading 'total evidence assembly filtering'.
Differential expression analysis was conducted in R (version 3.1.2) (R Core Team, 2013) using the edgeR package . Generalized linear models were used for data analysis on normalized count data. Initial normalization of data derived from wild collected samples (endosymbiont free and endosymbiont containing salamander cells, intracapsular algae, and intracellular algae), was performed by trimmed mean of M-values (TMM) library size scaling-normalization (Robinson and Oshlack, 2010). Incorporation of data from unialgal cultures into differential expression analysis required additional GC-content normalization of the libraries, due to differences in GCbias introduced by the two different library preparation methods (SMARTer cDNA synthesis followed by Nextera-XT library preparation for the wild collected samples and TrueSeq library preparation for the RNA preparation from unialgal cultures) (Figure 7). Normalization of GC-content and transcript length biases were accomplished using conditional quantile normalization (CQN) (Hansen et al., 2012). CQN resolves GC content and transcript length biases by fitting a model that incorporates observed read counts and a covariate such as GC content, and calculates an offset that is used to remove the covariation of these confounding factors. Following cqn normalization, differential analysis between unialgal cultures and wild collected intracapsular or intracellular algae was completed in edgeR. Salamander-only and salamander-plus-alga samples from the same individual embryo were considered paired samples for statistical analysis. Differentially expressed genes were considered as those with an FDR adjusted p-value less than 0.05.
Functional annotation
Functional annotation of A. maculatum and O. amblystomatis transcripts was accomplished by BLASTX of transcripts against the UniProt-SWISSProt curated database (Gasteiger et al., 2001). BLASTX results were filtered by 'homology-derived structure of proteins' (HSSP) score (Rost, 2002) such that annotations were retained for hits with an HSSP_DIST score greater than 5 (Burns et al., 2015). UniProt ID annotations were assigned based on the maximum HSSP score for each gene. This led to 14,761 functional annotations for A. maculatum transcripts and 3,850 functional annotations of O. amblystomatis transcripts. Further annotation of differentially expressed transcripts was accomplished by HHblits (Remmert et al., 2012) homology detection. Unannotated, differentially expressed transcripts were translated in all six reading frames and the translations were processed by HHblits. Significant hits were determined by manual inspection of the HHblits output. Transposable elements were categorized by homology to known transposons by BLASTX or HHblits, and through the use of the PASTEClassifier tool (Hoede et al., 2014). Multiple alignments were created with MUSCLE using default parameters (Edgar, 2004). Alignments were visualized in SeaView (Gouy et al., 2010). Hansen et al. (2012). This metric approximates bias in the sequence dataset by comparing read counts to expected models based on quantiles in the distribution of the GC content of the transcripts. The opposing trends in the two sets of lines shows that GC content bias between the two different libraries is vastly different. The reads obtained from SMARTer-cDNA synthesis-Nextera-XT libraries will tend to have more counts for low GC content transcripts, while the reads obtained from TrueSeq libraries will tend to have more counts for high GC content transcripts, systemically. (b) There is also some moderate transcript length bias differences between the two library prep methods visualized as the separation between the groups of red and blue lines. The methods implemented by the conditional quantile normalization (cqn) package in R handles both types of bias to make the gene count data from both library preparation methods comparable. DOI: 10.7554/eLife.22054.026
Functional grouping of differentially expressed genes
Due to the novelty of this symbiosis, the non-model organisms under consideration, and the multiorganism functional annotation obtained, an initial automated functional grouping, gene ontology (GO) term analysis, completed as in Burns et al. (2015) (Supplementary files 13 and 14 for algal and salamander GO term analysis, respectively) was determined to be insufficient to understand the likely biological function of over and under-expressed genes. Other tools such as REViGO (Supek et al., 2011) performed better (Supplementary files 15 and 16 for algal genes, and Supplementary files 17 and 18 for salamander genes), but did not catch large functional modules that were evident upon further manual inspection of the gene lists. For the small sets of differentially expressed genes observed between salamander cells with or without algae, and between intracapsular and intracellular algae, manual curation of each differentially expressed gene was implemented by performing an extensive literature search for each of the differentially expressed genes, based on the SWISSProt or HHblits annotation. Relevant functions associated with each gene in the scientific literature were noted, and those functions were grouped manually to give the final functional categories discussed in the text. For the larger set of differentially expressed genes observed between algae cultured in nutrient replete media and intracellular algae, only automated annotation and GO term grouping (REViGO) was used to define functional categories.
qPCR validation of gene expression patterns in the alga O. amblystomatis
Hypotheses concerning gene expression patterns associated with endosymbiosis in the alga O. amblystomatis were tested using culture strains of the alga. Validation experiments could not be conducted in the salamander A. maculatum due to the seasonal nature of the association and the lack of any laboratory stock of A. maculatum. O. amblystomatis cultures were maintained at 18˚C under a 12 hr light/12 hr dark cycle with an average light intensity of 34 mmolÁm À2 Ásec À1 , in AF-6 medium (Kato, 1982) with modifications as described previously ( et al., 2004). For examining the dependence of phosphate transporter expression on phosphate levels in the media, AF6 media was formulated without potassium phosphate. Appropriate quantities of concentrated potassium phosphate at a ratio of 1:2 K 2 HPO 4 : KH 2 PO 4 plus potassium chloride (to a final concentration of 130.8 mM K + ions, the concentration of K + ions present in normal AF-6 media) were added to the phosphate deficient AF-6 media to make AF-6 with the various phosphate levels used in the experiment. For examining the dependence of nitrogen-related transporters on nitrogen and glutamine concentrations, AF-6 media was formulated with various levels of nitrate and ammonia with the addition of NaCl to balance the loss of Na + ions from leaving NaNO 3 out of the media.
For phosphate limitation experiments, O. amblystomatis cells growing in AF-6 media were pelleted (1,000xg for 5 min) and washed three times with media completely depleted of phosphate. Following the third wash, cells were re-suspended in phosphate depleted media. Cells were counted and aliquoted into flasks at a concentration of 40,000 cellsÁmL À1 in 5 mL total volume per flask of phosphate deplete AF-6. For each phosphate concentration, 5 mL of AF-6 media with 2x phosphate was added to the appropriate flask to give the appropriate phosphate concentration for the experiment. Cultures were grown for 5 days prior to harvesting for RNA purification. Three flasks of algae were assayed for each phosphate concentration (from 100 pM to 10 mM phosphate in 10-fold intervals).
For nitrogen limitation and glutamine experiments, O. amblystomatis cells were prepared as described for the phosphate limitation experiments using nitrogen depleted AF-6 media. Cultures were grown for 5 days at nitrogen concentrations approximating observed nitrate and ammonia concentrations in salamander egg capsules (6.6 mM NO 3 À ; 17 mM NH 4 + ) prior to addition of glutamine.
Glutamine was added to a final concentration of 2 mM. Algal cells were harvested after 6 hr of incubation with or without glutamine and assayed for gene expression. Three flasks of algae were assayed for each condition.
To prepare cDNA, O. amblystomatis cells were harvested by centrifugation (1,000xg for 5 min), and 350 mL lysis buffer RLT-Plus with fresh b-mercaptoethanol (RNeasy mini Plus kit, Qiagen, Valencia, CA) was added, and the lysate was vortexed for 30s. RNA was purified from the lysate following the manufacturer's protocol. Purified RNA was converted to cDNA using the Quantitect RT kit (Qiagen) following the manufacturer's protocol. Resultant cDNA was diluted with three volumes of 10mM Tris buffer, pH 7.5, or RNase free water prior to qPCR reactions.
Quantitative PCR primers for four reference genes and five response genes (Table 3) were designed using conserved regions in multiple sequence alignments of cDNA sequences from the three O. amblystomatis cultured strains as well as the O. amblystomatis sequences obtained from the field material. Candidate reference genes were selected due to their utility in prior studies in the related chlorophycean alga Chlamydomonas reinhardtii, and were confirmed to have equivalent transcriptome expression levels in intracapsular and intracellular alga in this study. The reference genes were RPL32 & H2B1 (Liu et al., 2012), RACK1 (Mus et al., 2007), and YPTC1 (Lake and Willows, 2003). Several primer pairs were designed for each reference and response gene using the software tools GEMI (Sobhy and Colson, 2012), Primer3 (Untergasser et al., 2012), and Primer-Quest (Owczarzy et al., 2008). Primer pairs were validated by making standard curves using a cDNA dilution series. Primer pairs with the lowest Cq value for a given gene and PCR efficiencies between 0.9 and 1.1 in a standard curve of cDNA dilution series were validated for use in gene expression studies ( Table 3).
Quantitative PCR reactions used 1 mL of the diluted cDNA in 20 mL reactions with a 700 nM concentration of each primer using QuantiNova Sybr green (Qiagen) for amplification and detection. QPCR reactions were done in duplicate. Reactions were performed on a RotorGeneQ instrument (Qiagen) with a 2-step cycling program of 5s at 95˚C and 10s at 60˚C followed by melting curve analysis. Raw data was exported from the RotorGeneQ and per run-per amplicon efficiency correction was implemented in LinRegPCR (version 2015.3) (Ramakers et al., 2003;Ruijter et al., 2009). Differences in expression were analyzed using ANOVA with contrasts in R. | 2017-12-23T07:15:06.807Z | 2017-05-02T00:00:00.000 | {
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252718554 | pes2o/s2orc | v3-fos-license | Interfacial engineered iron oxide nanoring for T2-weighted magnetic resonance imaging-guided magnetothermal-chemotherapy
Due to no penetration depth limitation, low cost, and easy control, magnetic nanoparticles mediated magnetic hyperthermia therapy (MHT) has shown great potential in experimental and clinal treatments of various diseases. However, the low heating conversion efficiencies and short circulation times are major drawback for most existing magnetic-thermal materials. Additionally, single MHT treatment always leads to resistance and recurrence. Herein, a highly efficient magnetic-thermal conversion, ferrimagnetic vortex nanoring Fe3O4 coated with hyaluronic acid (HA) nanoparticles (Fe3O4@HA, FVNH NPs) was firstly constructed. Additionally, the doxorubicin (DOX) was successfully enclosed inside the FVNH and released remotely for synergetic magnetic–thermal/chemo cancer therapy. Due to the ferrimagnetic vortex-domain state, the ring shape Fe3O4 displays a high specific absorption rate (SAR) under an external alternating magnetic field (AMF). Additionally, antitumor drug (DOX) can be encapsulated inside the single large hole of FVNH by the hyaluronic acid (HA) shell and quickly released in response the tumor acidic microenvironments and AMF. What’s more, the non-loaded FVNH NPs show good biocompatibility but high cytotoxicity after loading DOX under AMF. Furthermore, the synthesized FVNH can efficiently reduce the transverse relaxation time and enhance negative magnetic resonance imaging (MRI). The impressive in vivo systemic therapeutic efficacy of FVNH was also proved in this work. Taken together, the results of this study demonstrate that the synthesized FVNH NPs offer the promise of serving as multifunctional theranostic nanoplatforms for medical imaging-guided tumor therapies.
Introduction
In recent years, with the vast development of nanotechnology, the biomedical field has been greeted with tremendous excitement, especially for antitumor applications (Mccarroll et al., 2014;Cryer and Thorley, 2019;Barani et al., 2021). Chemotherapy is one of the most common cancer treatments in clinical practice. However, most chemotherapeutics lack the property of tumor targeting, resulting in various side effects (Lindley et al., 1999;Wu et al., 2005). In order to improve the efficiency of the drug and reduce damage to the normal tissues, many researchers have significantly paid diligently (Zhang et al., 2020a;Yang et al., 2020;Cheng et al., 2021). Among various methods employed to overcome this limitation, designing smart nanocarriers, which can load small molecule antitumor drugs, has shown great promise in precise treatments (Vines et al., 2019;Farzin et al., 2020). Thus, carrying material, encapsulating material, and targeting material is essential for intelligent drug transport (Ahn et al., 2018;Yoo et al., 2019). The porous material is one kind of ideal material for drug loading because of the inherent free space, such as hollow SiO 2 (Zhu et al., 2010;Hu et al., 2015), mesoporous carbon (Hussain and Guo, 2019;Zhang et al., 2020a), liposomes (Al-Jamal and Kostarelos, 2011;Zhao et al., 2019), and metal-organic frameworks Lawson et al., 2021;Ni et al., 2022). For the extra encapsulating material, easy to decorated organic polymers, like the polypyrrole, polyethylene glycol, and polydopamine, are commonly used as a gatekeeper to control the drug release (Wen et al., 2017). The implementation of targeted delivery, small molecule ligands, such as folic acid, Arginine-Glycine-Aspartic acid, and specific base sequences (Steichen et al., 2013;Bazak et al., 2015). Thus, the approach of establishing smart drug delivery nanocarriers can be achieved by integrating all the above-mentioned components.
Conventional medical management is two independent operations in cancer diagnosis and therapy (Litwin and Tan, 2017;Waks and Winer, 2019). Thus, usually, two different drugs are needed for different purposes. This may cause some potential problems: the time interval between the diagnosis and treatment process is long, which may miss the best treatment opportunity; additionally, the treatment depends on the initial diagnosis and can not keep pace with the progression of the disease. So it will be much more meaningful for clinical practice if the diagnosis and treatment can be integrated into one activity, which will help the physician to improve the accuracy of the disease treatment and cut the odds of trouble (Kelkar and Reineke, 2011;Lim et al., 2015). Among all approaches presented in the literature, the multifunctional nanomaterials mediated cancer therapy shows great promise (Janib et al., 2010;Zhang et al., 2019). In the past decades, numerous theranostic nanoplatforms have been developed for combing imaging and therapy in one nanocomposite (Madamsetty et al., 2019). The lipid-based nanoparticles, polymeric nanocarriers, and inorganic nanoplatforms are three major classes of basic materials . Among them, iron oxide is usually used for multiple purposes due to its biocompatibility and low prices, such as magnetic resonance imaging (MRI) T1 and T2 enhancement, photoacoustic imaging enhancement, near-infrared imaging, and magnetic hyperthermia (MHT), magnetic targeting drug delivery and ferroptosis induced cell death (Semkina et al., 2015;Ge et al., 2016;Zhou et al., 2018). In addition, the iron oxide nanoparticles will show active targeting, intelligent controlled drug release, and extra multimodal imaging after a simple modification.
In this work, we designed and prepared the ferrimagnetic vortex nanoring Fe 3 O 4 @HA nanoparticles (FVNH NPs) enclosing Doxorubicin for T2 MR imaging-guided hyperthermia therapy and chemotherapy. The base nanoring material has a single huge hole at the nanometer size, giving FVNH unique magnetic properties. These include extremely low coercivity and high saturated magnetization, which are prerequisites for high-performance negative MRI imaging and magnetothermal purpose. At the same time, the weak dipoledipole interactions of the ring structure at the microscopic level behave as a stable colloidal state at the macroscopic level. The properties of synthesized FVNH were fully characterized, and its antitumor therapeutic efficacy was evaluated in vitro and in vivo. The preliminary findings in this work suggested that DOXloading FVNH nanocomposite can be used as a novel drug delivery nanocarrier with the potential for cancer theranostic use.
Nanoring α-Fe 2 O 3 synthesis
The based single porous material, nanoring α-Fe 2 O 3 , was prepared through a hydrothermal approach according to Frontiers in Bioengineering and Biotechnology frontiersin.org previous studies (Jia et al., 2008;Bao et al., 2022). Briefly, 1 ml 0.5 mol/L FeCl 3 , 500 μl 0.01 mol/L NH 4 HPO 4 , 500 μl 0.03 mol/L NH 4 SO 4 , and 78 ml water were added into the 100 ml Teflon and then stirred severally for 30 min. Then, the Teflon tank was transferred into the high-pressure reactor and heated to 220°C in the silicone oil bath. After the 24-h reaction, the orange product was collected using a centrifugal machine with 10,000°r/min for 10 min. Then the nanoring α-Fe 2 O 3 was washed three times with water and ethanol, separately, and then dried at 60°C overnight.
Reduction of nanoring α-Fe 2 O 3
The nanoring α-Fe 2 O 3 was reduced into Fe 3 O 4 by hydrogen. Briefly, the nanoring α-Fe 2 O 3 was flat out on the horizontal corundum pot and then put the pot in the tube furnace. The reduction reaction was performed with 5% H 2 / Ar mixture gas and kept at 480°C for 2 h. The color of nanoring changed from orange to black, indicating that the hematite has been changed into magnetite and that a magnet can easily collect the final product.
Coating the nanoring Fe 3 O 4 with hyaluronic acid and loading doxorubicin 250 mg of previously reduced nanoring Fe 3 O 4 was dispersed in the de-ionized 100 ml water and then placed in an ultrasonic tank for 30 min at 85°C. Then 50 mg PEI in 10 ml water was added to the above solution, stirring the mixture for another 24 h. After that, the HA was conjugated to the Fe 3 O 4 @PEI surface according to previous work (Li et al., 2014). For coating the HA, 200 mg HA was dissolved in 20 ml of water at a temperature of 70°C. After cooling to room temperature, 20 ml DMSO with 20 mg carbodiimide and 10 mg N-hydroxysuccinimide was added to the HA solution with vigorous magnetic stirring for 3 h. Then activated HA mixture was put into the previous Fe 3 O 4 @PEI solution using the drop-by-drop mode with stirring for another 3 days in the dark. Finally, the ring ferrimagnetic vortex nanoring Fe 3 O 4 @HA nanoparticles (FVNH NPs) was separated from the waste solution using a magnet and dried using a freeze-dryer. For loading the DOX, the 5 mg DOX was added into the nanoring Fe 3 O 4 solution in the PEI coating step and with the same following reactions.
Characterization of FVNH NPs
The synthesized FVNH NPs were fully characterized. The microtopography was observed by scanning electron microscopy (SEM, JSM−5600LV) and electronic transmission microscopy (TEM, JEOL-2100). The crystal structure of obtained samples was determined from x-ray powder diffraction (XRD, D8 Advance Bruker) data. The dynamic light scattering (DLS, Malvern Nano-ZS90) instruments were used to measure the zeta potential and average particle size. The magnetic properties of all samples were measured on a SQUID magnetometer (Quantum Design, MPMS-XL7). An infrared thermal (IR) camera was used to record the thermal images and temperatures (Testo-875). The Nanodrop 2000 spectrophotometer was used to measure the UVvis spectrum (Thermo Scientific). The cells were evaluated by ELISA plate reader (DNM-9606, PuLang New Technology Ltd) at 490 nm and laser confocal microscopy (FV1000, OLYMPUS).
Magnetic hyperthermia assessed
The alternating magnetic field (AMF) was generated by using an inductive heating device (ASPG-10A-II, Shuangping). Different concentration (0-100 μg/ml) of FVNH dissolved in the water was put in the center of the coil of the AMF. After charging with fixed currents and frequency (273 kHz, 600 G), the temperatures of different samples were recorded using an IR camera. What's more, the heating stability and the efficiency of FVNH (100 μg/ml) were also accessed with several circles of onoff of AMF (273 kHz, 600 G) and the specific absorption rates (SAR) under different field strengths (200-800 G) were calculated according to a previously reported method (Quinto et al., 2015). The SAR was calculated using following formula: where Cm is the medium heat capacity, ΔT/Δt is the rate of temperature increase in 60 s, m v and m np are the mass of the suspension and the iron content.
T2 relaxation and magnetic resonance imaging
In this experiment, a clinical 3.0 T MRI scanner was used for magnetic resonance experiments. To measure the T2 relaxation time, different concentrations of FVNH samples were dissolved in the water with different concertation (0.1-2 mg/ml) and then fixed with 0.5% agarose gel in 2 ml centrifuge tubes. In order to accurately measure the R2, the amount of Fe in the PBS solution before relaxation measurement was quantified by ICP-MS. T2 relaxation time was measured using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with the following parameters: repetition time (TR) = 10,000 ms, echo time (TE) = 20, 40, 60, 80, 100, 120, 160, 200, 240 ms and the relaxation rate was obtained by fitting data points to calculate slope. For T2-weighted scan, following parameters were adopted: TR = 5,000 ms, TE = 60 ms, slice thickness = 5 mm, field of view = 50 × 50 mm, matrix = 128 × 128.
Frontiers in Bioengineering and Biotechnology frontiersin.org
Evaluation of the doxorubicin loading and doxorubicin releasing
To take it one step further, the obtained FVNH was explored as the drug-carrying to deliver DOX due to the large hole in the center of the nanoring. The absorption value directly measured the content of the DOX at 490 nm by a UV-Vis spectrophotometry. As previously reported (Zhang et al., 2012), the standard curve of DOX content was fitted using least squares with several known concentrations (1-200 mg/L) of DOX and corresponding absorption values at 490 nm. As mentioned above, the loading nanocarriers were collected with a magnet, and the free DOX in the supernate was separated from the nanocarriers. The AMF (273 kHz, 600 G) as an external stimulus was applied for 10 min to explore whether it would enhance the DOX release. The concentration of DOX was determined by measuring the light absorption intensity of the upper layer liquid, and the drug loading efficiency of DOX was further determined according to Eq. 1
DOX loading efficiency (%)
Amount of encapsulated DOX Amount of FVNH × 100% (1) And the cumulative release was calculated according to Eq. 2 Where N is the number of samplings, C is the DOX concentration and M is the total content of DOX. The volume for the whole system and each sampling is 50 and 5 ml, separately. Three parallel tests obtain all the measurements.
Cytotoxicity experiments
In vitro cytotoxicity tests of free DOX, FVNH, and FVNH-DOX were performed using 4T1 breast cancer cells with the standard MTT method as follows: 1×10 4 4T1 cells, which were in the logarithmic phase, were seeded to 96-wells plate and then different samples were added into the wells with various concentration (0-50 μg/ml). After 4 h of incubation, the previous medium was replaced by a fresh medium, and the cells were cultured for another 24/48 h. Then, 10 μl, 5 mg/ml MTT in the PBS was added to each well and incubated with the cells for extra 3 h at 37°C. The medium was abandoned, and 200 μl DMSO was added to each well to dissolve the cells. After shocking gently for 10 min, the 96-well plate was put into a microplate spectrophotometer (PULANG, DNM-9606, Beijing, China), and the absorbance values at 490 nm were recorded. Cell viability was further assessed by different treatments 1) DOX; 2) FVNH-DOX; 3) FVNH + AMF; 4) FVNH-DOX + AMF, and each group has three repetitions, and the mean value was used for analyzing. Calcein-AM/PI staining inspections were applied, to directly observe the therapeutic efficiency. Briefly, 4T1 cells were seeded to a 24-well plate at pH = 6.0 for 4 h, and then different treatments were performed as mentioned above. Subsequently, the calcein-AM and PI solution was added to each well for the double-staining, and then the dyes were incubated with cells in an incubator for another 20 min before washing off with PBS. Then the CLSM was used to observe and record the live/dead cells in the plate.
In vivo experiments
The 4T1 mouse breast cancer cells (1×10 7 per mouse) were injected into the right hind limb of 30, 4-week-old BALB/c mice. When the tumor volume reached 100 mm 3 , the mice were divided into five groups. The mice in the experimental group were intratumorally injected with 100 μl FVNH + DOX 1 mg/ml in PBS suspension and then placed in an AMF, for 10 min. For the other four control groups: 1) injected with the same volume of PBS; 2) injected with the same volume of FVNH-DOX; 3) injected with 100 μl free DOX, 4) injected with 100 μl FVNH + AMF, 5) injected with 100 μl FVNH-DOX + AMF (the amount of DOX was 5 mg/kg, AMF: 273 kHz, 600 G, 10 min). The treatment was repeated the day after the first treatment to enhance the effect. Five major organs and tumor tissue were excised for H&E staining. Twenty days later, all tumor-bearing mice were sacrificed. The length (L) and width (W) of the tumors were recorded every 2 days using vernier calipers, as well as the weight of the mice was monitored. The tumor volume (V) was estimated by the following formula: V= (L×W 2 )/2. The relative volume of the tumor was calculated as V/V 0 (V 0 was the initial tumor volume before treatments for each mouse).
Statistical analysis
The measurement values were presented as mean ± standard deviation (SD). All statistical analysis was performed by using t-test analysis. *p < 0.05 and **p < 0.01 were accepted as significant and high significant level.
Synthesis and characterization of FVNH
The ring shape of nanoparticles forms progressively as expected through a classical nucleation-aggregation-dissolution process under high pressure and temperature condition. Specifically, the nano hematite disks were first formed by the FeCl 3 . The electronegative ions, SO₄ 2 ⁻ and PO₄³⁻, will coordinate competitively with the surface iron and replace the oxygen. Finally, as previously reported, the ring hole is formatted after Frontiers in Bioengineering and Biotechnology frontiersin.org the reaction duration can control dissolution accrued on the exposed crystal plane and the hole size (Jia et al., 2008). The morphology of ferrimagnetic nanoring is observed by SEM ( Figure 1A), and the uniform ring shape of nanoparticles can be clearly seen. The average external and inner diameters are around 119 ± 11 nm and 69 ± 7 nm. The ring thickness and height are about 32 ± 4 nm and 82 ± 6 nm. The TEM image and high-resolution TEM image of the ferrimagnetic nanoring are shown in Figures 1A,B. Besides the ring shape of the synthesized nanoparticles being further confirmed, the d-spacing of lattice fringes is 0.265 nm ( Figure 1C), which is in line with (311) plane of Fe 3 O 4 . After coating with the HA, no significant changes were observed in SEM images ( Figure 1D). However, a layer of a mistlike slice, about 11 nm thick, can be identified on the TEM image ( Figure 1E). The fast Fourier transforms electron diffraction patterns ( Figure 1F) display well-defined sharp diffraction spots and thus further verifying that the synthesized nanoring Fe 3 O 4 particles are single crystals. The XRD technique was used further to analyze the nanoring crystal structure and its derivatives. As shown in Figure 2A, all the diffraction peaks belong to the crystal peaks of Fe 3 O 4 . It should be noted that there were no other peaks, which further showed that the compositions of both samples are pure. For the FVNH, the XRD pattern is very similar to the naked nanoring Fe 3 O 4 , except for a slightly elevated envelope between the 20-30degree region caused by the PEI and HA, which was caused by the PEI and HA together. Since the surface charge is one factor influencing the materials entering cells, the zeta potential of different samples was measured. What's more, consistent with previous studies (Jiang et al., 2008;Yeh et al., 2013), HA was successfully decorated to the nanoring Fe 3 O 4 -PEI, and the zeta potential dropped down to about -13.5 mV from 18.2 mV accordingly. After the FVNH was dispersed in water, the average hydrodynamic diameter was 181.2 nm measured by DLS ( Figure 2B), much higher than the size measured from the SEM image. This discrepancy can be attributed to the invisible shell layer for the latter. What's more, the colloidal stability of FVNH was explored by monitoring the hydrodynamic diameter over 1 week and the result demonstrate the suitable stability (Supplementary Figure S1). As shown in Figure 2C, the zeta potential for naked nanoring α-Fe 2 O 3 and Fe 3 O 4 is −3.3 mV and −13.5 mV. After the polymerization of PEI and HA, the zeta potential decreased to −22.6 mV. Theoretically, the negative charge of FVNH will aid the NPs enter cells (Asati et al., 2010;Prijic et al., 2010). As shown in Figure 2D, the hysteresis loops of FVNH were evaluated. FVNH show relatively large saturation magnetization, 69.8 emu/g, which means that the FVNH may have great potential to enhance the MHT and MRI. What's more, both coercivity and remanence of FVNH are very low. Thus stable magnetic sol-gel can be realized by reducing the dipoledipole interaction like the superparamagnetic iron oxide nanoparticles. In addition, as shown in the photo, FVNH can be easily absorbed by a magnet, further indicating that the synthesized material has excellent magnetic properties, which can be used for tumor magnetic target treatment. Subsequently, the magnetic-thermal property of FVNH under the AMF was explored, and the results are shown in Figure 3. The temperature rose rapidly in the initial several minutes and then reached to flattened phase gradually within 10 min. Figure 3A shows the temperature-increasing profiles of AMF-caused heat generation for different FVNH concentrations (0-100 μg/ml). Furthermore, the temperature changes were recorded with fixed strength of 600 G, as shown in Figure 3A, the faster rate of temperature increases with the increase of FVNH concentration. For the same FVNH concentration (100 μg/ml), the higher magnetic field strength will increase a magnetic heating effect, and higher SAR values can be achieved ( Figure 3B). The high SAR values of FVNH can be attributed to two factors: 1) the magnetic vortex structure generates large hysteresis loss, and 2) the magnetization reversal process of vortex-to-onion. The magnetothermal stability of FVNH dispersion was also investigated, as can be seen in Figure 3C, the temperature of FVNH dispersion raised from room temperature (~26°C) to the maximum balance temperature (~55°C) during the five cycles of AMF on-off. It can be preliminarily determined that the proposed FNVH has high magnetothermal stability, which may be suitable for multiple treatments with only one injection. What's more, the thermal images of different times (100 μg/ml, 600 G) are shown in Figure 3D. When the AMF duration was increased from 0 to 10 min, the thermal images became much hotter, which is in line with the temperature-increasing curves. Preliminarily, the obtained FVNH shows excellent magnetic heating characteristics and may have potential application in MHT.
T2 weighted magnetic resonance images
For modern medicine, MRI plays an essential role in tumor diagnosis. Besides the morphological examination, MRI is the primary way to evaluate benign and malignant conditions through the injection of the contrast agent. Various gadolinium-based small molecules are currently used as T1W contrast agents, which will make the tissues much brighter but have biological security problems. On the other hand, the T2W contrast agent developed slowly. Superparamagnetic nanoparticles, well known for their good biocompatibility, are considered an ideal contrast agent that can efficiently shortenthe T2. As a similar magnetic material, the potential of obtained FVNH as T2 contrast agents were preliminary explored in this work at 3 T. As shown in Figure 4A, the T2W images become dark gradually as the concentration of the FVNH increase.
Additionally, the transverse relaxivity (r2) value of the FVNH NPs reached 56.25 mM −1 S −1 , which indicats that the synthesized material has a great ability to shorten T2. The amount of relaxation change depended on the distance between the Fe of nanoring Fe 3 O 4 core and the nearby proton of water. Due to shielding effects from the PEI-HA shell, the saturation magnetization decreased slightly, and the transverse relaxation rate decreased accordingly for the same reason, which is consistent with previous studies. It should notice that the FVNH also shows higher T2 values than conventional spherical iron oxide nanoparticles, which can be attributed to the vortex property. T2W images are shown in Figure 4B, with the increase of FVNH concentration from left to right tubes, the image becomes darker. Then the sample was injected into a tumor-bearing mouse, as can be seen in Figure 4C, after the injection, the tumor (red dash circles) showed much lower MRI signal intensity, which means FVNM NPs have been concentrated in the tumor site. Additionally, MR signal intensity is significantly lower after 6 h injection than that of 2 h injection ( Figure 4D), which indicates the sample concentration is higher for the former time point. The results Frontiers in Bioengineering and Biotechnology frontiersin.org revealed that FVNH NPs had an outstanding performance for enhancing T2W MRI both in vivo and in vitro, and could serve as a potential contrast agent for MR image-guided MHT.
Drug loading and release
The UV-Vis spectrophotometer evaluated the DOX loading in FVNH by the absorption value at 490 nm. Moreover, the calculated loading capacity of DOX for the synthesized NPs is 31.5%. The relatively high value of the drug loading capacity can be attributed to two factors: 1) There is a very large hole in the center of the ring shape basic nanomaterial; and 2) The other may be the intermolecular interactions, like the hydrogen bonding interaction. The in vitro DOX release behaviors were explored at different pH (7.4, 6.0, and 5.0) using dialysis bags. As shown in Figure 5A, the release profiles were plotted for 48 h duration according to the absorbance values. The fluorescent intensity was much higher for pH 5.0 than 6.0 and 7.4, which means the lower value environments will accelerate the DOX release. The cumulative released amount in 48 h is 13.0%, 26.6% and 41.2% for pH 5.0, 6.0, and 7.4, separately. It should be noticed that the efficient release under acidic conditions is very adaptable to the tumor microenvironments. The results are in line with previous similar studies (Fang et al., 2019;Wu et al., 2019). This phenomenon is attributed to the interaction effects between DOX and nanocarrier disappearing and the dissociation of carboxylic acid groups. The responses of drug release excited by the magnetothermal thermal were assessed subsequently. As shown in Figure 5B, the fluorescence intensity shows a leaping increase when applying AMF, while the leaping phenomenon disappears when the AMF is absent. The smart "on-off" effect may be that the increased temperature will debilitate the interaction between DOX and nanocarrier, weakening the electrostatic force.
Cell viability assay
Subsequently, we studied the cell viability with various treatments by the standard MTT method, and the results are shown in Figures 6A,B. FVNH has a negligible effect on the 4T1 cell viability even at a concentration of 500 μg/ml and 48 h incubation, which preliminary evidence it is excellent biocompatible nature. The amount of iron internalized in the cells was quantified using ICP-MS. As shown in Supplementary Figure S2, higher cellular iron levels were observed with higher FVNH concentration from 0 to 0.2 mM [Fe] as expected. While for different treatments (free DOX, FVNH-DOX, FVNH + AMF, FVNH-DOX + AMF), with the decrease in the concentrations of Frontiers in Bioengineering and Biotechnology frontiersin.org samples (conversion of DOX content), the cytotoxicity for 4T1 cells increased accordingly. As expected, for FVNH-DOX + AMF group, the cell viability is significantly lower than in any other group. That is to say, the synergistic therapy is better than each independent treatment. In order to observe the cell killing, a live-dead cell stain was further applied as well. As shown in Figure 6C, the live cells were stained with green color, and the dead cells were stained with red color. The largest red fluorescence area appeared in the synergistic group, which is consistent with the MTT experiments. Altogether, these results proved that the designed FVNH-DOX had a very good combined MHT-chemo effect for in vitro antitumor cell efficiency.
The in vivo therapeutic efficacy
In vivo combined antitumor tests were performed on tumorbearing mice using FVNH-DOX as a therapeutic reagent. To monitor the magnetothermal effect of synthesized FVNH in vivo, temperature changes in the tumor site under the AMF were recorded using an IR camera. As shown in Figure 7A, the temperature of the tumor increased rapidly under the AMF, rising significantly from approximately 37°C to about 45°C within 1 min. In contrast, the temperature of the control tumors remained almost unchanged under the same AMF. As tumor cells are more heat sensitive than normal cells, these results further demonstrate the promise of FVNH-DOX as an effective agent for both chemo-magnetothermal therapy. Although the magnetic nanorings have been used as magnetothermal agents to treat tumors, the potential use of it encapsulated with DOX for synergistic cancer treatment has not been reported. The changes in relative tumor volumes in 4T1 tumor-bearing mice further demonstrated the anti-tumor capacity ( Figure 7B). All treatment groups showed a slight overall increase in body weight over the 18-day study ( Figure 7C), further indicating few side effects of the FVNH and the magnetothermal treatment. The FVNH-DOX + AMF treatment group showed significant tumor suppression after 18 days of treatment, but for the other four groups, PBS, FVNH-DOX, free DOX, and FVNH + AMF groups still had various degrees of tumors growth. The FVNH-DOX + AMF treatment group had the smallest tumor volume, even disappeared ( Figure 7D), of the five groups, confirming that the combination treatment was much better than the other single treatment groups. Furthermore, as seen in the H&E-stained tumor tissue sections, there was more necrosis and apoptosis in the tumor tissue of the FVNH-DOX + AMF treated group ( Figure 7E), but no significant necrotic cells in other major organs (Supplementary Figure S3). Histological analysis of diferent organs in tumor-bearing mice stained with H&E are displayed in Supplementary Figure S3. All the organs from five gruops were normal, preliminary indicating that less toxicity of FVNH and treaments to the major organs. Overall, the FVNH loading with DOX nanocomposites in the present of AMF could be a promising nanoplatform for combined magnetothermalchemotherapy of cancer in future clinical trials.
Due to the lack of depth limitation, low cost, and suitability for remote control, magnetothermal therapy, and chemotherapy, based on magnetic nanoparticles in combination with controlled drug delivery systems, have been extensively studied in recent years. To improve the biocompatibility of magnetic nanoparticles and to achieve superior diagnostic and therapeutic purposes, scientists have made a number of material-based strategies. Fan et al. (2010) and our previous study demonstrated that the ring shape nano magnetite has magnetic vortex properties and thus Frontiers in Bioengineering and Biotechnology frontiersin.org excellent magnetothermal properties for magnetothermal tumors therapy as well as enhancing MRI (Bao et al., 2021); By modifying HA on graphene and attaching the composite to iron oxide nanoparticles after wrapping DOX, Pramanik et al. found that this nanoplatform could not only control the drug release by an external magnetic field, but it would kill much more breast cancer cells than the non-magnetic thermal control group (Pramanik et al., 2019). Recently, Zhang et al. (2020b) synthesized an iron oxide nanoparticle with super magnetothermal efficiency through a green biomineralization process, Frontiers in Bioengineering and Biotechnology frontiersin.org 10
SCHEME 1
Schematic illustration of the nanoring Fe 3 O 4 @HA-DOX for MRI imaging-guided synergistic MHT-chemo cancer therapy.
Frontiers in Bioengineering and Biotechnology frontiersin.org 11 with a specific absorption rate of 2390 W/g, and they demonstrated that the NPs could be used as an excellent magneto-thermal agent and nanoenzyme as well for liver tumor inhibition. Despite the great progress made in the field of nanomediated magnetothermal therapy, more time may be needed for its application in the clinic, and there are still challenges to be addressed, such as the low thermal efficiency compared to photothermal and biosafety issues.
Conclusion
In this paper, the one-pot solvothermal method combined with the following reduction reaction was used to prepare ring shape and mono-dispersion Fe 3 O 4 nanoparticles. The core-shell nanoring Fe 3 O 4 @HA loaded with DOX was formed for magnetothermalchemo cancer therapy and enhancing T2W MRI imaging. Notably, the FVNH shows significantly extremally high magnetothermal conversion efficiency and shoring the transverse relaxation time. Additionally, a thin HA coating was formed on the nanoring surface, which endowed the NPs with a high-capacity of drug loading and triggered releasing abilities. The preliminary results demonstrated that the established vortex nanoring Fe 3 O 4 -based nanoconstruct might hold great potential for DOX delivery and cancer theranostics.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Ethics statement
The animal study was reviewed and approved by The Ethics Committee of the First Affiliated Hospital of Zhengzhou University. | 2022-10-06T13:06:23.788Z | 2022-10-06T00:00:00.000 | {
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53334404 | pes2o/s2orc | v3-fos-license | Application of Simulated Annealing in Water Resources Management: Optimal Solution of Groundwater Contamination Source Characterization Problem and Monitoring Network Design Problems
[Extract] Estimating various characteristics of an unknown groundwater pollutant source can be formulated as an optimization problem using linked simulation-optimization. Meta-heuristics based optimization algorithms such as Simulated Annealing (SA), Genetic Algorithm (GA), Tabu Search etc. are now being accepted as reliable, faster and simpler ways to solve this optimization problem. In this chapter we discuss the suitability of a variant of traditional Simulated Annealing (SA) known as the Adaptive Simulated Annealing (ASA) in solving unknown groundwater pollutant source characterization problem.
Introduction
Estimating various characteristics of an unknown groundwater pollutant source can be formulated as an optimization problem using linked simulation-optimization. Meta-heuristics based optimization algorithms such as Simulated Annealing (SA), Genetic Algorithm (GA), Tabu Search etc. are now being accepted as reliable, faster and simpler ways to solve this optimization problem. In this chapter we discuss the suitability of a variant of traditional Simulated Annealing (SA) known as the Adaptive Simulated Annealing (ASA) in solving unknown groundwater pollutant source characterization problem. Growing anthropogenic activities and improper management of their impacts on groundwater quality has resulted in widespread contamination of groundwater worldwide. Coupled with ever increasing water demand leading to increased reliance on groundwater, it has resulted in a widespread recognition of public health risk posed by contaminated groundwater. This has triggered massive efforts for better management of groundwater quality in general and remediation of contaminated aquifers in particular. The sources of contamination in groundwater are often hidden and inaccessible. Characteristics of these pollutant sources such as their location, periods of activity and contaminant release history are often unknown. Groundwater contaminant source identification problem aims at estimating various characteristics of an unknown groundwater pollutant source using measured contaminant concentrations at a number of monitoring locations over a period of time. It has been widely accepted that for any remediation strategy to work efficiently, it is very important to know the pollutant source characteristic. A detailed account of different categories of source identification problems and various approaches to solve them has been presented in Pinder [26].
Typically, groundwater contamination is first detected by one or more arbitrarily located well or monitoring location. Unknown Groundwater source identification problem specifically attempts to ascertain the following source characteristics: Source type is often obvious. In some cases, information on groundwater contaminant source location may be available from preliminary investigations. If an exhaustive record of pollutant inventory and industrial activities of the area is available, it may be possible to infer start time. Release history of the source, however, is difficult to ascertain as the source is not physically accessible for measurements and hence it is unlikely that any accurate temporal record of contaminant fluxes released from the source exists.
Release history reconstruction problem is one of the most widely studied groundwater source identification problems. Ascertaining release history of the contaminant sources from available contaminant concentration measurements is an inverse problem as it requires solving groundwater flow and transport equations backwards in time and space. The process of solving this inverse problem is essentially the process of finding various unknown characteristics of source using observed information about the transport media and the effects caused by the source. In such circumstances, a solution cannot be guaranteed, especially when observed information is sparse. Even if the solution exists, it may not be unique. This is because different combinations of various source characteristics can produce the same effect at a monitoring location. Moreover, the solution of this problem is highly sensitive to measurement errors either in the observation data or model parameters and hence this problem has been classified as an ill-posed inverse problem [33] . When this inverse problem has to be solved by using inaccurate values of media parameters such as hydraulic conductivity and porosity and contaminant concentration observed at arbitrarily placed monitoring wells, it becomes even more challenging to obtain a reliable solution.
Methods proposed in the past to solve this ill-posed inverse problem can be broadly classified as optimization approaches, analytical solutions, deterministic direct methods and probabilistic and geo-statistical simulation approaches. A detailed review of these methodologies can be found in Atmadja & Bagtzoglou [2]; Michalak & Kitanidis [25]; Bagtzoglou & Atmadja [3] and Sun et al. [31,32]. The most effective of all suggested methods seems to be those based on optimization or probabilistic and geo-statistical simulation. Of the optimization methods, linked simulation-optimization approaches have been established as one of the most efficient methods. In this approach, a numerical groundwater flow and transport simulation model is linked to the optimization model. All the linked simulation-optimization approaches aim at solving a minimization problem with an objective function representing the difference in measured concentration and simulated concentration at various monitoring locations. The optimization model generates candidate solutions for various source characteristics. This is used as an input for the simulation model to generate estimated contaminant concentration observations at designated monitoring locations. The optimization algorithm then calculates the value of objective function by calculating the difference in contaminant concentrations estimated by simulation model and actual observed values at the same monitoring locations over a period of time. Over a number of iterations, the optimization algorithm minimizes the objective function value. Most prominent approaches in this category are linear programming with response matrix approach [9], nonlinear optimization with embedding technique [21][22][23], artificial neural network approach [28][29][30], constrained robust least square approach [31,32], classical optimization based approach [5][6][7], genetic algorithm based approach [1,24,27] etc.
In recent past, heuristic global search approaches such as Genetic Algorithm [12], Harmony Search, Tabu Search, Ant-Colony Optimization, Simulated Annealing [19] have developed rapidly and have been applied to a wide range of optimization problems. One of the major reasons for their popularity is the fact that these optimization methods do not easily get trapped in the local optima, thereby maximizing the probability of achieving a global optimal solution. Genetic algorithm (GA) and its variants, in particular, have been widely applied for solving unknown pollutant source identification. [11,24,27]. Genetic Algorithms are computational optimization algorithms that simulate the laws of natural genetics and natural selection and use it to search for the optimal solution.
Apart from GA or its variants, Simulated Annealing has also been used in solving inverse problems in groundwater management. Simulated annealing is inspired by the physical process of annealing in metallurgy which involves heating and controlled cooling of a material to reduce defects in crystal structure. The atoms are excited by heat and they become agitated while getting into higher energy states. The slow cooling allows a better chance for these atoms to achieve lower energy states than the ones they started with. In simulated annealing, a current solution may be replaced by a random "neighborhood" solution chosen with a probability that depends on the difference between corresponding function values and on a global parameter T (called temperature) that is gradually decreased in the process. Implementations of simulated annealing has been relatively limited because the traditional simulated annealing algorithm is reported to converge slower compared with GA or its variants. However, faster variants of simulated annealing have been developed and one of the most promising variants in terms of convergence speed is Adaptive Simulated Annealing (ASA) [14,15]. The ASA code was first developed in 1987 as Very Fast Simulated Re-annealing (VFSR) [13]. Ingber & Rosen [16] showed that VFSR is at least an order of magnitude superior to Genetic Algorithms in convergence speed and is more likely to find the global optima during a time limited search.
Linked simulation-optimization based approaches are computationally intensive as the simulation model has to be run many thousands of times before an acceptable solution is produced. This has been a deterrent to any desktop based implementation of the simulation-optimization approach. Faster convergence can reduce the computational burden significantly and thereby enhance the possibility of a desktop based implementation of linked simulation-optimization approach. This paper investigates the applicability of ASA to unknown groundwater contaminant source release history reconstruction problem and compares its performance to genetic algorithm based solution. The performance evaluation of competing simulation-optimization approaches are based on a realistic scenario of missing measurement data, where contaminant concentration measurements are available a few years after the sources have ceased to exist. Apart from the convergence speed, the two algorithms are compared for their ability to produce accurate source release histories with moderately erroneous data and with uncertainty in estimation of hydro-geological parameters. One of the most important factors that affects the execution time and accuracy of solutions generated by linked simulation-optimization approaches is the choice of observation locations. Poorly chosen contaminant observation locations often produce misleading results and hence it becomes important that after the initial estimation of the contaminant sources, a monitoring network is designed and implemented. In this study we use a monitoring network designed specifically to enhance the efficiency of source identification. However, a detailed discussion of the methodology used for monitoring network design is beyond the scope of this book.
Methodology
The linked simulation-optimization approach consists of two parts. An optimization algorithm generates the candidate solutions corresponding to various unknown groundwater source characteristics.
The candidate solutions are used as input in the numerical groundwater transport simulation model to generate the concentration of contaminant in the study area. The generated concentration at designated monitoring locations is matched to the observed values of contaminant concentrations at various time intervals at the same locations. The difference between simulated and observed concentration is used to calculate the objective function value which is utilized by the optimization algorithm to improve the candidate solution. The process continues until an optimal solution is obtained. A detailed schematic representation of this process of using SA as the optimization algorithm in a linked simulation-optimization model is presented in Figure 1. The classical simulated annealing (SA) algorithm has many associated guiding parameters such as the initial parameter temperature, annealing schedule, acceptance probability function, goal function etc. Effective application of the classical simulated annealing to a particular optimization problem normally involves a lot of trials and adjustments to achieve ideal values for all or most of these parameters. ASA, which is a variant of classical SA, helps overcome this difficulty to a certain extent by automating the adjustments of parameters controlling temperature schedule and random step selection thereby making the algorithm less sensitive to user defined parameters compared with classical SA. This additional ability of ASA combined with inherent ability of classical SA to find the global optimal solution even when multiple local optimums exists, makes it a natural choice for solving the groundwater pollutant source identification problem.
Governing equations
The three-dimensional transport of contaminants in groundwater can be represented by the following partial differential equation [17] Where C is the concentration of contaminants dissolved in groundwater,ML −3 ; t is time, T; x i , x j is the distance along the respective Cartesian coordinate axis, L; θ is the porosity of the porous medium, dimensionless; N is the number of chemical species considered; ∑ N k=1 R k is the chemical reaction term for each of the N species considered, ML −3 T −1 .
In order to solve this transport equation, linear pore water velocity needs to be known for the study area. Hence, it becomes necessary to first calculate the hydraulic head distribution using a groundwater flow simulation model. The partial differential equation for groundwater flow is given by the following equation: Where K xx , K yy , and K zz are the values of hydraulic conductivity (LT −1 ) along the x, y and z co-ordinate axes respectively; H is the potentiometric head (L); W is the volumetric flux per unit volume representing sources and/or sinks of water (T −1 ); S s is the specific storage of the porous media (L −1 ); and t is time (T).
The flow equation describes transient groundwater flow in three dimensions in a homogeneous anisotropic medium, provided the principal axes of hydraulic conductivity are aligned with the co-ordinate directions. A computer code called MODFLOW is used to solve this groundwater flow equation. MODFLOW was developed by United States Geological Survey (USGS) and is one of the most popular computer programs being used to simulate groundwater flow today. MODFLOW is based on modular finite-difference method which discretizes the study area into a grid of cells. The potentiometric head is calculated at the center of each cell.
To solve the three dimensional ground water transport equation, another computer code called MT3DMS is used. This is also a very popular computer program developed by the USGS and uses modular finite-difference just like MODFLOW. The transport simulation model (MT3DMS) utilizes flow field generated by the flow model (MODFLOW) to compute the velocity field used by the transport simulation model. [34]
Formulation of the optimization problem
It is assumed in this study that information on a set of potential source locations are available. The objective of simulation-optimization method then reduces to regenerating the source release histories at these potential source locations. Spatial and temporal contaminant concentration(C) is known at specific monitoring locations at various point of time. Candidate source fluxes are generated by the optimization algorithm. These values are used for forward transport simulations in MT3DMS. The difference between simulated and observed contaminant concentrations are then used to calculate the objective function. The objective function for this optimization problem is defined as: Where, cest k iob = Concentration estimated by the identification model at observation well location iob and at the end of time period k. nk = Total number of concentration observation time periods; nob= Total number of observation wells; cobs k iob = Observed concentration at well iob and at the end of time period k; w k iob = Weight corresponding to observation location iob, and the time period k.
The weight w k iob can be defined as follows: Where n is a constant, sufficiently large, so that errors at low concentrations do not dominate the solution [18]. It is possible to include other forms of this weight.
Optimization algorithms
Of the various simulated annealing implementations, it is evident in literature that the adaptive simulated annealing algorithm converges faster [16] while maintaining the reliability of results and hence it was preferred over traditional Boltzmann annealing implementation [19]. Its application to the unknown pollutant source identification has been limited but it is potentially a good alternative because its convergence curve is steep, thereby producing better results when execution time is limited.
Currently, the most widely used optimization algorithm for solving groundwater source identification problem using linked simulation-optimization model is Genetic Algorithm and its variants. The effectiveness of ASA in solving this problem is compared against the effectiveness of GA. Genetic algorithms (GAs) are population based search strategies which are popular for many difficult to solve optimization problems including inverse problems. GAs emulate the natural evolutionary process in a population where the fittest survive and reproduce [12]. GA-based search performs well because of its ability to combine aspects of solutions from different parts of the search space. Real coded genetic algorithm was used with a population size of 100, crossover probability of 0.85 and a mutation probability of 0.05. The values were chosen based on a series of numerical experiments.
Performance evaluation
In order to evaluate the performance of two different optimization algorithms involving comparison of solutions obtained, it is vital to first ensure that only one solution exists. In other words, a unique solution has to be guaranteed. This is possible only under the following idealized assumptions [33]: 1. The numerical models used for simulation of groundwater flow and transport are able to provide exact solution of the governing equations in forward runs.
2. All the model parameters and concentration measurements are known without any associated errors.
3. The unknown parameter is piecewise constant.
The first assumption is valid for cases where grid size and time step used in the numerical solution tends to zero. However since the groundwater simulation models used in this study have been proven to be stable and convergent, this assumption approximately holds. The second assumption however, cannot hold in real life scenarios. Hence it becomes necessary to use synthetically generated observation values initially which can be considered free of measurement errors. The third condition is implemented by assuming that the unknown fluxes are constant in every stress period. In such conditions it approximately resembles a well posed problem. Therefore these evaluations are initially carried out for synthetic data (simulated data) with known parameter values. There is another related issue of unique solutions. Whenever numerical simulation and optimization models are used, the convergence of the solutions may be another issue related to unique solutions. These issues are discussed in [4]. In this study the use of synthetic observation data, with known hydro-geologic parameter values reduces the ill-posed nature of the problem. The uniqueness of the solution cannot be guaranteed. However, sufficient iterations were allowed to ensure convergence to the optimal solution. Performance of the source identification methodology is evaluated using synthetic data from a three dimensional aquifer study area. The synthetic contaminant concentration data is obtained by solving the numerical flow and transport simulation models.
Simulating errors in concentration measurement data
Once the global optimal solution has been obtained for the idealistic assumption, the performance evaluation of developed methodology can take into account the effects of contaminant concentration measurement errors as well as uncertainty associated with the determination of hydro-geological parameters. To test the performance for realistic scenarios, concentration measurement errors are incorporated by introducing varied amounts of synthetically generated statistical noise in the simulated concentration values. The perturbed simulated concentrations represents erroneous measurements and is defined as follows: Where,
C ns = Simulated Concentration
S ud = a uniform random number between -1 and +1 a = a fraction between 0 and 1.0.
Performance evaluation criteria
The execution time of the algorithms is compared based on convergence curves which represent the value of objective function achieved versus time. To compare the ability of competing linked simulation-optimization approaches to produce accurate source histories, the errors in estimating source fluxes accurately is also used as a performance criterion. Normalized absolute error of estimation (NAEE) is used as the measure of errors in estimation of the sources. It can be represented as:
Discussion of solution results
The developed methodology was applied to a hypothetical illustrative study area with synthetically generated concentration measurements over space and time. Advantage of using a hypothetical study area lies in the fact that unknown data errors do not distort the performance evaluation of the methodology. This helps in understanding the drawbacks of developed methodology and improving it further.
Study area
The hypothetical study area is a heterogeneous aquifer measuring 2100m x 1500m x 30m and consisting of three unconfined layers as shown in Figure 2.
The East and west boundaries are constant head boundaries, whereas north and south boundaries are no flow boundaries. There are two sources (S1 and S2) of contamination. S1 is located in the top layer and S2 in middle layer. Five monitoring location (M1 through M5) are located in the first layer as shown in Figure 3. A grid size of 30m x 30m x 10m is used for finite difference based numerical calculation of groundwater flow and transport equations. Transport time step used for MT3DMS is 36.5 days. Other model parameters are listed in Table 1. Only a conservative contaminant is considered. There are two point sources of contaminants. One in the top layer and another one in the middle layer. A time horizon of 16 years is considered. Entire time horizon is divided into 5 different stress periods. The first four stress periods are each 1.5 years long and the final stress period is of 10 years duration. Sources are assumed to be active only in the first four stress periods or in the initial 6 years. Original source fluxes are presented in Table 2. It is assumed that groundwater contamination is detected at five different locations in the study area at the
Release history estimation with error free data
A set of error free observation data is generated. These observations are then used to evaluate the developed linked simulation-optimization methodology based on both GA and ASA. Input parameters used for GA and ASA are presented in Table 3. Every iteration of ASA based method uses one run of the groundwater transport simulation model (MT3DMS) whereas every generation of GA based method uses 100 (population size) runs of the same simulation model. Irrespective of the method, one run of the groundwater transport simulation model takes 4.281 sec to run on a Dell Optiplex®running an Intel®Core™2 Duo Processor at 2.93GHz. The execution time for one transport simulation run is however dependent on the computing platform. In order to keep the comparison independent of computing platform, both the methods were compared based on number of transport simulation runs which is directly proportional to the execution time. Both the methods were used to estimate source release histories using the error free data. In order to verify the convergence of each optimization method, time of run was made practically unconstrained. It was found that eventually both the optimization algorithms were able to achieve an objective function value very close to zero and identified the release history accurately. The objective function convergence profile as well as estimated fluxes were plotted at the end of 40,000 simulation runs of the groundwater transport model. Minimum value of objective function achieved is plotted against number of runs of the transport simulation model. The estimated flux values for both the sources in each stress period is also plotted against actual source fluxes. Convergence profile and source flux estimates are shown in Figure 4. Convergence profile shows that the objective function value for source identification model converges to a value very close to zero with about 5,000 simulation runs. However, further convergence is accelerated when using ASA algorithm. From these results, it can be concluded that the developed methodology is able to achieve optimal solution for an ideal error free scenario which resembles a well-posed problem.
Release history estimation with erroneous data
Five sets of erroneous observation data are generated with the formulation described in Equation 5. The value of fraction 'a' is specified as 0.1. These erroneous observations are used to reconstruct the release histories of contaminant sources. Linked simulation optimization method using ASA is compared with the one using GA as the optimization algorithm. Parameters used for both the optimization algorithms is presented in Table 3. Unlike the case with error free measurement data, in this case both the methods were used Table 3. Parameters used in Optimization Algorithms to reconstruct source release histories using the erroneous data with a limit on execution time.
In order to make the comparison consistent by ensuring same number of simulation runs in the ASA and GA based methodologies, the number of simulation runs are restricted to 40,000. This restriction was based on the fact that increasing the number of simulation runs even to 80,000 resulted in very little improvement in the objective function value. Minimum value of objective function achieved is averaged over five solutions and is plotted against number of runs of the transport simulation model. The plot is presented in Figure 5. This Figure 5. Convergence Plot plot clearly shows that ASA based method converges much faster in the beginning and the GA based method is able to achieve comparable objective function values only after a much larger simulation runs. Because of the erroneous measurement data this problem may be ill-posed and the solution may not be unique. Therefore, lower objective function values do not always mean accurate reconstruction of the release histories.
In order to test the effectiveness of the competing methods based on accuracy of solutions produced, reconstructed release histories were compared to the actual release history after every set of 10,000 transport simulation runs. The results are shown in Figure 6. It can be seen that ASA based method is more efficient compared to GA based method after 10,000 and 20,000 simulation runs. However, as the execution time increases further with increase in number of simulation runs, the release histories produced by both methods become similar. This is also confirmed from the calculated values of NAEE presented in Table 4. As the execution time increases, the NAEE of ASA based method appears to increase only slightly. This could be due to statistical variation in the five different solutions and may be attributed to the input data error. Averaging over larger number of solutions may modify this inference. NAEE of GA based method consistently improves. However, the NAEE values obtained using ASA is still better in comparison.
Application of simulated annealing for monitoring network design
Monitoring network design in the context of groundwater quality management essentially means specifying the spatial location of monitoring wells and frequency of sampling. Since this is one of the most cost intensive part of most contaminated groundwater remediation problems, an efficient and cost effective design of monitoring network is essential. Monitoring of groundwater quality may be necessitated by a variety of objectives such as: 1. Unknown groundwater source characterization 2. Compliance monitoring for limiting the effects of groundwater contamination 3. Better aquifer characterization 4. Hydro-geological parameter estimation Irrespective of the various objectives, the problem of monitoring network design can be formulated as an optimization problem [8,20]. While designing a monitoring network for estimating unknown groundwater source characteristics, the objective of optimization can be to maximize the reliability of estimated source characteristics or to minimize the total number of monitoring locations in the network or both. Compliance monitoring is aimed at minimizing the area of contamination when the contamination is first detected at monitoring network or maximizing the probability of detection of contaminant in groundwater. Often, only the average values of hydro-geological parameters of the aquifer are known. This results in uncertainty in the modeling results. In order to better characterize an aquifer, spatial distribution of hydro-geological properties should be specified. This objective can be achieved by sampling hydro-geologic parameter at sufficient locations such that the interpolated values can represent actual hydrological parameters accurately. The objective of optimization in this case is to find the minimum number of samples required to accurately represent a population of random hydro-geological parameter values. In all such cases, Adaptive Simulated Annealing can be efficiently used as the tool for optimization. Our attempts to develop classical simulated annealing algorithm for optimal design of a dedicated monitoring network for enhancing the efficiency of source identification was successful to a large extent. However, the mixed integer nature of the decision variables in a monitoring network design problem makes the application of classical simulated annealing algorithm a bit constraining. Adaptive Simulated Annealing is more suitable to solve this monitoring network design problems.
Suitability and sensitivity of adaptive simulated annealing
In the application discussed here, Simulated Annealing is utilized for finding the global minimum of a cost function that characterizes large and complex systems such as transport of pollutants in groundwater.Simulated Annealing, as an algorithm, is very efficient in solving non-convex optimization problems by ensuring that it does not always move downhill on a complex non-convex search space and hence avoids getting trapped in local minimum. Simulated annealing also differs significantly from conventional iterative optimization algorithms in that gross features of the final state of the system are seen at higher temperatures whereas the finer details of the state appear at lower temperatures [10]. The fact that simualted annealing ensures a global optimal solution enhances its suitability for solving ill-posed inverse problems in general and the problem of unknown groundwater pollutant source characterization in particular.
Its ease of use and remarkable efficiency in handling complex objective functions and constraints has made simulated annealing an attractive choice for solving a wide range of complex optimization problems. However, the slow convergence and hence long time of execution of standard Boltzmann-type simulated annealing has been a constraint. Adaptive Simulated Annealing removes that constraint by making the annealing schedules decrease exponentially in annealing-time, thereby making the convergence much faster. A major difference between ASA and traditional Boltzamnn Annealing algorithms is that the ergodic sampling takes place in terms of n parameters and the cost function. In ASA the exponential annealing schedules permit resources to be spent adaptively on re-annealing and on pacing the convergence in all dimensions, ensuring ample global searching in the first phases of search and ample quick convergence in the final phases [15].
Another major advantages of using Adaptive Simulated Annealing is also the fact that the parameters of algorithm are adjusted adaptively and hence the solutions do not vary widely if parameter values are changed within reasonable limits. This is in contrast with Genetic Algorithm where even minor changes to parameters such as mutation probability, cross over probability or population size causes a significant difference in the solutions.
Conclusion
A linked simulation-optimization method for source identification was developed based on adaptive simulated annealing. It was applied to an illustrative study area. The results obtained were compared with those obtained using genetic algorithm, a more commonly used optimization approach. It is evident from the limited numerical experiments that adaptive simulated annealing algorithm based solutions converge to the actual source fluxes faster than genetic algorithm based solutions. This results in substantial saving in computational time. The source fluxes identified by using adaptive simulated annealing are closer to actual fluxes when compared to the results obtained using genetic algorithm, even when the observation data are erroneous and the hydro-geological parameters are uncertain. It can be concluded that adaptive simulated annealing is computationally more efficient for use in simulation-optimization based methods for identification of unknown groundwater pollutant sources, specially in a time constrained environment. Use of ASA has the potential to reduce CPU time required for solution by an order of magnitude. However, with very large number of iterations in the linked simulation-optimization approach, it is possible that the solutions obtained using GA could converge to a marginally better solution compared to that ASA based algorithm. However, it appears that ASA based solutions converge very close to the optimal solution using only a small fraction of iterations required while using GA. The relevance of contaminant monitoring locations is demonstrated. Further studies are required to develop dedicated monitoring networks which can increase the efficiency of source identification.
Author details
Manish Jha and Bithin Datta James Cook University, Townsville and CRC CARE, Adelaide, Australia | 2018-10-23T15:44:07.007Z | 2012-10-17T00:00:00.000 | {
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250547423 | pes2o/s2orc | v3-fos-license | Spatial Analysis of the Occurrence and Spread of Wildfires in Southwest Madagascar
: The island of Madagascar, located in the southern hemisphere between the equator and the Tropic of Capricorn in the Indian Ocean, 450 km from the African continent, is particularly affected by wildfires. The vegetation of the phytogeographic Western Domain of the island consists largely of savannas, wooded grassland, and secondary grassland, maintained by the repeated action of fire operating each year on a seasonal cycle. Rural populations employ fire to manage land use. Depending on the burning practice and the environment in which the fires happen, the impacts vary. This paper supplement the studies that have so far located and quantified wildfires by taking into account their different behaviors, particularly their spread. We analyzed the modalities of the relationship between the two fire products, active fire and burned area, derived from Moderate Resolution Imaging Spectroradiometer (MODIS) data to establish a typology based on fire spread patterns. We identified three general patterns of fire behaviors, as well as their locations in the studied area. Spatial analysis of this patterns enabled us to understand spatial logics better. Type 1 fires are the least frequently observed and have many active fires, but little or no burned area. Type 2 fires are the most common and have areas that burn like a mosaic. Type 3 fires are observed slightly less frequently than the previous type and have few active fires and large burned areas. An inter-annual analysis reveals the spatial stability or variability of these fire types.
Introduction
The island of Madagascar is almost fully covered by fires [1,2], with nearly 4 million hectares burned each year (mean of 2001-2018), (Figure 1). These fires occur most frequently on the western side of the island, particularly during the dry season, which runs from May through to November [3]. These fires largely correspond to the definition of "bushfires" proposed by Fournier et al. [4], affecting the savannas. However, as is the case in many African nations, the Malagasy authorities have a very strict policy against "bushfires". For the country's environmental management agencies, particularly the forestry department, efforts to suppress such fires are primarily justified by the detrimental impact they have on the environment [5]. Nevertheless, fire is widely employed to manage land use by rural populations, with a diverse array of practices employing various different types of fire. Rajaonson et al. [6] have established a typology of these fires, categorized on the basis of the intentions behind them. Depending on the practices employed and the nature of the fire, the behavior and environmental impact of this burning can vary considerably. In addition to climate factors, topographic variables, such as altitude, slope, and aspect, can also affect the behavior and speed of forest fires' spread [7].
The pastoral burning used by livestock farmers leads to large-scale fires used to increase the quality and quantity of pasture land, stimulating grass growth. They also serve to reduce bush cover. These fires can range over large areas, clearing space and removing view obstacles, making it easier to keep track of grazing herds. "Dahalo" (zebu thieves) fires are connected with insecurity, opening up passages in the savanna which allow for easy escape. Agricultural fires, meanwhile, are restricted to specific plots of land, as are clearing fires linked to land appropriation and fires set to produce charcoal. "Burning off" fires are slightly more complex. They may be restricted to small piles of dry material in direct proximity to residential areas or farmland, or else they may be used to clear out a whole plot of land, or an even larger area. The areas cleared by such fires thus vary considerably. We might also cite the fires set in the hunting and capture of wild zebus [8], or the mining-related fires set to locate lodes and deposits of precious and semi-precious stones. In the general literature, fires are often regarded as a major factor in the deterioration of the environment, particularly flora [9]. They are accused of destroying forests, causing a serious decline in biodiversity and leading to erosion and soil loss. In many cases, these processes have been reasonably well-documented in scientific studies, but research has also demonstrated how the use of fire is an integral part of land management, as well as being governed by socio-cultural traditions [5,8]. For example, "doro tanety", a type of agricultural and pastoral fire used in savanna zones, has been described as practical, protective, and useful [6]. Other studies have illustrated the historic roots of fire and its uses in the Malagasy environment [10][11][12][13].
In a bid to clarify the ambiguous status of wildfires in savanna landscapes, much research, particularly in Africa, has sought to identify burned areas, connect that data with plant cover, and analyze the cycles at work, looking in particular at their interannual frequency and whether they occur earlier or later in the year [3,[14][15][16][17][18][19][20][21][22]. Among other sources, this research is based on data derived from remote sensors. Due to the important role that fire plays in the country, Madagascar provides the ideal terrain in which to pursue such studies in greater depth. In Madagascar, the forestry agents of the former Water and Forestry Directorate (DEF), now the Regional Directorate for the Environment, Ecology, and Forests (DREFF), gather information on the location and extent of wildfires, and the nature of the material consumed by these fires. These data are gathered in the field, with all of the constraints associated with the territory, and are not sufficiently exhaustive to give a comprehensive, satisfactory overview of fire systems. This creates real problems with regard to data representativity, and the validity of drawing general conclusions for the whole territory from the cases thus reported is doubtful [23]. Satellite estimates are far superior to those derived from official statistics [24]. In order to overcome these problems, we make use of fire data derived from satellite images, which allow for an overhead view spanning the entirety of the zone in question. Nonetheless, satellite data are not without their own problems and lacunae.
In the 1980s, the first fire detections were made using satellite data from sensors originally designed to observe and forecast meteorological events [25]. Sensors with infrared channels have proved to be very useful for fire detection [26,27]. Since 1999, with the launch of Terra satellite of the Earth Observing System (EOS) program, the moderate resolution imaging spectroradiometer (MODIS) is used to monitor and characterize the fire processes [28,29]. Other authors exploited the 15 min temporal resolution of Spinning Enhanced Visible and Infrared Imager (SEVIRI), aboard Meteosat Second Generation (MSG) geostationary satellites, to monitor fires with an increased frequency of observation. More recent instruments, such as Visible Infrared Imaging Radiometer Suite (VIIRS) and Sea and Land Surface Temperature Radiometer (SLSTR) offering data at higher spatial resolution in the infrared bands (e.g., up to 375 m for VIIRS), have further improved the detection capabilities of active fires [30][31][32]. Moreover, thresholding methods based on temperature or photosynthetic activity measurements can lead to over-or underestimation errors. For example, in tropical areas subject to alternating wet and dry seasons, photosynthetic activity decreases during the dry season. Thus, an area may be identified as having burned when in fact it has not. The temperature of a fire is not the same depending on the type of vegetation burned. Fires burning a grassy stratum are lower in temperature than fires burning woody vegetation. In addition, the presence of clouds, which are particularly prevalent during the wet season, can compromise the reading of data from optical sensors [33,34].
Errors of commission and omission are a recurring problem for algorithms/products using satellite data to investigate and detect fires [35]. With regard to MODIS fire data in particular, frequently used in small-scale contexts, the 500 m resolution threshold for "burned areas" runs the risk of over-or underestimating the surface area of burned vegetation. Underestimates, caused by errors of omission, may also arise from the algorithm used to identify burned areas, which tends to count only those pixels which it considers to be "burned" with a high degree of certainty, thus failing to count many burned pixels [36]. Problems also arise when the spatial distribution of the fire is too small or too fragmented to be identified as burned vegetation in the satellite images [37]. Clouds and thick smoke can obscure large fires [38]. Overestimation of burned areas, caused by errors of commission, may arise from the fact that individual pixels are counted as entirely burned up, even if the burned surface on the ground is actually smaller than the size of a pixel [39]. To prevent the occurrence of false alarms caused by bright/reflective surfaces (e.g., metal factory rooftops), the MODIS algorithm tries to account for the effects of sun glint [40]. Hot volcanic features and gas flares may also lead to false positives.
Due to the aforementioned issues, these biases that negatively impact data quality make it difficult to precisely determine the number of active fires and hectares burned. The method proposed in this paper does not require exhaustive or even very precise data on fires and burned areas. In this work, we combine information on active fires and burned areas to identify different fire spread patterns in the South-western Madagascar. By analyzing the modality of the relationship between the number of pixels of active fires and the number of pixels of burned areas over a given region, we can identify different fire spread patterns in Sections 2.4.1-2.4.5. Based on these spatial patterns we can establish a typology of different modes of fire spread on portions of regions in Section 2.4.4. This approach allows us to analyze not only the spatial dynamics of wildfires, but also their temporal variation in South-western Madagascar in Section 2.4.6. To this end, our analysis spans an extended period ranging from 2001 to 2018. We begin by identifying the different behaviors of fire observed during this 18-year period in Section 3.1, before focusing specifically on areas in transition, i.e., those areas where the pattern of wildfires has changed most dramatically in Section 3.2. We will then be able to compare these changes with any changes in land use. This step is currently underway and will be the subject of a future publication.
Study Region
The area covered by our study spans 75,947 km² in South-western Madagascar. This region includes the watersheds of the Fiherenana and Onilahy rivers ( Figure 1). This relatively large area spans multiple climates. The southern and western zones have an arid climate, the northern zone has a tropical climate and the easternmost zone has a temperate climate ( Figure 2) [41].
Average annual rainfall in the north of the western regions is 2000 mm/year, which falls to just 500 mm/year in the south. Average annual temperatures here fluctuate between 25 and 27°C. The dry season lasts for 7 months, on average. The Southern region is drier, with an average annual rainfall of 300-500 mm/year and a longer dry season of up to 8 months, which has been known to go on for as long as 12 or even 18 months. The east of the Onilahy drainage basin also includes a portion of the eastern region, characterized by higher altitudes and much greater precipitation, with average annual rainfall of 1300 to 1500 mm/year and a shorter dry season of five to six months. Temperatures here are also lower than in the west, with annual averages of between 17°C and 20°C [42].
South-western Madagascar is mostly part of the "western" region in terms of flora, with 80% of its surface area covered by various types of savanna, secondary grassland, or wooded grassland, as reported by White [42]. There are also two main types of primary vegetation in South-western Madagascar: dry deciduous forests and deciduous thicket. Dry deciduous forests are very localized, or else form patchwork formations with the savannas. More often than not, they seem to be transitional with thicket land. Deciduous thicket generally exists on the fringes of dry forests. The transition between the two is often very gradual. Thicket results from ecological, climatic, or edaphic conditions, which are not conducive to the development of forests, resulting in a predominantly xerophytic vegetation characterized by high numbers of succulent plants, particularly Didiereaceae and tree species of Euphorbiaceae [43].
As in other tropical regions, fire is the variable which best explains the coexistence of trees and grasses [44]. The frequent burning leads to wide-open savanna landscapes [45]. The wildfire season here stretches from May through to November, broadly corresponding to the dry season [3].
Data Used
In this study, we used both Aqua and Terra-MODIS data. The Terra satellite crosses the equator at 10:30 a.m. (local time) on a descending node, and Aqua passes the equator at 1:30 p.m. on an ascending node. The MODIS sensors have a viewing swath width of 2330 km, thus providing a complete image of the surface of the Earth every one to two days, in 36 spectral bands ranging from 0.4 µm to 14.4 µm. The spatial resolution varies depending on the spectral bands: 250 m for bands 1 and 2, 500 m for bands 3 to 7, and 1000 m for bands 8 to 36.
The MODIS data are used to generate "fire products"-active fires and burned areas. User manuals are available for both the "active fire" and "burned area" products [46,47].
These data present a number of advantages for our study. The temporal resolution of the data-grouped into 8-day periods and available from the 2000 s onwards-devoted to interannual analysis in order to track developments over a relatively long period of time. The spatial resolution is also the most suitable option for the scale of our analysis, which spans some 75,947 km². Our aim is to identify fire systems on a small scale, and study their specific patterns. Both products "active fire" and "burned area" products can be complementary [17]. By analyzing the two fire products, we can ascertain patterns of fire behavior.
The "Fire products" used in our analysis are available online: https://ladsweb. modaps.eosdis.nasa.gov/ (accessed on 22 May 2022). For "active fire" products, we downloaded the products MOD14A2 (Terra) and MYD14A2 (Aqua) in raster format GeoTIFF. The same format was selected for "burned areas" product MCD64A1.
The processing algorithms and daily data output for the planet as a whole are different for the "active fire" and "burned area" products [48].
Active Fire Products
Fire Products: MOD14A2 (Terra) and MYD14A2 (Aqua) Level 3 include 46 raster images per year (8-Day Summary), in which each pixel is assigned to a class (Table 1), 3 of which (classes 7, 8 and 9) indicate the presence of fire, with varying degrees of confidence. The finished product detects fires burning at the time of overpass under relatively cloudfree conditions, the energy emitted and the flaming-to-smoldering ratio, for pixels with a resolution of 1 km [49]. The algorithm is entirely automated, and builds on the heritage approach used for the Geostationary Operational Environmental Satellites (GOES) and Advanced Very High Resolution Radiometer (AVHRR) sensors [29,50,51]. It is used to process MODIS images from both Terra and Aqua, where the apparent temperature is observed from band 22 upwards (or band 21 if 22 is saturated or data is missing), from 3.929 to 3.989 µm and band 31 from 10.78 to 11.28 µm. Band 2, in the near-infrared band of 841 to 876 nm (Table 2), is also used to identify highly reflective surfaces, liable to be mistaken for fires and thus provoke false reports [48].
The first processing step eliminates all pixels not considered to represent potential fires, with a temperature threshold judged to be too low, and adapted for day and night. The detection of fire is then determined by absolute fire detection, which is to say areas where the temperature is above the threshold values set for day and night in band 22, or else above a specific threshold for the value of band 22 minus band 31. This absolute detection only concerns fires of sufficient intensity. For low intense fires, detection depends on the thermal emissions from surrounding pixels. Pixels representing high temperatures are no longer identified using an absolute threshold, but instead in relation to the background thermal emissions detected from the surrounding pixels [49].
Burned Area Products
The burned area mapping algorithm handles the MODIS imagery with a spatial resolution of 500 m, seeking to identify persistent changes caused by the combustion of biomass in pixels. The daily surface reflectance values in Bands 5 (1230 to 1250 nm) and 7 (2105 to 2155 nm) atmospherically corrected are employed to derive a burn-sensitive vegetation index (VI). Composite imagery summarizes the persistent changes in the time series of the VI. Indeed, the index shows a significant decrease rapidly after burning. In this step, the absence of clouds on the reflectance values is verified with the internal cloud mask (MO/YD09) and the absence of active fires with the MODIS active fire observations (MO/YD14). A cumulative mask is processed with active fire data to guide the selection of burned and unburned training samples and specify the fire date in each grid cell. The algorithm then performs a classification based on training samples of burned and unburned areas. Burned training grid cells are created from the cumulative mask of active fires and to reduce the impact of under-sampling of spatial fire extent by active fire observations. It is expanded through a process of region growing, constrained by a maximum growth distance of 10 km. Unburned training grid cells must meet the following selection criteria: they must be "a-priori" unburned, i.e., identified as unburned based on the VI time series and they must contain valid data, i.e., not be in the burned training mask. This MODIS burned area collection 6 is described in Ref. [47] and the production algorithm is fully detailed in Ref. [52].
Differences between the Data for These Two Products
The main difference between the "active fire" and "burned area" products is the ephemeral nature of active fires and the lasting changes to burned areas. Fires of short-term duration may be undetected by MODIS owing to the daily temporal coverage. Burned areas, on the other hand, can be observed over longer periods of time, since it takes more than two days for vegetation to grow back. Repeated flyovers by satellite thus allow the algorithm to identify burned areas which may be concealed by clouds or thick smoke, or which may not have been burning during the first flyover. "This implies that active fire and fire-affected area products may be used in conjunction to improve fire information" [37].
Pre-Processing the Data
All of the active fire and burned area data retrieved for the period 1 January 2001 to 31 December 2018 were processed using the R Core Team software program [53].
We began with a few pre-processing operations in order to prepare the data for our calculations (Appendix A, Figure A1).
For the MODIS "active fire" data, we conducted the same processing operations for the Terra and Aqua data.
To begin with, the active fire pixels were reclassified: a value of 1 was assigned to all pixels declared to contain an active fire. We selected all fires, regardless of confidence level [54]. NA was assigned to fireless pixels (Class 5 in the MOD14A2 and MYD14A2 data), and a value of 0 for pixels where fire is not possible (Classes 3, 4, and 6 in the MOD14A2 and MYD14A2 data) ( Table 1). A distinction is thus made for pixels in Class 5, because even if the algorithm does not detect a fire, these pixels are at least capable of containing fire, compared with Classes 3, 4, and 6, which cannot, because they either represent areas covered by water or else a degree of uncertainty that is too high, for example as a result of cloud cover.
The 46 raster images from a given year were then combined with each pixel given a value (0-46) equal to the number of images in which they were classified as active fire. The active fire pixels, which have an initial resolution of 1 km, were then disaggregated to 500 m, corresponding to the spatial resolution of the pixels used to detect burned areas. Finally, data taken from Terra were combined with those from Aqua to obtain a single raster image of active fires over the course of the year.
The procedure applied to the burned area data is essentially the same, with the exception of the disaggregation operation and the merging of the Terra and Aqua data, since the data are directly provided in a single raster file. We used the data layer "burn data" to which we assigned the value of 1 for each burned pixel, i.e., pixels with values greater than 0 (from 1 to 366). We assigned the value of 0 to the unburned pixels, i.e., those less than or equal to 0, 0 being the unburned land, −1 the unmapped area due to insufficient data and −2 the water surfaces.
Heatmap of Active Fires and Burned Areas
The first step of data processing involves the spatialization of active fires and burned areas. In order to represent the concentration of active fires and burned areas, we used Quantum Geographic Information System (QGIS) to create a density raster (heatmap) using point vector layers for active fires and burned areas for all years between 1 January 2001 and 31 December 2018. To do this, all raster files for active fires and burned areas derived from the pre-processing phase were combined in advance. Through vectoring, we thus obtained a point file whose value corresponds to the sum total of detected active fires and burned areas. We then calculated a heatmap using the Kernel Density estimation, based on the value of these points for specific locations, and using a radius of 10,000 m and a cell size of 1000 m for the output raster. Since both burned areas and active fires vary from one year to the next, performing this density calculation over the whole period enabled us to broadly determine their spatial distribution.
Our results ( Figure 3) reveal a clear difference between the spatial organization of active fires and burned areas. We thus set about questioning and attempting to better comprehend the relationship between these two variables.
Relationship between Active Fires and Burned Areas
We thus set out to document the relationship between active fires and burned areas for each year between 2001 and 2018. Our objective was to determine whether or not there is a linear relationship between fires and burned areas, which could be defined in the form of a pattern.
First, it is important to take into account certain characteristics of the data used, particularly the ephemeral nature of active fires and the persistence of burned areas. The Terra and Aqua sensors are thus capable of detecting a certain number of active fires, perhaps even over several days, which will correspond to a resulting burned area. There will therefore logically be more burned areas than active fires detected, or at the very least an equal number, unless some burned areas prove to be too small for detection [55]. All previous studies utilizing the MODIS "burned area" data have observed a high rate of omission of pixels corresponding to burned areas. However, observing the ratio between the number of active fires observed and the corresponding quantity of burned areas could reveal interesting information, since the passage of the satellite at regular intervals should make this ratio relatively stable. For this reason, we have chosen to observe whole-year data.
By accumulating the data for a whole year, we are left with a certain number of active fires and a certain surface area of burned land. We can identify very extensive burned areas for which few active fires have been detected, which in theory would suggest that the fires spread rapidly and over a great distance. At the other end, when the sensor has not identified any burned areas (as they are too small), but numerous active fires have been reported, we can infer the presence of frequently recurring fires. Analyzing the interrelationship between active fires and burned areas thus allows us to identify different fire systems. To this end, we use a hexagonal grid with cells of 15 km wide and 17.32 km high (giving a surface area of just under 200 km²). There are 389 cells. The size of these cells-not too small, not too large-is compatible with the area covered by this study, and allows us to identify all different types of wildfire occurrences. The choice of a hexagonal grid allows us to minimize boundary effects. The grid is based on information taken from the raster of active fires and burned areas used in the previous stages, covering the years 2001 through 2018. A function then counts the number of active fire pixels within each cell, then the number of burned area pixels (Appendix A, Figure A2).
The graphs generated by this process (Figure 4) show the relationship between the two variables "active fires" (x-axis) and "burned areas" (y-axis) for each year. Each point represents a cell within the grid.
For all of the years studied, the scatter plot appears to be split between various different linear relationships. It is quite a challenge to identify and interpret any clear relationship, but it is equally difficult to declare that the two variables are totally independent.
Measure of Relationship
To assess the independence or intensity of the relationship between the two variables, we thus use a test of independence which allows us to compare the observed frequencies with a theoretical distribution, by measuring the disparity between the two. In this case, the theoretical distribution represents the hypothesis that active fires and burned areas are entirely independent of one another. To simulate this, we calculate a contingency table for the frequency distribution of the active fires and burned areas, and another, theoretical, contingency table representing active fires and burned areas that are mathematically independent of one another. We then calculate the sum of the deviation between the observed situation and the theoretical situation, in each cell of the contingency table, using the Chi2 or χ 2 method [56]. The result is then compared with a χ 2 table for a risk of error (α) set at 0.05. Since the degree of freedom (df ) corresponding to our observations is greater than 30, the corresponding χ 2 is calculated as follows.
where u = 1.6449 as per the normal distribution function, centered for (α) = 0.05 ; d f = degree of freedom.
Since these results rule out independence as a viable hypothesis, it seems pertinent to calculate a coefficient that will allow us to measure the intensity of the relationship between the number of active fire pixels and the number of burned area pixels. Tschuprow's coefficient (T) allows us to measure the strength of the association, where the value indicated is a non-dimensional number between 0 (independence) and 1 (perfect dependence) [56].
Linear Model and Residuals
Based on these initial results, it appears that there are different types of fire that can be identified with reference to the correlation between active fires and burned areas. To accurately identify and characterize these fires, and establish a typological classification, we will look more closely at the correlation between the number of burned area pixels and the number of active fire pixels detected. The idea is to identify, using a regression line, those cells in the grid which correspond to the linear model derived from the correlation between active fires and burned areas observed in the previous stages, and those which deviate from this model.
To do this, we begin by replacing the active fire and burned area data with their logarithms, which allows us to transform (anamorphosis) the curve into a line. We then calculate the regression of the number of burned area pixels (dependent variable) against the number of active fire pixels (explanatory variable). We use the generalized linear model (GLM) in order to discard the large number of 0 values contained in the statistical series.
Regression is a means of breaking down the information contained in the statistical series. On one side, the regression line represents the estimated variable, where the dependent variable (number of burned area pixels) is linearly dependent in linear fashion on the explanatory variable (number of active fire pixels). On the other side, we have the residuals, which are independent of the explanatory variable. This residual information represents the disparity between the observed situation and the constructed model, which is of interest to us because these residuals inform us of the gap between each cell in the grid and the projections of the model, giving us a measure of specificity ( Figure 5).
Coefficient of Determination
Calculating the coefficient of determination allows us to measure the proportion (in %) of information taken into account by the regression model. Since the total sum of distribution information is equal to the information taken into consideration in the regression model plus the residual information, this coefficient of determination allows us to determine how much information corresponds to the model and how much is regarded as residual. The coefficient of determination is equal to the square of the coefficient of correlation.
Taking this analysis further by graphically representing the values in the form of a box plot (Figure 6), showing the number of cells corresponding to each type of fire in a given year, allows us to observe year-on-year variation.
Range of Residual Values between 2001 and 2018
To distinguish between areas where fire patterns did not change over the years we studied, and those where change did occur, we extracted the maximum and minimum residual values for each cell in the grid and for each year between 2001 and 2018, then calculated the range between these maximum and minimum residual values. This allowed us to divide the statistical series into four classes, based on mean values and standard deviation, and subsequently map these categories (Figure 7).
Taking this analysis further, for each year in the period 2001-2018 we counted the number of cells corresponding to each type of fire and also falling within the category "fairly stable." We then did the same for those in the "significant change" category. This gave us a table showing, for each type of fire and each year, the number of stable cells and the number of cells experiencing significant change. We then calculated these values as a percentage of the total for each type of fire. This allowed us to compare different types of fires, without worrying about the differences in the number of cells corresponding to each type of fire.
Graphically representing these values in the form of a box plot ( Figure 8) allows us to fine-tune the results and the conclusions to be drawn from them.
Analysis of Heatmap of Active Fires and Burned Areas
The first observation to emerge from our heatmaps (Figure 3) is that fires occur across nearly the whole study area, the South-western Madagascar, with the exception of its southern tip. This is most likely due to the local climate, which is too arid to provide sufficient combustible material.
The active fire heatmap (Figure 3a) shows two zones at the western and eastern fringes of our area of study that are particularly prone to active fires, with far greater intensity in the west than in the east. These two zones can also be clearly distinguished on the burned area heatmap (Figure 3b), but here the intensity appears to be equal in east and west. Moreover, the high-intensity burned areas in the west cover display spatial disparities with the active fire heatmap. These results show a difference between areas marked by the active fire products and those affected by burned area products, similar to the findings of Devineau et al. [54] in their study in Burkina Faso: "The ability of active fire data and of burned area data to account for areas affected by fire is somewhat different".
Relationship between Active Fires and Burned Areas
As regards the relationship between burned areas and active fires (Appendix C), for each of the years studied, the scatter plots reveal different linear relationships. Two groups show weak correlation. In one, the correlation line is very steep, as visible in the first column of graphs (Figure 4), with a fairly small number of active fire pixels and a number of burned area pixels that varies considerably. This indicates a situation of statistical independence, whereby the number of active fire pixels has little influence on the number of burned area pixels. In the second case, on the contrary, the number of burned area pixels is fairly small while the number of active fire pixels varies considerably, as visible in the third column of the graph (Figure 4). In this case, the number of active fire pixels has little influence on the number of burned area pixels, which remains low regardless of the number of active fire pixels. Between these two extremes, a third trend emerges for the correlation between active fires and burned areas, characterized by a more linear statistical relationship, but one where the number of burned area pixels is always greater than the number of active fire pixels. This case is visible in the second column of the graph (Figure 4).
In all years the calculated χ 2 is greater than the χ 2 values in the table (Appendix B, Table A1), so the hypothesis that the number of active fire pixels does not depend at all on the number of burned area pixels can be rejected. There is indeed a relationship between these two variables.
The calculated Tschuprow coefficient varies between 0.63 and 0.74 for the period 2001-2018 (Appendix B, Table A2), which indicates the existence of a connection, but not a very strong one. This suggests that the number of active fire pixels is just one of several factors which determine the number of burned area pixels.
Drawing upon studies of fire patterns conducted in other parts of the world, we can put forward the hypothesis that the land cover of burned areas is one of those contributing factors that determine the extent of burned areas [54]. In this respect, the work of Monnier [57] is particularly instructive, especially the three conditions required for fire to spread: a sparking event, the presence of combustible material, and the continuity of the latter. This reflects the decisive impact that the nature of vegetation has on the way fires behave.
These results remain fairly ambivalent, with some cases which resemble statistical independence. However, the statistical calculations reveal the existence of a certain relationship nonetheless, albeit a weak one. This suggests that there are different mechanisms at work.
Linear Model and Residuals
Residuals are results that are independent of the explanatory variable, i.e., the number of active fire pixels. The further the residual value is from 0, the less likely it will be that the dependent variable (in this case the number of burned area pixels) is determined by the explanatory variable. In short, the further away we move from the linear regression, the less directly the extent of burned areas will depend on the number of active fires detected. A positive residual indicates that the extent of burned areas in the cell in question is higher than the models would lead us to expect, meaning that the number of burned area pixels is greater than expected, while a negative residual indicates a number of active fire pixels greater than that predicted by the model. Residual values close to 0 indicate that the figures correspond closely to the model. This allows us to split the residual results into three categories, using the first and third quartiles of the statistical series as threshold values (Table 3). These three categories correspond to three distinct patterns of fire spread, which we can map for all of the years in our period (Appendix D). Table 3. Typology of different fire spread patterns. Although some subtle disparities can be observed over the years analyzed, there is a certain invariance of behavior in space, which allows us to identify two particular zones. Broadly speaking, this mapping exercise allows us to divide the area spanned by our study into two distinct zones. In the east, and in all years, we find many more Type 3 fires, i.e., a small number of detected active fires that leave a large amount of burned land. The west, on the other hand, sees far more Type 1 fires, i.e., a large number of active fires are detected, but they leave behind them little or no burned land. Zones characterized by Type 2 fires, i.e., with a linear distribution between active fires and burned areas, consistent with the regression model, are more or less located between these two main zones.
Results of the Coefficient of Determination
According to the coefficient of determination (Appendix B, Table A3), our regression accounts for between 25 and 40% of the total information. This means that 60 to 75% of the information falls into our residual categories. This high proportion of residual information indicates that, in the area covered by our study, many zones function in a manner which does not correspond to the predictions of the model. The large number of residual values, both positive and negative, are poorly represented by the model, and are thus determined by one or more explanatory variables other than the number of active fire pixels, as discussed above.
This distribution can be observed in the box plot showing the number of cells corresponding to each type of fire ( Figure 6). It is worth noting that Type 1 fires are the category least frequently observed in the area covered by our study, and that the year-on-year variation in their number is less substantial than that seen in the other categories. Type 2 fires are the most common, and the year-on-year variation in their number is much greater. Type 3 fires are observed slightly less frequently, and their year-on-year variation is also slightly below that recorded for Type 2.
Analysis of Residual Values
We built upon this initial observation by calculating and spatializing the range of residual values for each cell in the grid, for all of the 18 years in our period (Figure 7). This allows us to observe the evolution of our different types of fires, and to identify areas where fire patterns have remained stable, i.e., the residual values are stable across the 18 years, and others which have witnessed frequent changes, i.e., where the residual values vary considerably.
Cells identified as stable may correspond and stick to one of the three types of fire defined above , whereas those which vary significantly will have witnessed a change among this fire type.
Two large areas appear to have remained relatively stable, one to the east and the other to the north-west, as well as the whole of the coastline. The southern regions, particularly the south-west, are those where the fire patterns have undergone the greatest change.
Discussion
After having discriminated three different patterns of fire spread, we will first discuss this typology and its spatial distribution, and then address the dynamics of evolution and potential transformations of the different patterns identified. First, it is necessary to show the relevance of the approach that consists in developing a typology based on the relationship between the number of fire pixels in a given area for each of the fire products.
There is a clear difference between the maps of active fire and burned area products. Although active fire products are present in the burned area processing algorithm, as described by Giglio et al. [52], they serve as a guide for the training samples that support the final burned area classification but do not determine the final burned area detection results. The burned area data retrieved from MODIS imagery are not the result of the accumulation of data from the active fire products, so they can be used to identify unique spatial dynamics and to distinguish areas that behave differently.
Analysis of the Spatial Distribution of Fire Patterns
Previous work [58][59][60][61] has already shown that fire use practices and the composition of the landscape can explain the spatial distribution. Based on these results, we can advance the hypothesis that Type 3 fires, with extensive burned areas but only a small number of active fires, appear to correspond to the use of fire by pastoralists, miners, or "dahalo". These fires affect savanna landscapes, with very wide-ranging burning used to clear land and remove obstacles [6,8,23].
The fires identified here as Type 2 leave behind a patchwork of burned areas. The very high number of active fires recorded indicates the continued presence of combustible material in the periods between satellite flyovers. However, fires burning over large areas would not leave any combustible material in their wake. These fires correspond to controlled agricultural fires limited to specific plots of land, or land-clearing and charcoal-making fires, which are also highly localized and limited in scale [62]. They reflect controlled burning practices deployed in strategically selected locations. The composition of some landscapes may also prevent fires from spreading to engulf larger areas [63].
For fires identified as Type 1, where active fires are detected but few or no burned areas are observed, we can surmise that a fire did indeed occur, but that the resulting burned area was too small to be picked up in the 500 m pixels used for MODIS images. For example, the fires used to cook yams directly after harvesting, a fairly widespread practice in forest areas, may generate such results [62]. So too may certain forms of "burning off", especially when the combustible materials are gathered into a single pile before being set alight.
Among the factors which may explain these initial results, the nature of the vegetation present is very important, as discussed above. A lack of dry, combustible material will stop forest fires from spreading, restricting them to the dry vegetation at floor level [8,59,63].
Analysis of Dynamics Transformation of Fire Systems
As numerous studies have already demonstrated, the evolution over time of the distribution of fires and burned area is very instructive [1, 20,37,64,65]. Here we have chosen to focus rather on the evolution, not only quantitative but qualitative, of our fire types. We could propose a number of hypotheses to explain the spatial changes in the typology. They might be the result of changing practices, of transformations in the landscape, or both, since the two are closely linked. For example, the transformation of pastures into agricultural land is highly likely to be accompanied by a change in fire use practices, as well as changes to the landscape.
Areas which have seen "significant change" may reflect changes in fire types resulting from the "rotation" of burned areas. We will explore these explanatory factors in our study.
An analysis of these changing fire patterns, focusing exclusively on those cells which have changed significantly and those which have remained stable (Figure 8), demonstrates that those corresponding to Type 1 fires tend to change frequently over the years. Cells characterized by this type of fire show a clear tendency to vary from year to year. Type 2 cells, while more likely to be stable, may also display frequent changes. Cells corresponding to Type 3, however, have a tendency to remain stable from one year to the next.
On this basis, we can further develop our hypotheses regarding the different patterns of fire and their spatial and temporal evolution. Type 1 fires are localized burnings which do not spread. They generally correspond to fires used for cooking or burning off waste. These cooking fires are not domestic in nature, corresponding instead to the fires set in forest areas to cook and prepare produce such as yams. These fires are rarely set in the same place twice, and their location tends to vary from year to year. The fires identified as Type 2 correspond to a patchwork of small burned areas. These patterns are associated with controlled burning, generally limited to specific plots, most often for agricultural purposes. The proportion of such spaces that remain unchanged from year to year, and thus see regular fires, is broadly the same as the proportion of spaces affected by irregular fires, which vary from year to year. Type 3 corresponds to fires which spread rapidly over wide areas. Such fires are typical of pastoral land management. The areas affected by fires of this type change relatively little from year to year. The way in which the different types of fires evolve provides information on their nature.
Conclusions
Processing the MODIS data-combined with a fairly strong empirical understanding of the terrain, local practices, and the types of landscape present-has enabled us to identify three broad patterns of wildfire distribution, defined by the number of active fires detected and the extent of burned areas. This typology can be correlated to the characteristics of the landscape and different fire practices. This study shows the potential in using fire data to differentiate geographic areas with different characteristics. MODIS fire products, active fires, and burned areas are proving to be good tools for spatial distinction. These initial results allow us to determine the functional characteristics of different patterns of fire and identify the spaces affected. In some areas, fire patterns have remained relatively stable over the 18 years spanned by our study. We also identified those areas that witnessed the biggest changes during this period.
Our approach was focused on identifying the occurrence and spread of fires, and tracking their interannual evolution. By dividing the zone into cells we were able to identify areas in which fire patterns were stable or variable. We are now working to build up a more accurate profile of the vegetation in these fire-affected areas. A further study focusing on specific cells and using data with a higher resolution would be useful in this respect, enabling us to analyze the relationship between fire patterns and vegetation. Plans are being made for an approach based on the analysis of spatial forms and structures using landscape metrics, allowing us to identify the key characteristics of these different fire patterns in greater detail. This would also help us to better understand the reasons behind changing patterns and functions. The methodology applied here to South-western Madagascar would not necessarily be pertinent for other parts of the country. Madagascar is home to a large and diverse array of fire practices, particularly in the highlands and the east of the island, and our method may not be appropriate in those areas.
It seems highly likely that the three patterns of fire identified here also differ in terms of their environmental impact. This raises questions regarding the suitability of fire-fighting measures that fail to distinguish between different types of fires. Outlawing all types of fire appears to be entirely ineffective as a preventive measure. It is also ineffective as a means of protecting the environment, and fosters resentment among rural communities who use fire with considerable expertise and skill. It is also worth noting that, in spite of such repressive measures imposed by the government, the total surface area of burned zones has not decreased over the past 20 years. Adopting a differentiated approach to fires would allow for more rational management of such practices. By improving our understanding of fire patterns and their causes in south-west Madagascar, our hope is that this study will contribute to the creation of an effective strategy of fire management as recommended by the Food and Agriculture Organization of the United Nations (FAO) [66]. The results of this study must be further explored in different directions, in particular by identifying and comparing the different types of vegetation concerned, generally savannas and woodlands.
Conflicts of Interest:
The authors declare no conflict of interest.
Abbreviations
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257240060 | pes2o/s2orc | v3-fos-license | Prevalence of normoalbuminuric chronic kidney disease among individuals with type 2 diabetes mellitus from India
Background & objectives: A subset of diabetic individuals are known to develop progressive renal insufficiency without albuminuria, referred to as normoalbuminuric chronic kidney disease (NACKD). There is, however, a paucity of studies regarding this condition in India. So, this study, aimed to find the prevalence of normoalbuminuric renal dysfunction and its clinical associations in diabetic Indian population. Methods: Medical record search of patients with type 2 diabetes mellitus at a tertiary care centre was done. Based on the urinary albumin:creatinine ratio (>30 mg/g creatinine) and estimated glomerular filtration rate (e-GFR) (<60 ml/min/1.73m2), individuals were classified as having, (i) no kidney disease (NKD), (ii) chronic kidney disease (CKD), (iii) albuminuria alone (ALB), (iv) normoalbuminuric low e-GFR (NACKD) and (v) albuminuria with low e-GFR albuminuric CKD (ACKD). Furthermore, the clinical and biochemical parameters of these groups were also compared. Results: Data from 3534 diabetes patients with a mean age of 53.8±10.9 yr and mean duration of diabetes of 10.3±7.5 yr were available for the analysis. NACKD constituted 39.1 per cent of the patients with reduced e-GFR, NACKD was found in 2.1 per cent and ACKD in 3.4 per cent of all diabetic patients. Compared to NKD patients, was found an independent association between NACKD and higher age, male sex, lower body weight and statin intake but not with glycated haemoglobin, fasting and postprandial plasma glucose. Patients with NACKD were found to be older than those with ACKD. Retinopathy was found to be more prevalent in the ACKD patients, whereas the rates of macrovascular complications were found to be similar between the groups. The prevalence of NACKD relative to ACKD decreased in CKD stages 3b, 4 and 5. Interpretation & conclusions: The results of this study suggest that NACKD constituted greater than one third of patients with diabetes and decreased e-GFR, which showed a strong association with age but not with duration or severity of hyperglycaemia or the presence of retinopathy. Both NACKD and ACKD showed similar associations with macrovascular disease.
Diabetes mellitus accounts for around 50 per cent of cases of end-stage renal disease (ESRD) in the developed world 1 . Other than the progression to ESRD, diabetic kidney disease can lead to cardiovascular disease and predispose to infections and hence contribute to mortality 2 . Diabetic nephropathy is classically described by the clinical triad of proteinuria, hypertension and ultimately, low glomerular filtration rate (GFR) 3 . It is known that ESRD or doubling of serum creatinine generally occurs within 10 yr of detection in around 20 per cent of micro-albuminuric individuals and in 60 per cent of macro-albuminuric individuals 4,5 . Recently, the traditional concept about the natural history of diabetic kidney disease has been challenged with emerging evidence citing progressive decline in GFR without significant albuminuria in a subset of patients with type 1 or type 2 diabetes (T2D), i.e. normoalbuminuric chronic kidney disease (NACKD) 3 . There is, however, a paucity of data from India on the relative frequency of normoalbuminuric compared to albuminuric renal dysfunction. This study attempted to find the prevalence of normoalbuminuric renal dysfunction and its clinical association among Indian patients attending a tertiary diabetes care centre.
Material & Methods
Medical record search was done to find out the proportion of NACKD among diabetes patients attending the Indian Institute of Diabetes, Thiruvananthapuram, India, a tertiary diabetes care centre. The data were obtained from the electronic record of the clinical, biochemical and medication profiles of patients newly registered with the hospital between June 2017 to January 2020, after approval of the Human Ethics Committee of Government Medical College, Thiruvananthapuram.
Inclusion/exclusion criteria: The study included all newly registered T2D patients attending the hospital and whose records had a clinical history, weight recording, serum creatinine, urine albumin : creatinine ratio and glycated haemoglobin (HbA1c) estimation during the study period. The presence of poorly controlled hypothyroidism (serum thyroid-stimulating hormone [TSH] >10 μIU/ml)/hyperthyroidism (serum thyroxine >upper limit of normal), liver disease, chronic infections like tuberculosis and recurrent or active urinary tract infections were kept as criteria for exclusion.
Sample size: A study by Laranjinha et al 6 , had identified the prevalence (P) of non-albuminuric CKD in T2D patients as 46.6 per cent. The minimum required sample size was estimated to be 3250 patients using the formula: 4P×(100−P)/d 2 wher, P=46.6 per cent, (100−P) = 53.4 per cent, d (desired precision) was set at 1.75 per cent. For the current study, a total of 3615 patients were screened and 81 patients were left out after subjecting to exclusion criteria. The data of 3534 patients were available for final analysis.
Study procedure: The anthropometric measurements were done by staff nurses and the clinical details, diagnoses and medications were recorded into the electronic database by the treating physicians themselves. Biochemical reports were entered into electronic records directly from the laboratory. Trained staff were utilized to fill these details in a separate pro forma for each patient, after retrieving them from the records. Demographic parameters, duration of diabetes and blood pressure were noted. History of addictions, comorbidities and treatment were also noted from records. Smoking and alcoholism were defined as any inhaled tobacco use or any ethanol ingestion within the past three months. Details of prior treatments were specifically checked for diagnosis liver disease, chronic infections, treatment with angiotensin-converting enzyme inhibitors (ACEI)/ angiotensin receptor blockers (ARB) and statins was also assessed. Laboratory data were collected from the patients' last visit details, including HbA1c, fasting plasma glucose (FPG) and 2 h postprandial plasma glucose (2 h PPPG), fasting lipid profile, thyroid profile and renal function test. For those patients with a low estimated GFR (e-GFR) (Cockroft and Gault method), records were further searched for an alternative cause of CKD. Those who did not have a mention of CKD or its reasons in records were contacted to get their treatment records pertaining to kidney disease, including ultrasound abdomen and urine sediment. Details about the presence of coronary artery disease, cerebrovascular accident, peripheral arterial occlusive disease and retinopathy were obtained from hospital records. Details of fundus fluorescein angiography testing were not available in all patients.
Study parameters: All biochemical investigations were done using the same analyzers under a National Accreditation Board for Laboratories, India, certified quality control programme as part of existing hospital protocol. From the available parameters body mass index (BMI) and e-GFR (Cockcroft and Gault) were calculated for each patient.
The proportion was found out for albuminuric (urine albumin: creatinine ratio more than 30 mg/g creatinine) and normoalbuminuric fall in e-GFR (<60 ml/min/1.73 m 2 using the Cockroft and Gault formula) and that of albuminuria without low e-GFR. These categories were labelled as albuminuria alone (ALB), NACKD and albuminuric CKD (ACKD), respectively. Other patients without the above renal abnormalities were labelled as no kidney disease (NKD).
Statistical analysis: Data were tabulated Microsoft Excel 2010.
IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, NY, USA) was used for the analysis of data. The variables were tested for their distribution and classified as normally distributed or not. Continuous variables were expressed as mean±standard deviation (normal distribution) or as median interquartile range (IQR) if not normally distributed. Categorical variables were expressed as frequencies (N) and percentages (%). Analysis of variance (with post hoc Bonferoni's correction for subgroup comparison) was used to make the comparison of normally distributed continuous variables between the multiple groups of NKD, NACKD and ACKD. Chi-square test was used for categorical variables for finding the significance of the association. For the non-normally distributed variables Mann-Whitney U test was used. Logistic regression was employed to find out the independent association of co-varying parameters (e.g. age, duration of diabetes, weight, statin intake) with NACKD. P<0.05 was considered as significant.
Results
A total of 3615 patients satisfying the inclusion criteria were included in the study. Patients with poorly controlled hypothyroidism (TSH >10 μIU/ml) or other comorbidities mentioned in the exclusion criteria (n=81) were excluded, with 3534 patient's data being available for final analysis. The clinical characteristics of the study participants are shown in Table I whereas three patients (0.1%) were found to have a non-diabetic cause of renal function decline (Fig. 1).
Hence, among patients with diabetes reaching a stage 3a CKD or worse (e-GFR< 60 ml/min/1.73 m 2 ), 38.2 per cent (75 of 196) were normoalbuminuric. The proportion of patients with normoalbuminuria across CKD stages is shown in Fig. 2.
A comparison was made between patients with NKD and NACKD (Table II) to find the associations of NACKD with other clinical variables. Higher proportion of NACKD patients were males (70.6%) when compared to those with NKD (59.2%) (P<0.05). Similarly, subjects with a history of hypertension and dyslipidaemia, also were more likely to have NACKD. Significantly higher number of subjects with NACKD was on statins and ACEI/ARBs when compared to those with NKD. Furthermore, subjects with lower weight, lower BMI, a lower diastolic BP were found to have a significantly higher chance of NACKD. On binary logistic regression, higher age, male sex, lower body weight and statin intake were found as risk factors for NACKD. Interestingly, none of the glycaemic parameters (FBG, PPBG and HbA1c) showed an association with NACKD.
A comparison was also made between the ACKD and NACKD groups (Table II). It was found that NACKD patients had a significantly higher age, lower duration of diabetes, lower body weight, lower systolic and diastolic blood pressures, lower fasting (FPG) and postprandial (PPPG) plasma glucose and lower HbA1c levels. Hypertension was more prevalent in ACKD patients. On binary logistic regression, among the above associations, only a higher age was identified as the risk factor. The proportion of patients with retinopathy was significantly higher in those with ACKD when compared with NACKD patients, whereas the proportion of patients having coronary artery disease, cerebrovascular accidents and peripheral vascular occlusive disease was found to be equal among both the groups.
Further analysis was performed to find the prevalence of NACKD in more severe stages of renal dysfunction and it was seen that, as the e-GFR level decreases, the proportion of patients with the normoalbuminuric variant of CKD comes down. The proportion of patients with normoalbuminuria in CKD stage 3A (eGFR 45-59 ml/min) was 45.1 per cent, whereas in patients with CKD stage 3B (eGFR 30-44 ml/min) was 31.3 per cent and that for stages 4 and 5 (eGFR <30 ml/min) was 10.0 per cent (Fig. 1).
Discussion
Till the last decade, albuminuria was considered the sine-qua-non of CKD caused by diabetes; it was believed to be a marker which shows up much before the fall in e-GFR 7 . However, recent studies have shown that significant proportions of patients with T2D have a fall in e-GFR before the onset of micro-albuminuria. Albuminuria and decreased GFR were identified as complementary manifestations of diabetic kidney disease and not obligatory or temporally related ones 8 . Decline in GFR, unlike albuminuria is progressive and spontaneous regression is unlikely. Although 30 years have passed since the first reports of NACKD in T2D mellitus 3 , there are scarce data from India regarding its prevalence or associations. The present study was done to determine the prevalence and clinical association of NACKD in Indian patients with T2D.
In the present study, 3534 patients with T2D having a mean age of 53.8±10.9 yr and a mean duration of diabetes of 10.3±7.5 yr, attending a tertiary diabetes care centre in South India were studied. The prevalence of NACKD in this diabetic cohort was 2.1 per cent which constituted more than one-third of all patients with CKD. Other previous studies report a contribution The significance of association (P value) between the variables computed using * Chi-square test (categorical variables), ** ANOVA with post-hoc Bonferoni's correction for normally distributed continuous variables marked with, *** Mann-Whitney U test for non-normally distributed variables. ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; UACR, urine albumin: creatinine ratio, NKD, no kidney disease, NACKD, normoalbuminuric chronic kidney disease, ACKD, albuminuric chronic kidney disease; NA, not available of 22-73 per cent by NACKD to the population of patients with compromised GFR.
A large retrospective study in Sweden, including 94,446 participants with diabetes, showed that 17 per cent of participants had renal impairment, with a high proportion of (62%) normoalbuminuric subjects 9 . Similarly, among the patients with T2D and eGFR <60 ml/min/1.73 m 2 included in NHANES III, 30 per cent had NACKD. Another study (n = 15 773), conducted across 19 diabetes clinics in Italy, found that among patients with e-GFR less than 60 ml/min per 1.73 m 2 , 56.6 per cent were normoalbuminuric 10 . A follow up study including the patients having low eGFR in the UKPDS cohort with 1132 participants had shown that 51 per cent had no albuminuria 11 . Other studies have shown a lower proportion (23.7% and 28.4%) of NACKD 12,13 , but patients with reninangiotensin blocker therapy were excluded from these analyses. The recent rise in the proportion of NACKD in diabetic patients was thought to be contributed by RAS inhibitor therapy, but a Japanese study 14 showed that even after adjusting for RAS inhibitor therapy, 73 per cent of patients among those with low e-GFR had normoalbuminuria. In the present study also, the proportion of patients taking ACEI/ARB therapy was found to be statistically similar among NACKD and ACKD groups.
Literature search did not reveal any study from India done exclusively in a large cohort of T2D subjects to find the proportion of patients with NACKD. A study on non-communicable diseases, which included 53 diabetic patients showed a prevalence of NACKD to be around 11.3 per cent 15 . Another study done on 75 diabetic patients from Pune, India, showed that the proportion of NACKD among all patients with low e-GFR was 50 per cent 16 . The prevalence of NACKD was reportedly higher in hypertensive individuals as compared to individuals with diabetes mellitus.
In the current study, males were found to be more likely to have NACKD when compared to females, which is in contradiction to studies done elsewhere 9,17 . Other associations found were age, lower body weight and statin intake. Interestingly, none of the glycaemic parameters, including HbA1c showed any association with normoalbuminuric fall in e-GFR. A Swedish study found that normoalbuminuric participants had better metabolic control and lower BMI as compared to those with ACKD, similar to the findings of the current study 8 . In this study, patients having ACKD were found to have a longer duration of diabetes, higher HbA1c as well as other glycaemic parameters than those with NACKD, whereas higher age was the only independent association favouring NACKD.
In the present study, only 26.5 per cent of patients with NACKD had retinopathy. By contrast, 49.3 per cent of patients with ACKD had retinopathy. This is in accordance with the RIACE study 18 where advanced retinopathy was twice as common in the albuminuric CKD group compared to the group with NACKD. The reduced prevalence of retinopathy in normoalbuminuric fraction might exclude microangiopathy as the major causative factor. The degree of macroangiopathy in albuminuric and non-albuminuric CKD appears similar, as evidenced by the comparable proportions of coronary artery and cerebrovascular diseases among both groups. A large prospective study 6 from Portugal demonstrated that increased urinary albumin excretion and reduced eGFR are both independently associated with the risk for both cardiovascular outcomes and microvascular complications in patients with T2D. This would mean that reduced GFR is an independent risk factor for cardiovascular disease in all patients with T2D, irrespective of albuminuria concentrations. The statin use identified to be associated with NACKD could be taken as surrogate evidence for the link between coronary artery/cerebrovascular disease or dyslipidemia with NACKD.
The significance of NACKD has been doubted by some previous studies, which showed a similar fall in age-matched non-diabetic patients as seen in the present study. Furthermore, the current study could not demonstrate a relation of NACKD with any parameter related to hyperglycaemia. Moreover, the prevalence of normoalbuminuric renal dysfunction when considering a lower e-GFR cut-off of 45 ml/min/1.73 m 2 and 30 ml/min/1.73 m 2 comes down significantly and albuminuria becomes more frequent as the e-GFR declines. Similar observations have been made by Singh et al 19 who found that the proportion of patients with proteinuria increased as eGFR decreased and almost all participants with eGFR <45 ml/min/1.73 m 2 had evidence of dipstick proteinuria. From these observations, it could be hypothesized that, although the lesser degrees of renal dysfunction may be normoalbuminuric, albuminuria is essential for the development of severe stages of CKD in diabetes. Alternatively, a fall in e-GFR to low values is often accompanied by albuminuria due to secondary glomerular dysfunction irrespective of the cause or mode of initial e-GFR fall.
The progression of renal dysfunction from the initial moderate normoalbuminuric fall in e-GFR in patients with diabetes is an aspect which is not well studied in a prospective fashion. Conflicting reports on accelerated fall in e-GFR as well as those showing lack of it exist [20][21][22] . Koye et al 13 in their study observed that the individuals with non-albuminuric variant of diabetic kidney disease had a lower risk of rapid decline of GFR and progression to ESKD compared to those with albuminuria. The diagnosis of diabetic kidney disease could be delayed in a significant proportion of subjects if albuminuria alone is assessed without GFR for screening and monitoring renal dysfunction in diabetes mellitus.
The strength of the study is its large sample size. As per the authors' knowledge this may be the first large study reporting the relative prevalence of normoalbuminuric fall in eGFR in Indian patients with T2D compared to the albuminuric subjects. Limitations of this study include an absence of detailed investigations or follow up for patients who had NACKD, to rule out other causes of renal dysfunction. These were due to the retrospective nature of the study. We were also not able to repeat the urinary sampling in albuminuric subjects. Being a study from a tertiary care centre there could also be a referral bias whereby the higher proportion of subjects with severe hyperglycaemia, longer duration of diabetes and hence the complications of diabetes could also have been included in the study. However, since the prevalence of NACKD was not found to be related to the duration of diabetes or degree of hyperglycaemia, this bias is unlikely to affect the conclusions of this study.
Overall, NACKD accounts for more than one third of subjects with CKD among South Indian patients with T2D. The proportion of NACKD decreases in more severe stages of CKD. The normoalbuminuric decline in e-GFR was associated with higher age, male sex, lower body weight but not with the duration or severity of hyperglycaemia. NACKD group has a similar proportion of patients with macrovascular complications as ACKD, but fewer of them have retinopathy in contrast to ACKD. Longitudinal studies are hence required to delineate the progression of NACKD, if any, and its long-term implications to cardiovascular health and mortality in patients with T2D mellitus. | 2023-03-01T16:02:06.310Z | 2022-10-01T00:00:00.000 | {
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237842991 | pes2o/s2orc | v3-fos-license | The Cold Fusion Phenomenon – Nuclear Reactions in the CF Materials at Around Room Temperature
: Since the discovery of nuclear reactions in PdD x alloys at around room temperature in 1989, there have been accumulated very many experimental data sets showing existence of nuclear reactions in solid materials composed of transition metals and occluded hydrogen isotopes (let us call them the CF materials , for short) resulting in various nuclear products such as neutrons, tritium, transmuted nuclei, and others accompanied with large excess energies at relatively low temperatures up to 1000°C (let us call these whole events the cold fusion phenomenon ( CFP ), for short). As the cause of these nuclear reactions in the CFP, we have to accept the existence of the interactions between nuclei in the CF material through the nuclear force (let us call this interaction the nuclear-force interaction, for short) recognized its existence in the nucleus in the nuclear physics. We can classify the CF materials, i.e. materials where CFP occurs, into three groups: (1) metallic material including transition-metal hydrides (e.g. NiH x , AuH x ) and deuterides (e.g. PdD x , TiD x ), (2) carbonic material including hydrogen graphite (HC x ) and XLPE (cross-linked polyethylene) and (3) biological material including microorganisms, microbial cultures and biological tissues or organs. We will explain the characteristics of the CFP observed in each group in this paper. The nuclear reactions in the CF material gives rise to production of new particles from neutron, triton, and new nuclei with proton numbers Z up to 83 accompanying enormous excess energy. In addition to these events, there occurs the stabilization of unstable nuclei, including the decay-time shortening of radioactive nuclei, which is especially interesting to apply it to treat hazardous nuclear waste produced by the nuclear power plant. Finally, we give an overview of the CFP in relation to the solid state-nuclear physics and the solid state-nuclear chemistry where the nuclear-force interaction may play important roles to explain the riddles found but not given appropriate explanations in these old sciences hitherto.
Introduction -Nuclear Reactions in the CF Materials
In more than 30 years since the discovery of nuclear reactions in PdD x alloys in 1989 ( [1], and e.g., [2,3,4]), there have been accumulated very many experimental data sets showing existence of nuclear reactions in solid materials composed of transition metals and occluded hydrogen isotopes (let us call them the CF materials, for short) resulting in various nuclear products such as neutrons, tritons, transmuted nuclei, and others accompanied with large excess energies at relatively low temperatures up to 1000°C (let us call these whole events the cold fusion phenomenon (CFP), for short). It will be persuasive to cite several experimental data obtained in recent years (e.g., [5,6,7,8]) to show that the field of the CFP is progressing even now after 30 years of its discovery.
In contrast to the large quantity and the excellent quality of the evidence of the nuclear reactions in the CF material, there are too low evaluations for them in the world of the natural sciences. The causes of this social phenomenon might be various but the most important one in them is, in our impression, the lack of possible unified explanations for the wide variety of events in the CFP outside of established fields of sciences developed in 20 th century.
To explain these nuclear reactions related to the events in the CFP, it is clear that we have to accept the existence of the interactions between nuclei in the CF material through the nuclear force (let us call this interaction the nuclear-force interaction, for short) recognized its existence in the nuclear physics.
In this paper, we introduce the whole field of the cold fusion phenomenon (CFP) and a possible approach to give a unified explanation of the whole experimental data observed there to promote the investigation of the possible new field of the science between two big fields, nuclear physics and solid state physics, developed in these almost 100 years. In Section 2, we give a brief summary of the events of the CFP. In Section 3, we introduce our phenomenological approach with the TNCF model and give short references for its quantum mechanical foundations. The nuclear transmutations in the CF materials composed of metals and alloys are discussed in Section 4 and those composed of polymers and biological systems are discussed in Section 5. In Section 6, we give a short introduction to the new field of solid state-nuclear sciences where the nuclear-force interaction discovered by the CFP will contribute to develop in near future. There are many riddles found but not given appropriate explanations hitherto. The nuclear-force interaction which has not noticed its existence until now will play decisive role to resolve these riddles.
Summary of the Cold Fusion Phenomenon
We summarize the nuclear transmutations observed in the cold fusion phenomenon (CFP). The CF materials, i.e. materials where CFP occurs, are classified into three groups: (1) metallic material including transition-metal hydrides (e.g. NiHx, AuHx) and deuterides (e.g. PdDx, TiDx), (2) carbonic material including hydrogen graphite (HCx) and XLPE (cross-linked polyethylene) and (3) biological material including microorganisms, microbial cultures and biological tissues or organs. We will explain the characteristics of the CFP observed in these groups using our phenomenological approach with the TNCF model in this paper.
In the explanation of the CFP in this paper, we put a special emphasis on the following characteristics of the CFP observed in the third group. The stabilization of unstable nuclei and the decay-time shortening of radioactive nuclei are especially interesting from the viewpoint of application to treat hazardous nuclear waste produced by nuclear power plant. A characteristic of biological systems, wherein selective adsorption of specific ions occurs, seems especially useful for this purpose. If we have a microorganism or microbial culture absorbing an ion of a radioactive element selectively, we can remediate the radioactivity of the ion by biotransmutation.
Even if the neutrons and tritons measured in the experiments by Fleischmann et al. [1] are not persuasive due to their unsatisfactory detection techniques, the observation of excess energy by them and the detection of helium in their samples by experts [2] had shown clear evidence of the nuclear reactions generating helium and accompanying large excess energy inexplicable by other sources in their experiments.
The nuclear reactions generating tritium and neutron in the CF materials had been confirmed by many investigators in one or two years after the first experiment by Fleischmann et al. We can cite several papers where are detected tritium in deuterated CF materials as follows [3,9,10,11] and [12].
We tabulated some characteristics of the CFP in Table 1. In the table, the CF materials are divided into host solids (in the row 1) and agents (in the row 2) to indicate clearly possible opponents of the reaction with the "trapped neutrons" assumed in out TNCF model [13,14]. Dissipative observables Excess energy Q, neutron energy spectrum n(ε), energy spectrum of neutrons Nn, Gamma ray spectrum γ(ε), X-ray spectrum X(ε), It is interesting to see the ubiquitous appearance of the CFP in various CF materials as shown in the row 1 (host solids) of Table 1.
The almost all experimental data obtained in the CF materials, having a huge variety shown in Table 1 have consistently been explained by the phenomenological approach using the TNCF model as shown in our books [13,14].
Phenomenological Approach and Its Foundation
There are many routes of approaches to the problem showing apparent contradiction with the principles of the established sciences. One of the effective approaches to the cold fusion phenomenon showing several riddles inexplicable by the principles in the quantum physics is the phenomenological one using a model based on the experimental facts.
The premises of the TNCF model have been investigated quantum mechanically using the idea of the neutron energy bands generated by the super-nuclear interaction between neutrons in different lattice nuclei mediated by interstitial protons/deuterons ( [14], Appendix A3 in [16]).
We could find out the necessary and sufficient condition for the generation of the neutron energy bands explained above using the idea of self-organization in the complexity developed by the nonlinear dynamics [17]. The regular superlattice XY of host elements X at lattice points and occluded protons/deuterons Y at interstices is generated by the self-organization revealed by the nonlinear dynamics even if the average composition of the CF material is X x Y y with x ≠ y. in the situation where the material is an open, nonequilibrium, and multi-component systems ( [17], Sections 3.8 and 3.9 in [18]).
The explanation of the characteristics of the experimental data in the CFP has been given by the TNCF model as follows.
Nuclear Reactions in Free Space and in the CF Materials
Reactions in free space supposed by Fleischmann et al. to be responsible to their experimental results are written down as follows [1,19]: The branching ratios of these reactions have been determined in the nuclear physics as 1:1:10 -7 down to low energies of a few keV.
Other nuclear reactions between light nuclei in free space are written down as follows.
t + d = 4 2 He (3.5 MeV) + n (14.1 MeV), Unified Explanation of the CFP by the Phenomenological Theory using the TNCF model is given by us in which the gamma in Eqs. (3), (4) and (5) in free space should be read as φ's (phonons excited by the reaction in contact with trapped neutrons) as explained below [13,14,15,19,20].
According to the discussions for the phenomenological approach given in these books and papers, we have assumed that the gammas in the reactions (3), (4) and (5) in free space should be read as φ's (phonons excited in the CF materials by the fusion reactions in contact with trapped neutrons) in the CF material: where Q = 23.8 MeV, Q' = 2.2 MeV and Q'' = 6.25 MeV.
Relations Between the Observed Number of Observables in the CFP Deduced by the TNCF Model
There are several evidence showing the effectiveness of the TNCF model to give a unified explanation of the whole experimental data obtained in the CFP. One of the strong evidence of them is the explanation of the ratios of the observed numbers N x /N y of observables x and y are explained consistently with the experimental values by assuming a value for the adjustable parameter n n of the model.
The lack of the quantitative reproducibility in the CFP has been explained by the concept of the complexity in the generation of the neutron energy bands [17].
The surface nature of the nuclear reactions in the CFP is explained by the nature of the neutron Bloch waves in the neutron energy bands. Neutrons in a neutron energy band in perfect superlattice do not interact neither with a lattice nucleus nor with a proton/deuteron but do interact with specific nuclei at disordered site in the CF material. The most popular disordered lattice nuclei exist at boundaries of the CF material which result in nuclear transmutations observed very often there in CF experiments [13,14,21].
Explanation of the distribution of transmuted nuclei in the CFP over the atomic number concordant with the natural abundance in the universe has been formulated as the First Law (Appendix A10 in [16,22]).
Quantum Mechanical Foundations of the TNCF Model
The premises used to construct the TNCF model have been investigated quantum mechanically. The essence of the justification of the premises in the TNCF model has been given in our papers and books [14,20,21]. There appears a possibility to realize the nuclear-force interaction between nucleons in the material when the wavefunctions of the occluded protons/deuterons have extension to overlap with the lattice nuclei as in the CF material such as NiH x and PdD x (x ≲ 1).
Nuclear Transmutations in the CF Materials (1) -Metals and Their Alloys
The nuclear transmutation, one of the characteristics of the nuclear reactions in the CFP, has shown peculiarity of the nuclear reactions in the CF materials inexplicable by simple two-body reactions like the d -d fusion reactions assumed by the pioneers of the "cold fusion" reaction in 1989.
Nuclear Transmutations
The evidence of the nuclear reaction in the CF material resulting in the nuclear transmutation generating new nuclei with large nucleon numbers A and emission of tritons and neutrons have been observed since 1989 until now as tabulated Tables 2 and 3 for the data until 1989 [13]. Table 2. Pd/D(H)/Li Systems. Neutron density nn and relations between the numbers Nx of the event x obtained by theoretical analysis of experimental data on the TNCF model (NQ ≡ Q (MeV)/5 MeV). Typical value of the surface vs. volume ratio S/V (cm -1 ) of the sample is tabulated, also (Table 11.2 in [13]). References in this table, e.g., Fleischmann et al. 1) , are posted at the CFRL website: http://www.kozima-cfrl.com/Books/bookse/bookse01/refer1.htm.
Another extensive compilation of experimental data in the CFP had been given by E. Storms in his book published in 2007 [23].
There are several kinds of CF materials which we did not take up specifically until 2019. They include two kinds of materials; (1) the composite materials including alloys, ceramics, and polymers and (2) the compound materials with interfaces between several parts (materials supported by substrates, multi-layered materials, etc.) composing the system. The materials in the second group are (a) CF materials surrounded by liquids and gases, (b) those supported by substrates, (c) and those have multilayered structure. The experimental data obtained in these CF materials had been analyzed taking into the characteristics of these materials and published in the Proceedings of JCF21 [23,24].
Stabilization of Unstable Nuclei in the CFP
In addition to the nuclear transmutations in the CF materials, there are observed changes of radioactivity of unstable nuclei in the CFP. We have given explanation on the stabilization of unstable nuclei (including decay-time shortening) in the CFP as follows (Sec. 2.5.1.1 Decay Time Shortening in [14]).
Decay-Time Shortening
In general, the explanations of the mechanisms of the nuclear transmutations by decay (NT D ) with absorption of a neutron drop A Z ∆ composed of Z protons and (A -Z) neutrons are given by the following reaction formulae: A' + m Z' X*→ A' + m Z'+ 1 X' + e -+ ν e + Q, (β) (13) A' + m Z' X*→ A' + m -4 Z'-2 X" + 4 2 He + Q, (α) (14) Where α and β in parentheses designate types of the decay. In these nuclear transmutations, there are several cases where the decay-times τ d of the intermediate compound nuclide A Z X* in free space are very long of orders of 10 6 to 10 9 years (10 12 -10 15 s). The time elapsed in experiments is at most several months (≈10 5 s). Therefore, if the decay products with such long decay-times are observed, there should be drastic shortening of the decay-times (or stabilization of unstable nuclei).
Following compound nuclei correspond to this case:
Nuclear Transmutation in Actinoid Hydrides and Deuterides
Experimental data sets on actinoid hydrides and deuterides prepared by electrolysis and glow discharge [26,27,28] have been analyzed and explained using the TNCF model [29]. The experimental data have shown that these hydrides and deuterides are classified as the CF materials where occurs the cold fusion phenomenon (CFP) and the observed events are understood by nuclear reactions common to other reactions observed in CF materials, mainly transition-metal hydrides and deuterides.
The changes of radiation properties of actinoids occluding hydrogen isotopes are explained by formation of the cf-matter similar to the free neutron sea in neutron star matter [30] and then by the interaction of actinoid nucleus and the cf-matter. We have explained the experimental results [26,27,28] on the acceleration of the alpha decay of 238 92 U into 234 90 Th, the so-called the decay-time shortening, by the change of the boundary layer between the nucleus 238 92 U and the cf-matter. The change of the boundary layer is induced by the increase of the density ratio n o /n i of neutrons (n i and n o are neutron densities inside and outside the nucleus, respectively) due to the formation of the cf-matter (Sec. 2.9 in [29]).
A possible application of this phenomenon to A 94 Pu (A = 238 -244) is hopeful. If we can handle plutonium isotopes properly, which are produced in atomic plants as hazardous waste, to accelerate their decay, it is the savior of our age from the dangerous nuclear waste waiting its resolution. As shown in Table 4, plutonium isotopes have very long decay-times and therefore the decay-time shortening as shown in the CFP will be applicable to transmute them into other nuclides easy to treat. Instead of neutron bombardment in free space, we can use the cf-materials to give an effect of neutrons on target nuclei (cf. [31]). This fact may be interesting in science and technology of the neutron-nuclear interaction in low energy region. It is a pleasant fantasy to imagine a machine simultaneously remediates the radioactive waste and generates excess energy.
Nuclear Transmutations in the Composite and Compound CF Materials
In the recent papers presented at JCF21, we have summarized and analyzed nuclear transmutations observed in the composite (alloys and ceramics) and compound (including boundaries between different components) CF materials [16,24,25]. Several of most interesting works in these genres are works by Miley et al. [32,33], by Kitamura et al. [5] by Celani et al. [7], and by Iwamura et al. [8] which have been extensively treated in our papers cited above.
Nuclear Transmutation in the CF Materials (2) -Polymers and Biological Systems
The nuclear reactions in the CFP are not confined in the CF materials composed of transition metals/alloys and hydrogen isotopes but also in polymers and biological systems composed of regular arrays of carbon and hydrogen. We have given explanations of a few events of nuclear reactions in polymers and biological systems [34,35,36] using the same idea used to analyze the CFP analyzed in Section 3.
One of the characteristics of these materials is the beforehand existence of the superstructure of C and H in the system different from the self-organization of the superlattice of transition metals/alloys and hydrogen isotopes in the transition metal/alloy hydrides/deuterides discussed in Section 3. This characteristic of the polymers and biological systems seems an advantage in realizing the necessary and sufficient condition for the CFP over the CF materials considered in Section 3.
A characteristic of biological systems wherein selective adsorption of specific ions occurs seems very useful for the CFP especially in its application for remediation of hazardous nuclear wastes. If we have a microorganism or microbial culture absorbing an ion of a radioactive element selectively, we can effectively remediate the radioactivity by biotransmutation.
Nuclear Transmutation in XLPE
Similarly, we can contemplate appearance of neutron bands in XLPE shown in Figure 1 [37]. The rather regular structure of the carbon-hydrogen array in XLPE suggests similar mechanism to form the neutron bands as discussed in our recent papers [34,37]. The atomic arrays in biological cells are more complex compared with that in XLPE as seen in Sec. 5.2 but we may be able to use the same idea used in the CFP general in biological systems.
Lattice structure of XLPE. [34,37]. For the illustration of nuclear transmutations in their systems including XLPE, we show a data in Table 5 from our paper ( Table 2 in [37]).
Characteristics of Biotransmutation
Allowing application of the TNCF model to the problems of biotransmutation, we can use following reactions between a trapped neutron n and a nucleus A Z X at or in the surface of a bacterium or a tissue [35]: In this reaction formula, A+1 Z X* is an excited state of the nucleus A+1 Z X which will decay through following several channels in free space: where ν e is an anti-particle of the electron neutrino, γ is a photon (in free space) and Y, Y' and Y'' are daughter nuclides of the reactions. In the CF materials, the photon γ in the free space is supposed to be absorbed by the cf-matter formed of neutrons in the neutron band and its energy dissipates in phonons to heat the system as a whole (Section 3.7.5 in [14]).
Recent Experimental Data on the Biotransmutation Obtained by Vysotskii et al V. I. Vysotskii and his collaborators have performed sophisticated experiments and obtained refined data sets in
biological systems in these about 20 years mainly by as cited in our paper [34]. To realize the complex structure of microorganisms and microbial cultures used in their experiments, we give their fundamental structures in Figure 2. The structure of cell walls in microbial cultures depicted in Figure 2 (c) show complex but similar regular structures to that in XLPE shown in Figure 1.
There are data sets showing (1) production of 57 T-8. Selection of these cultures was motivated either by their experimentally proven ability to grow in the heavy water based media or by the prospect of using the radiation-stable culture Deinococcus radiodurans M-1 in transmutation processes given the presence of powerful radioactive fields, as was noted earlier [41].
25 Mn
The first example is the production of 57 26 Fe in a CF material containing 55 25 Mn. The nuclear reaction responsible to this case is suggested by (17) and (16). We can explain the production of 57
Decay-Time Shortening in Biological System
The observed acceleration of the decay process of 137 55 Cs isotope is shown in Figure 3. The behavior of the decay-time shortening in transition-metal hydrides had been noticed before and discussed in our paper already [42]. Another data of the decay-time shortening in the biological system is obtained in 140 56 Ba and 140 57 La in pure reactor water with presence of metabolically active microorganisms as shown in Figure 4. and 60 27Co in the experiment on transmutation (activity Qcultures in pure reactor water with presence of metabolically active microorganisms) and in the control one (activity Qcontrol in the same pure reactor water without microorganisms) (Figure 3.21 in [41] and Figure 8 in [43]).
Explanation of Stabilization of Unstable Nuclei (or Decay-Time Shortening) in Biological System
The sophisticated experiments performed by Vysotskii et al. on the nuclear processes in biological systems revealed existence of the decay-time shortening observed already in inorganic CF systems as discussed recently in our paper [44]. There are two examples of the decay-time shortening in biological system: one for 137 55 Cs in electrolytic liquid, MCT + electrolyte (KCl, NaCl, or CaCO 3 ), and another for 140 56 Ba and 140 57 La in electrolytic liquid (water + metabolically active microorganisms). Explanation of MCT used in the experiment is given elsewhere (Appendix 2 in [35]).
(i). 137 55 Cs
The second example is the decay-time shortening of radioactive isotope 137 55 Cs which decays in free space according to the following reaction shown in Figure 3: Assuming the existence of the trapped neutron in the TNCF model, we can apply the equation (17) to this case: where 137 55 Cs nuclei are adsorbed and their decay characteristics are drastically influenced by the density of the trapped neutron in samples from our point of view. Thus, the electrolyte seems to have large effect on the adsorption characteristics of 137 55 Cs by MCT. This fact reminds us the effect of K and Li on the CFP in Ni and Pd discussed by us for many years (e.g., Sec. 2.2.1.2 in [14] and Sec. 4 in [45]).
"It should be emphasized here that there is preference for combination of a cathode metal (Pd, Ni, Ti, --), an electrolyte (Li, Na, K, or Rb) and a solvent (D 2 O or H 2 O) to induce CFP. " (p. 45 in [45]). Now, let us investigate the characteristics of the decay-time shortening of 137 55 Cs in these systems. The temporal evolution of the number of a radioactive nuclide with a decay constant τ is described by following equations: On the other hand, decrease of the number of a nucleus A Z X due to absorption of thermal neutrons described by Eq. (28) and the relation (29) assumed in our model (Sec. 11.1 in [13], Sec. 3.2 in [14]): In the above equations, n n is the density of the trapped neutron, v n is the thermal velocity of the assumed trapped neutron, N X is the number of the nucleus A Z X, and σ nX is the absorption cross section of thermal neutrons by the nucleus X by the reaction (28) (σ nX = 0.113 b for 137 55 Cs) assumed to be the same as the thermal neutron absorption cross section in free space. We use a value 2.2 × 10 5 cm/s for v n according to our premises of the TNCF model.
If an 137 55 Cs nucleus is adsorbed by the MCT granules to be reacted by the trapped neutron, the reaction (28) The reaction occurs with a probability P in a unit time interval for a nucleus 137 55 Cs as expressed in Eq. (29): Let us determine the density n n of the TNCF model assuming that the observed decay-time shortenings of 137 55 Cs in electrolytic liquids depicted in Figure 3 are the results of the neutron absorption described by Eq. (30).
For an example of calculation, we take up the case of In this calculation, we notice that the exponential factor exp (-t/τ*) ≈ 1 and does not essentially contribute to the final result.
On the other hand, the equation (31) gives n n through the relative number of transmuted 137 55 Cs nuclei δN/N as: This value is compared with the values 10 7 -10 12 cm -3 obtained in inorganic CF materials tabulated in Tables 2 and 3 in Section 4.
As the Eq. (27) (or Eq. (32)) shows that the decrease of the number of radioactive nuclei is proportional to the decay time τ* inversely and it is also proportional to x and n n as shown by Eq. (38) (where x is the ratio of adsorbed nuclei). The differences of τ* observed in different electrolytic liquids are explained as follows.
If the density of trapped neutrons n n is not influenced by the kind of electrolyte in the liquid, the difference of τ* depend only on the value of x which may depend on the electrolyte. The values of τ* = 310 d, 380 d, 480 d, 10 y in the liquid with CaCO 3 , non, NaCl, KCl, respectively, show that the ratios x in these electrolytic liquids are given by 1 Figure 4 are consistently explained by the TNCF model.
Other Biotransmutations
About the data of biotransmutations known since 18 th century, let us quote some sentences related with our investigation from page 25 of Michio Kushi's book [46]. The elemental transmutation (ET) in biological systems is considered as "most likely taking place at the cellular level." "---it was concluded that, granted the existence of transmutations (Na to Mg, K to Ca, Mn to Fe), then a net surplus of energy was also produced." "A proposed mechanism was described in which Mg adenosine triphosphate (MgATP) played a double role as an energy producer." "The MgATP, when placed in layers one atop the other, has all the attributes of a cyclotron." "It was concluded that elemental transmutations were indeed occurring in life organs and were probably accompanied by a net energy gain." (p. 25 in [46]).
This summary of the biotransmutations by M. Kushi is based on many observations by Vauquelin and others which are introduced in our paper [47] and book (Sec. 10.1 Biotransmutation in [13]).
Characteristics of Biotransmutations Applicable to the Remediation of the Hazardous Nuclear Waste
In inorganic systems used in the CFP, it is known that there are specific favorable combinations of an electrode and an electrolyte to realize the CFP in electrolytic experiments: Pd-Li and Ni-K are the most outstanding pairs used in positive experiments for the CFP [45].
It might be possible to have a biological system in which the CF material (e.g. a bacterium) adsorbs selectively a specific element on the surface by the nature of the surface or of the material. Then if the bacterium works as an agent similar to the MCT in Figure 3, the system is applicable to remediate effectively hazardous radioactive waste.
Solid State-Nuclear Sciences It should be noticed that the nuclear reaction in the CF materials at around room temperature observed in the CFP has its counterpart in the physics and chemistry of the CF materials even if there have no nuclear reactions but fingerprints in the behavior of protons/deuterons and also in the states of lattice nuclei. These problems have only its preliminary investigation now (e.g., [20,24,48]) but show profound and fruitful future developments.
Conclusion
The nuclear reactions in the specific solids having a superlattice composed of a sublattice of some elements having characteristic nuclear structure and another sublattice of hydrogen isotopes at around room temperature have been discovered by chance in 1989 in a research of the excess energy generation in PdD x alloys (x ≲ 1) with an excessive or rather erroneous expectation of the d -d fusion reactions there [1]. Very many research works followed the pioneering paper by Fleischmann et al. have revealed reality of the nuclear reactions in the CF materials (various transition metal/alloy hydrides/deuterides) at around room temperature up to 1000°C with events generating transmuted nuclei from small masses as neutron, triton, helium-4 to large masses 209 83 Bi, 208 84 Pb, ---. The experimental data obtained in these more than 30 years since 1989 show clearly there are some mechanisms not noticed before inducing the nuclear reactions observed in the CFP.
One of the approaches successful to give a consistent explanation for the whole heap of data has been given by our TNCF model constructed phenomenologically using several premises in which is an adjustable parameter n n , a density of the "trapped neutrons" in the CF material. As summarized in Section 3, the model has been successful to give qualitative, sometimes even more semi-quantitative explanations for events observed in the CFP.
The premises including the existence of the trapped neutrons have been investigated and verified quantum mechanically using such knowledge obtained in nuclear physics as the exotic nuclei and in transition metal hydrides/deuterides as super-diffusivity. The TNCF model successfully applied to the original CF materials composed of transition metal/alloy hydrides/deuterides have been used in the investigation of the CFP in the CF materials composed of polymers and biological systems with hopeful results to explain the events observed in them consistent to the explanation given in the original CF materials.
Then, we can extend our investigation using the TNCF model to other physical and chemical phenomenon than the CFP, i.e. the cold fusion phenomenon. There are several such big genres investigated for long times as the super-diffusion of hydrogen (in transition metal/alloy hydrides), the hydrogen electrode reaction (HER) and the underpotential deposition (UPD) (in electrochemistry), the exotic nuclei with large excess of neutron numbers over the proton numbers isolated in free space (in nuclear physics). The nuclear reactions in the CFP as explained by our model using the super-nuclear interaction between neutrons in different lattice nuclei (cf. [14] [ 17,20], and [29]) might be useful to investigate the unsolved riddles encountered in these research fields listed up above and also to develop a new phase in the situation where are interactions with other nuclides.
The nuclear-force interaction revealed its existence in the CF material will induce a great impact on many sciences including materials with similar properties to the CF material where the occluded protons/deuterons have extended wavefunctions to overlap with the lattice nuclei. | 2021-09-28T01:16:13.783Z | 2021-06-08T00:00:00.000 | {
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233208307 | pes2o/s2orc | v3-fos-license | Chelating Fabrics Prepared by an Organic Solvent-Free Process for Boron Removal from Water
A chelating fabric was prepared by graft polymerization of glycidyl methacrylate (GMA) onto a nonwoven fabric, followed by attachment reaction of N-methyl-D-glucamine (NMDG) using an organic solvent-free process. The graft polymerization was performed by immersing the gamma-ray pre-irradiated fabric into the GMA emulsion, while the attachment reaction was carried out by immersing the grafted fabric in the NMDG aqueous solution. The chelating capacity of the chelating fabric prepared by reaction in the NMDG aqueous solution without any additives reached 1.74 mmol/g, which further increased to above 2.0 mmol/g when surfactant and acid catalyst were added in the solution. The boron chelation of the chelating fabric was evaluated in a batch mode. Fourier transform infrared spectrophotometer (FTIR) was used to characterize the fabrics. The chelating fabric can quickly chelate boron from water to form a boron ester, and a high boron chelating ability close to 18.3 mg/g was achieved in the concentrated boron solution. The chelated boron can be eluted completely by HCl solution. The regeneration and stability of the chelating fabric were tested by 10 cycles of the chelation-elution operations. Considering the organic solvent-free preparation process and the high boron chelating performance, the chelating fabric is promising for the boron removal from water.
Introduction
The boron in seawater is about 4.5 mg/L, while in rivers it ranges from 0.3 to 100 mg/L. In Japan, the boron concentration in some hot-spring water even exceeds 100 mg/L. In natural water, it exists in the form of undissociated boric acid, which is a weak acid as shown in Scheme 1. The pKa of boric acid at room temperature is 9.24. Boric acid in natural water comes from the leaching of soils and rocks, as well as the wastewater from the glass/ceramic and metallurgy industries [1]. In an alkali solution it forms a borate salt, while in vicinal diol compound solution it forms boron ester.
The boron in water is of special concern because of its possible toxic effects on humans, animals, and plants. According to the WHO guidelines in 2011, boron in drinking water must not exceed 2.4 mg/L. Considering its impact on crops, boron in irrigation water is strictly regulated by many countries to be less than 0.5 mg/L [2][3][4].
The low-cost removal of boron from water has attracted considerable scientific interest [5,6]. Chemical precipitation is not suitable for removing trace boron from water. Ion-exchange resin is ineffective because boric acid exists as an unassociated compound in water. Membrane separation technologies, such as reverse osmosis membranes, are inadequate for rejecting boron because of the small size and neutral properties of the boric acid [7,8]. Adsorption methods using activated carbon, metal oxides, and other adsorbents for boron removal are also inefficient because of their low boron-adsorption [9]. Recently, chelating resins having vicinal diol groups, such as Amberlite ® IRA743 and DIAION ® CRB05, have been specifically designed for boron removal [10]. These chelating resins are composed of polystyrenic microporous frameworks and N-methyl-D-glucamine (NMDG) moieties. The boron removal is based on the chelation reaction shown in Scheme 1, Equation (3), where boric acid is chelated by the vicinal diol to form a stable five-or six-membered ring boron ester and release a proton [11][12][13]. The other frameworks, such as poly(glycidyl methacrylate-co-methyl methacrylate) [3], poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) [14], poly(chloromethyl styrene-codivinylbenzene) [7,15], and cellulose [16], have also been attached with the NMDG moieties to prepare the chelating resins for boron removal. These chelating resins are in the form of beads or particles, and are suitable for use in the column mode. Their boron chelating ability is strongly affected by the chelating capacity of the resins [13,17], and is hardly affected by the pH in a wide region of 4.0-8.0 and ionic strength of the aqueous solution [15,18]. In an acidic medium with a pH lower than 2.0, the chelated boron can be eluted according to the reverse reaction shown in Scheme 1, Equation (3). However, owing to their high porosity and crosslinking, these chelating resins are fragile and should be transported and stored in a water-swollen state.
On the other hand, nonwoven fabrics are easy to manufacture and have been widely used in the industry. As a fibrous porous medium nonwoven fabric has a high specific surface area. The phenomenon of liquid or gas transport through it has been well studied [19]. The polymeric nonwoven fabric can be chemically modified and converted into functional materials for water treatments [20]. Herein, a chelating fabric with NMDG moieties was designed. The chelating fabric can be used in both batch and column modes and transported and stored in the dry state [21]. For this purpose, glycidyl methacrylate (GMA)-grafted fabric is used as a framework to attach the NMDG moieties. The grafted fabric was prepared by radiation grafting the GMA onto a polyethylene-coated polypropylene (PE/PP) nonwoven fabric. Radiation grafting is an effective method to modify existing materials in any form of fiber, film, and fabric, giving them new properties such as reactivity, hydrophilicity, and conductivity [22,23]. The PE/PP nonwoven fabric has many advantages, including low cost, high mechanical strength, and easy radiation grafting [23][24][25]. The GMA monomer has vinyl groups and active epoxy rings. The former gives it high graft polymerizability, while the latter gives it high reactivity to introduce various new functions [26][27][28][29][30].
The attachment of NMDG moieties onto the GMA-grafted fabric is based on the substitution reaction of amine with the epoxy ring. The reaction was carried out in a heterogeneous system, wherein NMDG is dissolved in the solution, and the GMA-grafted fabric is in the solid state. Therefore, the reaction is significantly affected by the diffusion of NMDG into the fabric and the steric structure of the reactants [31,32]. Acids and bases can accelerate the reaction or increase its selectivity [33]. Until now, the reaction of GMAgrafted fabric with NMDG was carried out in mixture solvents such as 1,4-dioxane and water [22]. The organic solvent 1,4-dioxane enhances the affinity of the reaction solution to the fabric, while water improves the solubility of NMDG in the solution. In this solution more than 80% of the epoxy rings can be converted to NMDG moieties [34][35][36]. However, the use of organic solvents is harmful to the environment and human health. In this study, we carried out the attachment reaction in NMDG aqueous solution without the use of organic solvents. This attachment reaction in an organic solvent-free process has rarely been reported. This may be due to the low affinity of the GMA-grafted fabric to the aqueous solution and the low reactivity. To address these concerns, we used surfactants and acid/base catalysts to accelerate the reaction. The boron chelation of the resulting chelating fabric was evaluated in batch mode in a boron aqueous solution.
Materials
The PE/PP nonwoven fabric was obtained from Kurashiki Textile Manufacturing Co., Ltd., Kurashiki, Japan. The thickness of the fabric was about 0.5 mm, and the diameter of the fabric fiber was about 13 µm. The chemical reagents of GMA, NMDG (also known as meglumine or methylglucamine), and boric acid (B(OH) 3 or H 3 BO 3 ) were purchased from Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan, and were used as-received. Three surfactants, polyoxyethylene sorbitan monolaurate (POSM, or Tween ® 20), sodium ndodecyl sulfate (SDS), and n-octyl trimethylammonium bromide (OTBr), along with KOH and HCl solutions, methanol, and 1,4-dioxane, were purchased from Kanto Chemical Co., Inc., Tokyo, Japan. The resistivity of water used for the washing and preparation processes was higher than 18.0 MΩ cm. The concentrations of monomer and NMDG solutions were presented in the form of weight percentage, and the concentrations of the boron aqueous solutions were presented as mg/L.
Preparation of Grafted Fabric
Scheme 2 shows the preparation process and the boron chelation image of the chelating fabric. For the preparation of GMA-grafted fabric, the PE/PP nonwoven fabric (14.06 g) was cut into 3.5 cm × 6.0 cm slices and placed into a three-necked reaction flask. The flask was vacuumed, filled with nitrogen, and then placed in a Co-60 irradiation room for gamma-ray irradiation. The oxygen was completely removed to avoid the generation of peroxide during irradiation. The irradiation dose rate was 1.5 kGy/h, and the total irradiation dose was 24 kGy. After irradiation, the flask was filled with GMA emulsion to completely immerse the fabric slices. The GMA emulsion was prepared by ultrasonic dispersion of a mixture of 40 g GMA, 4 g POSM surfactant, and 756 g water for 30 min before use. The flask was then placed in a bath at 40 • C for 4 h to initiate the graft polymerization. During grafting, nitrogen was bubbled into the emulsion at a rate of 5 mL/min to prevent oxygen from entering. After grafting, the fabric slices were thoroughly washed with water and methanol, and then dried in an oven at 60 • C for more than 24 h. Finally, a GMAgrafted fabric was obtained. The weight of the dry GMA-grafted fabric was 39.30 g, which corresponds to a degree of grafting of 179.5% and an epoxy density of 4.52 mmol/g.
Preparation of Chelating Fabric
For the preparation of chelating fabric, the GMA-grafted fabric was immersed into an NMDG solution at 80 • C to bring about a substitution reaction between the epoxy rings and NMDG molecules. The reaction was fixed at 80 • C and was investigated for up to 20 h. After reaction, the fabric was removed and washed thoroughly with methanol and water, and dried in an oven at 60 • C for 24 h. Thus, a chelating fabric with NMDG moieties was obtained. The conversion of epoxy rings to NMDG moieties and the chelating capacity (density of NMDG moieties) of the chelating fabric were calculated using the following equations.
where W a (mg) and W g (mg) are the weights of the GMA-grafted fabric after and before the attachment reaction, respectively; M NMDG (195.2 g/mol) is the molecular weight of NMDG; and D e (4.52 mmol/g) is the epoxy density in the GMA-grafted fabric. We confirmed that the difference is very small for the chelating capacity under a same reaction condition. Therefore, only one experiment was performed for each condition to obtain the chelating capacity.
Characterization
Fourier transform infrared spectrophotometer (FTIR) in attenuated total reflectance (ATR) mode (Spectrum One, PerkinElmer, Inc., Tokyo, Japan) was used to analyze the fabric samples. The scanning range and resolution were 600-3800 cm −1 and 4.0 cm −1 , respectively.
Evaluation of the Chelating Fabric
For the evaluation of the chelating fabric, the chelating fabric (about 100 mg) was immersed in a boron aqueous solution (35 mL) in a glass bottle and was shaken at a rate of 150 rpm at 25 • C, during which the boron was chelated by vicinal diol of the fabric. The chelating capacity of the chelating fabric is 2.15 mmol/g. From the viewpoint of practical use, boron aqueous solution was prepared by dissolving boric acid in deionized water without further adjusting the pH and ionic strength [10,30]. After chelation, the chelating fabric was immersed in 0.1 M HCl for boron elution. The chelating fabric was then washed thoroughly with 0.1 M KOH and water for regeneration.
The boron concentrations were detected by an ICP-OES system (Optima 8300, PerkinElmer, Inc., Tokyo, Japan). The samples were filtered through a 0.45 µm filter and then diluted with a 10-fold volume of 0.5% HNO 3 . The amount of boron chelation was calculated using the following equation.
where C 0 and C i are the boron concentrations (mg/mL) of the solution before and after chelation, respectively; V a (mL) is the volume of the boron aqueous solution; and W a (g) is the weight of the chelating fabric. It was found that the boron chelation detected in each experiment under the same conditions was slightly different. This may be due to the structure of the chelating fabric and the stirring effect. Even so, the error of the boron chelation was still less than 1.0%, and the chelating results showed a good and reasonable trend with the chelating time.
The reusability and stability of the chelating fabric were tested through 10 cycles of chelation-elution operations. This series of operations was performed under shaking at 25 • C. For each cycle, the chelating fabric is immersed in 30 mL boron solution (20.42 mg/L) for 30 min for chelation, immersed in 15 mL HCl solution (0.1 M) for 15 min for elution, and then water washed for 5 min, treated with 0.1 M KOH for 5 min, and water washed again thoroughly to regenerate the chelating fabric for the next cycle. The elution time was sufficient for the release of the chelated boron. We confirmed that more than 98% of the boron was eluted within 5 min.
To evaluate the stability, the chelating fabrics were immersed in water, 0.1 M HCl and 0.1 M KOH solutions, respectively, at 80 • C for 48 h. The weights of chelating fabrics before and after immersion in these solutions were weighed to evaluate their stability.
Preparation of GMA-Grafted Fabric
The GMA-grafted fabric was used as a framework for the attachment of NMDG moieties to prepare a chelating fabric. The GMA-grafted fabric was prepared by a preirradiation grafting technique [35].
In the first step of preparation, the starting fabric is irradiated with gamma rays. Under irradiation, some C-H and C-C covalent bonds split into free radicals. In the amorphous phase, the radicals can re-bond to form crosslinked structures or small molecules. However, in the crystal phase, they are quite stable and can survive for several days at room temperature [37]. When the irradiated fabric comes in contact with the GMA monomers, the radicals can initiate the graft polymerization. Table 1 summarizes the grafting conditions and properties of the resulting GMAgrafted fabric. The high grafting of 179.5% and the corresponding epoxy density of 4.52 mmol/g can be expected to yield a fabric with high chelating capacity. When each epoxy ring is converted to one NMDG moiety, fabric with a high chelating capacity of 2.40 mmol/g can be obtained. The GMA-grafted fabric with a high grafting of 179.5% has sufficient strength in both wet and dry states. This can be attributed to the high mechanical strength of PP in the PE/PP fibers. A degree of grafting higher than 200% can be obtained easily by changing the reaction conditions. However, the stability of the resulting chelating fabric decreases due to the separation of the graft phase from the fibers. After grafting, the fabric becomes stiff and somewhat hydrophilic.
Preparation of Chelating Fabric
The chelating fabric was prepared by immersing the GMA-grafted fabric in an NMDG aqueous solution with different concentrations up to 40%, which is close to the saturation concentration at room temperature. The reaction temperature was 80 • C and the reaction time was 3 h. No organic solvents were added to the reaction solution. Figure 1 shows the chelating capacity of the resulting fabrics as a function of the NMDG concentration. In the 40% NMDG solution, the chelating capacity reaches 1.74 mmol/g, which corresponds to 58.4% conversion of the epoxy rings to NMDG moieties. Because of steric hindrance and side reactions, a large number of epoxy rings are not converted to the NMDG moieties [38]. Prolonging the reaction time to 24 h or increasing the reaction temperature to 100 • C had almost no effect on the further increase in conversion. Nevertheless, considering the trade-off between performance and cost, the chelating fabric with a chelating capacity of 1.74 mmol/g is still acceptable for practical use.
In Figure 1, a threshold concentration appears between 10 and 20% concentrations. The chelating capacity is quite low at the 10% NMDG concentration and increased linearly above the 20% concentration. The low reactivity in the low NMDG concentration solution is because of the low affinity of the fabric toward the NMDG solution. When the GMAgrafted fabric was immersed into the 10% NMDG solution, initially, it was slightly white and was not completely wetted by the solution. In contrast, in the solution with an NMDG concentration of more than 20%, the GMA-grafted fabric was quickly wetted and completely swollen by the solution, indicating good affinity and reactivity between them. Increasing the NMDG concentration increases the possibility of contact between the NMDG molecules and epoxy rings, and decreases the polarity of the solution. This leads to an increased affinity between the solution and the fabric and thus increases the reactivity.
Enhancing the Attachment Reaction by Surfactants
We added surfactants to the NMDG solution to improve its affinity to the fabric. Three surfactants, cationic (OTBr), anionic (SDS), and non-ionic (POSM), were added at a concentration of 1.0% in 40% NMDG aqueous solutions. The reaction was controlled at 80 • C for 3 h. After adding the surfactants, the GMA-grafted fabric was fully wetted by the solutions quickly. As shown in Figure 2a, when there is no surfactant, the chelating capacity is 1.74 mmol/g, and it increases to 1.85, 2.00, and 2.08 mmol/g, with the addition of the surfactants of OTBr, SDS, and POSM, respectively. The increase in reactivity is caused by the improved affinity of the GMA-grafted fabric to the solution. The commercial Amberlite ® IRA743 chelating resin has an NMDG moiety density of 0.70 mmol/mL and a water content of about 50%, corresponding to a chelating capacity of about 1.9 mmol/g in the dry state [39]. The chelating capacity of 2.08 mmol/g of the resulting chelating fabric is close to that of the Amberlite ® IRA743 chelating resin. Among the three surfactants, the addition of POSM resulted in the highest chelating capacity of 2.08 mmol/g. Furthermore, POSM has excellent properties such as low toxicity and good solubility in water. Figure 2b shows the effects of the POSM concentration on the chelating capacity. As expected, the chelating capacity is almost independent of the surfactant concentration. The critical micelle concentration (CMC) of POSM in water at room temperature is about 0.01%, which is much lower than that used in this study. Even a small amount of surfactant (less than 0.5%) can make the fabric well wetted by the solution. Therefore, the surfactant concentration used in this study is sufficient to enhance the affinity of the fabrics in solution [40].
Accelerating the Attachment Reaction by Base and Acid Catalysts
The attachment of NMDG onto the GMA-grafted fabric is an SN 2 substitution reaction between the epoxy rings and secondary amine of NMDG [27,41,42]. Therefore, inorganic bases and acids can act as catalysts to accelerate the reaction. Figure 3a shows that, at a low concentration of 10% NMDG solution, the chelating capacity for the reaction without the catalyst is only 0.13 mmol/g, whereas by adding KOH and HCl it increases to 0.68 and 0.76 mmol/g, respectively. However, when a high concentration of 40% NMDG solution was used, the chelating capacity for the system without the catalyst was 1.74 mmol/g, and it slightly increased to 1.81 and 1.92 mmol/g, respectively, when KOH and HCl were added. Thus, at high NMDG concentrations, the catalyst effects on the reaction are relatively low. HCl is a more effective catalyst than KOH. Figure 3b shows the effect of the HCl concentration on the chelating capacity. Here, 1.0% of the POSM surfactant was added to the reaction solution. As shown in Figure 3b, in the absence of HCl, the chelating capacity is 2.04 mmol/g. When HCl is added, it increases to 2.15-2.20 mmol/g and is almost independent of the HCl concentration. Thus, a low HCl concentration of 0.1 M is sufficient to catalyze the reaction. Scheme 3 shows the mechanism of reaction catalyzed by KOH and HCl. Owing to the strain of the three-membered rings, the epoxy rings on the GMA-grafted fabric are highly reactive. In the SN 2 substitution reaction between the epoxy rings and NMDG, the secondary amine of NMDG attacks the carbon of epoxy rings and forms a new fabric with NMDG moieties [27,33]. The NMDG moiety is usually attached to the less hindered terminal carbon of the epoxy ring due to steric effects. When HCl is used as a catalyst, the oxygen of the epoxy ring is protonated by the acid, thereby increasing the reactivity of NMDG to attack the carbon of the epoxy ring. Conversely, when KOH is the catalyst, the secondary amine of NMDG is activated by the hydroxide ion, which is more conducive for attacking the carbon of epoxy ring [43,44]. Here, the GMA-grafted fabric is a solid material that is insoluble. The attachment reaction of NMDG onto the GMA-grafted fabric is a solid-phase reaction or a heterogeneous reaction. Compared with the general solution reaction, the reaction of the GMA-grafted fabric with NMDG is more complex, and the reaction rate and conversion are relatively low.
Reaction Kinetics
The reaction was confirmed to be enhanced by adding surfactants and catalysts to the reaction solutions. The reaction kinetics in the four conditions of the NMDG aqueous solution, (1) without any additives, (2) adding HCl as a catalyst, (3) adding POSM as a surfactant, and (4) adding both HCl and POSM, are summarized in Figure 4a. For comparison, the kinetics of the traditional attachment reaction in a 1,4-dioxane/water mixed solvent is shown in Figure 4b. Figure 4a shows that there is an initial induction period during which the reaction is quite slow for all four conditions. This is because of the relative hydrophobicity and microporous structure of the fabric, which makes the NMDG diffuse slowly into the GMAgrafted fabric. After the fabric is well wetted by the solution, the NMDG molecules diffuse into the fabric and react with the epoxy rings quickly. The chelating capacity increased dramatically and reached a plateau level.
The rate of the reaction increased when the HCl catalyst and surfactant were added. Without any additives, the reaction plateaus after 5 h, whereas on adding both the catalyst and surfactant, the chelating capacity attains a high level of 2.0 mmol/g within 2 h. The surfactant improves the affinity of the fabric to the solution, and HCl catalyzes the substitution reaction by protonation of the oxygen atoms on the epoxy rings [41]. For comparison, the kinetics of the traditional reaction using a mixed solvent of 1,4-dioxane and water is shown in Figure 4b. The chelating capacity increases linearly with the increasing reaction time and reaches a value of 2.0 mmol/g within 2 h. No induction period was observed for this system. Consequently, the reaction in the aqueous solution containing the catalyst and surfactant is comparable to that in the solution with an organic solvent [36]. Figure 5a shows the kinetics of boron chelation of the chelating fabric in boron aqueous solutions with the initial concentration of 10, 50, and 100 mg/mL. The chelating fabric with high chelating capacity of 2.15 mmol/g was used. The boron chelation is very fast, reaching a steady-state plateau within 20 min. The higher the initial boron concentration of the aqueous solution, the higher is the boron chelating ability obtained by the chelating fabric. The boron chelation of the chelating fabric reaches 17.8 mg/g in the 100 mg/L solution, 12.0 mg/g in the 50 mg/L solution, and further reduced to 4.3 mg/g in the 10 mg/L solution. In the 10 mg/L solution, all the boron is captured by the chelating fabric, while in the 100 mg/L solution, the chelating fabric is saturated to bond with boron. The chelating kinetics of boron chelation is similar to the results reported by Ting et al., where the attachment reaction was performed in a 10% 1,4-dioxane solution [45]. Therefore, the preparation of the chelating fabric without organic solvent is more environmentally friendly, and the resulted chelating fabric can be used to remove trace boron in drinking and irrigation water and effectively reduce boron concentration in wastewater. Figure 5b shows the relationship between the boron chelation of the chelating fabric and the boron concentration of the remaining solution at 25 • C, recorded at a chelation time of 24 h, which is sufficient to establish an equilibrium state. For a low equilibrium of boron chelation of 4.3 mg/g, the equilibrium boron concentration in the remaining solution is too low to be accurately detected, indicating that the chelating fabrics can remove trace boron from water. In contrast, in the remaining solution with a boron concentration higher than 70 mg/L, a high boron chelating ability of 18.3 mg/g or 1.69 mmol/g is reached.
Boron Chelation in Aqueous Solution
Considering the NMDG density (2.15 mmol/g) of the chelating fabric, it can be concluded that about 50% of NMDG moieties were bonded with boron to form the mono-chelated structures and the remaining 50% were bonded in the form of bis-chelated structures (Scheme 2) [46,47].
FTIR Characterization
FTIR was used for characterization of the chelating fabrics before and after the boron chelation, the corresponding GMA-grafted fabric, and the starting nonwoven fabric. Figure 6 shows that the nonwoven fabric presents a simple spectrum because of the olefine chain of polyethylene. After grafting, the spectrum of the GMA-grafted fabric becomes complex and presents a strong peak around 1721 cm −1 assigned to the C=O structure, and three peaks at 986, 903, and 839 cm −1 assigned to the epoxy rings. After the attachment reaction with NMDG, very broad and strong peaks around 3324 cm −1 (assigned to the O-H bond) and 1053 cm −1 (assigned to C-O and C-N bonds) appear in the spectrum of the resulting chelating fabric. These results indicate that the GMA was grafted onto the fabric, and the chelating fabric was obtained by the attachment reaction of NMDG to the grafted fabric. The boron-chelated sample was obtained by immersing the chelating fabric (chelating capacity, 2.15 mmol/g) in concentrated boron aqueous solution for 30 min, followed by drying in oven at 60 • C for 24 h. A new peak around 930 cm −1 , assigned to asymmetric B-O stretching, can be observed on the curve of boron-chelated sample (Figure 6d) [22,48].
Stability of the Chelating Fabric
The chelating fabric is expected to be used repeatedly as a commercial chelating resin. Therefore, the chelating fabric should possess regeneration ability and high mechanical/chemical stability [49]. The regeneration of the chelating fabric includes HCl elution, KOH neutralization, and full water washing. As shown in Figure 7, 10 cycles of chelation-elution operations were performed, and the amount of boron chelated and eluted was determined. As shown in Figure 7, the amount of the chelated boron on the new chelating fabric at the first cycle was 0.23 mg, increased to more than 0.31 mg after the second cycle, and then remained at a high level. The initial low chelating is due to the direct use of the dry chelating fabric without any pretreatment, where the boron diffusion is limited because the fabric is incompletely swollen. After the first cycle, the fabric is well wetted by the solution and the boron chelation is improved, resulting in a stable and high boron chelation.
In each chelation-elution cycle, the eluted boron is almost equal to that of the chelated boron. This indicates that the chelated boron can be completely eluted by the 0.1 M HCl acid. According to the reaction shown in Scheme 1, Equation (3), at a low proton concentration, the reaction of boric acid with vicinal diol tends to form boron ester, while at a high proton concentration, boric acid tends to get released. During the elution of boric acid, the amine on the chelating fabric reacts with HCl. Therefore, in addition to water washing, alkaline washing is needed to convert the amine [13].
After 10 cycles of chelation and elution, the chelating fabric was dried in an oven at 60 • C for 24 h. It was found that the weight of the chelating fabric dropped from 55.2 to 53.1 mg after the ten-cycle operations. This may be due to the strong shaking during the chelation-elution operations, which causes the fiber and the graft layer to separate from the fabric. Conversely, the chelating fabric was found to be very stable in water, 0.1 M HCl, and 0.1 M KOH solutions at 80 • C for 72 h, and there was no change in weight under the above treatments. Therefore, the chelating fabric with high boron-chelating ability can be durable in acid elution solution and alkali washing solution at high temperature, and can be reused to remove boron from water.
Conclusions
A chelating fabric was successfully prepared by radiation grafting GMA onto a nonwoven fabric, followed by an attachment reaction with NMDG using an organic solvent-free process. Addition of surfactant enhances the affinity of the fabric to the solution, while acid catalyzes the substitution reaction. Both the reaction rate and the conversion of epoxy rings to NMDG moieties of the reaction in aqueous solution are comparable to those utilizing organic solution.
The boron chelation of the resulting chelating fabric in water is very fast, reaching steady-state chelation within 20 min. The chelated boron can be quantitatively eluted with 0.1 M HCl solution within 5 min. The boron chelation reaches a high level of 18.3 mg/g in the concentrated boron aqueous solution. Furthermore, the chelating fabric is chemically stable in acid and alkali solutions, showing good durability and reusability for at least 10 cycles of chelation-elution operations. Therefore, it can be concluded that the chelating fabric is promising for boron removal from water. In future work, we will further optimize the process for the mass production of the chelating fabric, and investigate the cost feasibility of actual use in industry. | 2021-04-12T17:21:56.516Z | 2021-04-01T00:00:00.000 | {
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214347493 | pes2o/s2orc | v3-fos-license | EFFECT OF OIL FLUCTUATION ON STOCK MARKET RETURN: AN EMPIRICAL STUDY FROM INDIA
This paper examined the impact of oil price on Indian stock market. Data used for the study are monthly datasets from the period January 2000 to November 2018 obtained from Bombay Stock Exchange (BSE) and World Bank. BSE index is taken as dependent variable whereas oil price, inflation, exchange rate and real interest rate taken as independent variable. Augmented Dickey-Fuller (ADF) unit root test is used to make it certain that variables should be stationary as most of the economic data are non-stationary in nature. The Auto Regressive Distributed Lag (ARDL) model is used here and bound testing test method to cointegration is employed to test for long-run association among our time series dataset. Our finding shows that oil price changes have a significant as well as positive impact of Indian stock market return in both long run and short run. Impact of inflation and real interest rate have negative relationship with Indian stock market but insignificant for long run. Log of exchange rate have positive relation with Indian stock market, but it is also insignificant.
INTRODUCTION
Oil is considered the world's dominant fuel, and its global market is the most important of the world energy markets (BP Statistical Review of the World Energy, 2016). Its prices have shown great instability in recent times, caused by economic, financial and geopolitical factors. The variations in its prices have a great impact on the overall economy, inflation, the exchange rate, corporate earnings, and other economic variables due to its extensive use as a crucial input in the production process, and as a final consumption good (Muhtaseb andAssaf, 2017and Aggarwal andSaqib, 2017). A considerable volume of work has emerged examining the connections between oil price shocks and stock returns and between oil price shocks and stock market volatility (Kang et al., 2015;Lee et al., 2017;Aggarwal and Saqib, 2017;Smyth and Narayan, 2018). It has been seen that fluctuation of prices of oil has dominant effect on economy of the world. But it is still not clear how much fluctuation of oil price are affecting the market.
Hence the objective in this paper is to investigate how structural oil price shocks drive the contemporaneous stock market return and volatility relationship.
LITERATURE REVIEW
In 1970's the only thing we really understood about the oil market was its role in contributing to recessions in the United States (Hamilton, 1983). But post that decade, large amount of literature has emerged that studies the effect of oil price changes on a range of macroeconomic variables (Hamilton, 2003;Cunado and Perez de Garcia, 2005;Bachmeier, 2008;Chiu, 2011a, 2011b;Lee et al., 2017;Aggarwal and Saqib, 2017;Smyth and Narayan, research (Narayan and Narayan, 2014;Narayan et al, 2017). Putting forward the theoretical validation proffered by Jones and Kaul (1996), most of the literature has sought to test the cash flow hypothesis, which states that asset values are determined by expected discounted cash flows (Fisher, 1930 andWilliams, 1938). The cash flow hypothesis suggests that there could be a negative or positive relationship between oil prices and stock returns. There are two approaches for the negative relationship. First, oil being a major input for most firms, higher oil prices increase the cost of production, reducing future cash flows, earnings and dividends and, hence, stock returns. Second, higher oil prices can lead to an overestimation of expected inflation and higher nominal interest rates. Because interest rates are used to discount expected future cash flows, this will depress earnings, dividends and, hence, stock returns (Kling, 1985;Jones and Kaul, 1996;Gjerde and Saettem, 1999;Sadorsky, 2008;Papapetrou, 2001;Basher and Sadorsky, 2006;Driesprong et al., 2008;Park and Ratti, 2008;Henriques and Sadorsky, 2008;Chen, 2009;Filis, 2010;Arouri et al., 2011;Basher et al., 2012;Diaz and De Gracia, 2017;Anyalechi et al., 2019). However, some studies have found a positive relationship between the responses of stock returns to oil prices; higher oil prices, resulting from an unanticipated global expansion, has a positive effect on stock returns. Oil price volatility can influence the effect of sensitivity of changes in oil prices on the risk premium component of the discount rate and on cash flow through demand side consequences (Narayan and Narayan, 2010;Zhu et al., 2011;Zhu et al., 2014;Silvapulle et al., 2017).
Another possible reason for a positive association between oil prices and stock returns is, as Kollias et al. (2013. p. 744) note, "investors may well associate increasing oil prices with a booming economy. Thus, higher oil prices could reflect stronger business performance and the concomitant impact on stock markets." Along these lines, Chen et al. (2017) suggest that oil price volatility and stock market momentum are positively correlated. Using China as a case study, they suggest that this relationship is driven by time-varying investor sentiment, in which investors respond to oil return volatility associated with uncertainty through putting upward demand pressure on winner stocks. Using a two-stage Markov-switching approach, Zhu et al. (2017) also finds that the relative importance of demand and supply shocks varies between low and high-volatility regimes. However, Ciner (2013) argues the positive correlation can be surprisingly persistent. As Ciner (2013. p. 13) writes: "oil price increases do not always suggest negative stock returns. In fact, prolonged periods of joint increases in oil and stock markets may be observed" (emphasis ours). Narayan and Narayan (2010) modeled the impact of oil prices on Vietnam's stock prices using daily data for the period 2000-2008 and include the nominal exchange rate as an additional determinant of stock prices. The results showed that stock prices, oil prices and nominal exchange rates are cointegrated, and oil prices have a positive and statistically significant impact on stock prices. This result is inconsistent with theoretical expectations. From the emerging market standpoint, Basher and Sadorsky (2006); Henriques and Sadorsky (2008) and Arouri et al. (2011) evaluated the impact of oil price changes on a large set of emerging stock market returns. The approach adopted in the paper was an international multi-factor model that allows for both unconditional and conditional risk factors to determine the relationship between oil price risk and emerging stock market returns. In general, the findings revealed strong evidence that oil price risk impacts stock price returns in emerging markets. Further, Diaz and De Gracia (2017) examined the impact of oil price shocks on stock returns of four oil and gas corporations listed on NYSE over the period January 1974-December 2015. The novelty evidence supports a significant positive impact of oil price shocks on stock returns in the short run. Also, Anyalechi et al., 2019 revealed that changes in oil price have had positive but insignificant impact on stock market returns both in the long-run and the short-run. Impact of inflation was positive and insignificant in the long-run but positively significant in the short-run. Real interest rate and log of exchange rate exerted negative influence on the stock market returns, where the short-run effect of real interest rate was significant, the long-run impact was found to be insignificant.
Thus, it can be concluded that oil is one of the important commodities of the world. It has been seen that fluctuation of prices of oil has dominant effect on economy of the world. But it is still not clear, how much fluctuation of oil price are affecting the market. Hence this paper is examining the effect of oil price on Indian stock market return, where other variable like inflation, exchange rate and real interest rate are also included to refine our research.
DATA AND METHODOLOGY
Indian stock market data is considered for our study. Since Bombay stock exchange (BSE) index is the true representative of the Indian economy, so BSE Index is taken as dependent variable, where oil price, inflation, exchange rate and real interest rate are taken as independent variables. Data for this study are monthly data for the period January 2000-November 2018 which consist of 227 observation. BSE index (stock market) data were obtained from BSE and oil price was obtained from World Bank. Our major study focused on oil price (brunt crude index) and stock market returns. Inflation rate, exchange rate (USD/INR rate) and real interest rate are other independent variables, which are sourced from Reserve Bank of India and Bloomberg.
Augmented Dickey-Fuller (ADF) unit root test is used to make it certain that variables should be stationary as most of the economic data are non-stationary in nature. The auto regressive distributed lag (ARDL) model is used here and bound testing test method to cointegration is employed to test for long-run association among our time series dataset.
The baseline equation function for our study expressed as below: Here t denotes time, BSERET=BSE index return (Indian stock market return), CRUDRET=Brent crude return, INFLATION=Inflation in India, RINTRATE=Real interest rate, LNEXCHRATE=Log of exchange rate (USD/INR) and ε=Error term.
We can represent equation 1 in error correction model in line with Engle and Granger (1987), where this equation can be modified and allow short-run dynamic adjustment.
Here m i represent the number of lags where represents differencing operator. ε t−1 is the lagged error after one period and derived from the residuals of equation 1. Engle and Granger (1987) suggested that variables must be I(1) and error term I(0) for co-integration relationship. Pesaran et al. (2001) suggested that if the variables are either I(1) or I(0) then equation 2 can be represented as in equation 3. This approach is called as ARDL (Autoregressive Distributed lag) and replaces ε t−1 in equation 2.
Equation 3 is represented as below: The equation 3 is the representation of ARDL to co-integration which are widely employed in the long run relations when data generation process underlying the time series variables are integrated of order one or order zero.
We can also formulate an "unrestricted" error-correction model (ECM). This will be a particular type of ARDL model and perform a "Bounds Test" to see if there is evidence of a long-run relationship between the variables. If the outcome is positive, we can estimate a long-run "levels model", as well as a separate "restricted" ECM where lagged level variables in equation 3 can be replaced by ECT t-1 in Equation 4.
RESULTS AND DISCUSSIONS
The ADF test results has been shown in Table 1. It clearly shows that the null hypothesis of unit root is rejected in the case of INFLATION, RINTRET and LNEXCHRATE variables in the first difference at 1% level of significance and these variables are integrated of order one [I(1)]. Variables BSERET and CRUDERET are stationary and rejected the null hypothesis at 1% level of significance but these variables are integrated at order zero [I(0)].
As our variables are integrated at order I(0) and I(1),therefore our model ARDL model will be good estimator for finding the longrun association among our variables.
Model Selection
The Akaike information criterion (AIC) is applied here in process of selection of best model and optimum number of lags in the ARDL model. Among various model, we selected ARDL (1, 1, 1, 1, 1) based on AIC.
Bound Testing for Long Relationship
Our result of ARDL long-run form has been presented in Table 2. The value of F-statistic 55.33583 from F-bounds tests and the value of t-statistic value = −16.38036 from t-bounds tests indicates that these values are significant at 1% level and it clearly show that there is a long relation exist between the variables. So, our interest of variables BSE index and oil price have long run relationship. The t-statistics on the coefficient of the lagged error-correction indicates that there is Granger-causality.
Here we applied the cumulative sum (CUSUM) test to analyze the stability of selected ARDL model specifications. This Figure 1 shows that model is stable.
Regression Results
The ARDL long-run form is presented in Table 3. It shows that change in oil price are positively related to stock market return (BSERET) and significant. Other variables like inflation and real interest rate have negative influence on the regress and whereas log of exchange rate have positive influence, but all these variables are not significant.
But the results indicate that there are variables that are jointly significant and long relationship exist between the variables and there is cointegration between the variables.
Here, Table 4 (error correction model) shows that oil price (CRUDERET) is positively related to stock market return. Whereas inflation, real interest rate and log of exchange rate are negatively related and also significant. The coefficient of error term is negative and statistically highly significant at 1 percent level. So, there is cointegration between oil price and stock market return (BSERET). The magnitude of error term indicates that nearly 96% of any disequilibrium between crude oil and Indian stock market is corrected within 1 month. Table 5 shows the Breusch-Godfrey serial LM test data. The results indicate that our model has no auto correlation problems.
Correlation Test
We applied the cumulative sum (CUSUM) test to analyze the stability which is shown in below Figure 2. The model seems stable and correctly specified as it didn't exceed the bounds of the 5% level of significance.
CONCLUSION
The current empirical study analyses the impact of oil price on Indian stock market. We also took some other macro variable like interest rate, exchange rate and inflation for our study. But major focus emphasized on the Indian stock market return fluctuation due to movement in oil price.
Our finding shows that oil price changes have a significant as well as positive impact of Indian stock market return in both long run and short run. Impact of inflation and real interest rate have negative relationship with Indian stock market but insignificant for long run. Log of exchange rate have positive relation with Indian stock market, but it is also insignificant.
The bound test result showed that long run relationships exist between the oil price and stock market returns during the period under study. The magnitude of error term indicates that 95.8% of any disequilibrium between crude oil and Indian stock market is corrected within 1 month. | 2020-01-30T09:05:03.963Z | 2020-01-23T00:00:00.000 | {
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224946432 | pes2o/s2orc | v3-fos-license | COVID-19 and haematology services in a cancer centre from a middle-income country: adapting service delivery, balancing the known and unknown during the pandemic
The COVID-19 pandemic has caused major disruptions in multiple spheres of healthcare delivery in the world. Developing nations have had to tackle this unanticipated crisis in the midst of various other healthcare delivery issues and resource constraints. As a tertiary level cancer care provider located in an eastern Indian city, a COVID-19 hotspot, we share our experience from the perspective of haematology and haematopoietic stem cell transplantation (HSCT) services. The primary challenges related to infection control included infection screening and decreasing exposure among patients and healthcare workers. Logistic challenges include maintaining essential patient care services, personnel redeployment, blood bank inventory constraints and maintaining the supply chain for a continuum of care. Clinical management challenges were dealt with by rationalising treatment delivery by modification of treatment regimens, risk-based deferral of HSCT, management of COVID-19 in patients, and staggering the follow-up schedules in survivors and those on maintenance therapies, among other strategies. These challenges were compounded by the restrictions imposed by a countrywide lockdown in the initial period of the pandemic, which also affected the socio-economic aspects of treatment delivery. As a training institution, this period also impacted academics and research activities. This overview details our response to these challenges during the COVID-19 pandemic, which has many unknowns.
Institutional challenges
At the outset, problems were created by logistic challenges which emerged due to the strict countrywide lockdown imposed by the government from 23 March 2020 [2]. This severely affected the supply chain of food essentials, drugs, laboratory reagents, radio-diagnostic agents, maintenance requirements and personal protective equipment (PPE) in the initial days. Infection prevention in a pandemic has a two-fold challenge of preventing infection in patients visiting the hospital, and among healthcare providers. Our hospital has an active infection control policy which primarily focuses on preventing hospital acquired infections. The challenge that a pandemic pose is that healthcare settings act as potential amplifiers in spreading an infection which is primarily community acquired. This hospital is exclusively for cancer (and related conditions) with most of its patients being severely immunocompromised. This increases the risk of transmission to care providers as well, as immunocompromised hosts are more likely to have a high viral load. The lack of adequate information in the initial phase, of the actual rate of spread in the community, was also affecting the ability to make policy decisions.
Maintaining safe distancing was a challenge with an outpatient division (OPD) footfall of 1,000-1,200 cancer patients per day, accompanied by attendants. A screening facility for patients and attendants to look for symptoms and fever was necessary and crowd control was essential especially since an average patient is accompanied by two attendants (at least). Protecting healthcare workers (median age at our institution: 27 years (18.8-70.4 years)) anxious by the fear of a yet unknown virus, maintaining staff morale, facilitating employee attendance and ensuring adequate coverage of services, were among many challenges the administration had to grapple with it. Exposing the entire workforce to this potential pandemic would have deleterious consequences, including maintenance of hospital services for patients. There were Government guidelines in this regard, by mid-April 2020 [10].
Challenges in the management of COVID-19 patients with haematological cancers
The SARS-CoV-2 COVID-19 infection was expected to have deleterious outcomes among patients with haematological malignancies and hematopoietic stem cell transplant recipients. The Haematology unit registers 2,500 to 2,700, and the Paediatric division 500 to 600 new patients annually. The average daily in-patient strength of the two units, was 80 in the month before lockdown and was scaled down to less than 30. This was done to provide a 'holding area' for COVID suspects, isolation rooms for quarantining clinically stable COVID-19 infected patients and healthcare workers. The availability of high-dependency unit and intensive care unit beds were limited, and this was further complicated by the need to set aside a reserve number in the eventuality of a large outbreak. There was a major shortfall in volunteer blood donors coming to the hospital which led to a significant stretch in the resources of the blood bank and affected transfusion services across the hospital. In a Haematology service, decisions on intensive chemotherapy including admissions to a bone marrow transplant (BMT) facility also depend on blood product availability, and patient families had to provide donors as replacement blood banking. Being a not-for-profit private trust medical centre, we are dependent on funding agencies to provide for many of our patients undergoing curative treatments and who most often require financial assistance for intensive chemotherapy and supportive care. Offices of government agencies, non-governmental organisations and corporate social responsibility liaisons were either closed or constrained (with partial staffing), resulting in cash flow challenges initially, for patient funding and treatment delivery. Psychological comorbidities are common in any chronic medical illnesses including cancer. For many patients visiting our hospital, fear of cancer progression mattered more significantly than fear of COVID-19 [11]. The patients were seen to be additionally distressed by the constraints imposed by the lockdown, during this pandemic.
The therapy for COVID-19 has no standard of care and there are conflicting reports on the efficacy of drugs like hydroxychloroquine (HCQ) monotherapy (including prevention), HCQ combinations, Remdesivir, dexamethasone and many others in treatment [12][13][14][15]. Availability of generic drugs is not a compelling constraint here, as India has one of the largest production facilities for manufacturing generics [16]. Our challenge of formulating a treatment policy was complicated by the presence of multiple treatment guidelines and recommendations from several professional organisations, societies and Government authorities regarding COVID-19 infection, and the management of cancers during the COVID-19 pandemic [17].
Institutional response
The problem posed by the COVID-19 pandemic was a challenge to the living memory of most personnel at our institution. The hospital administration reviewed best practices from published literature, cross consultation and experience from institutional memory [18]. A central command centre was established and made functional from 17 March 2020 in the hospital led by the chief executive and senior medical administrator. The command centre and COVID management team included all verticals of the administration (including Finance, Human resources, Quality, Customer care, engineering, staff health, security, etc.), infection control, laboratory services, imaging services, clinical staff, nursing and para-medical lead personnel, etc. In this unusual scenario, the clinical leadership consisting of all subspecialties of cancer care and diagnosis decided to organise themselves into a team led by a senior oncology physician, by 23 March 2020. The team's mandate was to distil current information and draft (or modify) clinical pathways for the care of cancer patients and healthcare workers (HCW) who would be COVID-19 infection suspected or confirmed. This team was given the acronym TMC CLINCH team.
The COVID management team inclusive of the command centre, infection control team and CLINCH team formulated the administrative, diagnostics and infection control, and clinical pathway standard operating procedures (SOPs). Isolation facilities and levels of isolation were identified and earmarked, a physical space for the command centre was commissioned; and a hospital mock drill was conducted in 48 hours. Training of staff members began immediately once the draft administrative protocols were prepared by 27 March 2020. The clinical pathways and management protocols were formulated and issued in a week. Considering the dynamic situation in the community and rapidly changing directives from various agencies, the SOPs needed frequent modifications. They are currently in version 3 as this paper is being finalised.
The shortage of PPE in the initial phase of the outbreak took a couple of weeks to rectify. To overcome the shortage of the full complement of PPE, we had to ration its use to provide for staff at the highest risk of infection, e.g., nurses and doctors involved in the care of suspected or proven COVID-19 patients. Furthermore, the full complement of PPE was provided to staff involved in emergency, isolation and surgeries and other aerosol generating procedures. This challenge was mitigated once production of PPE was ramped up in the country. All patients and relatives were required to wear 3ply mask, N95 mask or cloth masks at all times while inside the hospital. Staff were divided into batches and rotational workforce practices were implemented on a weekly basis, to reduce the risk of a large number of staff falling sick at the same time and disrupt services and limit the use of PPE. At individual departmental levels, the departmental heads (or lead consultants) were advised and empowered to make necessary administrative and work-related changes in order to prepare for the scenario and a potential surge in the number of COVID-19 affected patients and HCW, considering the best and worst situations.
Infection control
In addition to the existing infection control policy in the hospital, we set up protocols to guide patients and staff in preventing COVID-19 transmission. As early as January 2020, the infection control team initiated weekly monitoring of the COVID situation. Patients were screened at the time of entry into the hospital with a standard questionnaire and all deemed at risk for COVID-19 infection were shifted to a 'suspect' designated area, and later to isolation ward suites, for sample collection and testing. Testing kits validated by the National Institute of Virology and approved by Indian Council of Medical Research were procured to start RT-PCR for COVID-19 [19]. This included simultaneous detection of screening E-gene, and confirmatory S or RdRP gene as per the available kit. Patient with either of 1 gene positive were reported as 'Indeterminate' , while Samples showing no PCR amplifications were tried two times before finally reported as 'Inconclusive' . Patients with definitive symptoms or radiological findings as per the screening protocol, presenting to out-patient or emergency services also followed the same pathway. The isolation suites were re-organised to care for patients with intensive care support as well. Confirmed cases continued to be in isolation while others were shifted to the appropriate wards, cared for or discharged. The testing pathways were issued by 4 April 2020; as our centre was designated as a COVID-19 testing centre by the Indian Council of Medical Research (ICMR). Protocols for tracing and assessment of high-risk contacts were. A rapid response code to safely identify and transfer suspects to isolation areas was also implemented simultaneously. Testing criteria and protocols underwent periodic revisions based on directions and advisories from Government and professional health agencies. To ensure testing went on unhindered, staff were redeployed from different clinical and research areas and appraisal of testing kits and advance orders were fast-tracked. The research staff from the haematology departments also volunteered for these services.
Clinical service and treatment policies
In order to understand the impact on services, we reviewed data on patients availing adult and paediatric haematology out-patient, inpatient, day-care and BMT services in our institution between March and May 2020 and compared it with the same time period in 2019, where applicable. The outpatient services saw a significant drop (47%), and the day care (12%) a less significant one. Inpatient bed-occupancy dropped by 50%. The paediatric services functioned unchanged initially. Transfusion medicine services saw a significant drop (n = 2,519 versus 1,877, 25%) in donations and blood products prepared, with consumption remaining more or less the same. Haematological malignancies were categorised into potentially curable, controllable and palliative. Potentially curable leukaemia and high-grade lymphoma continued to receive treatment with a curative intent using dose adjustments for age and myelosuppression. Growth factors as well as supportive care were advised to be used generously during therapy. In patients with early-stage Hodgkin's lymphoma, the baseline and interim PET scan were utilised to minimise the number of chemotherapy cycles required. PET-directed therapy was used to avoid starting intensive BEACOPP regimen upfront for advanced disease. These regimens were used for patients with suboptimal response after the first two to three cycles of standard ABVD therapy. Radiation therapy was avoided and alternative regimens used to reduce repeated hospital visits. In case of relapsed leukaemia and lymphoma, outpatient-based salvage regimens such as GDP were used in place of more intensive ICE/ESHAP/DHAP therapies, where feasible. For indolent and controllable diseases, treatment in the outpatient clinic was encouraged with minimum OPD visits. Oral therapies replaced injectable management wherever possible. Maintenance therapy with rituximab in indolent lymphomas was postponed or temporarily withheld. Patients with multiple myeloma were advised to continue oral and subcutaneously administered therapy at home under supervision of house physicians, and hospital visits were reduced. This practise was in line with guidance from reputed professional societies and consensus statements [20][21][22][23][24][25][26]. All procedures such as high-dose chemotherapy and autologous hematopoietic stem cell procedure were conducted with due thought and postponed where feasible, after discussion within the multi-disciplinary team. Decisions on Allogeneic transplantation were similarly decided depending on the disease risk status, based on recommendations [21]. Among the patients tested or screened for COVID-19 from April 2020 to June 2020, infection was confirmed in 10 and indeterminate/inconclusive in 6, in the adult services. They COVID-19 positive patients were in various phases of remission induction therapies or initial evaluation phase for acute leukaemia (n = 3), aggressive non-Hodgkin lymphomas (n = 4), indolent lymphoma (n = 2) and multiple myeloma (n = 1). Two patients succumbed. Paediatric haematology section did not record any case positivity during this period, and had three indeterminate/ inconclusive reports. As a part of a global effort, our departments are collaborating in the ASH research Collaborative initiative on COVID-19 in patients with haematological disorders and reporting cases to this registry [27]. Adult Haematology, Paediatric Haematology-Oncology and BMT clinical services have a staff strength of about 181 personnel. Across adult and paediatric haematology and BMT services, 52 members of staff were quarantined post exposure, and underwent COVID-19 testing prior to re-joining the clinical services. Two nurses working in the adult haematology service tested positive and asymptomatic, while all others tested negative. At the time of finalising this paper, the departments have reverted to working full hours.
Patient follow-up and continuum of care
Patients continued to have difficulty in reaching the hospital though ambulances and private vehicles carrying patients to hospitals were allowed to ply. To overcome these difficulties at least partially, telephonic consultations, messenger services like WhatsApp® and emails were used for maintaining communication with patients. These services provided a channel for answering patient queries and providing treatment information. Online consultations between patients and clinicians in the institution have been predominantly informal in our institution as there were no clear laws regarding this form of patient interaction in India, for a long time. The medical council of India and ministry of health and family welfare, Government of India on 25 March 2020 issued legal sanction to online consultations by issuing guidelines for the same [28]. This pandemic, social distancing norms and legal sanction re-energised our institution to begin secure formal online and teleconsultations for patients from 18 June 2020.
Academic and research activities
The social distancing norms within the hospital meant cancelling most of our formal academic activities, including multidisciplinary meetings. All scheduled conferences planned to be conducted at the hospital were cancelled. Staff involved in research activities were advised to work from home, and recruitment of patients to clinical trials dropped consistent with the drop in hospital footfall. From no recruitments in the first month of the lockdown, trial screenings restarted from the second month and have recorded recruitments at 100% pre-lockdown levels. Active biobanking was withheld. Interactions with trial sponsors and research organisations shifted to internet web-based platforms. A revised online format of institutional review board meetings was notified and conducted at the time of manuscript preparation, and the departments had presented three studies on the new online platform. By mid-May 2020, academic activities and multi-disciplinary meetings in the haematology department were restarted through a popular internet platform for web-based team activities. This has been running successfully, as this paper was being finalised. We have two accredited and three institutional post graduate training programmes in adult and paediatric haematology which undergo periodic evaluations. As these are time bound fellowship programmes, we did not create disruption in the assessment schedules and conducted these assessments while practicing social distancing norms.
Conclusion
Faced with the threat of COVID-19, our administration first made local changes, put their acts into practice at a record speed to provide all logistics for the functioning of the hospital, staff protection and most importantly patient care. The first step was to shed our fear which initially gripped the staff. Care of cancer patients with COVID-19 needed special nursing and medical care, and constant revisions of the CLINCH document which provided information on PPE usage, screening of patients and their relatives, isolation, testing of suspects, management of the primary malignancy as well as SARS-CoV-2 in patients, treatment of the staff affected by COVID-19 and managing the dead. The care of haematological malignancies at our centre has been altered to mitigate the risks of COVID-19 in our vulnerable patient population and also to balance resource utilisation at this economically difficult time. Common themes include to continue the curative including allogenic stem cell transplants with all supportive care, use oral and outpatient medications in controllable disease, defer treatments known to be associated with high risk of viral infections in maintenance phases and increase Telemedicine facilities for patients on 'wait and watch' policy for low-grade malignancies as well as survivors on follow up, wherever possible our recommendations were evidence based and represented a consensus opinion. | 2020-09-25T09:28:00.262Z | 2020-09-24T00:00:00.000 | {
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129309961 | pes2o/s2orc | v3-fos-license | Increased loss of continental crust during supercontinent amalgamation
The volume of Earth's continental crust depends on the rate of addition of continental crust from the mantle compared to the rate of continental loss back to the mantle, which at present is roughly balanced. Models for the growth rate of continental crust vary, with isotope data suggesting various episodes of increased growth rate throughout Earth's history; these episodes have been correlated with the supercontinent cycle, but may be a consequence of preferential preservation of continental crust during these cycles. The global balance between addition and loss of continental crust is controlled by: 1) the extent of internal orogens versus exterior orogens, with the latter favouring continental addition, and 2) the balance between exterior orogens in retreating mode versus those in advancing mode, with the latter favouring continental loss. A greater balance of continental addition versus loss should exist during supercontinent break-up, due to a high magmatic flux in retreating accretionary orogens, whereas the amalgamation of supercontinents should involve increased continental loss due to increased sediment subduction and tectonic erosion. Zircon U–Pb and Hf isotopes provide insight to models of crustal growth rate since they sample the continental crust at their time of formation. Using the distribution of data within eHf(t)-time space of a global zircon database, it is demonstrated that the data are in accord with the concept of increased continental loss during supercontinent amalgamation. Periods featuring increased continental addition relative to continental loss, and hence increased continental crust growth rate, occur at ~ 1.7–1.2 Ga, ~ 0.85–0.75 Ga, and ~ 0.45–0.35 Ga, and follow the formation of the Columbia (Nuna), Rodinia and Gondwana supercontinents respectively. Distinct increases in continental loss compared to continental addition, i.e. decreased continental growth rate, occur at ~ 1.0–0.9 Ga, and ~ 0.6–0.55 Ga, correlating with the periods of Rodinia and Gondwana amalgamation respectively. Formation of Pangea by introversion rather than extroversion, means that continental addition in exterior orogens was concurrent with continental loss in interior orogens; a similar process may have been responsible for formation of the Columbia supercontinent. Peaks in the compilation of U–Pb zircon ages correlate with the timing of supercontinent amalgamation, and are likely to be a consequence of preferential preservation of continental crust during this part of the supercontinent cycle.
Introduction
The growth rate of Earth's continental crust remains a topic of much debate; two models that are in accord with the isotope record are: 1) that the average growth rate of continental crust has decreased since the Archaean (Belousova et al., 2010;Hawkesworth et al., 2010), and 2) that continental growth rate has episodically increased (e.g. Condie, 1998Condie, , 2004Kemp et al., 2006;Iizuka et al., 2010). Recently it has been established that apparent episodic increases in growth rate are probably a result of preservation bias during the supercontinent cycle Condie et al., 2011;Lancaster et al., 2011). The apparent episodic nature to continental growth is indicated by compilations of zircon U-Pb and Hf isotope data from individual continents (Rino et al., 2004(Rino et al., , 2008Iizuka et al., 2005Iizuka et al., , 2010Kemp et al., 2006;Wang et al., 2009Wang et al., , 2011Yang et al., 2009). A recent compilation of zircon-Hf data with near-global continental coverage reveals a more protracted history of continental growth during the last 3,000,000,000 years (Belousova et al., 2010); however, peaks in the U-Pb age data indicate a possible role for bias during the supercontinent cycle with preferential preservation of continental crust during supercontinent amalgamation (Condie et al., 2011;Lancaster et al., 2011). Overlap between peaks in both U-Pb ages and Hf model ages have been shown to overlap with periods of supercontinent amalgamation (e.g. Kemp et al., 2006;Iizuka et al., 2010), which has been used in support of a model whereby increased continental growth occurs during supercontinent amalgamation (Condie, 1998;Rino et al., 2004).
Addition of continental crust largely occurs via magmatism driven by subduction in accretionary orogens, with addition from plumes being comparatively minor; continental destruction occurs via subduction of sediments, subduction erosion, and lower crustal delamination (e.g. Clift et al., 2009a,b;Keppie et al., 2009Keppie et al., , 2011Hawkesworth et al., 2010;Stern and Scholl, 2010;Stern, 2011). The growth rate of continental crust relies on the balance between addition and loss of continental crust; a concept referred to as Yin and Yang by Stern and Scholl (2010). Mass balance calculations indicate that at present-day, the amount of loss is balanced by that of continental addition (Clift and Vannucchi, 2004;von Huene, 2007, 2009;Clift et al., 2009a,b), indicating that the crustal volume is not presently increasing; although a more recent estimation suggests that continental loss outweighs continental addition, implying that the continental crust is currently shrinking (Stern, 2011). The balance between continental addition and loss, and thus continental growth rate, is not a steady-state process, and is controlled by the supercontinent cycle. Continental crust growth rate should increase during supercontinent break-up due to an increased magmatic flux, and conversely, this growth rate should decrease during supercontinent amalgamation due to increased sediment-subduction and tectonic erosion (Santosh, 2010;Stern and Scholl, 2010;Yoshida and Santosh, 2011). To evaluate the dichotomy between previous models of episodic crustal growth and the concept of increased continental growth during supercontinent break-up, this study applies a novel approach to the analysis of a recently published global zircon-Hf database; rather than using estimated model ages, the distribution of Hf data within time-intervals is calculated as an average, and it is the trend of this average that is examined with respect to supercontinent cycles.
The zircon record
Detrital zircons, especially those from large rivers, provide a means of sampling continental crust to obtain its age and isotopic composition (e.g. Bodet and Schärer, 2000;Iizuka et al., 2005Iizuka et al., , 2010Wang et al., 2009;2011). A recent compilation of~15,000 analyses, including those from major rivers on most continents, provides a dataset of igneous and detrital zircon data with near-global continental coverage (Belousova et al., 2010); although this dataset has an Australian bias, it has been demonstrated that this doesn't significantly change the general distribution of data (Belousova et al., 2010;Condie et al., 2011). These data reveal that juvenile continental crust has been forming continuously since the Hadaean, and that 70% of the presentday volume of continental crust has existed since 2.5 Ga (Belousova et al., 2010;Hawkesworth et al., 2010).
Previous models for continental growth typically rely on calculated depleted mantle Nd and Hf model ages; however, rather than being geological events, these are typically mixed ages that average different components within a magma, and for Hf-in-zircon rely on an estimation of the magma's Lu/Hf ratio. Combining zircon-Hf analyses with oxygen isotopes allows for a better estimation of Lu/Hf (Wang et al., , 2011, and can also be used to screen out non-mantle signatures that derive from sedimentary recycling (Kemp et al., 2006;Lancaster et al., 2011). However, it has been demonstrated that recent magmas in young arcs can have a large spread in Hf model ages even when exhibiting an oxygen isotope signature of mantle-derived zircon (Nebel et al., 2011). In this study, rather than using model ages, the relative distribution of Hf data within individual time intervals is examined, i.e. the extent to which the population of Hf data has departed from the depleted mantle curve within each time interval. Fig. 1 shows a compilation of global Hf data (data from Belousova et al., 2010 andLancaster et al., 2011) plotted as εHf (t) versus time, with the mean, median and interquartile range shown and based on 50 m.y. intervals and a 100 m.y. moving window. The mean and median are similar throughout, only in the Phanerozoic where there is greatest variation, does the median depart from the mean, indicating only moderate skew to the population. Over time the depleted mantle curve increases into positive εHf (t) space, and the minimum value of εHf (t) (since a magma should not have a model age older than~4.56 Ga) decreases into negative εHf (t) space; thus, the possible range in εHf (t) for any one time interval will increase over time. For this reason, the mean curve is recalculated so that it represents the percentage of depleted mantle input within an available range, with this range being based on the depleted mantle value plus two epsilon units for the maximum, and an evolution line from 4.56 Ga using a crustal Lu/Hf value of 0.015 for the minimum (mantle-input curve in Fig. 1); this mantle input curve is plotted with the mean of the Hf data as zero. The population density in the Palaeoarchaean and Hadean is suggested to be too small and biased by too few samples to represent a reliable average of global continental crust, thus, the mantle-input curve is only shown up to 3300 Ma.
An oscillating growth curve
The Hf data is used as a proxy for continental growth in the following ways: positive excursions (from the mean) indicate increased mantle input and continental addition, negative excursions indicate decreased mantle input and increased continental loss. The population of Hf data is limited in the Hadaean and Palaeoarchaean, but from~4.5 to~2.0 Ga, can be described as a broad triangular swath that is rather symmetrical about the CHUR evolution. Although the median and mean trends show variation during this period, the record for Mesoarchaean and older is likely biased by sampling, with most samples coming from only a few localities; thus, no significant interpretation of the shape of the mantleinput curve is made for this period. After~2.0 Ga the record is more varied with significant excursions of the data into positive and negative εHf
Controls on continental growth rate
Addition and loss of continental material are controlled by both the type of orogen, and by the geodynamic regime within the orogen. Internal orogens, i.e. continental collision belts such as the Alpine-Himalayan-Indonesian system, feature both addition and loss of continental crust, but with recycling of pre-existing crust probably more dominant than addition of juvenile crust. This is represented in the zircon Hf record as deviation to both positive and negative εHf values that progressively fan out over time (Fig. 2), due to lower crust and lithospheric mantle being replaced by continental lithosphere from accreting continents (Collins et al., 2011). External orogens, i.e. accretionary or circum-Pacific orogens, feature a trend to positive εHf values from their onset until termination of the orogenic system; this is related to removal of lower crust and lithospheric mantle by newly formed crust which melts to produce rocks with juvenile εHf signatures (Collins et al., 2011). The pattern of zircon-Hf data from across the globe at any one time can therefore be thought of as representing the balance between crustal production in internal orogens versus that in external orogens. Internal orogens will outbalance external orogens during periods of supercontinent amalgamation, whereas external orogens will dominate when the continents are dispersed. The balance between internal versus external orogens during the supercontinent cycle thus provides a first order control on the continental crust growth rate. Accretionary (i.e. external) orogens can be divided into retreating-or advancing-types, based on the relative movement of the subduction trench relative to the over-riding plate, and will typically oscillate over the timespan of a subduction-zone margin (e.g. Collins, 2002). Isotope evidence from arc magmas in the Tasmanides indicate that retreating-mode favours increased mantle input, i.e. continental crust addition, whereas advancing-mode features decreased mantle input Phillips et al., 2011); this is due to a variety of factors, such as attenuated thinner crust in retreating mode, allowing greater influx of mantle material; and in advancing mode, thicker crust, increased sediment input and increased subduction erosion leading to increased crustal contamination. Examples of this relationship have been documented for Andean and Japanese subduction zones using various isotopic systems (Takagi, 2004;Kay et al., 2005;Stern et al., 2011); in all cases the isotope data point to an increased crustal signature when the volume of subducted continental material is increased. Since only b10 to 20% of subducted material is recycled back into arc magmas (Clift et al., 2009a,b;Stern, 2011), deviation from depleted mantle values for isotopes within arc magmas can be regarded not only as continental recycling, but as a proxy for continental loss. The balance between accretionary orogens in retreating mode versus those in advancing mode provides a second order control on continental growth rate.
Controls on preservation of continental crust
It has been argued that the record of preserved continental crust is biased by variation in preservation during the supercontinent cycle Condie et al., 2011;Lancaster et al., 2011). Hawkesworth et al. (2009) suggest that crust formed preceding supercontinent formation has a moderate preservation potential, crust formed during supercontinent break-up has a weak preservation potential, and crust formed during supercontinent amalgamation has the greatest preservation potential; this is due to capturing of crust during continent-continent collisions and stabilisation within cratons. This process may be responsible for peaks in U-Pb age histograms, i.e. an apparent episodic nature to continental growth (e.g. Rino et al., 2008; (Griffin et al., 2000) and a crustal evolution line with Lu/Hf 0.015 from 4.56 Ga. The data are recalculated as a percentage mantle input (see text for explanation) shown as the mean (thick black line), median(grey line), and interquartile range (thin black lines). Positive and negative excursions of this curve can be thought of as increased continental addition and continental loss respectively. The zircon data are also plotted as a histogram of U-Pb ages. Periods of supercontinent assembly are shown by the paler grey bars (from Campbell and Allen, 2008). Overlap between U-Pb maxima, supercontinent formation and negative εHf excursions, indicate a link between the supercontinent cycle and changes in continental crust growth rate, whereby decreased growth rate occurs during supercontinent amalgamation, along with a possible increase in preservation of crust during these periods. Fig. 2. The pattern of zircon-Hf data for internal and external orogens (based on Collins et al., 2011). External orogens feature a positive trend to radiogenic εHf values, whereas internal orogens feature a fanning out of data to both radiogenic and non-radiogenic εHf values. Condie and Aster, 2010). The effect of preservation during the supercontinent cycle is conceptualised in Fig. 3. If continental addition equals continental loss throughout Earth history, then the Hf data will comprise an array to increasingly positive and negative εHf values over time (Fig. 3, 1A). If continental addition versus continental loss is always balanced, but preservation of continental crust is increased during supercontinent amalgamation, then the data will fill the same array, but will be concentrated in time periods of supercontinent amalgamation (Fig. 3, 1B). The same array will occur if continental addition versus continental loss is balanced, but addition of continental crust is increased during supercontinent amalgamation. If supercontinent amalgamation increases the preservation of 'recycled' crust compared to 'juvenile' crust (i.e. low εHf crust compared to high εHf), but continental addition equals continental loss, then an array like that in Fig. 3 (4a) can be envisaged; in this case, the mantle input curve will vary over time, as is displayed in Fig. 1. As has been previously discussed, continental addition has likely outweighed continental loss during certain times within earth history; on the εHf-time plot, this will be represented as an array that oscillates over time (Fig. 3, 2A-3B). Increased continental addition during supercontinent amalgamation will produce positive excursions in the mantle-input curve during these periods, and vice versa. The combination of the U-Pb histogram, the Hftime array, and the mantle input curve, can thus be used to make inferences about the role of preferential preservation during the supercontinent cycle, and the balance between continental addition versus continental loss. The examined zircon-Hf dataset exhibits a scenario similar to that depicted in Fig. 3 (3B), whereby continental loss outweighs continental addition during supercontinent amalgamation, Fig. 3. The concept of increased preservation potential during supercontinent formation, and the concept of continental addition versus continental loss, are displayed using four hypothetical supercontinents at 0.5, 1.0, 2.0 and 3.0 Ga, with darker shading representing a greater population of data. 1A, 2A and 3A feature no preferential preservation of crust; 1B, 2B and 3B feature preferential preservation of all crust ('juvenile' and 'recycled'); 4A features preferential preservation of 'recycled' crust compared to 'juvenile' crust. The impact on a U-Pb age histogram, and a calculated mantle input curve similar to that in Fig. 1 is also depicted. The mantle input curve is similar in both 3B and 4A, but the shape of the data array in εHf-time space is indicative of the processes involved. and whereby these periods feature either preferential preservation of continental crust, or an increased production of continental crust; the data do not allow discrimination between the latter two processes.
The supercontinent cycle
Evidence for a single supercontinent existing in the Archaean is lacking (Senshu et al., 2009), although there is evidence for formation of 'supercratons' around 2700 Ma (Rogers, 1996;Bleeker, 2003;Eriksson et al., 2009). The first true supercontinent is that of Columbia (Rogers and Santosh, 2002); the maximum packing of this supercontinent based on timing of collisional orogenies is around 1.9-1.85 Ga (Zhao et al., 2004;Rogers and Santosh, 2009;Yakubchuk, 2010). The next supercontinent to form was Rodinia at~1.1-1.0 Ga (e.g. Dalziel, 1991;Hoffman, 1991;Li et al., 2008). Later supercontinents, although well studied, still feature debate on their timing in regard to true supercontinent formation. General consensus is that Gondwana formed around 550 Ma, and that Pangea formed around 250 Ma (e.g. Veevers, 2004;Collins and Pisarevsky, 2005;Meert and Liebermann, 2008;Murphy et al., 2009). Senshu et al. (2009) define Gondwana and Pangea as a single supercontinent, with Gondwana only being a significant landmass that formed prior to later supercontinent amalgamation; based on mantle dynamics these workers advocate the timing of supercontinent formation to be 340 Ma.
According to an idealised model of a supercontinent cycle, i.e. the Wilson cycle, the time period between the aforementioned supercontinents should see a dispersal of continents referred to as supercontinent break-up (e.g. Nance et al., 1988). In reality, the formation of supercontinents does not seem to fit such a pattern. Supercontinents can form by two end-member processes: introversion (i.e. 'inside-in'), where oceanic spreading along interior orogens is transformed to collision along the same orogens, and extroversion (i.e. 'inside-out') where exterior accretionary orogens are transformed into interior collisional orogens (Hoffman, 1991;Nance, 2003, 2008;Murphy et al., 2009). The latter has been proposed for formation of Columbia, Rodinia and Gondwana (e.g. Hoffman, 1991;Rogers and Santosh, 2002;Murphy and Nance, 2003;Rino et al., 2008), whereas introversion is indicated for formation of Pangea Nance, 2003, 2008;Murphy et al., 2009). The degree that continents were dispersed prior to the maximum packing of supercontinents has varied over time. For example, there appears to be a lack of evidence indicating break-up of Columbia; although some large radial dyke swarms suggest rifting amongst continental blocks (e.g. Ernst et al., 2008;Hou et al., 2008;Goldberg, 2010), palaeomagnetic evidence allows for the continents to be connected throughout most of the~1.8-1.2 Ga period (Yakubchuk, 2010). Rather than breaking-up completely, it appears that large continental blocks rotated from their position within Columbia to new positions within Rodinia, a process that transformed exterior orogens into interior orogens such as the Grenville Orogen (e.g. Johansson, 2009;Li et al., 2008), i.e. the process of extroversion. The break-up of Rodinia appears to have seen more significant dispersal of continents prior to formation of Gondwana and subsequently Pangea , than that of Columbia, although this may be a consequence of more reliable palaeomagnetic data from this time period. The break-up of Gondwana involved separation of several continental blocks via spreading in the interior Rheic ocean, but was halted by a geodynamic change that led to reamalgamation along this interior orogen (e.g. Murphy and Nance, 2008;Murphy et al., 2009); again, only part dispersal of large continental blocks occurred during supercontinent break-up. The break-up of Pangea has been occurring since 200-180 Ma (e.g. Veevers, 2004;Santosh et al., 2009), and has led to the current dispersal of continents that we see today.
It appears that the supercontinent cycle has not involved complete separation of continents prior to reamalgamation into single supercontinents, but rather a cycle whereby large groups of continents are rifted, rotated and reamalgamated. Even with this being the case, the effect on global addition and loss of continental crust will still be the same; interior orogens will be most prominent during the maximum packing of supercontinents, and exterior orogens will be most prominent during maximum dispersal of supercontinents. During supercontinent breakup, the balance between exterior orogens that are retreating compared to those that are advancing, may be largely controlled by the rate of oceanic spreading, i.e. a global increase in oceanic spreading should lead to a greater dominance of advancing accretionary orogens. Whether retreating or advancing orogens are more dominant will therefore depend on mantle convection rates as well as plate movements.
Peaks in the U-Pb histogram occur at 2700, 2450, 2000, 1850, 1600, 1150, 1000, 500, and 250 Ma. These may reflect increased preservation of continental crust at these times, or increased production of crust. These peaks largely overlap periods in time where supercontinents are amalgamating, particularly the more significant peaks at 2700, 1850, 1100-1000, 500 and 250 Ma. The εHf array, and associated mantleinput curve (Fig. 1), suggest increased continental addition (i.e. increased continental crust growth rate) during the periods subsequent to supercontinent amalgamation, i.e. at~1.7-1.2 Ga,~0.85-0.65 Ga, and 0.45-0.35 Ga. Periods of increased continental loss (i.e. decreased continental crust growth rate), occurring at~1.1-0.9 Ga, and~0.65-0.50 Ga overlap the periods of supercontinent amalgamation for Rodinia and Gondwana respectively. The amalgamation of Columbia features significant crustal loss, as indicated by the low εHf values, but also features substantial concurrent juvenile growth (high εHf). This pattern is also seen during amalgamation of Pangea at 250 Ma. A possible explanation for this irregularity in the pattern of continental growth, is that of the different processes of supercontinent formation. Exterior orogens that initiated during Gondwana formation, i.e. the Terra Australis orogen (Cawood and Buchan, 2007), allowed significant continental growth during break-up of Gondwana, but rather than being shutdown, these orogens continued to host continental growth during formation of Pangea, since they were still on the exterior of the supercontinent. Based on the εHf array, it is suggested that introversion may have been responsible for the formation of Columbia at~1.9-1.85 Ga. The supercontinents that are well documented to have occurred by extroversion, i.e. Rodinia and Gondwana (e.g. Hoffman, 1991;Li et al., 2008), have the greatest amplitude in variations in the εHf array, suggesting there may be a greater imbalance between continental loss and continental addition during the process of extroversion compared to that of introversion.
Conclusions
Based on the assumption that εHf of detrital zircons provide a proxy record for the amount of continental addition versus continental loss, the zircon-Hf data presented here, indicate that the balance of addition and loss of continental crust has varied throughout Earth's history. Periods of supercontinent amalgamation feature increased continental loss, whereas increased continental addition occurs in the periods subsequent to supercontinent amalgamation; these are partly correlative with supercontinent break-up, but this term is avoided since complete break-up appears to be rare. The data confirm the non steadystate nature of continental growth, and are compatible with the concept of Yin and Yang, i.e. continental growth rate increases during supercontinent break-up (Stern and Scholl, 2010); however, it should be noted that since the overall growth rate of continental crust has likely been decreasing since the Archaean Belousova et al., 2010), the variations in growth rate indicated in this study do not confirm actual increases in continental crustal volume, but changes in the balance between addition and loss of continental crust.
The balance between continental loss and addition is controlled by: 1) the balance between interior orogens and exterior orogens, with the latter hosting greater continental addition, and 2) the balance within exterior orogens between advancing and retreating modes, with the latter also hosting greater continental addition. The process of supercontinent formation also controls the balance between these orogens, introversion allows continuous continental growth along exterior orogens concurrent with continental loss within interior orogens, whilst extroversion features termination of growth along exterior orogens as they are transformed into interior orogens. A greater imbalance between continental addition and continental loss seems to occur during cycles of extroversion, i.e. during the formation and breakup of Rodinia and Gondwana. Pangea formed by introversion Nance, 2003, 2008;Murphy et al., 2009), and by analogy, the concurrent continental addition with continental loss during amalgamation of Columbia, suggests that this supercontinent may also have formed by introversion. Preferential preservation of continental crust during the supercontinent cycle has likely affected the detrital U-Pb and Hf record, with increases in U-Pb crystallisation resulting from greater preservation potential during supercontinent amalgamation ; however, by examining the distribution of Hf data, it is suggested that this bias has not significantly affected the general pattern of positive and negative εHf excursions, and therefore the interpretation of continental loss versus continental loss remains applicable. Although the interpretation of data in this study uses a qualitative approach, it presents a novel analysis of continental growth; more rigorous testing of crustal growth models in the future will be amenable with the constantly increasing detrital zircon database. | 2019-04-24T13:10:34.270Z | 2012-05-01T00:00:00.000 | {
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56512980 | pes2o/s2orc | v3-fos-license | Are Audits of Financial Statements Born Unequal ?
Audit is getting more and more importance in assuring the reliability of financial information including financial statements for all parties. Commonly, quality of an audit is viewed as probability that financial reports are free from material irregularities. In the previous literature, there is a positive relationship between size of audit firm and audit quality that has long been understood. This has resulted in many publications to gather evidence of differential audit quality relating to audit firm size. In consequence, the conclusion has been focused that bigger audit firms produce higher audit quality than smaller ones. However, the collapse of many big international audit firms, typically Arthur Andersen has reduced the statement that large audit firms have higher audit quality than small ones. Therefore, this study looks into audit quality basing on the extent of compliance with disclosure requirements pertaining to goodwill impairment of large listed firms in Hong Kong context in the second year transition to IFRS. We found that audit firm identity appears to be a substantial variation, in which compliance levels changed significantly among auditors in the context of Hong Kong.
Introduction
Quality of an audit is viewed as one of the most important issues in the audit activities and is defined as probability that financial statements are fairly presented and disclosed when an unqualified opinion is given (Kit, 2005;Simunic, 2003).The acceptance of big audit firm associated with high audit quality for a long passage of time is given in a huge literature (Krishnan & Schauer, 2000;Simunic, 2003).However, that view is much influenced by bankruptcy of Enron, Worldcom and Andersen collapse in the 2002.This makes users of financial statements have different views of audit quality of financial services and non financial services.
In order to have high audit quality, material irregularities should be detected basing on technical competence of auditor and reported basing on auditor independence.However, it is not easy to see clearly the call of technical competence and independence.How to quantify competence and independent is too challenging in accounting and auditing field.So it makes audit quality more complicated and difficult to evaluate on the side of financial statement users and even auditors as well.
A large number of countries and territories, including Hong Kong, switched to International Financial Reporting Standards (IFRS) based financial reporting framework.Adoption of IFRS views as the most revolutionary financial reporting development and makes very difficult for financial statement preparers and also difficult for very auditors as well (Hoogendoorn, 2006).
For the harmonization and convergence of IFRS, Hong Kong has designed an own version of accounting framework, i.e.Hong Kong Financial Reporting Standards (HKFRS), that came into effect from 1 st January 2005.Because of over-complexity and challenged requirements in HKFRS, there is a high possibility to have inherent misstatements in the financial statements of reporting firms in the early years after HKFRS implementation.So, this also makes more difficult for an auditor to detect material misstatements including fraud and errors in a client's business and accounting system.Because of this, quality of an audit may be influenced in these circumstances together with combination of increased market, regulatory and technical pressures, that all have some implications for variation in audit quality of financial statements.
Impairment of assets including goodwill impairment and its disclosure is one of the most difficult issues in practices.Preparation of financial statements aligning with the requirements of HKAS 36 "Impairment of Assets" requires reporting firms to apply some financial principles drawn from discounting, forecasting and valuating models under potentially uncertain conditions and events in the future.With different subjective assumptions relating on future cash flows, discount rates, growth rates and forecast periods produce different outcomes of present values (using value in use method) that are discounted from future cash flows and to evaluate which is the best result is too complicated and potential controversy in the world.
Because audit quality of financial statements conducted by independent audit firms is likely to be positively related with compliance with accounting standards & regulations (Copley et al., 1994), variations in disclosure of goodwill impairment testing regime in practice are likely to be the result of variations in quality of an audit.Thus, the audit quality measurement employed in this paper is the extent of compliance with requirements of HKAS 36.So the extent of technical compliance with disclosure requirements views as a surrogate for audit quality relating to challenged and over complicated provisions of the goodwill impairment testing regime.
Literature Review
Quality of an audit of financial reports is an important phenomenon of corporate governance can be understood as the probability that an auditor detects and reports material irregularities during the course of an audit of financial statements of auditee.
There is a fact that audit quality of financial statements is not public information and cannot be directly seen by financial statement users.So, evaluating it is one of the most controversial issues that many scientists have investigated.Auditor size is one of the most frequent item employed as a proxy for audit quality of financial reports in previous studies.Much empirical evidence is ostensibly consistent with large audit firms provide better audit quality than small ones.
Consistent with the given assumption that large audit firms provide higher audit quality.Capital market publications have shown that stock market reacts more strongly when a client shifts to a big audit firm and reports higher earnings response coefficients in comparison with client of a non big auditor.Other studies on the market reaction to the initial public offerings (IPO) of stocks experience less under-pricing from a big audit firm client than that from a non big audit firm client (Firth & Smith, 1992).
According to Krishnan & Schauer (2000), compliance levels of GAAP reporting requirements of big audit firm clients are higher than that of non big audit firm clients and propose that extent of compliance with GAAP is likely to be concerned to the probability of detecting material misstatements.
More and more other huge literature also provides much empirical evidence for assuring that auditor size is a proxy for audit quality of financial statements.However, events of the bankruptcies of Enron, Worldcom and others, and the related collapse of many large audit firms, typically Anderson have both reduced the good images of audit industry and confidence of financial statements users in audit quality.
As a result, to some extent, this fact has reduced the assertion that has been prevailing for a long time that large audit firms provide higher audit quality.Thus, a small number of recent researches have begun to investigate the possibility of differential audit quality among big 4 auditors, rather than supposing that there is a homogeneous audit quality among big 4 auditors.
Whereas audit quality literature is propensity to support the proposition that audit quality of financial statements conducted by large audit firms exceeds that conducted by small audit firms, there is a little evidence to show audit quality variation among big audit firms.The evaluation of audit quality on an indirect method tends to stem from a process of comparing observed values for some accepted surrogates for quality among audit firms.
The issue of audit quality variations among large audit firms as well as large audit firms vs. non big audit firms seems very crucial and needs to be answered.Some studies conducted by Laili and Khairil (2013), Carlin et al. (2010) and others investigated the audit quality differences in different jurisdictions and found the big variations among auditors.The question is that in the context of Hong Kong, the result is the same or difference.Moreover, in countries where implementation of IFRS based reporting framework has coincided with other types of structural shifts impacting much on audit services, substantial emphasis has been directed toward auditors (Carlin et al., 2009).So the adoption of IFRS represents a good point to be looked into, in which goodwill impairment is the one of the most difficult issues in practice (Hoogendoorn, 2006;Tran, 2011).
Data Collection and Research Methodology
Hong Kong Financial Reporting Standards form compulsory legal frameworks for all listed reporting firms to apply on or after 1 st January 2005.In this study, data from the second year to adopt HKFRS, including HKAS 36 have been collected and used for evaluating the changes of audit quality of financial statements through a surrogate of compliance levels with disclosure requirements of goodwill impairment testing regime.In designing the final sample, a number of steps were conducted.First, firms were required to be the members in Hong Kong Stock Exchange (HKEx) and of the second year implementation of IFRS.As a result, there were 975 firms with total market capitalisation of $13,248 billion at the year end.Then 500 biggest market value firms with $13,131 billion (making up 99.10% of total market capitalisation) were chosen.251 firms were excluded because of no having goodwill as comprising an element of their asset base in the financial statements.As a result, this process designed a commencing sample of 249 firms with market value of $8,349 billion, representing 63.02% of total market value in HKEx at the end of the second year adoption of IFRS in financial reporting.The aim of this study is to evaluate audit quality variation among auditors based on the reporting firms' compliance with disclosure requirements pertaining to goodwill impairment so different currencies used in the annual reports reported by listed firms are accepted for inspection.The reason for accepting various currencies is that the nature of this research focuses much on compliance levels of reporting firms bearing on methods used, cash generating unit aggregation, goodwill allocation to cash generating units (CGUs), and key assumptions of goodwill impairment rather than currencies.The percentages of firms used HK$, Renmibi and US dollars are 70%, 23% and 7% of the sample, respectively.However, in order to compare the values of items in the financial statements, converting currencies other than HK$ (US dollars and China Yuan Renmibi) into HK$ is necessary.For the items in the consolidated Balance Sheet which reflect the financial position of the firm at a point of time, a foreign exchange (forex) rate at the year end has been used to convert into Hong Kong dollars.For the items in the consolidated Income Statement (profit and loss account) which reflect the operational results of the firm for a period of time, so a 12 month average foreign exchange rate has been used for translating into Hong Kong dollars.
To allow for industry segmentation, all firms were allocated to one of five industry groupings comprising entities with related to business sectors, namely, Consumer Goods and Conglomerate; Financials; Telecommunications and Services; Materials and Industrial Goods; and Utilities, Energy and Construction.For the purpose of analysis in this study, auditors are classified basing on big four auditors including Deloitte, Ernst & Young (EY), KPMG, PricewaterhouseCoopers (PWC) and Others (non-big 4 auditors).At the date of sampling, 249 firms included in the final sample have asset values of $23,499 billion, which contained goodwill of $503.14 billion.An overview of the sample consists of assigned sectors; Hong Kong dollar value of firm assets and dollar value of goodwill within each sector are presented in Consistent with Carlin et al.(2009), six techniques were employed to the sample, as below: First, sample firms were sorted by audit firm, according to whether they employed a value in use method to estimation of CGU recoverable amount, a fair value less costs to sell method, a combination of methods (i.e. the use of value in use in some CGUs and use of fair value in others), or failed to report method disclosure.This data supported the development of insight into compliance level with basic disclosure requirements stipulated in HKAS 36.
Second, the firms in the research sample were classified by audit firm identity, according to whether they allocated all goodwill values to the specified CGUs, or whether they allocated partially goodwill values to CGUs, or whether their disclosures were not given so it was impossible to determine how or if value of goodwill had been allocated to defined CGUs.It is a basic requirement that for the purpose of impairment testing, goodwill should be allocated to each of CGUs or groups of CGUs that are expected to benefit from the synergies of the combination.So this data help financial users have insights into the compliance level with basic disclosure requirement prescribed in HKAS 36.
Third, the sample firms were filtered by audit firm, according to the relationship between the number of CGUs specified for the purpose of goodwill impairment testing and the number of business segments for the purpose of segment information reporting.So this data provides evidence of appropriate CGU aggregation on the part of reporting firms.
Fourth, the firms in the sample were classified by audit firm, according to calculated ratio of CGUs to business segments.This data provides more evidence of CGU aggregation on the part of reporting firms and combines with procedure prescribed in the third step for assuring whether CGU aggregation is appropriate.
Fifth, the sample firms were sorted by audit firm, according to the quality of discount rate disclosure in the goodwill impairment testing process.Data was stratified into four types, namely, multiple discount rates, single discount rate, range of discount rates and no effective disclosure.Firms categorized in the first category appeared to fully comply with the disclosure requirements of HKAS 36 by disclosing unique rates applicable to each of their various CGUs.This type of disclosure fully complies with the standard requirements and provides a higher assurance of process quality through different discount rates to each specified CGU.
Firms in the second category, i.e. "single discount rate", showed that they defined blanket whole of firm discount rate for all specified CGUs for measuring CGU recoverable amount in the discounted cash flow model.This did not appear to comply with the requirements that a discount rate unique to each specified CGU and each CGU risk was arguably different.
Firms were sticked in the third category disclosed a range of discount rates which had been employed for estimating CGU recoverable amount.Because of lacking specific discount rate to each specified CGU, it is questionable whether disclosure of this category meets the requirements of HKAS 36.
Finally, allocation of firms in the fourth category signified that the firms failed to provide inadequate discount rate disclosure, and in consequence provided no meaningful information for financial statement users to evaluate the robustness of goodwill impairment testing process.Therefore, these firms were judged to have poor disclosures and not to comply with the disclosure requirements of HKAS 36.
Sixth, the sample firms were filtered by audit firm, according to the quality of growth rate disclosure in the regime of goodwill impairment testing.Data was stratified according to a very similar taxonomy to that described bearing on discount rates, i.e. multiple growth rates, single growth rate, range of growth rates and no effective disclosure.The first category represented the highest level of disclosure, the fourth the poorest.
Results and Discussion
The interest of this paper concentrates on variation of audit quality among auditors based on the reporting firms' compliance with disclosure requirements bearing on goodwill impairment under HKAS 36.The first issue in understanding the process of goodwill impairment testing is the selection of valuation method for measuring recoverable amount of assets assigned to CGUs.
Under HKAS 36, recoverable amount of an asset or a CGU is the greater of its fair value less costs to sell, determined basing on market based evidence, and its value in use, determined basing on a discounted cash flow model.Table 3 shows the frequency of approach used for estimating recoverable amount of an asset or a CGU, either fair value or value in use or mixed method (combination of the fair value and value in use), and no effective disclosure.
The data denotes that the dominant approach to the estimation of recoverable amount was the approach of value in use.A total of 213 firms (accounting for 85.5% of the sample) employed this method as the exclusive basis for determining CGU recoverable amount.Only 6 firms chose fair value as the sole basis for estimating CGU recoverable amount.A further 10 instances disclosed that they applied a mixed method (employed value in use in some CGUs and fair value in others).
Up to 20 cases (about 8% of final sample) failed to report method employed for determining CGU recoverable amount.Specifically, clients of other audit firms provided no effective information relating to method used with highest percentage of 10.7%.Followed by clients of EY, PWC and Deloitte were at 9.8%, 8.3% and 7.5%, respectively, and with clients of KPMG at lowest percentage in total, about 3.2%.Under the requirements of HKAS 36, goodwill balance is subject to an impairment testing.Even if, whether the value of goodwill are immaterial compared with the values on the balance sheet.So the firms with failure of disclosing method used were judged not to comply with the disclosure requirements of the HKAS 36.However, based on only this analysis, it is not possible to reach robust conclusion as to possible variation in quality by audit firm.
The next analytical tool adopted was to compare the reported value of goodwill on the financial reports with the sum of the amounts of goodwill allocated to defined CGUs of reporting firms.As presented in Table 4, the majority of firms fully complied with the disclosure requirements, accounting for 71.9% of the total sample (in case of it was possible to have matched data between value of goodwill on the balance sheet and the sum of goodwill allocated to CGUs).Only two cases belonging to client of EY and KPMG that goodwill value did not allocate completely to specified CGUs and discrepancies between goodwill value and the sum of goodwill allocated to CGU were immaterial.
There have been 68 cases (about 27.3% of the final sample) provided no effective disclosure relating to goodwill allocation to defined CGUs.Clients of PWC failed to disclose the effective disclosure pertaining to goodwill allocation to CGUs with highest percentage of 37.5%.Followed by clients of EY, KPMG, other auditors were at 31.4%, 25.8% and 25%, respectively, and with clients of Deloitte at lowest percentage in total, about 15%.From an audit firm identity, there was little evidence of cross sectional variation in practice.In the first two analytical procedures applied to the sample data, however, it appeared that Deloitte's clients had the lowest percentage of noncompliance levels, whereas clients of remaining auditors had insignificant variation of non-compliance levels with the accounting standard.The next analysis procedure produces more evidence of compliance levels of audit firm clients relating to CGU aggregation, which is set out in the next table.
Table 5 reveals that clients of non big 4 auditors have a greater tendency to define fewer CGUs than business segments or report no meaningful disclosure of CGU definition than clients of big 4 firms, especially PWC.Under HKAS 36, each CGU or groups of CGUs to which the goodwill is so allocated will present the lowest level within the entity, and not be larger than a segment of the firm.So, clients of all audit firms violated the provision with different levels.
The data show that about 82% of other auditors specified fewer CGUs than business segments or provided no effective disclosure relating to relationship between number of CGUs and number of business segments.In contrast, this happened in only 62.5% of PWC clients, with EY, KPMG and Deloitte clients at about 80%, 71% and 69%, respectively.This suggests a higher risk of CGU aggregation belonging to non big audit 4 clients than that in clients of Big 4 firms, especially PWC.The data also show that clients of audit firms employed unusually low discount rates.Specifically, Deloitte clients adopted a rate of 3.8%, through to clients of EY at 4%, KPMG clients at 4.2%, PWC clients at 4.5% and other audit firm clients at 5.50%.Applying lower mean discount rates in the model of discounted cash flow would result in overestimating present values (recoverable amounts), and consequently reduce the chance to recognize impairment expenses in the accounting period, and to increase accounting profit recognized in the consolidated financial statements.Average estimated growth rates used by other auditor clients (about 4.3%) were higher than that chosen by big 4 auditor clients, particularly Deloitte (about 2.5%).By using higher growth rates in the model of discounted cash flow, other things being equal, would increase the determined recoverable amount of CGU assets, and reduce the chance of recognizing goodwill impairment charges, and increase the possibility of reporting accounting profit in a given year.
Conclusion
This research is conducted for accumulating evidence which might show variation in audit quality among auditors (Deloitte, EY, KPMG, PWC and non big 4 auditors) during the course of financial statement audit including audit of goodwill impairment.The approach employed in this research focussed on the nature and quality of disclosures pertaining to goodwill impairment testing regime under the accounting standard of "Impairment of Assets".
Basing on evidence collected from listed firms for the second year after HKFRS adoption.By testing the approach employed, CGU aggregation and variables of discount rates and growth rates, it appears that the systematically noncompliance levels in relation to goodwill impairment were on the part of clients of all audit firms.In which, clients of non Big 4 auditor have higher non compliance levels than clients of big 4 audit firms do.
Out of big 4 audit firms, clients of Deloitte were judged, in general, to be the best practice disclosure of goodwill impairment.Meanwhile, clients of EY, KPMG, and PWC were evaluated to have significant variations of practice disclosures pertaining to approach implemented, CGU aggregation and discount rates and growth rates.Interestingly, all financial statements of listed firms in the final sample were stated truly and fairly in all material respects on the eyes of independent auditors including big 4 auditors.So it raised the issue that quality of financial statement audit is really questionable in the context of Hong Kong.
Clearly, the extent of compliance with HKFRS including HKAS 36 is likely to be related to the probability of detecting and reporting material irregularities during the course of financial statement audit of listed firms in the context of Hong Kong where accounting framework is evaluated fairly well.Changes in disclosure of goodwill impairment of audit firm clients are likely to be the result of quality variations of financial statement audit.So evidence got in this study supports the viewpoint that audit quality of big 4 audit firms is regarded to be higher than that of non big 4 auditors and audit quality among big 4 auditors is not equal, but is unequal.Further research on variations in audit quality of financial statements among audit firms when compliance of goodwill impairment in the time series and legal framework improved are identified and discussed.
Table 1 .
Overview of research sample
Table 2 .
Table2shows the number of firms audited by auditors, and by sectors.The number of clients for each auditor is uneven, with PWC dominating at 28.9% of the firms in the sample, followed by Deloitte, EY, and KPMG at 26.9%, 20.5% and 12.5%, respectively, and other auditors with minimal share of 11.2% in the sample.Number of firms audited by sectors An important question is the extent to which the clients of audit firms align with over complex technical provisions of a new and challenged standard during the course of financial statement audit.Potential interests of CGU issue, discount rate and growth rate disclosures should be investigated under HKAS 36.
Table 3 .
Approach adopted to determine recoverable amount of CGUs
Table 4 :
Compliance of CGU allocation by auditors
Table 5 .
Relationship between CGU & business segments by auditorsOther technique of analytical procedure is used for identifying audit firm pertaining to the quality of discount rate disclosure for estimating CGU recoverable amount.As shown in Table6, clients of PWC emerged to provide less effective disclosure pertaining to discount rates than clients of remaining big 4 auditors and non big auditors, particularly Deloitte.
Table 6 .
Discount rates adopted for testing impairment of CGUs data to growth rates employed in the discounted cash flow model for estimating recoverable amount of each CGU.Table7illustrates a different pattern in comparison with the pattern shown in the discount rate disclosure in practice.Highest percentage of non-compliance level with the disclosure requirements belongs to clients of Deloitte, accounting for 80.3%.Followed by clients of other auditors, PWC, EY and KPMG were at about 71.2%, 68.2% and 57.1%, respectively.
Table 7 .
Growth rates adopted for testing impairment of CGUs | 2018-12-15T03:51:05.740Z | 2017-11-03T00:00:00.000 | {
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227249685 | pes2o/s2orc | v3-fos-license | Origins of Relative Age Effects in Youth Football—A Nationwide Analysis
Introduction: Relative age effects (RAEs) refer to the overrepresentation of players born earlier in the selection year compared to late-born players within the same age category. To date, the origins and mechanisms of RAEs are still unclear. To evaluate the development of RAEs in terms of age group and selection level, we analyzed data of all registered child and adolescent football players in Switzerland. Methods: Age category, selection level, and birthdate from all licensed 101,991 Swiss child and youth football players assigned to a specific team [9,149 girls (9.0%) and 92,842 boys (91.0%); age range: 4.6–19.6 years] were analyzed. Additionally, out of 1,128 clubs, 54 clubs provided their documented waiting lists (1,224 players). Birthdate distributions were split by age category, sex, and birth quarter (Q1 = January to March, Q4 = October to December). RAEs were calculated using odds ratios (Q1 vs. Q4) with 95% confidence intervals (95% CI). Results: We found small RAEs among U8 players (OR 1.44 [95% CI 1.31, 1.59]) and U10 (OR 1.24 [95% CI 1.16, 1.32]). The RAE was negligible in all other age categories, independent of gender. In children's football, 5,584 (71.3%) teams performed selections. In teams without selection, there were no obvious RAEs. However, teams with selections for the same age category showed small RAEs with an overrepresentation of Q1 athletes in the first team (OR = 1.29 [95% CI 1.24, 1.35]) and inverse RAEs with an underrepresentation of Q1 athletes in the last team (OR = 0.85 [95% CI 0.82, 0.89]). Only small RAEs were observed on the waiting lists for the U8 (OR = 1.48 [1.13, 1.95]). Discussion and Conclusion: RAEs have a small, but consistent effect on participation in Swiss children's football at the grassroots level. Contrary to expectations, no inverse RAEs were found on the waiting lists. Nonetheless, first time coach selections seem to be the origin of RAEs. To protect young athletes from discrimination, RAE biases should be analyzed and eliminated at all stages of sport participation, selection, and dropout situations. Modifications to the organizational structure of sport and athlete development systems are recommended to prevent RAE-related discrimination in youth sports.
INTRODUCTION
The practice of annual age grouping occurs throughout and across youth sport. In football (soccer), players are typically categorized into annual age groups to reduce developmental differences during childhood and adolescence. However, there is a potential gap of up to 12 months in chronological age between individuals in the same age category. Relative age effects (RAEs) are defined as the overrepresentation of chronologically older participants within one selection category (Cobley et al., 2009).
RAEs have been demonstrated in a wide variety of sports. Cobley et al. (2009), for instance, report RAEs in 14 different sports (ice hockey, volleyball, basketball, American football, Australian rules football, baseball, soccer, cricket, swimming, tennis, gymnastics, netball, Rugby Union, and golf) in male and female youth athletes from 4 years of age to adulthood. RAEs are present in all age categories and increase with age. The largest effects are observed in basketball and football. In a populationbased study, RAEs in all youth athletes who were registered in the Swiss talent development program in the year 2014 were analyzed . A large variability in RAEs was found across different types of sports and between girls and boys. Overall, RAE was present for athletes of both sexes, with considerably larger RAE in male athletes competing at national level or in Olympic sports than male athletes competing at a lower selection level or in non-Olympic sports, respectively. Similarly, population-based analyses in France showed RAEs in basketball and football (Delorme et al., 2010(Delorme et al., , 2011. In Switzerland, population-based analyses of athletes aged 10-20 years showed RAEs in men's Swiss football and athletics (sprinting) (Romann and Fuchslocher, 2013;Romann and Cobley, 2015), as well as in women's alpine skiing, tennis, football, athletics, fencing, snowboarding, and inverse RAEs in table tennis Fuchslocher, 2011, 2014).
RAEs can occur during the early development of youth athletes (Cobley et al., 2009;Smith et al., 2018) and may lead to a biased view of the potential of children in a particular sport. As athletes born earlier in the competition year may have more advanced physical and cognitive abilities compared to their lateborn opponents, they are likely to be identified as more talented (Cobley et al., 2009;Gil et al., 2014;Wattie et al., 2015;Smith et al., 2018). Furthermore, RAEs can lead to an increased dropout from sports, with an underrepresentation of dropouts among athletes born early in the competition year and an overrepresentation among those born late in the year shown in a sample of almost 75,000 young (9-15 years) basketball players (Delorme et al., 2011). A very similar observation, i.e., an over-representation of dropouts born late in the competitive year, was reported in an analysis of more than 360,000 male French football players in the U9 to the U18 age categories (Delorme et al., 2010).
It is generally assumed that physiological growth and maturation are major underlying causes of RAEs (Cobley et al., 2009). However, there is evidence that RAEs are present before puberty and, therefore, prior to maturity-associated selection biases. Furthermore, RAEs are also found, in academia, where physical capabilities are negligible (Musch and Grondin, 2001). However, playing experience, cognitive, emotional, behavioral, motor, and social development are more likely to follow age than maturity. Therefore, such differences in development are more likely to be the underlying causes of RAEs than (differences in) maturity in athleticism (i.e., speed, power, and strength). Furthermore, several aspects, such as the popularity of a particular sport, the number of active participants, the importance of physical development and the competitive level may affect the magnitudes of RAEs (Musch and Grondin, 2001). RAEs are likely larger with high rates of participation and high selection pressure.
Additionally social agents like parents, coaches, and athletes (Cobley et al., 2009) influence RAEs. They falsely associate physical maturity with actual skill differences, and through accelerated RAEs, which have been explained with the Matthew effect, the Pygmalion effect, and the Galatea effect (Hancock et al., 2013).
However, to date, little is known about the origins of RAEs. In football, it may be speculated that RAEs increase with age and are larger in teams of higher playing levels, based on increasing selection pressure. Thus, RAEs can be expected to be smaller or even inexistent in low-level youth teams in young age groups. Accordingly, the aim of this study was to address the effect of playing levels, age groups, and selection approaches (selfselection, waiting lists, and selection by team coaches) on the occurrence of RAEs, by analyzing a population-based sample of youth football players in Switzerland.
The objectives of this study therefore were (i) to investigate RAEs in a nationwide analysis of football players (5-20 years of age), who are registered as active players assigned to an individual team in the database of the Swiss Football Association (SFA) and (ii) to evaluate the effects of different selection procedures on RAEs.
Participants
Following ethical approval from the Swiss Federal Institute of Sport (App. No: 082_LSP_150819), RAEs of 101,991 junior football players [9,149 girls (9.0%) and 92,842 boys (91.0%), age range: 4.6-19.6 years] participating in one of 1,128 football clubs were analyzed. Age categories ranged from under-(U) 8 to U20 for male and from U12 to U19 for female football (FF) players. Including cases where players played in two teams (different levels or different categories), we analyzed 109,469 single data points in Swiss Football Juniors' leagues during the year 2019 ( Table 1).
Data were taken from the national football database with permission of SFA. In addition, all Swiss football clubs with children football's sections (U8-U12, N = 1,128 clubs) were contacted via email and asked whether they have (a) no waiting list; (b) anonymous waiting list, but not documented; or (c) anonymous documented waiting list. Under the term waiting list, we understand that some children sometimes cannot enter a club due to a lack of infrastructure or coaches, for example, and have to wait for a place in a football team. Out of the 1,128 clubs, 556 (49%) responded and 54 delivered their documented waiting lists, which included 1,271 players. Due to the low number of girls on waiting lists in general and of boys in the age groups older than the U12 age category, only the waiting lists of boys in the categories from U8 to U12 were analyzed (N = 1,224 boys).
Procedures and Data Analysis
In Switzerland, the cutoff date for all sports is January 1st. The players were categorized into two semesters (S) and four relative age quarters (Q) according to their birth month independently of birth year (i.e., S1 = January to June; S2 = July to December and Q1 = January to March; Q2 = April to June; Q3 = July to September; and Q4 = October to December). The observed birthdate distributions were calculated for every relative age quarter. The expected birthdate distributions were obtained from the actual corresponding distributions (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013) as the number of live births registered with the Swiss Federal Office of Statistics (2020). The relative age quarters of the Swiss population were as follows: Q1 = 24.5%; Q2 = 25.2%; Q3 = 26.1%; and Q4 = 24.2%. Self-selection in children's football was calculated using the distribution of the corresponding Swiss population and the distribution of registered players. Birthdate distributions were split according to gender, age category, selection level, and birth quarter. There were up to seven selection levels possible in the same age category, from the best league to the last team of each age category within the same club. Age categories were analyzed as given by the SFA as follows: U8, U10, U12, U14, U16, U18, and U20 for the boys' categories in which girls are allowed to play, and FF-12, FF-15, and FF-19 for the only-girls' categories.
RAEs were calculated using odds ratios (Q1 vs. Q4 and S1 vs. S2) with 95% confidence intervals (95% CI). The OR for the Q1 vs. Q4 comparison was interpreted as follows: we assumed the existence of an RAE if the CI did not include 1 and interpreted an OR < 1.22, 1.22 ≤ OR < 1.86, 1.86 ≤ OR < 3.00, and OR ≥ 3.00, as negligible, small, medium, and large, respectively (Olivier and Bell, 2013). If the OR was <1 and the CI did not include 1, this finding was interpreted as an inverse RAE. As population-based data were analyzed, inferential statistics were not applied (Gibbs et al., 2012).
RESULTS
The full dataset (both sexes across all age categories) shows a negligible RAE. When analyzing specific age categories, there are small RAEs in children's football (U8 and U10) and negligible RAEs in all other age categories, including FF-12 and FF-15. The FF-19 category shows an inverse RAE ( Table 2). Across all 1,128 clubs in childrens' football, 925 (82.0%) select multiple teams and 203 (18.0%) only select one team. Across all clubs 5,584 (71.3%) teams were built by selections and 2,250 (28.7%) were created without selection. Overall, there is a small RAE in the highest-level/first/top teams (OR = 1.29 [95% CI 1.24, 1.35]) and an inverse RAE for the lowest-level teams (OR = 0.85 [95% CI 0.82, 0.89]). Regarding specific age categories, there is a small RAE that decreases with age in the first/highest-level/top U8 to U14 teams. There is no RAE in older age categories for the first/highest-level/top teams, although OR is larger in the top/first/highest-level teams over all age categories (Figure 1). An inverse RAE with an overrepresentation of players born at the end of the selection year is found in the lowest-level teams in the U14, U16, and U18 age categories. Super, Challenge, and Promotion League Clubs show small overall RAE in their children's football teams (OR = 1.26 [95% CI 1.16, 1.36]), which decrease with age (Figure 1). None of the corresponding girl's categories show a relevant RAE.
Out of 556 children's football clubs that responded, 424 (76.3%) have no waiting list, 54 (9.7%) have a documented waiting list, 54 (9.7%) have a waiting list, but not documented, 16 (2.9%) have a periodic waiting list and 8 (1.4%) can offer an alternative program. All 54 clubs that delivered their waiting lists were included in the analysis. In total, the documented waiting FIGURE 1 | First and last level teams OR with confidence interval for each age category (male). Empty squares represent the last level teams. Full squares represent the first-level teams. Light gray color, small inverse effect; gray color, negligible inverse effect; blue color, negligible positive effect; green color, small effect; yellow color, medium effect.
DISCUSSION
The main findings of this study are: (i) no relevant overall RAE, (ii) a small RAE in the youngest age group, i.e., at entry into the official football system, which decreases with age, (iii) a small effect of the current playing level of the team within the club, but not necessarily of the first adult team's playing level, and (iv) a small RAE on the waiting list of the youngest age category (U8).
Self-Selection
The results of our study show that RAEs already exist in the youngest category for both sexes. In other words, children born at the beginning of the selection year are more likely to participate in children's football. Older children are generally biologically more mature, taller, and heavier, with more body mass for body height than younger children. This leads to advantages in athletic performance and psychological aspects, which are referred to as the maturation-selection hypothesis (Cobley et al., 2009). Therefore, it could be speculated that children born at the end of the selection year may self-select prior to playing organized football. Nationwide analyses of male French football players show similar RAEs at entry level of youth football (Delorme et al., 2010) emphasizing/reinforcing our observation. Furthermore, Smith et al. (2018), who found small RAEs (OR = 1.25 [95% CI 1.21, 1.30]) in female athletes across all sports, including football, revealed that RAE magnitude is highest in preadolescent (U11) and at higher competition levels. These findings indicate a self-selection effect (first hurdle) for children of both sexes prior to participating in organized sport. Furthermore, the advantage of being born at the beginning of the selection year is more pronounced the younger the children. For example, in the youngest category of children's football (U8), the age difference between two children in the selection year can be up to 12.5%, whereas this difference is reduced to about 6% in the U16 age group (Cobley et al., 2020). Fundamentally, sport participation requires opportunities to engage in sport, and the motivation to engage in such opportunities. Children born earlier in the selection year are more likely to initiate early age group participation. This may be based on a combination of early sporting experiences and encouraging interactions with others (e.g., parents and peers; Balish et al., 2014). At a young age, role models, stereotypes of the sport (women's sport vs. men's sport), popularity, positive experiences, and existing opportunities are important for sport participation, although there is a need for more in-depth examination and refinement of important correlates at various levels (intra-and interpersonal, biological, institutional, community, and policy levels; Balish et al., 2014). Therefore, peers and or parents, who are present during a child's first sports experiences, may influence the decision to join a club. Furthermore, pre-selection by parents may occur if they do not support the child's sport club participation. Therefore, a child will only join a sports club, if these two obstacles (self-selection and pre-selection by parents) are overcome.
Coach Selection
Following club entry, further selection is conducted by coaches in over 80% of clubs, starting in the youngest age category. Coach selection seems to have a large influence on the magnitude of RAEs, with negligible RAEs due to self-selection greatly increasing as a result of first-time coach selection. This is highlighted by the fact that RAEs are higher in first teams compared to the following teams. Younger athletes may fail to meet selection requirements, as they are often biologically less developed. Therefore, their performance is likely to be poorer in age-based competition compared to athletes born earlier in the selection year. The RAEs disappear in girls in the U15 and in boys in the U14. These results differ from the results of Delorme, who found RAEs in all age categories of French basketball and football (Delorme and Raspaud, 2009). However, Delorme et al. (2010) used inferential statistics, which are not appropriate for population studies. If the data are transferred to ORs, no relevant RAEs (all ORs <1.22) were observed in U10 to adult categories for both female and male athletes. This means that RAEs at the grassroots level should be counteracted particularly in the first age categories (i.e., U8, U10).
Underlying Mechanisms
The underlying mechanisms of RAEs in young elite athletes are well-known. In performance-oriented sport, RAEs increase with higher participation numbers, higher selection pressure, and during the growth spurt in puberty (Cobley et al., 2009). Therefore, when only assessing performance-oriented sport, peak RAEs are often associated with the ages during puberty, between the ages of 11 and 15 years. However, RAEs may be highest in children's sport and decrease consistently until the beginning of the growth spurt, which leads to a second increase in RAEs (Cobley et al., 2020). Furthermore, coaches often focus on short-term success by selecting first teams in children's football that can win matches for the club (Jimenez and Pain, 2008). However, differences in global performance, as well as success during selections of youth players mainly depend on biological and physiological variables (Coelho et al., 2010), which are inaccurate in predicting later success in adolescence and adulthood. Continued selection into better/higher teams, extra training, and elite coaching could provide first-team athletes with a performance advantage (Wattie et al., 2015). Therefore, coaches contribute significantly to the increase of RAEs in youth sports. Additionally, success in youth sport can lead to increased effort and motivation due to positive feedback from coaches, parents, and peers, and, therefore, increase performance of an athlete (Cobley and Till, 2017). Conversely, de-motivation, due to a lack of participation and selection, or the perception of a selection bias, could lead to increased levels of dropout among children and adolescents (Delorme et al., 2011;Cobley et al., 2020). Furthermore, dropouts due to demotivation are enhanced in less psychosocially supportive environments, such as larger cities (Fraser-Thomas et al., 2008). Additionally, during the talent development pathway, following previous selection, coaches (for athletes aged 10-14 years) must perform selections using a pool of players, which is already unequally distributed due to RAEs from previous selections. This process increases RAEs and dropouts even more and has been linked to the Matthew effect, the Pygmalion effect, and the Galatea effect (Hancock et al., 2013). However, there are several measures that could reduce RAEs during entry in sport, selections, and dropouts.
Strategies to Counteract RAE Inequalities in Football
It is important to protect young athletes from age-related discrimination. To ensure this, every child should have the right to participate in sport without any age discrimination (Bergeron et al., 2015;Unicef, 2018). Therefore, RAE biases should be analyzed and eliminated at all stages of sport participation, more specifically (i) at the entry into grassroots sport participation (e.g., self-selection and club entry), (ii) in all selection procedures (e.g., waiting lists, selection into higher-performing teams), and (iii) in all dropout situations (e.g., transitions, de-selections). Previous recommendations addressing RAE-specific inequalities are well-documented and include the creation of shorter (e.g., 6-month) and/or rotating age-group cutoff dates (Hurley et al., 2001), birthday-banding, whereby young athletes move up to their next age group on their birthday (Kelly et al., 2020), and grouping based on biological classifications (e.g., height and weight; Cumming et al., 2017). Furthermore, selection procedures at the grassroots level should be avoided. If they are indispensable, they should be improved so that no RAEs exist. However, more research and innovative approaches are needed to avoid self-selection. In regard to waiting lists, new innovative game, and training formats have been proposed that allow more players to participate (Hintermann et al., under review;Rüeger et al., 2020). During selection procedures, time points for tiers of selective representation should be delayed to post-maturation (Cobley et al., 2020). If these selections are absolutely needed, individual data can be adjusted to normal growth curves or compared with corrective adjustment procedures (CAPs) to interpret interindividual differences in performance (Votteler and Honer, 2014;Romann and Cobley, 2015;Cobley et al., 2020). Furthermore, rather than testing physical parameters, more technical and tactical competences should be tested, as they reduce RAEs and show higher prognostic validity for the transition from youth to elite adult football (Höner and Feichtinger, 2016). An improvement of the psycho-social climate that emphasizes "personal learning and development" and longer-term tracking of athletes beyond maturation have been recommended to avoid RAE-related dropouts (Cobley et al., 2014).
Methodological Considerations
This study has several limitations. Firstly, this study simply examines RAEs in Swiss soccer during the 2018/2019 season, which is not necessarily a reflection of the general situation over a longer period of time. Secondly, the data did not include specific match-based values (e.g., the number of matches played). Such data may provide a more complete picture of RAEs (Coelho et al., 2010). Nevertheless, this is the first study to include the complete nationwide database of both sexes across all age categories and selection levels. Therefore, this study highlights the origins of RAEs and how different selections influence the evolution of RAEs in youth soccer. Future research should address how waiting lists could be avoided and how relative age influences talent development pathways of athletes in the long run.
CONCLUSION
RAEs have a small but consistent effect on participation in Swiss children's football at the grassroots level. The effect is highest at entry level and decreases from U8 to U20. Contrary to expectations, no inverse RAEs were found on the waiting lists. As U8 is the first possible club entry, RAEs could partly originate from self-selection. Nonetheless, clubs using a selection system seem to increase RAEs. The first-time coach selection at the grassroots level is the origin of RAEs and increases the magnitude of RAEs during following selection processes. To protect young athletes from discrimination, RAE biases should be analyzed and eliminated at all stages of sport participation, selection, and dropout situations. Modifications to the organizational structure of sport and athlete development systems are recommended to prevent RAE-related inequalities.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics review board of the Swiss Federal Institute of Sport Magglingen. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. | 2020-12-03T14:18:26.363Z | 2020-12-03T00:00:00.000 | {
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235852444 | pes2o/s2orc | v3-fos-license | Evaluation of Daily Behaviors Related to Health Risks of the Ger Residents in Ulaanbaatar, Mongolia
: The capital city in Mongolia comprises two types of dwellings: apartments and Gers. The Ger is a traditional Mongolian house. Residents of Ger districts in Ulaanbaatar use coal as a source of fuel for daily activities such as heating and cooking in winter due to lack of central heating systems. After coal burning, the ash deposited at the bottom of the oven converts into a powder containing heavy metals that are toxic to human health. Through questionnaires at sites where concentrations of heavy elements were identified in our previous study, we examined the heavy metal exposure pathways of coal ash, which has potentially adverse effects on the health and quality of life of Ger district residents. Survey results indicate that residents have to store the coal ash in packing bags and metal garbage cans near their household fences for at least two weeks because of poor waste management. Owing to the weak seals of the packaging material and open metal cans that contain the coal ash, it is easily transported into surrounding areas by the wind. Adults come into contact with contaminated soil when they are cleaning areas outside and inside the fences, and children are exposed when they play outside the fences. Such activities enhance the dermal exposure as well as inhalation and ingestion pathways of the contaminants for adults and children.
Introduction
Currently, approximately 45% of the total population (3.1 million people) of Mongolia resides in the capital city, Ulaanbaatar. According to a report by the National Statistics Office of Mongolia in 2018, 60% of the residents of Ulaanbaatar live in Ger districts [1]. Owing to the lack of heating systems in the Ger districts, the residents use coal for their daily activities such as heating and cooking in the wintertime [2,3]. Numerous public health problems are caused by coal usage. In particular, coal ash has detrimental effects on air pollution and the health of people living in both Ger districts and apartments in Ulaanbaatar [4]. Although the studies concluded that there was no significant difference in health-related quality of life between both settlements during the warm season, healthrelated quality of life was worse for Ger residents in winter than for apartment residents owing to solid fuel combustion.
In addition, So et al. analyzed the lifestyle and living environment factors influencing the health of residents in Ger districts [5]. They observed that in self-reported surveys, residents cited symptoms such as eye irritation, heart-related disorders, nose and throat irritation, breathing difficulties or asthma, depression, and skin problems; illustrating the impact indoor air pollution had on the health of residents. Previous studies focused on the relationship between air pollution and public health. Likewise, soil contamination by coal ash could also have adverse effects on public health in the Ger areas.
Coal ash contains the heavy metals arsenic, lead, mercury, cadmium, chromium, selenium, aluminum, antimony, barium, beryllium, boron, chlorine, cobalt, manganese, molybdenum, nickel, thallium, vanadium, and zinc. Such heavy metals can cause cancer and have adverse impacts on the nervous system, including cognitive deficits, developmental delays, and behavioral problems if they are eaten, drunk, or inhaled [6][7][8]. They can also cause heart damage, lung disease, respiratory distress, kidney disease, reproductive problems, gastrointestinal illness, birth defects, and impaired bone growth in children that live near coal ash dumpsites [9,10]. Furthermore, such heavy metals can be easily transported through the air, and exposed humans can easily be affected through ingestion, inhalation, and dermal contact [11,12]. Consequently, the concentrations of heavy metals in soil in the Ger areas have been measured by several researchers [13,14] including our previous work [15].
Numerous studies have investigated the demographic aspects of the environmental pollution in Ger districts in Ulaanbaatar [3,[16][17][18][19][20][21][22][23]. Most of them explored the effects of air pollution on the health and quality of life of Ger residents. For instance, Y. Ishihara et al., studied the effects of particulate matter pollution on respiratory health, and reported the exacerbation of respiratory symptoms and worsening of health status in individuals aged more than 40 years in Ger districts in winter [22].
In 2016, Mongolia reported 38,699 cases of respiratory infections, which is the highest number recorded over the last five years [24]. Approximately 120-130 children and 1250 adults died of pneumonia last year; this is an increase of 8715 cases compared to the number in 2015. However, there is no assessed exposure to contaminated soil, especially soil polluted by coal ash near Ger residents and the correlation of the exposure to their daily lifestyles.
In our previous work, potential human health studies were performed based on the concentration of heavy metals in the samples collected from 42 sites in the Ger area of Ulaanbaatar city, Mongolia. In total, 28 heavy metals were identified using the ICP-OES and ICP-MS methods [15]. The carcinogenic risk was estimated to exceed the permissible limit for both adults and children at all of the sample sites. The total estimated noncarcinogenic risk exceeded the permissible limit or exceeded 1 at all of the sample sites, especially for children. Therefore, it is necessary to identify their daily behaviors in relation to contaminated soil.
The present study focuses on exposure pathways based on the daily activities of Ger residents, using a questionnaire survey administered in face-to-face interviews with Ger residents who live near areas where soil samples were collected in our previous study [15]. A questionnaire survey was used because it is simple and facilitates the identification of exposure pathways and their potential effects. The aim of the present study was to explore the role of exposure pathways, including frequency and duration of exposure in the course of residents' daily activities. Figure 1 is an illustration of a household living environment in the Ger, which is surrounded by fences. Ger residents can be exposed to coal ash pollution via dermal and inhalation pathways during the following daily activities:
1.
Removal and cleaning of coal ash from ovens 2.
Packing and disposal of coal ash inside and outside the fence
Methodology
The present study employed a social survey approved by the Tokyo Institute of Technology to collect information and assess the heavy metal exposure of Ger area residents in Ulaanbaatar, Mongolia. This study recorded exposure pathways by using a questionnaire survey to collect information on the daily activities of Ger residents who live near the areas contaminated by heavy metals based on soil samples gathered in the our previous study, as mentioned in the introduction [15].
In the survey, 126 representative households (three households in each of the 42 soil sampling sites) were interviewed. The participants' answers were marked on a questionnaire sheet in an Excel file.
Study Area
The study area covered five districts in Ulaanbaatar city: Songinokhairkhan, Chingeltei, Bayanzurkh, Sukhbaatar, and Nalaikh. Triangles in Figure 3 indicate the questionnaire survey sites. The resident survey was conducted between November 2019 and January 2020.
Methodology
The present study employed a social survey approved by the Tokyo Institute o Technology to collect information and assess the heavy metal exposure of Ger are residents in Ulaanbaatar, Mongolia. This study recorded exposure pathways by using questionnaire survey to collect information on the daily activities of Ger residents wh live near the areas contaminated by heavy metals based on soil samples gathered in th our previous study, as mentioned in the introduction [15].
In the survey, 126 representative households (three households in each of the 42 so sampling sites) were interviewed. The participants' answers were marked on questionnaire sheet in an Excel file.
Study Area
The study area covered five districts in Ulaanbaatar city: Songinokhairkhan Chingeltei, Bayanzurkh, Sukhbaatar, and Nalaikh. Triangles in Figure 3 indicate th questionnaire survey sites. The resident survey was conducted between November 201 and January 2020.
Methodology
The present study employed a social survey approved by the Tokyo Institute of Technology to collect information and assess the heavy metal exposure of Ger area residents in Ulaanbaatar, Mongolia. This study recorded exposure pathways by using a questionnaire survey to collect information on the daily activities of Ger residents who live near the areas contaminated by heavy metals based on soil samples gathered in the our previous study, as mentioned in the introduction [15].
In the survey, 126 representative households (three households in each of the 42 soil sampling sites) were interviewed. The participants' answers were marked on a questionnaire sheet in an Excel file.
Study Area
The study area covered five districts in Ulaanbaatar city: Songinokhairkhan, Chingeltei, Bayanzurkh, Sukhbaatar, and Nalaikh. Triangles in Figure 3 indicate the questionnaire survey sites. The resident survey was conducted between November 2019 and January 2020.
Structure of Questionnaire Survey
In this subsection, we elaborate on the structure of the questionnaire, which is illustrated in Figure 4. The questionnaire survey consists of 36 questions divided into five major sections. The first section of the questionnaire focuses on personal information such as the age, education, and occupation of the respondent. The subsequent three sections of the questionnaire survey focus on the daily activities and health status of the residents, as well as potential exposure pathways. The final section is focused on social aspects such as willingness to improve lifestyle and modify or change their living area.
Data Analyses
Data analyses were performed using SPSS v15.4 (SPSS Inc., Chicago, IL, US). Descriptive and multivariate analysis results were used to describe data. Principle Component Analysis (PCA) and factor analysis methods were used to reduce large sets
Structure of Questionnaire Survey
In this subsection, we elaborate on the structure of the questionnaire, which is illustrated in Figure 4. The questionnaire survey consists of 36 questions divided into five major sections. The first section of the questionnaire focuses on personal information such as the age, education, and occupation of the respondent. The subsequent three sections of the questionnaire survey focus on the daily activities and health status of the residents, as well as potential exposure pathways. The final section is focused on social aspects such as willingness to improve lifestyle and modify or change their living area.
Structure of Questionnaire Survey
In this subsection, we elaborate on the structure of the questionnaire, which is illustrated in Figure 4. The questionnaire survey consists of 36 questions divided into five major sections. The first section of the questionnaire focuses on personal information such as the age, education, and occupation of the respondent. The subsequent three sections of the questionnaire survey focus on the daily activities and health status of the residents, as well as potential exposure pathways. The final section is focused on social aspects such as willingness to improve lifestyle and modify or change their living area.
Data Analyses
Data analyses were performed using SPSS v15.4 (SPSS Inc., Chicago, IL, US). Descriptive and multivariate analysis results were used to describe data. Principle Component Analysis (PCA) and factor analysis methods were used to reduce large sets
Data Analyses
Data analyses were performed using SPSS v15.4 (SPSS Inc., Chicago, IL, US). Descriptive and multivariate analysis results were used to describe data. Principle Component Analysis (PCA) and factor analysis methods were used to reduce large sets of variables into smaller sets named principal components, which account for most of the variance in the original data.
Survey data generated 5 main sections in the questionnaire survey sheet, as mentioned earlier. The correlation between each family member (Section 1), and daily behavior and social aspects (other sections) was analyzed through PCA. The PCA used Varimax rotation and Kaiser normalization. After grouping in PCA, each factor identified the relationship between pairs of values by scattering using Excel to determine the correlation.
Results and Discussion
In this section, the main findings of the questionnaire survey are reported and discussed according to the questionnaire framework.
Personal Information
The results of the descriptive analysis are listed in Table 1. The total number of participants in the survey was 571. The number of men and women that participated in the survey was 267 (47%) and 304 (53%), respectively. Figure 5 describes the distribution of all the participants in 17 age groups. Most of the participants were aged between 0 and 16, which was 64% of the total, while groups aged 16 and over made up 36%. All participant information was collected from housewives and other householders during the questionnaire survey. of variables into smaller sets named principal components, which account for most of the variance in the original data. Survey data generated 5 main sections in the questionnaire survey sheet, as mentioned earlier. The correlation between each family member (Section 1), and daily behavior and social aspects (other sections) was analyzed through PCA. The PCA used Varimax rotation and Kaiser normalization. After grouping in PCA, each factor identified the relationship between pairs of values by scattering using Excel to determine the correlation.
Results and Discussion
In this section, the main findings of the questionnaire survey are reported and discussed according to the questionnaire framework.
Personal Information
The results of the descriptive analysis are listed in Table 1. The total number of participants in the survey was 571. The number of men and women that participated in the survey was 267 (47%) and 304 (53%), respectively. Figure 5 describes the distribution of all the participants in 17 age groups. Most of the participants were aged between 0 and 16, which was 64% of the total, while groups aged 16 and over made up 36%. All participant information was collected from housewives and other householders during the questionnaire survey. In total, 61% of respondents are labor force age, with 20% employed and 41% unemployed. The remaining 39% of participants were children and retirees. Figure 6 describes the occupation and income of all participants in the questionnaire survey. The average salary of about 76% of the particiapnts was between ₮450.000 and ₮850.000 ($157-$297). This is lower than the Mongolian average salary, which is around $682. The other 24% made closer to the average salary of Mongolia. This means the majority of In total, 61% of respondents are labor force age, with 20% employed and 41% unemployed. The remaining 39% of participants were children and retirees. Figure 6 describes the occupation and income of all participants in the questionnaire survey. The average salary of about 76% of the particiapnts was between of variables into smaller sets named principal components, which account for most of the variance in the original data.
Survey data generated 5 main sections in the questionnaire survey sheet, as mentioned earlier. The correlation between each family member (Section 1), and daily behavior and social aspects (other sections) was analyzed through PCA. The PCA used Varimax rotation and Kaiser normalization. After grouping in PCA, each factor identified the relationship between pairs of values by scattering using Excel to determine the correlation.
Results and Discussion
In this section, the main findings of the questionnaire survey are reported and discussed according to the questionnaire framework.
Personal Information
The results of the descriptive analysis are listed in Table 1. The total number of participants in the survey was 571. The number of men and women that participated in the survey was 267 (47%) and 304 (53%), respectively. Figure 5 describes the distribution of all the participants in 17 age groups. Most of the participants were aged between 0 and 16, which was 64% of the total, while groups aged 16 and over made up 36%. All participant information was collected from housewives and other householders during the questionnaire survey. In total, 61% of respondents are labor force age, with 20% employed and 41% unemployed. The remaining 39% of participants were children and retirees. Figure 6 describes the occupation and income of all participants in the questionnaire survey. The average salary of about 76% of the particiapnts was between ₮450.000 and ₮850.000 ($157-$297). This is lower than the Mongolian average salary, which is around $682. The other 24% made closer to the average salary of Mongolia. This means the majority of 450.000 and of variables into smaller sets named principal components, which account for most of the variance in the original data.
Survey data generated 5 main sections in the questionnaire survey sheet, as mentioned earlier. The correlation between each family member (Section 1), and daily behavior and social aspects (other sections) was analyzed through PCA. The PCA used Varimax rotation and Kaiser normalization. After grouping in PCA, each factor identified the relationship between pairs of values by scattering using Excel to determine the correlation.
Results and Discussion
In this section, the main findings of the questionnaire survey are reported and discussed according to the questionnaire framework.
Personal Information
The results of the descriptive analysis are listed in Table 1. The total number of participants in the survey was 571. The number of men and women that participated in the survey was 267 (47%) and 304 (53%), respectively. Figure 5 describes the distribution of all the participants in 17 age groups. Most of the participants were aged between 0 and 16, which was 64% of the total, while groups aged 16 and over made up 36%. All participant information was collected from housewives and other householders during the questionnaire survey. In total, 61% of respondents are labor force age, with 20% employed and 41% unemployed. The remaining 39% of participants were children and retirees. Figure 6 describes the occupation and income of all participants in the questionnaire survey. The average salary of about 76% of the particiapnts was between ₮450.000 and ₮850.000 ($157-$297). This is lower than the Mongolian average salary, which is around $682. The other 24% made closer to the average salary of Mongolia. This means the majority of 850.000 ($157-$297). This is lower than the Mongolian average salary, which is around $682. The other 24% made closer to the average salary of Mongolia. This means the majority of residents make a lower salary than the Mongolian average and most of them are not working while living in poor quality conditions. Approximately 46% of participants reported that they were settled in the area for ≤10 years, while the remaining 54% resided in the area for more than 10 years. The average size of land plots per Ger household is 700 m 2 , which is entitled to by law for individual households. The majority of land plots in Ger districts range between 470 m 2 to 589 m 2 per household. Private ownership of land and houses is generally high at around 99%.
Exposure Factors
According to a health risk assessment that was performed in our previous study, the dermal exposure pathway was associated with the highest non-carcinogenic and carcinogenic risks following exposure to contaminated soil in Ger residents. Such exposure can occur when residents come into contact with contaminated media such as soil or coal ash. Questions 13-21, which focused on the daily activities of respondents, explored potential dermal exposure. According to the survey results, Ger residents (specifically adults) often come into contact with soil and coal ash when they are cleaning areas in and outside the fence. Figure 8 shows the daily behaviors of adults. Around half (51%) of the participants reported that they spent about 11-30 min cleaning Approximately 46% of participants reported that they were settled in the area for ≤10 years, while the remaining 54% resided in the area for more than 10 years. The average size of land plots per Ger household is 700 m 2 , which is entitled to by law for individual households. The majority of land plots in Ger districts range between 470 m 2 to 589 m 2 per household. Private ownership of land and houses is generally high at around 99%.
Exposure Factors
According to a health risk assessment that was performed in our previous study, the dermal exposure pathway was associated with the highest non-carcinogenic and carcinogenic risks following exposure to contaminated soil in Ger residents. Such exposure can occur when residents come into contact with contaminated media such as soil or coal ash. Questions 13-21, which focused on the daily activities of respondents, explored potential dermal exposure. According to the survey results, Ger residents (specifically adults) often come into contact with soil and coal ash when they are cleaning areas in and outside the fence. Figure 8 shows the daily behaviors of adults. Around half (51%) of the participants reported that they spent about 11-30 min cleaning areas inside their fences. Another 49% reported that they spent more than 30 min and up to 2 h cleaning areas outside their fences. In addition, the frequency of cleaning is a key parameter influencing dermal exposure, and 53% of participants reported that they cleaned their fences once a week. Nevertheless, 88% of participants reported using safety items, such as masks, hats, and gloves, while the remaining 12% reported rarely using safety gear or not at all. areas inside their fences. Another 49% reported that they spent more than 30 min and up to 2 h cleaning areas outside their fences. In addition, the frequency of cleaning is a key parameter influencing dermal exposure, and 53% of participants reported that they cleaned their fences once a week. Nevertheless, 88% of participants reported using safety items, such as masks, hats, and gloves, while the remaining 12% reported rarely using safety gear or not at all. In the case of children, 118 children participated in the present study, and they did not have designated play areas. The child participants reported playing inside and outside their fences, as shown in Figure 9. Playing time inside and outside the fence was 30 min at minimum and 180 min at maximum. In the case of children, 118 children participated in the present study, and they did not have designated play areas. The child participants reported playing inside and outside their fences, as shown in Figure 9. Playing time inside and outside the fence was 30 min at minimum and 180 min at maximum. In addition, with regard to the ingestion pathway, 45% of participants reported th they always washed their hands before eating and preparing food, while 38% and 17 reported washing their hands often and sometimes, respectively.
We considered smoke as one of the main sources of heavy metal transfer from ha to mouth in Ger residents. This study is focused on explicating this aspect of t exposure pathways in relation to contaminated soil, and not about the toxicity of smo to human health. Two questions related to smoking were asked in the questionnai Eleven percent of the total respondents reported that they smoked a maximum of cigarettes per day. Each instance of smoking is a case of ingestion of contaminant fro hand to mouth. This implies that their habit of smoking after contact with contaminat soil is one of the main pathways of heavy metal ingestion. In addition, with regard to the ingestion pathway, 45% of participants reported that they always washed their hands before eating and preparing food, while 38% and 17% reported washing their hands often and sometimes, respectively.
We considered smoke as one of the main sources of heavy metal transfer from hand to mouth in Ger residents. This study is focused on explicating this aspect of the exposure pathways in relation to contaminated soil, and not about the toxicity of smoke to human health. Two questions related to smoking were asked in the questionnaire. Eleven percent of the total respondents reported that they smoked a maximum of 20 cigarettes per day. Each instance of smoking is a case of ingestion of contaminant from hand to mouth. This implies that their habit of smoking after contact with contaminated soil is one of the main pathways of heavy metal ingestion.
Inhalation exposure can occur when adults and children pass through or play in contaminated soil. In addition, inhalation exposure can occur when Ger residents are cleaning their stoves and disposing of coal ash without using appropriate masks, since it is easily transported by air. In the survey, 88% of participants reported passing near the soil sampling points two to four times per day. If they passed a high-risk area for 1.5 min per day, the duration of spent time at high risk could be 9.1 h per year. Figure 10 shows the results of questions length of living year and frequency of pass sampling points per day in questionnaire surveys. In addition, with regard to the ingestion pathway, 45% of participants reported t they always washed their hands before eating and preparing food, while 38% and 1 reported washing their hands often and sometimes, respectively.
We considered smoke as one of the main sources of heavy metal transfer from h to mouth in Ger residents. This study is focused on explicating this aspect of exposure pathways in relation to contaminated soil, and not about the toxicity of sm to human health. Two questions related to smoking were asked in the questionna Eleven percent of the total respondents reported that they smoked a maximum of cigarettes per day. Each instance of smoking is a case of ingestion of contaminant fr hand to mouth. This implies that their habit of smoking after contact with contamina soil is one of the main pathways of heavy metal ingestion.
Inhalation exposure can occur when adults and children pass through or play contaminated soil. In addition, inhalation exposure can occur when Ger residents cleaning their stoves and disposing of coal ash without using appropriate masks, sinc is easily transported by air. In the survey, 88% of participants reported passing near soil sampling points two to four times per day. If they passed a high-risk area for 1.5 m per day, the duration of spent time at high risk could be 9.1 h per year. Figure 10
Living Conditions
Based on the results of the questionnaire survey, coal ash is one of the main sources of soil pollution in the Ger area. At least 1565 kg of coal is generated by 126 Ger households per day. The residents keep the coal ash in packing bags and metal garbage cans ( Figure 5) outside and inside the fence for at least two weeks because of poor waste management. Owing to weak seals and open metal cans, coal ash waste could easily be transported by wind and air into surrounding areas. The respondents reported that briquette coal had reduced levels of air pollution in the capital city and increased thermal capacity; however, it increased the quantity of coal ash disposed of by more than around 3-5 kg per packing than in previous years. Coal is the main source of fuel for Ger residents in the wintertime. All the residents use coal from October to April. They use 21-30 kg of coal per day and generate the same amount of coal ash. Figure 11 shows coal usage and coal ash quantity.
Living Conditions
Based on the results of the questionnaire survey, coal ash is one of the main sources of soil pollution in the Ger area. At least 1565 kg of coal is generated by 126 Ger households per day. The residents keep the coal ash in packing bags and metal garbage cans ( Figure 5) outside and inside the fence for at least two weeks because of poor waste management. Owing to weak seals and open metal cans, coal ash waste could easily be transported by wind and air into surrounding areas. The respondents reported that briquette coal had reduced levels of air pollution in the capital city and increased thermal capacity; however, it increased the quantity of coal ash disposed of by more than around 3-5 kg per packing than in previous years. Coal is the main source of fuel for Ger residents in the wintertime. All the residents use coal from October to April. They use 21-30 kg of coal per day and generate the same amount of coal ash. Figure 11 shows coal usage and coal ash quantity.
Health Issues
Participants reported their health conditions according to the questions in the survey. According to the results, most of the respondents considered their health status as Good (51%), followed by Neither good nor bad (26%), Very good (17%), and Bad (6%). According to Figure 12, in the cold season, approximately 46%, 35%, and 19% of residents reported various symptoms such as rheum, lung disease, and sore throat, respectively. In winter, their children had similar diseases, however, the frequency of symptoms was higher in children than in adults. All the respondents reported none of Figure 11. Coal usage and quantity of coal ash disposal.
Health Issues
Participants reported their health conditions according to the questions in the survey. According to the results, most of the respondents considered their health status as Good (51%), followed by Neither good nor bad (26%), Very good (17%), and Bad (6%). According to Figure 12, in the cold season, approximately 46%, 35%, and 19% of residents reported various symptoms such as rheum, lung disease, and sore throat, respectively. In winter, their children had similar diseases, however, the frequency of symptoms was higher in children than in adults. All the respondents reported none of the symptoms mentioned above during the summer.
Health Issues
Participants reported their health conditions according to the questions survey. According to the results, most of the respondents considered their health as Good (51%), followed by Neither good nor bad (26%), Very good (17%), an (6%). According to Figure 12, in the cold season, approximately 46%, 35%, and 1 residents reported various symptoms such as rheum, lung disease, and sore t respectively. In winter, their children had similar diseases, however, the freque symptoms was higher in children than in adults. All the respondents reported n the symptoms mentioned above during the summer. Previous studies by Byambadorj, et al. [3] and Nakao et al. [4] mentioned the issues concerning health over the duration of winter. Moreover, So et al. reporte residents taking off their shoes when they came inside. As mentioned in these r some of them tried to wash the dust off their shoes when they came into the Ge Previous studies by Byambadorj, et al. [3] and Nakao et al. [4] mentioned the same issues concerning health over the duration of winter. Moreover, So et al. reported Ger residents taking off their shoes when they came inside. As mentioned in these results, some of them tried to wash the dust off their shoes when they came into the Ger. This could also have worsened their health issues as they wore the same set of shoes both in and outside of the Ger, and most of the roads in the Ger district in Ulaanbaatar are unpaved [5].
Social Aspects
The last part of the questionnaire explored the social aspects of residents, with participants answering questions related to quality of life and district issues needing to be resolved. According to most of the residents, waste management was the priority, which is shown in Figure 13. According to the residents, the frequency of waste collection should be increased, considering the waste is collected only two times a month. Other issues considered by the participants to be a priority in the Ger area included kindergartens, heating systems, toilets, schools, and drinking water. could also have worsened their health issues as they wore the same set of shoes both in and outside of the Ger, and most of the roads in the Ger district in Ulaanbaatar are unpaved [5].
Social Aspects
The last part of the questionnaire explored the social aspects of residents, with participants answering questions related to quality of life and district issues needing to be resolved. According to most of the residents, waste management was the priority, which is shown in Figure 13. According to the residents, the frequency of waste collection should be increased, considering the waste is collected only two times a month. Other issues considered by the participants to be a priority in the Ger area included kindergartens, heating systems, toilets, schools, and drinking water. In addition, primary school attendance is less in Ger districts than in other apartment areas. Most of the schools are located too far from the children's homes. Some of them travel to school at least 1-2 km by foot. Moreover, the duration of their stay is limited owing to the lack of school capacity. After 3-5 h, they must go back to their homes. This is a high-pressure issue for children. However, only about 4% of the adult participants were concerned because there are many problems in the Ger area other than lack of schooling.
Overall, each of the respondents reported a desire to improve their quality of life and strongly agreed that they would give up their land ownership for an apartment dwelling. Figure 6 shows the most pressing issues for Ger district residents. In addition, primary school attendance is less in Ger districts than in other apartment areas. Most of the schools are located too far from the children's homes. Some of them travel to school at least 1-2 km by foot. Moreover, the duration of their stay is limited owing to the lack of school capacity. After 3-5 h, they must go back to their homes. This is a high-pressure issue for children. However, only about 4% of the adult participants were concerned because there are many problems in the Ger area other than lack of schooling.
Overall, each of the respondents reported a desire to improve their quality of life and strongly agreed that they would give up their land ownership for an apartment dwelling. Figure 6 shows the most pressing issues for Ger district residents.
Principal Component Analysis (PCA) Results
This section reports the PCA results. Before performing PCA, Bartlett's test of sphericity and Kaiser-Meyer-Olkin (KMO) were performed to measure sample adequacy. The results of the Bartlett test of sphericity and KMO showed that the data is sufficient (p < 0.0) and adequate (p > 0.8) for PCA analysis. The total variance and rotated component matrix results are listed in Tables 2 and 3, respectively. The four factor analyses were extracted and grouped. Four components explained 72.7% of the variance. The rotated component score contained variables with strong correlation results based on bivariate analysis. Four components that explained 72.7% of the variance were extracted and grouped using PCA. The first principal component explained 46.9% of the total variance. Results show higher scores with the highest probability of their daily activity related to coal waste during time spent drinking water and cleaning fences. Therefore, Factor 1 was labeled "daily activity". The second component explained 9.3% of the variance, and it was related to the playground area and time spent staying at home. Therefore, Factor 2 was labeled "time of staying home". The third principal component explained 8.6% of the variance. It explains that the smoking quantity scored the highest values in component 3. Therefore, Factor 3 is labeled as "frequency of smoking". The fourth principal component explained 7.9% of the variance and was associated with frequency of family members passing through the sampling sites each day and hand washing. Consequently, Factor 4 is labeled "source of exposure".
Factor analysis results based on the first two principal components accounting for most of the variance are illustrated in Figures 14-16. Figure 14 illustrates the relationship between daily activity and time spent staying at home based on employment status and education level. According to Figure 14, more educated and employed individuals are more active than the unemployed and retirees, as well as children, who also spend more time at home.
From a health and safety perspective, relatively educated and employed people use safety gear such as gloves and masks when they are cleaning their fences. Moreover, they are eager to change and improve their living conditions and lifestyles. Conversely, in Figure 15, the relationship between daily activities and time staying at home are demonstrated in terms of age and duration of residence in the Ger districts. All respondents reported cleaning their fences. However, according to Figure 15, the residents aged more than 19 years tended to spend more time on daily activities and stayed home less when compared with children; which is reasonable because children in the Ger districts stay at home except during school time. In the case of infants, they stay home with their parents or grandparents due to inadequate nursery and kindergarten facilities. According to Figure 16, educated and employed people were at greater risk of exposure to contaminated soil compared to children, the unemployed, and retirees, who all spent most of their time at home (Figure 14). The employed and the relatively educated pass through the sample sites at least two times a day as they go to work and schools, according to the results of the survey.
Factor analysis results based on the first two principal components accounting for most of the variance are illustrated in Figures 14-16. Figure 14 illustrates the relationship between daily activity and time spent staying at home based on employment status and education level. According to Figure 14, more educated and employed individuals are more active than the unemployed and retirees, as well as children, who also spend more time at home. Factor analysis results based on the first two principal components accounting for most of the variance are illustrated in Figures 14-16. Figure 14 illustrates the relationship between daily activity and time spent staying at home based on employment status and education level. According to Figure 14, more educated and employed individuals are more active than the unemployed and retirees, as well as children, who also spend more time at home. From a health and safety perspective, relatively educated and employed people use safety gear such as gloves and masks when they are cleaning their fences. Moreover, they are eager to change and improve their living conditions and lifestyles. Conversely, in Figure 15, the relationship between daily activities and time staying at home are
Conclusions
In this study, we explored the potential heavy metal exposure pathways for Ger residents based on their lifestyles using face-to-face questionnaires. All respondents reported using coal as a source of heating and cooking, but only in wintertime. During the winter, each household burns approximately 25 kg of coal and an equivalent amount of coal ash is produced per day. All respondents answered that they dispose of the coal ash but it is left for two weeks in or outside of their fences until garbage trucks collect them. Most of the Ger residents do not dispose of the coal ash in sealed dust bins. Consequently, over the two-week period, the surrounding area becomes contaminated because coal ash is easily transported by wind, leading to the contamination of the soil in the Ger area.
Ger district residents could get exposure by coming into contact with the contaminated soil when they are cleaning areas both in and outside their fences. According to survey results, adult Ger residents' hands and noses often become dirty, indicating dermal and inhalation exposure pathways of coal ash pollution. In the case of children, dermal, inhalation, and ingestion exposure can occur when they play inside or outside the fence. Based on the disease history of respondents, rheum, lung disease, and sore throat were the major symptoms. According to factor analysis, when compared with unemployed individuals, retirees, and children, the employed and those with more education were more at risk of exposure because they are relatively more active in their fence cleaning activities, and in turn, more likely to come into contact with contaminated soil.
The capital city has expanded to 30 times larger than the original inhabited areas, especially by Ger districts. The Mongolian government has been developing strategic policies since 2013, such as Master Plans, to better manage the expansion of Ger areas. However, its improvements on urban services are slow. Ger areas have been continuing to expand until recently, and the local government's capacity to respond to these challenges is still limited [25].
As a consequence of the incomplete combustion in traditional inefficient stoves, coal combustion in Gers is the main source of air pollution not only because of particulate matter but also soil pollution. This makes Ulaanbaatar one of the most polluted cities in the world [26][27][28]. Environmental pollution of the capital city is mainly derived from incomplete coal combustion in Gers, and frequently exceeds 10 times the national permissible level (50 µg/m 3 ) in winter [28]. Therefore, coal combustion emissions may be one of the major sources of metal pollution in Ulaanbaatar soil.
We have identified the frequency and time of three exposure pathways from contaminated soil of the Ger areas. The main findings of this present study will be the basis of our future research relating to quantitative evaluation of exposure pathways to reduce adverse effects on human health from contaminated soil. This study can be a helpful source to reduce risk and improve the social, economic and legal environment. | 2021-07-16T00:05:58.622Z | 2021-04-25T00:00:00.000 | {
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1922415 | pes2o/s2orc | v3-fos-license | Intersection theorems for multisets
Let $k$, $t$ and $m$ be positive integers. A $k$-multiset of $[m]$ is a collection of $k$ integers from the set $\{1,...,m\}$ in which the integers can appear more than once. We use graph homomorphisms and existing theorems for intersecting and $t$-intersecting $k$-set systems to prove new results for intersecting and $t$-intersecting families of $k$-multisets. These results include a multiset version of the Hilton-Milner theorem and a theorem giving the size and structure of the largest $t$-intersecting family of $k$-multisets of an $m$-set when $m \leq 2k-t$.
Introduction
In this paper, we show that the method used in [13] to prove a natural extension of the famous Erdős-Ko-Rado theorem to multisets can be used to prove additional intersection theorems for multisets.
We prove a multiset version of the Hilton-Milner theorem; this result gives the largest family of intersecting multisets satisfying the condition that the intersection of all of the multisets in the family is empty. We determine the largest family of k-multisets having the property that no more than s of the multisets from the family can be pairwise disjoint. A related question that we answer is, what is the largest family of multisets that can be partitioned into two intersecting families? We also consider multisets that have intersection of size at least t, so are t-intersecting. We prove a theorem giving the size and structure of the largest t-intersecting family of k-multisets of an m-set when m ≥ 2k − t. Finally, we prove a version of the Hilton-Milner theorem for t-intersecting multisets.
In Section 1, we introduce notation and provide some background information on the Erdős-Ko-Rado theorem and the known results for intersecting families of multisets. Additional known results for intersecting set systems are stated in Section 2. In Section 3, we extend the results from Section 2 to families of multisets. Section 4 contains our results concerning t-intersecting families of multisets. In Section 5, we discuss some open problem for multisets.
Notation and definitions
Throughout this paper, small letters are used to denote integers, capital letters are used for sets (and multisets) of integers, and script capital letters are used for collections of objects. The set of integers from x to y inclusive is represented by [x, y]. If x = 1, this is simplified to [y].
A k-set (or k-subset) is a set of cardinality k and the collection of all k-subsets of [n] is denoted by [n] k . We say that a collection of sets is intersecting if every pair of sets in the collection is intersecting and that it is t-intersecting if every pair has at least t elements in common. Collections are said to be isomorphic if one can be obtained from the other by a permutation of the underlying set.
Background
The Erdős-Ko-Rado theorem is an important result in extremal set theory that gives the size and structure of the largest intersecting ksubset system from [n]. It appeared in a paper published in 1961 [5] which contains two main theorems. The first of these is commonly stated as follows: 1.1 Theorem (Erdős, Ko and Rado [5]). Let k and n be positive integers with n ≥ 2k. If F is a collection of intersecting k-subsets of [n], then Moreover, if n > 2k, equality holds if and only if F is a collection of all the k-subsets from [n] that contain a fixed element from [n].
A Kneser graph, denoted by K(n, k), is a graph whose vertices are the k-subsets of [n]. Two vertices are adjacent if and only if the corresponding k-subsets are disjoint. Thus an independent set of vertices is an intersecting set system and the cardinality of the largest independent set is equal to n−1 k−1 . (It is assumed that n ≥ 2k since otherwise the graph would be the empty graph.) A second theorem in [5] gives the size and structure of the largest t-intersecting k-subset system provided that n is sufficiently large relative to k and t. A later theorem due to Ahlswede and Khachatrian extends this result to all values of n, k and t. In virtually all cases, the families attaining maximum size are isomorphic to F (r) , where F (r) is defined as follows. For t, k, n ∈ N, let In the following statement of the theorem, it is assumed that n > 2k − t since for n ≤ 2k − t, the collection of all k-subsets of [n] is t-intersecting. [1]). Let t, k and n be positive integers with t ≤ k ≤ n and let r be a non-negative integer such that r ≤ k − t.
If
, then F (r) = F (r+1) and a system of maximum size will equal (up to isomorphism) F (r) or F (r+1) .
In this paper, we present intersection theorems for multisets. A multiset is a generalization of a set in which an element may appear more than once. We define the cardinality of a multiset as the total number of elements in the multiset including repetitions. A k-multiset is a multiset of cardinality k and the collection of all k-multisets containing elements from [m] is denoted by [m] k . The size of this collection is denoted by m k and is equal to m+k−1 k . Let m(i, A) denote the multiplicity of the element i in the multiset A. We define the intersection of two multisets, A and B, as the multiset C where m(i, This definition can be applied to the intersection of a multiset and a set by considering the set to be a multiset where each element has multiplicity one. For a multiset, A, the support of A, denoted S A , is the set of distinct elements of A. A collection of k-multisets from [m] is intersecting if every pair of multisets from the collection is intersecting and it is t-intersecting if the intersection of every pair of multisets is a multiset with cardinality at least equal to t. A graph homomorphism was used in [13] to prove the next theorem which extends a previous result of Brockman and Kay [4]. This graph homomorphism is used in Section 3 to prove additional theorems concerning intersecting collections of multisets. [13]). Let k and m be positive integers with m ≥ k + 1. If G is a collection of intersecting k-multisets of [m], then A result concerning the size of the largest t-intersecting families of k-multisets from [m] was recently proved by Füredi, Gerbner and Vizer [9] using an operation which they call down-compression. In the following theorem, AK(m + k − 1, k, t) represents the size of the largest t-intersecting collection of k-subsets from [m+k −1] given by Theorem 1.2.
Since m ≥ 2k − t ensures that m ≥ t + 2r where r is given by Theorem 1.2 for n = m + k − 1, this size can be attained by a family of multisets of the form In Section 4, we use the down-compression operation from [9] and a graph homomorphism to add the structure of the families that attain this size.
Theorems for set systems
In this section, we state some additional known results inspired by the Erdős-Ko-Rado theorem. These theorems are used in Section 3 to prove analagous results for multisets. Define an intersecting family of k-sets as follows: Then where n−1 k−1 counts the k-subsets of [n] that contain 1 and n−k−1 k−1 counts the k-subsets containing 1 that do not intersect with [2, k+1].
In [11], Hilton and Milner considered the size of the largest intersecting family of k-subsets of [n] where there is no element common to all subsets. They proved the following theorem which disproves a conjecture in [5]. [11]). Let k and n be positive integers with 2 ≤ k ≤ n/2. Let F be an intersecting family of k-subsets
In [10], Hajnal and Rothschild generalize the Erdős-Ko-Rado theorem to families of k-subsets of [n] that are not completely pairwise t-intersecting. These families are defined as having the property that, for s, t ≥ 1, no more than s of the k-subsets have pairwise fewer than t elements in common. We denote this property by P (s, t). A family with property P (s, t) can be constructed by choosing s disjoint t-subsets of [n] and taking all of the k-subsets that contain at least one of these t-subsets. Such a family is said to be fixed by the t-subsets. [10]). Let F be a family of ksubsets of [n]. Suppose F satisfies P (s, t). Then there is a function n(k, s, t) such that if n > n(k, s, t),
Theorem (Hajnal and Rothschild
Equality holds if only if F is the family of all k-subsets of [n] fixed by some s disjoint t-subsets of [n].
When t = 1, the number of k-sets of [n] fixed by s disjoint 1subsets of [n] is equal to the number of k-sets that intersect a given s-set. Thus If t = s = 1, a family satisfying P (s, t) is an intersecting family of ksubsets and the theorem is equivalent to the Erdős-Ko-Rado theorem. For t = 1 and s ≥ 1, a family with property P (s, t) corresponds to the vertex set of an induced subgraph of the Kneser graph that does not contain a clique of size s + 1. In particular, a family with property P (2, 1) will correspond to a triangle-free subgraph of K(n, k). A number of upper bounds for n(k, s, 1) when Theorem 2.2 is restricted to t = 1 have appeared in the literature. These include 2k 3 s in [3] and 3k 2 s in [12]. Most recently, Frankl, Luczak and Mieczkowska proved the following result. [8] A slightly different problem is addressed by Frankl and Füredi in [7]. They prove the following theorem giving the maximum possible size of the union of two intersecting families of k-sets of [n] where the two families are not required to be disjoint. This is equivalent to the number of vertices in the largest induced bipartite subgraph of the Kneser graph K(n, k). Any set that is in both of the intersecting families can be arbitrarily assigned to one partition or the other and will be an isolated vertex in the induced subgraph. A bipartite subgraph is triangle-free, that is, the vertices in the subgraph have property P (2, 1). However, property P (2, 1) does not imply that the induced subgraph in K(n, k) is bipartite. [7]). Let F 1 and F 2 be intersecting families of k-subsets of [n]. If n > 1 2 (3 + √ 5)k, then
Intersecting multisets
For positive integers k and m, let M (m, k) be the graph whose vertices are the k-multisets from [m] and where two vertices are adjacent if and only if the corresponding k-multisets are disjoint (i.e. the intersection of the two multisets is the empty set). The graph M (m, k) has m+k−1 k vertices and an independent set of vertices is an intersecting collection of k-multisets.
The following proposition is proved in [13].
Proposition. Let m and k be positive integers and set
k with the following properties: We will use this graph homomorphism to prove three new results for multisets based on the three theorems for set systems stated in Section 2.
The next proposition is used in the proof of Theorem 3.3. The term maximal refers to a collection of multisets, Proof. For a given A ∈ G, let B be any set of (k − |S A |) elements from [m]\S A and let C = B ∪ S A . Then |S C | = k and C will intersect with all multisets in G. Since G is maximal, it follows that C ∈ G.
Define an intersecting k-multiset system, G, as follows: Then We now show that the size of the largest intersecting collection of kmultisets with no element common to all of the multisets is equal to the size of G when m ≥ k + 1. (For 2 < m < k + 1, the largest intersecting families with no common element are the largest intersecting families as given by Theorem 1.3 in [13].)
Theorem. Let k and m be positive integers with
Proof. Let G be an intersecting family of k-multisets from [m] of maximum possible size such that This implies that k be a function with the properties given in Proposition 3.1.
Combining this with Equation 3.1 gives
Since B is an intersecting k-set system with no element common to all the sets and n ≥ 2k, the Hilton-Milner Theorem (Theorem 2.1) gives To prove the uniqueness statement in the theorem, let m > k+1 and let G be an intersecting multiset system of size m+k−2 With the function used above, the preimage of G will be an independent set in K(n, k) of size n−1 Specifically, there will exist some set X ⊂ [n] with |X| = k and some k : y ∈ B and B ∩ X = ∅} ∪ X. Note that X ⊂ [n]\{y} and, since n = m + k − 1, there must be at least one element from [m] in X.
Suppose that y / ∈ [m]. Then X must contain at least two elements from [m]. Call these elements x 1 and x 2 . Then the sets . Then there exists some x ∈ X such that x ∈ [m+1, n] and the set B = {y, m+1, . . . , n} will be in B since Thus X ⊂ [m] and y ∈ [m] and it follows from the properties of f that [m] k : y ∈ A and A ∩ X = ∅} ∪ X (i.e. G will be isomorphic to G).
If k = 3 or m = k+1, then families attaining the maximum size are not limited to those isomorphic to G. For example, G (1) will attain the maximum size even though it is not isomorphic to G.
and if m = k + 1, 3.4 Theorem. Let G be a family of k-multisets of [m] and suppose that G satisfies P (s, 1), that is, no more than s of the multisets are Equality holds if and only if G is the family of all k-multisets that intersect with a given set of size s.
Proof. Let G be a family of k-multisets from [m] of maximum possible size having property P (s, 1) and assume that m > 2k 2 s log k − k + 1. Set n = m + k − 1 and let f : k be a function with the properties given in Proposition 3.1.
Since m > 2k 2 s log k −k+1 > s, a family consisting of all k-multisets that intersect with [s] will attain this size and will have property P (s, 1). Since G was assumed to be as large as possible, it follows that From Theorem 2.3, we know that f −1 (G) is a family consisting of all k-subsets of [n] generated by some s disjoint t-subsets of [n]. Since t = 1, this is equivalent to the family consisting of all k-subsets that intersect with a given s-set from [n]. If this s-set, S, is a subset of [m], then it follows from the definition of f that G consists of all k-multisets that intersect S. If S is not a subset of [m], then there will be some y ∈ S such that y ∈ [m + 1, n]. Thus for all x ∈ [m], the set {x, m+1, . . . , n} will be in f −1 (G) and the multisets consisting of k copies of x will be in G. Since m ≥ s + 1, this contradicts our assumption that G has property P (s, 1). Therefore, if m > 2k 2 s log k −k+1, and G is a family of k-multisets of [m] with property P (s, 1) having the maximum possible size, then G consists of all k-multisets that intersect with a given set of size s.
If G 1 consists of all k-multisets containing 1 and G 2 consists of all k-multisets containing 2 but not 1, then the upper bound given in the theorem. Note that this is equal to the size of the family consisting of all k-multisets that intersect with {x, y} and that To prove the uniqueness portion of the theorem, assume that G 1 ∪G 2 is as large as possible. Then
t-intersecting multisets
The main result in this section is the following theorem which is a restatement of Theorem 1.4 with the addition of the structure of the t-intersecting multiset families that attain the maximum size. Recall that G (r) is defined as 4.1 Theorem. Let 1 ≤ t ≤ k and let r be a non-negative integer such that r ≤ k − t. Let G be a t-intersecting collection of k-multisets of [m] with m ≥ 2k − t.
If
then |G| ≤ G (r) . (By convention, t−1 r = ∞ for r = 0.) If r > 0, equality holds if and only if G is isomorphic to G (r) . If r = 0, equality holds if and only if G consists of all k-multisets containing a fixed t-multiset.
If
If r > 0, equality holds if and only if G is isomorphic to either G (r) or G (r+1) . If r = 0, equality holds if and only if G is isomorphic to G (1) or it consists of all k-multisets containing a fixed t-multiset.
Our proof uses a graph homomorphism between two graphs having the same vertex sets as K(n, k) and M (m, k) but where independent sets of vertices are t-intersecting families of k-sets and k-multisets respectively. We begin by defining these graphs and showing that a function with the properties given in Proposition 3.1 exists.
Let K(n, k, t) be the graph whose vertices are the k-subsets of [n] and where two vertices, A and B, are adjacent if and only if |A ∩ B| < t. An independent set of vertices in K(n, k, t) will be a t-intersecting family of k-subsets of [n].
Let M ′ (m, k, t) be the graph whose vertices are the k-multisets of
For any A ∈ [n]
k , the support of f (A) is equal to A ∩ [m]. 3. f is a graph homomorphism from K(n, k, t) to M ′ (m, k, t).
Proof.
The first two properties follow from Proposition 3.1. Let A 1 , A 2 be two adjacent vertices in K(n, k, t). Then |A 1 ∩ A 2 | < t and it follows from Property 2 that Hence the bijection k is a graph homomorphism from K(n, k, t)) to M ′ (m, k, t).
If f is a function with the properties given in Propostion 4.2 and G is an independent set in M ′ (m, k, t), then f −1 (G) will be an independent set in K(n, k, t). Thus Theorem 1.2 gives an upper bound on the size of the largest independent set in M ′ (m, k, t).
We also make use of the down-compression operation defined in [9]. This operation maintains or increases the size of the supports of the multisets in a t-intersecting family of multisets while maintaining the size and t-intersection of the family. Repeated applications of this operation will transform any t-intersecting family of multisets into a t-intersecting family of the same size but with the additional property that |F 1 ∩ F 2 ∩ [m]| ≥ t for all F 1 , F 2 in the family. We begin with some notation and definitions of concepts from [9].
Note that the multiset containing t copies of each of the integers in [m] will be a t-kernel for any t-intersecting family. Let K(G) denote the set of all t-kernels for G that contain [m]. For T ∈ K(G), let T >1 = T \[m]. Thus T >1 contains only elements that occur more than once in T .
A multiset can be represented by a set of ordered pairs where the first integer is an element of [m] and the second integer serves to differentiate among the copies of that element in the multiset. For example, the multiset {1, 1, 1, 4} can be written as {(1, 1), (1, 2), (1, 3), (4, 1)}. This ordered pair notation is used in the definition of the following shifting operation.
For s ≤ m(i, F ) and j ∈ [m] with j / ∈ F , let F (i,s)(j) denote the multiset formed from F by replacing all but s − 1 of the copies of i with j. That is, If s > m(i, F ), then F (i,s)(j) = F . Füredi et al. [9] define a shifting operation on G as follows: Provided that s ≥ 2, this operation increases the size of the support of any multiset F such that S (i,s)(j) (F ) = F . For example, if F = {1, 1, 1, 3, 4} then S (1,2)(2) (F ) = {1, 2, 2, 3, 4} and the size of the support increases by one.
The down-compression operation consists of combining this shifting operation with the concept of t-kernels. Given a t-intersecting family of k-multisets from [m] and a t-kernel, T , such that [m] ⊆ T , the down-compression operation consists of sequentially applying the shifting operation for some i ∈ [m] such that i ∈ T >1 and for all values of j from 1 to m with s = m(i, T ). The next lemma shows that applying the down-compression operation gives a t-intersecting family of k-multisets of the same size as the original family and proves that the new family will have T \{i, m(i, T )} as a t-kernel. The second statement means that the down-compression operation can be performed on G using T \{i, m(i, T )} as the t-kernel. Repeated applications of the operation results in a family that has [m] as a t-kernel. Thus any t-intersecting collection of k-multisets of [m] can be transformed into a collection of the same size in which [9] ). Let 1 ≤ t ≤ k and let
Theorem (Füredi, Gerbner and Vizer
We use a similar approach to prove Theorem 4.1. We first prove the result for families where |F 1 ∩ F 2 ∩ [m]| ≥ t for all F 1 , F 2 in the family and then show that this can be extended to all t-intersecting families using the down-compression operation.
4.5 Lemma. Let 1 ≤ t ≤ k and let r be a non-negative integer such
If
then |G| ≤ G (r) . (By convention, t−1 r = ∞ for r = 0.) Equality holds if and only if G is isomorphic to G (r) . Then F = f −1 (G) is an independent set in K(n, k, t) and so applying Theorem 1.2 gives |F| ≤ F (r) for some r ∈ [0, (k − t)]. Thus |G| ≤ F (r) and since F (i) = G (i) whenever both are defined, it follows that |G| ≤ G (r) . We now consider the structure of the families attaining the maximum possible size in the two statements in the lemma.
If
That is, F will consist of all k-subsets of [n] containing at least t + r elements of X, where X is a subset of [n] of size t + 2r. If X ⊆ [m], it follows from the definition of the function f that every multiset in G will contain t + r elements of X. Thus G is isomorphic to G (r) . If X [m], then there is some x ∈ X such that x / ∈ [m]. Since f −1 (G) will consist of all k-subsets of [n] containing at least t + r elements of X, there will be a pair of m, k, t) which contradicts our assumption that G is an independent set in M ′ (m, k, t). Therefore, any family satisfying the conditions in the lemma is isomorphic to G (r) .
Let
r+1 . Again using Theorem 1.2 we have that F attains the maximum possible size if and only if F is isomorphic to either F (r) or F (r+1) . The argument used in the first case can be used to show that G must be isomorphic to either G (r) or G (r+1) . Lemma 4.5 along with Theorem 4.3 are sufficient to prove the size of the largest t-intersecting families as given in Theorem 4.1. The next two lemmas are needed to extend the structure portion of Lemma 4.5 to all t-intersecting multiset families. The first deals with the case when down-compression of the family of maximum size results in a family isomorphic to G (r) for some r > 0. The second deals with the case when down-compression of the family results in a family isomorphic to G (0) . The need to treat r = 0 separately arises from the fact that a family consisting of all k-multisets containing a fixed t-multiset will have the same cardinality regardless of the multiplicity of each of the elements in the t-multiset. Thus there are families of multisets that have the same size as G (0) but that are not isomorphic to G (0) . For example, if m = 5, k = 4 and t = 2, the family consisting of all 4multisets containing {1, 1} is not isomorphic to G (0) but has the same size as G (0) , the family consisting of all 4-multisets containing {1, 2}. This does not occur when r > 0. If r > 0, a family consisting of all k-multisets containing at least t + r elements from a (t+2r)-multiset in which the multiplicity of some element is greater than one contains fewer k-multisets than G (r) . For instance, if m = 5, k = 4 and t = 2, then since there are only three distinct ways to select three elements from {1, 1, 2, 3}.
Recall that G is the family of k-multisets formed by applying the down-compression operation to G, a t-intersecting family of kmultisets from [m]. It is defined as where i ∈ T >1 for some t-kernel T ∈ K(G) and s = m(i, T ).
k be a t-intersecting family of multisets. For T ∈ K(G) and i ∈ [m] such that i ∈ T >1 , set s = m(i, T ). If G is isomorphic to G (r) for some r > 0, then so is G.
Proof. It is sufficient to show that if S (i,s)(j) (G) is isomorphic to G (r) for some r > 0, then so is G. Let S (i,s)(j) (G) = G ′ . We assume without loss of generality that G ′ = G (r) . Thus If B ∈ G ′ and j / ∈ B or m(i, B) = s − 1, then it follows from the definition of the down-compression operation that B ∈ G. Therefore, in the remainder of the proof we assume that j ∈ B and m(i, B) = s−1 for some B ∈ G ′ . With these assumptions, either B ∈ G or there is some other multiset A ∈ G such that S (i,s)(j) (A) = B. Then Note that j / ∈ A and that m(i, A) ≥ s. We consider two cases and show that in both B ∈ G.
We can further stipulate that m(j, B ′ ) = k − t − r + 1 and that i / ∈ B ′ . This last condition is possible even if i ∈ [t+2r] since r > 0 and i ∈ B. Clearly B ′ ∈ G ′ and since m(i, B ′ ) = s − 1, it must also be in G. But |B ′ ∩ A| = t − 1 and so A cannot be in G. Therefore B ∈ G.
containing a fixed t-multiset, then G is a collection of all of the kmultisets of [m] containing a fixed t-set.
Proof. It is sufficient to show that S (i,s)(j) (G) has a t-multiset common to all of its k-multisets only if G does. If k = t, the result is trivial so we assume that k > t.
Let S (i,s)(j) (G) = G ′ . Assume that there exists a t-multiset, X ∈ [m] t , such that As in the proof of Lemma 4.6, we assume that j ∈ B and m(i, B) = s − 1 for some B ∈ G ′ . Then m(i, X) ≤ s − 1 and either B ∈ G or 1. Case 1: j / ∈ X. Since m(i, A) > m(i, B), it follows that X ⊂ A and so A ∈ G ′ and A ∈ G. This implies that B ∈ G. Therefore G = G ′ .
Partition G ′ as follows. Let and let Then G ′ 0 ⊂ G since the multiplicity of i will be greater than s − 1 in all multisets in G ′ 0 . We now show that either all of the multisets in G ′ 1 are in G or all were changed by the down-compression operation. Define a graph, G, with the multisets in G ′ 1 as its vertex set and where two vertices are adjacent if and only if they exactly t-intersect. In other words, two vertices, B 1 and B 2 , are adjacent if and only if B 1 ∩ B 2 = X. Then if B 1 and B 2 are adjacent vertices in G, either both are in G or both were changed by the downcompression operation since if only one changed G would not t-intersect. From this it follows that if G is connected, either all multisets in G ′ 1 are in G or all were changed by the downcompression operation. Let B 1 and B 2 be any two multisets from G ′ 1 . Since k > t and m ≥ 2k − t, it follows that m > k, and thus there is some x ∈ [m] such that x / ∈ B 1 . Let C 1 = X ∪ {(x, 1), . . . , (x, k − t)}. Then C 1 ∈ G ′ 1 and B 1 ∩ C 1 = X. Therefore B 1 is adjacent to C 1 in G. If x / ∈ B 2 , then B 2 is adjacent to C 1 and thus there is a path between B 1 and B 2 . Suppose that x ∈ B 2 . Then there is some y = x ∈ [m] such that y / ∈ B 2 . Let C 2 = X ∪ {(y, 1), . . . , (y, k − t)}. Then B 2 and C 1 are adjacent to C 2 and thus there is a path between B 1 and B 2 . Since B 1 and B 2 were arbitrary vertices of G, it follows that G is connected. Therefore either all of the multisets in G ′ 1 are in G and G = G ′ or all were changed. Clearly, if all the multisets in G ′ 1 are in G then G = G ′ . If all of the multisets in G ′ 1 were changed by the shifting operation, we claim that G consists of all of the k-multisets containing the t-multiset (X\{(j, 1)}) ∪ {(i, m(i, X)+1)}. Suppose that all of the multisets in G ′ 1 were changed by the shifting operation. Let G 1 ⊂ G be the collection of multisets such that S (i,s)(j) (G 1 ) = G ′ 1 . Choose B ∈ G ′ 1 such that j / ∈ B\X and let A be the multiset in G 1 such that S (i,s)(j) (A) = B. Then j / ∈ A and m(i, A) = m(i, X) + m(j, X) ≥ m(i, X) + 1. Let Thus if S (i,s)(j) (G) consists of all the k-multisets of [m] containing some t-multiset X, then G consists of all the k-multisets of [m] containing X or G consists of all the k-multisets of [m] containing the t-multiset (X\{(j, 1)}) ∪ {(i, m(i, X)+1)}.
The homomorphism of K(n, k, t) to M ′ (m, k, t) can be used in conjunction with the down-compression operation and known results for set systems to prove a t-intersecting versions of Theorem 3.3. Let [k+1]\{i} .
Then F t is a t-intersecting family of k-subsets of [n] which does not have a t-set common to all k-subsets. We use the following theorem. [2]). Let 1 < t < k and let F be a t-intersecting family of k-sets from [n] such that F ∈F F < t.
We define a k-multiset system, G t , as follows: Then G t is a t-intersecting collection of k-multisets of [m] which does not have a t-multiset common to all k-multisets.
4.9 Theorem. For 1 < t < k and m ≥ 2k − t, let G be a t-intersecting family of k-multisets from [m] such that A∈G A < t.
Equality holds if and only if G is isomorphic to the larger of G (1) and G t .
When m ≥ 2k − t, the down-compression operation can be used to transform any t-intersecting family of k-multisets, G, into one in which |F 1 ∩ F 2 ∩ [m]| ≥ t for all F 1 , F 2 ∈ G. If the condition that |F 1 ∩ F 2 ∩ [m]| ≥ t for all F 1 , F 2 ∈ G is added to Theorem 4.9, the result can be proved using Theorem 4.8 and the graph homomorphism from K(n, k, t) to M ′ (m, k, t) in a manner similar to the proof of Theorem 3.3. By Lemma 4.6, we know that the down-compression operation will give a family isomorphic to G (1) only if the original family was isomorphic to G (1) . It only remains to be shown that a similar result holds when the down-compressed family is isomorphic to G t . This can be proved using the definition of the down-compression operation and the fact that G is t-intersecting. The proof is similar to that of Lemma 4.6 but requires the consideration of more cases.
Open questions
The graph homomorphism method used in this paper provides a convenient way to prove results for intersecting families of multisets based on known results for sets, but there are questions that cannot be approached in this manner. For t > 1, the graph homomorphism requires that the supports of the multisets t-intersect. Althought the downcompression operation can be used to transform any t-intersecting family into one that satisfies this condition, it requires m ≥ 2k − t. Also, the graph homomorphism cannot be used to give a multiset version of Theorem 2.2 for t > 1 since the down-compression operation does not preserve the P (s, t) property.
When n ≤ 2k−t, the collection of all k-subsets of [n] is t-intersecting. No similar result holds for collections of k-multisets of [m] for any m > 1. If m = 2, it is easy to see that any t-intersecting family will have a t-multiset common to all multisets in the family. Thus the question of the size and structure of the largest t-intersecting family of k-multisets of [m] with t > 1 is open for 2 < m < 2k − t. We conjecture that, for all values of m, k and t, the largest family is the collection of all k-multisets containing at least t + r elements from a (t + 2r)-multiset where 0 ≤ r ≤ k − t. We further conjecture that the cardinality of the support of the (t+2r)-multiset is equal to the lesser of m and t + 2r and that the multiplicities of the elements in the (t+2r)-multiset will vary by at most one unless the multiplicity of all of the elements is greater than or equal to r.
If we consider only those t-intersecting families of multisets where the supports are t-intersecting, the graph homomorphism method gives an upper bound on the size of the largest family for all m > k − t + 1. However, this bound may not be attainable. For example, consider the case when k = 4 and t = 2. Let G be a t-intersecting family of multisets such that |F 1 ∩ F 2 ∩ [m]| ≥ t for all F 1 , F 2 ∈ G. If m = 5, the graph homomorphism method gives |F (1) | as an upper bound on the size of G and this size is attained by G (1) . If m = 4, then |G| ≤ |F (2) | but a family isomorphic to G (2) cannot be constructed since m < t + 2r.
Another obvious question arises from Theorem 4.8. When k > 2t + 1, Theorem 4.8 gives two possible options for the largest family. However, Frankl proved in [6] that F t is the largest family when k > 2t + 1 provided that n is sufficiently large. Determining precisely how large n must be would give a bound on m for multiset systems above which G t is the largest family. | 2015-05-26T20:15:17.000Z | 2015-04-24T00:00:00.000 | {
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181863901 | pes2o/s2orc | v3-fos-license | A flexible Ti3C2Tx (MXene)/paper membrane for efficient oil/water separation
The scalable fabrication of flexible membranes for efficient oil/water separation is in high demand but still significantly underdeveloped. Here, we present a flexible membrane using Ti3C2Tx (MXene) as the functional layer on conventional print paper as the substrate. With a simple coating process using MXene ink, we developed a highly hydrophilic and oleophobic membrane with an underwater oil contact angle of 137°. Such a simple membrane shows outstanding flexibility and robustness, and demonstrates a facile approach for membrane scale-up using MXene ink on low-cost print paper. The membrane shows high separation efficiency for oil/water emulsions, of over 99%, and a high water permeation flux of over 450 L per m2 per h per bar. We demonstrate the excellent anti-fouling property of this membrane by cleaning the membranes without chemicals. These low-cost, highly efficient, anti-fouling membranes can provide new opportunities for industrial oil/water separation applications.
Introduction
A large quantity of oily wastewater is produced on a daily basis from various oil industries, including production, reneries, shipping facilities, etc. 1 Technologies for treating oily wastewater are in great demand. Conventional technologies, including gravity settlement, hydrocyclone and air otation, are not effective in separating emulsied oil/water mixtures, due to the small sizes of the oil droplets in these emulsions. 2 Membrane separation technologies, based on a "size sieving" mechanism, possess great potential in separating tiny oil droplets from water. However, most commercial membrane materials, like polyvinylidene uoride (PVDF), polysulfone (PS), polypropylene (PP), or polytetrauoroethylene (PTFE) are not hydrophilic, and thus suffer from serious membrane fouling issues, which inevitably cause a quick decline in permeation ux and separation efficiency. [3][4][5][6][7][8][9] This drawback leads to the limited adoption of these commercial membranes for treating large amounts of oily wastewater. Therefore, the development of new membranes with highly hydrophilic, underwater oleophobic and anti-fouling properties is crucial and in great demand for efficient oil/water separation in industry. [10][11][12][13][14] With advances in nanomaterials and nanotechnology, new nanocomposites have been suggested to improve the effectiveness of oil/water separation membranes by adjusting the surface chemistry, roughness, and pore size. [4][5][6][7][8]15 Recently, a number of fabrication methods, such as surface graing, [16][17][18] biomimetic adhesion, [19][20][21] and surface mineralization, 20,22,23 have been adopted to fabricate superwetting membranes with a hydrophilic (water-favoring) and underwater oleophobic (oilrepellent) layer on some substrates for oil/water separation. Several carbon-based nanocomposite membranes demonstrate promising efficiency in separation performance and antifouling property for various oil-in-water emulsion systems. [24][25][26] However, these newly-developed membranes are not suitable for scalable production due to their complexity and high-cost chemical preparation and membrane fabrication processes.
MXenes are a class of 2D nanomaterials from a large family of transition metal carbides, nitrides and carbonitrides. 27 Titanium carbide (Ti 3 C 2 T x ), where T x denotes surface terminal groups (-OH, -O, and/or F), is the most widely studied member of the MXene family in water purication and environmental remediation applications due to its high surface area, hydrophilicity, surface functionality, abundance, facile scale-up synthesis, and environmentally benign characteristics. [28][29][30] MXenes have been also used extensively in water treatment applications, including water purication membranes, 31-33 heavy metal removal, 34,35 capacitive deionization, 36 and antimicrobial coatings. [37][38][39] In this study, a new concept is explored to produce membranes with a simple and low-cost process by coating 2D Ti 3 C 2 T x (MXene) on commercial print paper, to achieve efficient separation of emulsied oil/water mixtures. These new membranes are produced by simple coating of Ti 3 C 2 T x MXene ink on commercial print paper as the substrate. These hydrophilic and densely packed 2D MXene nanoakes will act as a functional layer for the efficient separation of emulsied oils, while the print paper substrate provides mechanical exibility and strength. This very simple and yet novel membrane exhibits a good lifetime with less membrane fouling propensity and an easy membrane cleanup protocol. Moreover, the new concept demonstrates a simple and economic way for scalable fabrication of paper-based membranes for efficient separation of oil/ water emulsions.
Materials
The commercial white print papers and sunower oil that were used in this study were purchased locally. Hexane, silicone oil and petroleum ether were procured from VWR Chemicals (Radnor, PA, USA), Merck Millipore (Billerica, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Ti 3 AlC 2 (MAX) was purchased from Y-Carbon, Ltd., Ukraine. Other reagents are of analytical grade and were used as received. In all experiments, deionized (DI) water was used.
Synthesis of MXene
Ti 3 C 2 T x MXene was prepared from the MAX (Ti 3 AlC 2 ) phase using 12 M lithium uoride (LiF)/9 M hydrochloric acid (HCl) solution, following methods in the literature. The etching mixture was prepared by adding 0.8 g of LiF to 10 mL of 9 M HCl solution under continuous stirring for a period of ve minutes. 0.5 g of Ti 3 AlC 2 was added in small portions into the etching solution and kept under stirring for 24 h at room temperature to produce multi-layered (ML)-Ti 3 C 2 T x . The synthesized material was centrifuged at 3500 rpm for ve minutes and washed using deionized (DI) water in order to adjust the pH to within the range 4-5. The ML-Ti 3 C 2 T x solution was then probe sonicated in degassed water for one hour under inert conditions (using argon gas) at room temperature to produce delaminated (DL) Ti 3 C 2 T x . The resulting solution was then centrifuged at 3500 rpm for 30 min and, nally, the supernatant was collected and freezedried and stored for preparation of the composite membrane.
Preparation of the composite membrane
MXene ink suspension at a concentration of 2 mg mL À1 was prepared by homogeneously dispersing the DL-MXene nano-akes in deionized water using probe sonication for ve minutes. The stock ink was then further diluted as per the required MXene mass loadings for membrane coating. The composite membrane was prepared by depositing the solution on commercial white print papers using a vacuum-assisted ltration setup.
Oil-in-water emulsion preparation
A stable 1% v/v oil-in-water emulsion was prepared as per the protocol described by Wang et al. 40 10 mL of oil (e.g. sunower oil, diesel, hexane, petroleum ether or silicon oil) was sonicated in 90 mL of deionized water for a period of ve minutes using an 8510E-DTH ultrasonic bath (Danbury, CT, USA). The welldispersed emulsion was then further diluted with 900 mL of deionized water to achieve a nal concentration of 1% v/v. The prepared emulsion was stable over a long period, as no precipitation was observed even aer several months.
Characterization
Surface and cross-sectional morphologies of the MXene-based composite membrane were studied using an FEI Quanta 400 (Hillsboro, OR, United States) environmental scanning electron microscope (ESEM). For imaging the cross-section, the composite membranes were cryo-fractured in liquid nitrogen. Transmission electronic microscopy (TEM) images of the MXene akes were recorded using a bright eld transmission electron microscope (TEM) (FEI Tecnai G2 TF20 UT). The samples were dispersed in water and mounted on a lacey carbon formvar coated Cu grid. The surface roughness of the composite membranes was characterized using an atomic force microscope (AFM, Bruker, USA). The water contact angle (WCA) and underwater oil contact angle (OCA) on the surface of the membranes were measured at room temperature using an advanced contact angle (CA) goniometer (Rame-hart model 500, Succasunna, NJ, USA). The WCA and hydrophilicity of the membranes were studied by analyzing the spreading process of water into the membranes where a high-speed camera with a speed of 100 frames/second was used to record the spreading process. As for the OCA measurement, the membranes were rst immersed in distilled water. Oil droplets of 14 mL were then released from the bottom and allowed to rise to the surface of an MXene membrane immersed underwater. For all the contact angle measurements, an average of ve measurements for two membrane samples was reported. The mechanical strength of the membranes was investigated at room temperature using a burst tester (TruBurst4, Halifax, England, UK). Circular membranes with testing areas of 7.3 cm 2 and a clamp pressure of 50 kPa were used in this study. The MXene-based membranes were deformed until the breakage point using compressed air operated at a constant pressure rate of 0.5 kPa s À1 . The deection of the membrane center was recorded as a function of applied differential pressure.
Filtration performance testing
The oil/water separation efficiencies of the MXene-based composite membranes were evaluated using a glass ltration apparatus with an effective membrane area of 1.77 cm 2 . The net trans-membrane pressure was maintained at 50 kPa using a diaphragm vacuum pump. Operating ux was calculated by measuring the volume of permeate passing through the membrane at a xed ltration time. The oil content in the resulting permeate was measured using a total organic carbon analyzer (TOC-L, Shimadzu, Japan).
Results and discussions
The hierarchy of the deposited MXene nanoakes on the print paper substrate is illustrated in Fig. 1(a). The delaminated nanoakes are stacked on the substrate, forming overlapping lamellar sheets that resulted in interlaced porous nanostructures. These stacks are expected to create multi-channel passages for water to ow through the membrane. The TEM image of Ti 3 C 2 T x MXene is exhibited in Fig. 1(b). In this gure, it can be observed that Ti 3 C 2 T x MXene can be exfoliated to a single sheet/few sheets, showing that the exfoliation process was effective. 41 The average sheet diameter can vary between 400 and 600 nm. Fig. 1(c) highlights the exibility of the synthesized composite membrane. The stacked structures of the MXene lm enable the composite membrane to be folded without any cracking or disintegration. Surface morphological investigations of the composite membrane before and aer MXene coating using SEM are shown in Fig. 1(d-f). It can be observed that the cellulose framework of print paper in Fig. 1(d) was evenly wrapped and covered by MXene nanoakes, showing a more compact structure and continuous surface, as clearly shown in Fig. 1(e and f). Cross-section SEM images clearly show the enlarged interplanar spacing of the stacked DL-MXene nanoakes. The thickness of MXene lm on the print paper substrate was 1.2 mm (inset, Fig. 1(f)).
XRD patterns of Ti 3 C 2 T x MXene are shown in the ESI. † The sharp and intense peak located at 2q values of 7.2 can be assigned to the (002) plane of Ti 3 C 2 T x MXene. Other characteristic diffraction peaks at (004), (006), (008), and (0010) conrm the successful etching of aluminum layers from MAX and the delamination of multilayered MXene.
AFM analyses, shown Fig. 2, indicate that the print paper substrate had a root-mean-squared roughness (R q ) of 914 nm. With the deposition of MXene nanoakes on print paper via vacuum ltration, the R q decreased to 200 nm, suggesting the loss of microscale roughness for the print paper support. Lower surface roughness indicates a lower membrane fouling tendency aer coating with MXene.
The hydrophilicity prole of the composite membrane over the time is described in Fig. 3(a). The water contact angle swily decreased to zero within two seconds of water droplet dispersion on the surface of the composite membrane, thus highlighting the excellent hydrophilicity of the composite membrane. The rapid water wetting phenomenon could be due to MXene's high affinity towards hydroxyl groups and the presence of interlaced porous nanoakes that promote water passage. 31 The underwater oil contact angle, OCA, for the composite membrane, as shown in Fig. 3(b) was approximately 137 when tested using a sunower oil droplet. This shows the great underwater oleophobicity properties of the MXene composite membrane. Fig. 3(b) compares the mechanical strength of the MXenebased composite membrane versus the print paper substrate, which conrms that the composite membrane possesses better mechanical strength and ductility due to its higher breakage pressure and deection. The breakage pressure increased from 107.0 kPa to 165.1 kPa when the print paper was coated with delaminated MXene, which represents an increase of 54.3%. Similarly, the breakage deection increased by 24.2% in the MXene-based composite membrane. The enhanced mechanical property of the MXene-based composite membrane is attributed to its more compact structure, resulting from the integration of MXene nanoakes with cellulose papers that can also be observed from Fig. 1(f). The higher fracture response during mechanical loading could be due to effective interaction between the MXene nanoakes and cellulose microbers that beneted stress transfer and hence delayed the failure of the composite membrane. 42 The performances of the MXene-based composite membrane in separating oil-in-water emulsion were evaluated by measuring the oil content in the permeate and operating ux. The composite membrane was optimized by varying the mass loadings of MXene deposited on the print paper. For this optimization test, the emulsied oil solution was prepared from sunower oil due to its high viscosity. [43][44][45] The oil droplet size distribution of the emulsied sunower oil ranged from 1 to 18 mm with a mean droplet size of 3.92 mm. Additional details on the oil droplet size distribution can be obtained in the ESI. † From Fig. 4, it can be observed that both permeation ux and oil content in the ltrates were inversely proportional to the mass loadings of MXene. The poor oil rejection rate of the porous commercial paper substrate without MXene loading was unsurprising, despite displaying a maximum ux of 2717.6 L per m 2 per h per bar. By increasing the mass loading of MXene from 0.1 mg to 0.8 mg, the ux of the composite membrane decreased vefold from 1019.1 to 203.8 L per m 2 per h per bar. The oil rejection ratio in the ltrate improved by 98.5% as the oil content in the ltrate dropped from 102.5 ppm to 1.6 ppm. By comparing the morphologies in Fig. 1(d) and (f), it can be conrmed that higher MXene loading leads to a more compact structure and a denser selective layer that in return reduce the ux and increase the permeate quality. In addition, the excellent hydrophilicity and underwater oleophobicity, as well as the interlaced porous structures of the composite membrane, contribute to the enhanced oil rejection rate. As the MXene loading was increased to 0.4 mg, the oil content in the permeate dropped below the requirement enacted by the US Environmental Protection Agency (USEPA) that permits the discharge of oil and grease in wastewater to a maximum of 42 ppm for any one day and a daily average lower than 26 ppm for 30 consecutive days. The corresponding ux and permeate quality at 0.4 mg of MXene loading were 543.5 L per m 2 per h per bar and 11.8 ppm. Further additions of MXene loadings (higher than 0.4 mg) lead to poorer water permeate ux. For subsequent studies, the MXene composite membranes were prepared using a mass loading of 0.4 mg MXene as an optimal composite.
The optimized MXene composite membranes that were prepared using a mass loading of 0.4 mg were also tested with four other different types of oil-in-water emulsion: namely (i) diesel, (ii) silicone oil, (iii) petroleum ether, and (iv) hexane. Fig. 5 shows the ltrate oil content and corresponding ux of the MXene composite membrane when challenged with these emulsions. The permeate ux ranges between 543.5 L per m 2 per h per bar and 682.3 L per m 2 per h per bar, where the highest ux was recorded for hexane, followed by petroleum ether, silicone oil, diesel and sunower oil in a decreasing trend. As for the oil content in the ltrate, the levels for all the emulsions were less than 12 ppm, which is way below the allowable daily average limit of 26 ppm set by USEPA. It is worth mentioning that these high oil rejection ratios were achieved in a single pass with a low operating pressure of only 0.5 bar. Such separation performance is very attractive from the energy consumption perspective, since conventional oil/water membrane technologies operate at a higher transmembrane pressure. 46 The excellent permeation is most likely due to the synergy between hydrophilicity, underwater oleophobicity and the low oiladhesion property of the membrane surface induced by the integration of MXene nanoakes, as well as the interconnected porous structure.
The capability of an oil/water separation membrane to produce consistent water ux and permeate quality is of utmost importance for practical applications. In this study, we have investigated the reusability/recyclability of the MXene-based composite membrane in separating oil-in-water emulsion for several operating cycles. The membrane was ushed with hot water at the end of each ltration cycle to recover the ux. It can be observed in Fig. 6 that the operating ux of the composite membrane decreased only marginally aer eight cycles of ltration. As for the oil separation efficiency, the oil content in the permeate was consistently lower than 11 ppm throughout all the ltration cycles, hence demonstrating an oil rejection efficiency of more than 99%. At the end of the eighth cycle, the operating ux and permeate oil content of the MXene composite membrane were 472.1 L m À2 h À1 and 7.5 ppm, respectively. Surprisingly, even with commercial lter paper as substrate, the membrane remained intact with no sign of degradation even aer 8 cycles of operation/washing steps. This is most likely due to high mechanical stability imposed by MXene lm. Nevertheless, other substrates should be tested in the future to assure the long-term durability of the membranes. These results revealed that the MXene-based composite membrane featured excellent recyclability with anti-fouling property for emulsied oil-in-water separation, attributed to its high hydrophilicity and underwater oleophobicity properties. These favorable characteristics greatly mitigate fouling by facilitating the formation of a water layer that prevents direct interaction between the oil phase and the membrane surface during the separation process. 47,48
Conclusion
In conclusion, we have demonstrated the fabrication of an efficient oil/water separating membrane through a simple and scalable coating process by coating 2D MXene ink onto everyday print paper. The print paper functions as a substrate to provide mechanical exibility and strength, and the hydrophilic MXene layer functions as a selective layer to effectively separate emul-sied oil from water. The new membrane demonstrated over 99% oil rejection efficiency while maintaining a high operating ux of 472.1 L m À2 h À1 aer 8 consecutive cycles. Such MXene/ paper-based membranes are exible, robust and anti-fouling, and are excellent for oily wastewater treatment in various industries.
Conflicts of interest
There are no conicts to declare. | 2019-06-07T21:51:06.783Z | 2019-05-24T00:00:00.000 | {
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218772924 | pes2o/s2orc | v3-fos-license | Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma
Background Combinations of drugs can improve the efficacy of cancer treatment, enable the reduction of side effects and the occurrence of acquired drug resistance. Methods We approached this challenge mathematically by using the validated technology called the Therapeutically Guided Multidrug Optimization (TGMO) method. In a set of genetically distinct human renal cell carcinoma (RCC) cell lines, either treated chronically with sunitinib (−ST) or sunitinib-naive, we identified cell line-specific low-dose-optimised drug combinations (ODC). Results Six cell-type-specific low-dose drug combinations for three sunitinib-naive as well as three sunitinib pre-treated cells were established. These ODCs effectively inhibited the RCC cell metabolic activity while being ineffective in non-cancerous cells. Based on a single screening test and three searches, starting with ten drugs, we identified highly efficacious drug mixtures containing four drugs. All ODCs contained AZD4547 (FGFR signalling pathway inhibitor) and pictilisib (pan-phosphatidylinositol 3-kinase inhibitor), but varied in the third and fourth drug. ODC treatment significantly decreased cell metabolic activity (up to 70%) and induced apoptosis, independent of the pretreatment with sunitinib. The ODCs outperformed sunitinib, the standard care for RCC. Moreover, short-term starvation potentiated the ODC activity. The translation of the 2D-based results to 3D heterotypic co-culture models revealed significant inhibition of the spheroid growth (up to 95%). Conclusion We demonstrate a promising low-dose drug combination development to obtain drug combinations effective in naive as well as resistant tumours. Nevertheless, we emphasise the need for further mechanistic investigation and preclinical development.
BACKGROUND
The clinical benefit of using low-dose drug combinations to treat complex diseases has become a major interest in the medical community because of the advantages, such as enhanced efficacy, lack of intrinsic or acquired resistance and the limitation of doserelated toxicities. This particularly holds true in the case of cancer therapy, where in many cases, adequate treatments are still lacking due to intra-patient and/or inter-/intra-tumour heterogeneity. These issues together restrict the efficacy and overall success of many treatment strategies that are currently used for the clinical management of cancer. 1,2 The use of targeted anticancer agents and genotype-based treatments has provided attractive avenues for the improvement of therapy, and has shown important clinical successes. 3,4 For the treatment of renal cell carcinoma (RCC), targeted agents have generally shown good efficacy. Sunitinib (Sutent TM ) is one of the first-line treatment therapies for RCC. 5 Many preclinical and clinical efforts for this indication have focused on identifying effective combinations of existing targeted agents using either simultaneous or sequential administration to maximise their clinical benefits. Large studies, such as the BEST, RECORD, INTORACT, CALGB, TORAVA, Check-Mate 214 and Keynote-426 studies, [6][7][8][9][10][11] investigated first-line combination regimens and, in most cases, did not show superior outcomes over single agents. 12 Instead of enhanced efficacy, substantial toxicity has been a recurrent observation in these studies, 12 even when combinations are designed to target complementary pathways. Therefore, the application of appropriate methods to design optimal drug combination regimens with optimal drug selections and use at appropriate doses is of major importance.
Properly designed drug combinations may help to overcome some of the current limitations of cancer therapy by challenging the robust biological processes. 13,14 The limited success of clinically tested drug combinations thus far can largely be attributed to intolerable toxicities, lack of efficacy and/or acquired drug resistance. 15,16 Two of the main challenges in defining optimal drug combinations are patient heterogeneity and the broad number of possibilities, where one must consider the candidate drug selection together with their relative dose ratios. These will affect potential synergism or antagonism, as well as toxicity profiles of the drug combinations.
In order to solve these problems, we have developed and applied our technology called streamlined-feedback system control (s-FSC), a strategy for rapid in vitro identification of optimised, synergistic low-dose multidrug combinations. 13,14 In contrast to other approaches based on pharmacogenetics or www.nature.com/bjc high-throughput screening, this phenotypically driven approach based on the statistical design of experiment (DoE) identifies synergistic drug mixtures with minimal experimental effort. Moreover, it does not require background and mechanistic information about the system prior to the start of optimisation. 13,14,16 Drug combinations are tested in only three iterative searches, and the in vitro data are used to perform an analysis in 'search rounds'. Each search produces a second-order linear regression model and specified regression coefficients defining these models. This technique helps to discriminate synergistic, additive and antagonistic drug interactions. 14, 17 We have previously applied the s-FSC platform to navigate in the experimental parametric space of ten small-molecule drugs 13,14,17,18 applied at low doses. In all cases, the rapid, iterative approach of in vitro testing for cell metabolic activity, together with a second-order regression modelling, allowed for the identification of an optimal, synergistic low-dose drug combination.
In this study, we investigated the improvement of RCC treatment via the identification of synergistic drug combinations effective in sunitinib-naive and sunitinib pre-treated RCC cells. We used a new generation of the s-FSC method called the Therapeutically Guided Multidrug Optimization (TGMO), where drug combinations are tested simultaneously on both cancer (in this case RCC cells) and non-cancerous cells (e.g. non-malignant renal cells) at low doses (i.e. below maximal plasma doses, MPD, in humans). This strategy allows the identification of optimised multidrug combinations (ODC) acting with high efficacy and selectivity. 19 Starting with a set of ten small-molecule-based compounds, we have identified fourdrug combinations applied simultaneously specific for each cell line. The individually optimised regimen revealed different drugdose parameters for each cell type. The drug mixture optimised for A489 sunitinib pre-treated (A489-ST) cells contained AZD4547 (an inhibitor of the fibroblast growth factor receptor (FGFR) tyrosine kinase family), 20 AZD8055 (an ATP-competitive mammalian target of rapamycin kinase (mTORC1) inhibitor), 21 pictilisib (GDC-0941, a pan-phosphatidylinositol 3-kinase inhibitor), 22 and selumetinib (an ATP-independent inhibitor of mitogen-activated protein kinase (MEK or MAPK/ERK kinase) 1 and 2). 23 This drug combination was effective at inhibition of cell metabolic activity in both 2D and heterotypic 3D co-cultures, and induced G1 arrest, p21 expression as well as apoptosis.
Cell culture
Human clear-cell RCC cell lines A498, Caki-1 and 786-O were purchased at ATCC. All three cell lines are of epithelial origin, with A498 being isolated from a primary clear-cell RCC lesion, Caki-1, from a skin metastasis, and 786-O from a primary clear-cell adenocarcinoma.
Combinatorial drug screen and TGMO-based screen The inhibition of the cell metabolic activity was measured to analyse the activity of each drug combination using CellTiter-Glo solution (Promega, G7572). The therapeutic window was determined by comparing the efficacy of each drug combination between cancerous RCC and non-cancerous cell lines. Secondorder stepwise linear regression models using Matlab® were applied to model the results as described by Weiss et al. 14 Statistical measures-Cook's distance, regression coefficient, root mean square error and data point correlations-defined the accuracy and reliability of regression models for each iteration, with an ANOVA lack-of-fit test confirming the selection of a relevant model structure. After three sequential search rounds, the compounds alisertib, crenolanib and icaritin were excluded from further experiments based on the regression model elimination. The final dose-optimisation step has led to the selection of celltype-specific four-drug combinations (Table 1).
F-actin-nucleus double staining Glass inserts were placed in each well of a 24-well plate before seeding. After experimentation, cells, attached to the glass inserts, were fixed for 10 min in 4% PFA solution, washed twice with PBS and permeated in 0.1% Triton-X solution. Fluorescence double staining was performed by placing the glass slides upside down on a drop of staining solution that was positioned on a table area covered with parafilm. F-actin staining with 1:200 dilution of Alexa Fluor 488-conjugated phalloidin (Invitrogen, Carlsbad, California, United States, A12379) was performed for 20 min at RT following nuclear staining with 1:2500 dilution of Dapi for approximately 5 min. Images were taken with a Biotek Citation 3 (BioTek instruments) with corresponding software at the default settings. Pictures were analysed with CellProfiler TM and Adobe® Photoshop.
Cell cycle and cell fate analysis Induction of necrosis after combination treatment was analysed using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Kit (Promega, JA1011) following the manufacturer's instructions. Discrimination of the cell cycle distribution has been performed staining a cell pellet of approximately 5 × 10 5 cells with 500 µL of FxCycle TM PI/RNase staining solution (Invitrogen, F10797). p21 fluorescence imaging Cells were plated, fixed and permeabilised on glass slides as previously described, and stained with a 1:800 α-p21 antibody (CellSignalling, Leiden, Netherlands, 2947S) overnight at RT in the dark. Glass slides were dipped in PBS several times for washing and afterwards stained with a 1:400 dilution of DyLight488coupled secondary antibody (ImmunoReagents, Raleigh, NC, USA, Rb-003-V) for 1 h. Glass slides were washed thoroughly and further stained with DAPI as described above.
Western blot One-million RCC cells were grown in petri dishes and treated for 2 h either with the Ctrl or the optimised combination treatment. Following two washing steps with ice-cold PBS, cells were lysed in 1× RIPA buffer (Cell Signaling, 9806S) supplemented with phosphatase inhibitor (Roche, Basel, Switzerland, 04906837001) and protease inhibitor (Roche, 11836170001). Protein concentrations of lysates were quantified using Bradford assay (Sigma, Darmstadt, Germany, B6916-500ML). Protein separation of 30 µg of lysate was performed on 4-15% pre-casted polyacrylamide gels (Biorad, Hercules, California, United States, 456-1083). The wet transfer technique was used to blot the proteins to a nitrocellulose membrane. About 5% milk was used to block membranes following incubation with primary (overnight) and fluorochromecoupled secondary antibodies (30 min); bands from immunoreactive proteins were measured with an Odyssey infrared imaging system (700 nm for α-mouseand at 800 nm for α-rabbitstained proteins). We stained for α-tubulin (Cell Signaling, 3873S), MAPK (Cell Signaling, 4695S) and p21 (Cell Signaling, 2947S). Images were obtained with the Licor Odyssey CLx scanner at one default exposure setting. Image Studio TM Lite software was used to quantify the intensity of every single band by adjusting the brightness per blot row for sufficient band quantification.
Heterotypic 3D co-cultures Heterotypic RCC 3D co-cultures consisted of 1800 RCC cells cocultured with 900 NHDFα cells and 300 ECRF24 cells supplemented with 2.5% Matrigel (Corning, Root, Lucerne, Matrigel #354230). 24 All consumables were pre-cooled, and the mixing of cells and basement membrane was performed on ice to prevent early polymerisation of Matrigel. To promote the spheroid formation, 80 µl/well were seeded in a round-bottom 96-well plate with the cell-repellent surface (Cellstar® 650970), which was centrifuged at 4 o C for 2 min at 1200× rpm. Drug treatment on cocultured spheroids was performed for 72 h. Growth kinetics and cell metabolic activity (CellTiter-Glo® 3D Cell Viability Assay (Promega, #G9681)) were measured with a Biotek citation 3 with corresponding software at the default settings.
CellTracker staining Prior to staining, all cells were washed with PBS. Afterwards, RCC cells were stained for 20 min with 5 µM Green CMFDA, NHDFα for 40 min with 50 µM blue CMAC and ECRF24 cells for 20 min with 10 µM Red CMPTY (C7025 and C34552, Life Technologies) diluted in serum-free medium. Following another washing step with PBS, cells were harvested, and 3D co-cultures established. Spheroid formation was monitored by fluorescent imaging.
Statistical analysis
The data are presented as the mean of multiple independent experiments. Error bars represent the standard error, unless otherwise specified. Using either one-way ANOVA test with post hoc Tukey's multiple-comparison test or a student's t test (Graphpad Prism®), significance was determined. Statistically significant values were calculated vs. the control or monotherapeutic regimens, p values are specifically indicated in each figure legend and marked with ** or * according to graphs.
RESULTS
TGMO-based identification of synergistic four-drug combinations specific to each RCC cell line Starting with a set of ten small-molecule-based drugs that are well known and well-characterised, either FDA approved or in clinical evaluation or with existing clinical data (Supplementary Table S1), we performed an experimental search using a simple in vitro cell metabolic activity assay. The latter indirectly corresponds to cell viability. 25 All selected compounds bind to different extra-or intracellular components of growth factor receptors (GFR) ( Fig. 1a and Supplementary Table S1). The main targets are the EGFR, the FGFR and the VEGFR, as well as enzymes of the MAPK/Erk, 26 PI3K/ Akt 27 and Grb2/Nck 28 pathways. Three RCC cell lines, i.e. A498, Caki-1 and 786-O varying in their origin and mutation status were used (Supplementary Table S2). In order to mimic the clinical situation where RCC patients receive sunitinib as the first-line therapy, we chronically treated cells with 1 µM sunitinib in order to induce insensitivity to sunitinib. The response of cells to sunitinib treatment was experimentally verified every 2 weeks, until cells became unresponsive to sunitinib ( Supplementary Fig. S1A). Moreover, sunitinib-treated (ST) cells showed clear accumulation of sunitinib in lysosomes, in agreement with previously reported studies [29][30][31][32] (Supplementary Fig. S1B).
In the first step, single-drug dose-response curves were generated for cell metabolic activity in all cell lines (Supplementary Fig. S1C). Subsequently, the following doses were selected for use in the TGMO-based search ( Fig. 1b): (i) dose where 20% of the maximal response was observed; (ii) a dose representing half of the concentration of dose (i) and dose 0 (ED 0 ), where this drug was absent in the mixture. Moreover, the maximum doses of all compounds were limited to clinically attainable doses based on the calculation of maximum drug plasma doses obtained in Table 1. Cell line-specific four-drug low-dose combinations and their efficacy in cell metabolic activity inhibition identified in the TGMO-based search. patients in clinical applications. The optimisation was accomplished by performing a series of orthogonally designed multidrug experiments (Fig. 1b, c) simultaneously in RCC and non-cancerous cell lines (human embryonic kidney cells, HEK-293T) (Fig. 1d), developing a therapeutic window (TW, i.e. % control of nonmalignant cell metabolic activity-% control of cancer cell metabolic activity) for efficacy. Data obtained from these experiments (Fig. 1c) were used to build second-order linear regression models, which enabled the analysis and the elimination of non-synergistic drugs for a subsequent drug-dose search (Fig. 1d). In each regression model, an estimated regression coefficient was generated for significant terms. The latter were grouped as "single-drug first order", "two-drug interaction" and "single-drug second-order" terms. They describe the contribution of each drug as an individual agent and as a drug pair to the overall activity of the drug combinations. Appropriate data and model verification methods were implemented to ensure the accuracy and reliability of model-based predictions, including the verification of the main assumptions of the linear regression models i.e. weak exogeneity, linearity, constant variance, independence of errors and lack of multi-colinearity. This was achieved globally, for each cell line, through the analysis of the coefficient of multiple determination (R 2 ) and the plot of observed vs. fitted data points, and by residual analysis through visualisation of residual plots, Cook's distance plot, normal Q-Q plot and residual histogram ( Supplementary Fig. S2).
In search 1, 91 drug combinations were tested experimentally. Based on the stepwise regression analysis, three drugs were eliminated from further search due to the lack of synergy or antagonism (axitinib, crenolanib and icaritin for A498-ST cells). In the following search 2, we tested a set of 50 drug mixtures composed of up to seven drugs along with a set of 25 four-drug mixtures. Finally, search 3 was performed to evaluate the most promising four-drug combinations identified in search 2.
ODC in sunitinib-naive and sunitinib-ST cells is listed in Table 1. Cell line-specific ODCs impaired cell metabolic activity, after a single treatment, with 66-72% efficacy, respectively. Corresponding monotherapies were relatively inactive (Fig. 1e for A489-ST cells and Supplementary Fig. S3 for other cells). Data analysis releveled the cell line-specific drug-drug interactions. As shown in the example of A489-ST cells, the ODC was composed of AZD4547, AZD8055, pictilisib and selumetinib. The main synergistic activity was found between AZD4547 and selumetinib (Fig. 1d). The biological measures of drug interactions for other cell linespecific ODCs are presented in Supplementary Fig. S3A-F.
We also performed in silico analysis of possible alternative ODC mechanisms of action, see Supplementary Information. Using drug concentrations applied in each cell line-specific ODC, we minded ProteomicsDB repository for additional protein targets with an effective inhibition score of ≥50% and retrieved. Interestingly, for A489-ST-specific ODC at doses applied, targets for drugs used in the combination (Supplementary Fig. S4A, upper-left graph) do not seem to play a role in a particular network ( Supplementary Fig. S4A, bottom-left graph). Since the potential targets might be extended with firstand second-order interactors, we added up 20 proteins per each order interaction using STRING. As a result, we observed the induction of tight clusters and multiple interactions. For example, the FGFR1 connection with other networks affecting e.g. cyclin family, eukaryotic translation initiation factor 2-alpha kinase (EIF2AK) proteins and other essential survival factors such as PLK1, might be lethal in most of the cells when knocked down ( Supplementary Fig. S4A, right graph). Based on gene ontology (GO) analysis, the biological processes behind the targets were classified using beneontology.org as mostly related to phosphorylation of MAPK signalling regulation. The results for other cell lines are presented in Supplementary Fig. S4B-F. It is important to note that all RCC-specific ODCs contained two core compounds, i.e. AZD4547 and pictilisib, whereas 5 out of 6 drug mixtures also contained AZD8055. Therefore, this drug combination seems to be transversally potent in all tested RCC subtypes. Moreover, the A498-ST-specific ODC represented the best overall activity in all RCC cell lines with the lowest doses (Supplementary Table S3). Therefore, we selected the A498-STspecific ODC as the most promising one, and present the respective results in the main figures, whereas the results for the other cell lines are shown in the Supplementary Material.
Since RCC is a highly vascularised tumour, targeting tumour endothelium may potentiate the overall treatment effect. Therefore, we optimised a four-drug combination specific to the human endothelial cells (ECRF24). This drug combination was composed of axitinib, pictilisib, saracatinib and selumetinib. Moreover, the incubation of ECRF24 cells with A498-ST-specific ODC, led to 61% of reduction of cell metabolic activity (Fig. 2e).
ODCs maintain the activity in time in chronically treated cells with sunitinib Stable acquired insensitivity to 1 µM sunitinib was obtained after multiple weeks (cell line specific) of continuous retreatment of cells. To monitor the kinetics of this insensitivity in time, we separated the cells into two subpopulations, i.e. (i) cells exposed to chronic treatment where sunitinib was maintained over a period of 70 days, and (ii) cells in which the sunitinib treatment was discontinued. Over this period, the cells were treated once weekly, and the cell metabolic activity readout was performed. ODC activity persisted, inhibiting cell metabolic activity up to 80% in a subpopulation (i), see Fig. 2a for A498-ST cells and Supplementary Fig. S5A for other cell lines, as well as for (ii), see Fig. 2b for A498-ST cells and Supplementary Fig. S5B for other cell lines. This suggests that the mechanism of action of ODCs was not impaired by the presence of sunitinib.
The synergistic strength and the efficacy of the combination were evaluated in various ways. First, we evaluated the power of the final optimised four-drug combination for A498-ST cells in two ways. First, by reducing the dose of the four-drug combination by 25 and 50%, which subsequently reduced the efficacy in a linear fashion by 10 and 30% compared with the original ODC (Fig. 2c). Second, we removed the one drug that would add the least synergy to the overall efficacy of the drug combination, in this case, selumetinib, which resulted in a 20% decrease in cell metabolic activity.
Since in clinical conditions, the RCC patients are re-treated several times with a treatment regimen, which leads to an enhanced effect, we re-treated A498-ST cells three consecutive times for 72 h with the readout performed for 72 h, 144 h and 216 h post the first incubation, respectively. The overall efficacy of the ODC treatment increased specifically with the second treatment time point from 70 to 85% (Fig. 2d).
After establishing a cell line-specific ODC, we validated the efficacy in other RCC cell lines, as well as non-cancerous cell lines of different origin (HEK-293T and NHDFα, see Supplementary Table S2). In all RCC cell lines (A498, Caki-1 and 786-O), the cell metabolic activity was significantly diminished by 50-70%, independently of the sunitinib treatment (−ST) and oncogenic mutation status. Cell metabolic activity of epithelial cells and fibroblasts, making up a large proportion of the tumour stroma, was only partially influenced (50-60%), to a smaller extent than in the RCC cells. HEK-293T cells were insensitive to the ODC treatment (Fig. 2e). Each established four-drug combination exhibited the most potent and synergistic anticancer activity when applied at optimised doses, thus harming the noncancerous populations only marginally.
Starvation enhances the activity of ODCs in sunitinib-naive and pre-treated RCC cells Following metabolic differences between non-cancerous and cancerous cells, it is known that cell survival depends on the Identification of low-dose multidrug combinations for sunitinib-naive and. . . M Rausch et al.
supply of diver nutrients. 33 Therefore, it is possible to induce short-term starvation of cancer cells, while maintaining healthy metabolism in non-cancerous tissue, and potentiate the effect in cancer cells. 34 Our results show that starvation of cells by serum deprivation enhances the power of the ODC treatment by 10-15%, resulting in an overall efficacy of 94%, in A498 and A498-ST cells, respectively (see Supplementary Fig. S6 for all cell lines). Access to glucose seems to be of less importance, which is in agreement with other reports. 33,34 ODCs are active in heterotypic 3D co-cultures Subsequently, we validated the activity of the ODCs in heterotypic 3D co-culture systems. 24 They were composed of 70% RCC cells, 10% endothelial cells (ECRF24) and 20% fibroblasts (NHDFα), in order to mimic the human RCC tumour composition. The cell suspension was supplemented with 2.5% Matrigel to deliver the extracellular matrix (ECM), and also to promote ECM production by the cells themselves. 24 Staining of each cell population within the 3D co-culture with a cell tracker dye of A498-ST cells in green, NHDFα in blue and ECRF24 in red visualised the first 24 h of spheroid formation ( Fig. 3a and Supplementary Videos S1-S2). The results were obtained administering each treatment for 72 h at two treatment schedules, i.e. (i) day 2-5 after spheroid formation (Fig. 3b, left graph), or (ii) day 4-7 after spheroid formation (Fig. 3b, right graph). Representative spheroid bright-field images recorded in consecutive spheroid growth days, are shown in Supplementary Fig. S7A. The ODC treatment efficacy, when treating already after 48 h of culturing (day 2-5 treatment schedule), in the 3D A489-ST co-culture system reached 93% exceeding the measured efficacy of 70% in the 2D culture system (compare Fig. 1e). The ODCs of the other cell lines were similarly more effective in 3D co-cultures than in 2D, i.e. Fig. S7B). Independently of the treatment schedule, sunitinib treatment remained ineffective. Therefore, our 2D-identified cell line-specific ODCs could be translated into more complex heterotypic 3D systems.
in 786-O cells (94%) and 786-O-ST cells (91%), and to a lesser extent in Caki-1 and Caki-1-ST cells (Supplementary
Cell morphology after cell line-specific ODC treatment To identify possible changes in the cellular morphology after ODC and corresponding monotherapy treatments, the RCC cells were stained for f-actin (GDP-labelled phalloidin, green) and nuclei (Dapi, blue). The morphometric analysis of RCC cells 24 h post ODC treatment ( Fig. 4 and Supplementary Fig. S8) was performed and showed heterogenic results. We noted morphological changes between A498 and A498-ST ODC-treated cells vs. control (shamtreated cells), see representative images in Fig. 4a, where the alterations of the actin cytoskeleton are depicted for A498-ST cells, defining the cell body size, number of cell-cell connections and the nucleus size. The ODC treatment induced the formation of stress fibres visualised by polymerised f-actin (Fig. 4a, ODC). Moreover, ODC treatment increased the cellular size (Fig. 4a, c and d) and induced cell clustering ( Fig. 4a and Supplementary Fig. S8). Analysis of other cell lines can be found in Supplementary Fig. S8D, E. An analysis of nuclear events, such as the number of multinuclear cells, mitotic cells, micronuclei or other mitotic abnormalities in ODC-treated and control cells, was carried out. The results were cell-line dependent. No significant differences were observed in A489-ST cells (Fig. 4e, f), whereas a significantly increased number of multinuclear cells was observed in Caki-1-ST cells (Supplementary Fig. S8). Notably, we observed the presence of tunnelling nanotubes (TNTs) in A498-ST cells, which are actincontaining membrane protrusions (>10 µm), which are known to play an essential role in long-range intercellular communication. Treatment with particular monotherapies or the ODC impaired the formation of these protrusions as presented by the average TNT length ( Fig. 4b and Supplementary Fig. S8).
ODCs modulate cell cycle and induce cell death in RCC cells
Investigating the mechanism of action of ODCs, we looked at the cell cycle, necrosis and senescence. We monitored cell cycle distribution and the frequency of cell death by flow cytometry using propidium iodide staining. Exposure of A498-ST cells to the ODC for 24 h resulted in a significant arrest in the G1 phase (≥30% cells arrested in G1 vs. control, Fig. 5a), consequently leading to cell death (38%) measured after 72 h of cell incubation with ODC (Fig. 5b). Cell cycle results for other cell lines are presented in Supplementary Fig. S9.
Further, we evaluated the expression of MAPK (4695S) and the cyclin-dependent kinase (CDK) inhibitor protein p21 (2947S), molecules known for playing a role in cell cycle regulation and senescence. Western blot was performed 2 h and 24 h post ODC treatment ( Fig. 5c and Supplementary Figs. 10, 11). Independently, the expression levels of MAPK did not change significantly, independent of the duration of the ODC exposure. No significant alterations between sunitinib-naive and A498-ST cells were noted. In contrast, the expression of p21 was significantly altered after ODC exposure in a time-dependent manner (Fig. 5c, d).
Using annexin V staining of ODC-treated cells, we demonstrated that neither treatment with the ODC nor the corresponding monotherapies induced necrosis in treated cells. Sunitinib at a concentration of 10 µM (63%), as well as 400 µM cisplatin (100%), used as positive controls, leads to a significant increase in necrosis (Fig. 5e).
Our data suggest that the mechanism of action of the ODC treatment might be due to immediate interference in pathways promoting the G1 phase. It seems that this particular blockade of the cell cycle further leads to the induction of apoptosis-mediated cell death.
DISCUSSION
In this study, we used the s-FSC technology to identify four-drug combinations specific to sunitinib-naive and sunitinib pre-treated RCC cells. We experimentally tested only approximately 0.4% of all possible drug combinations containing 2-10 drugs in the mixture to determine cell line-specific synergistic drug combinations. This was done simultaneously in non-cancerous cells, allowing analysis to be performed based on a therapeutic window that selected against drug toxicity. Other standard approaches would require millions of drug combinations to be tested in order to reach the same outcome. Interestingly, our system identified cell-type-specific ODCs all composed of AZD4547 and pictilisib, known to target FGFR and phosphatidylinositol 3-kinase, respectively. FGFR signalling network consists of multiple pathways, such as ERK1/2 and PI3K/AKT/ mTOR differentially activated, depending on the cellular context. Of note, the inhibition of the aforementioned pathways has previously been reported to play an instrumental role in the treatment of various cancer types. [35][36][37] Although an in-depth investigation is required to identify the exact mechanism of action behind the drug-drug interaction and observed ODC activity, we can speculate that not only the known targets are involved. Based on the protein-interaction mapping analysis at the concentrations used in each cell line, the activity of each cell line-specific ODC may also act as well via indirect higher-order network interactions ( Supplementary Fig. S4). Similarly, we have recently discovered that crenolanib, a known PDGFR inhibitor, acts on tumour endothelial cells, a mechanism that is not always directly related to its designated targets. 38 Since resistance to sunitinib is a frequent event in the clinical management of RCC, one way to overcome this challenge is the optimisation of high-order drug mixtures to kill drug-resistant cancer cells. 19,39 Notably, treatment with relatively low-dosed ODCs was effective also in cells chronically pre-treated with sunitinib. Those ODCs were shown to be superior to non-optimised drug combinations (not shown) as well as to sunitinib. Our ODCs are active after multiple administration in sunitinib-treated cells, and their activity is even stronger under short-term starvation conditions ( Supplementary Fig. S9). Repeated drug intake following a well-designed schedule seems to result in a promising treatment strategy. Morphometric analysis of the cells chronically treated with sunitinib did not show hypertrophy as compared with sunitinibnaive cells. However, this phenomenon was observed by others in RCC cells treated with 10 µM sunitinib. 40,41 This difference might be due to the fact that long-term treatment with a lower dose of sunitinib, in our case 1 µM, which is sequestered in lysosomes of the cell, does not lead to any cell skeleton adaptation. We noticed, however, the appearance of hypertrophy in Caki-1-ST cells after ODC-and the corresponding monotherapy treatment. The exact mechanism of this phenomenon is worth pursuing, and it could be related to the impaired migratory properties of these cells. 42 Interfering with the EGFR-MAPK signalling pathway can lead to such morphologic changes by increasing cell-cell contacts profoundly stabilising focal adhesions. 43 Therefore, it seems that the loss of motility may be induced by blocking EGFR-MAPK signalling, further modulating the cell-matrix and simultaneously enhancing cell-cell interactions. 42 Malignant cells are capable of communicating with neighbouring cells, as well as the tumour microenvironment by forming tunnelling nanotubes (TNT). 44,45 Intercellular exchange of organelles, vesicles and signalling molecules can occur, which mediates the passage of resistance and the insensitivity to small-molecule drugs. 46 A498-ST (Fig. 4) and Caki-1, as well as Caki-1-ST cells (not shown), formed TNTs with an average length of ≤45 µm. Treatment with ODC destroyed already-existing tubes and blocked the formation of new connections. Our data suggest that applying the ODC blocks long-distance cell communication and interchange that might alleviate the aggressiveness of RCC and decelerate tumour progression.
The abnormal proliferation and cell cycle regulation are hallmarks of cancer cells. 47 These disordered processes are influenced after short-and long-term treatment with cell linespecific ODCs. The cell division, in particular, the progression from G1 to S phase of the mitotic cycle is impaired. It appears that this blockade is the leading cause driving RCC cells into apoptosis. Exceptionally, our results revealed that Caki-1 cells die via necrosis after ODC treatment. Senescence-mediated cell fate via p21 and MAPK is not increased after 2 h or 24 h of ODC treatment. However, senescence appeared due to chronic sunitinib treatment shown by western blot. 48,49 Another important finding of this study was that the potent anti-proliferative effects of the ODCs observed in simple 2D in vitro assays were also present in complex 3D co-culture systems where the crosstalk between RCC, fibroblasts and endothelial cells is present. In some cell lines, the activity of ODCs was even more potent in 3D conditions ( Fig. 3 and Supplementary Fig. S7), which might be related to the different target expression as compared with 2D models. 50,51 Since RCC is known to be chemo-and radio-resistant, 52,53 current clinical treatment is based on the use of angiostatic targeted tyrosine kinase inhibitors, administered individually or in sequenced combinations. 54 However, these drugs do not radically prolong the survival of RCC patients mostly due to the acquired drug resistance. 2,55,56 As targeted drugs were originally aiming for angiogenesis inhibition, we performed a dedicated TGMO-based drug optimisation in human ECRF24 cells. The ODC found for ECRF24 cells, although containing drugs such as pictilisib, which was present in all cancer cell line ODCs, was less effective, only inhibiting approximately 50% in ECR24. Interestingly, it was also found that all RCC-cell line-specific ODCs were potent in ECs (Supplementary Table S3). That indicated that both anticancer and anti-angiogenic mechanisms constitute the global activity of the ODCs. This is an expected advantage, considering the intrinsic sensitivity of RCC for angiogenesis inhibitors.
Targeting various molecules up-and downstream of GFR signalling pathways is a common strategy in cancer treatment. One approach to treat RCC is to use multi-kinase inhibitors, i.e. sunitinib, 57 pazopanib 58 or cabozantinib. 59 However, the doses applied are high, leading to enhanced off-target effects and the appearance of severe side effects. In the case of sunitinib, pazopanib and cabozantinib, the targets where sunitinib binds with the strongest affinity are located at an equal level within tyrosine kinase signalling. Combinations of drugs that have a specific targeting profile in different signalling pathways at lower doses will demonstrate a better safety profile, and are easier to adapt in a personalised fashion. 60 Previous studies have shown the importance of combining drugs that interfere with molecules up-61-63 and downstream [64][65][66] of a certain signalling cascade. However, the choice of targets and drugs is crucial and determines treatment success. Combinations of mTORC1 inhibitors with anti-VEGF agents have failed in the clinic the treatment of advanced RCC, 67 because of inaccurate composition. Nonetheless, this does not exclude the benefit of combined blockade of these targets for validation in experimental or clinical settings. [68][69][70] As a consequence, we suggest that our way of designing combinations of four drugs at low doses, inhibiting GFR signalling at multiple levels and within multiple pathways, is valuable in the development of innovative treatment options for RCC. We are confident that the ODCs validated through our study are of high quality, as it has been demonstrated that a triple combination of AZD8055, pictilisib and selumetinib can reduce tumour growth in vitro as well as in vivo settings. 66 The potency of the anticancer efficacy resulted from an underlying synergy between mTORC1/2 (AZD8055), PI3K (GDC-0941) and MEK1/2 (selumetinib) inhibitors. 66 In accordance with these studies, we are confident that our approach revealed ODCs with highly selective and synergistic anticancer activity.
We are aware that our model systems do not fully represent in vivo conditions. Several factors, such as drug metabolism, drug clearance, specific organ toxicities, the presence of stem cell involvement and immunity in the treatment response were not evaluated in this study. However, it has been demonstrated previously that a triple combination of AZD8055, pictilisib and selumetinib can reduce tumour growth in in vitro as well as in vivo settings. The potency of the anticancer efficacy resulted from an underlying synergy between mTORC1/2 (AZD8055), PI3K (pictilisib) and MEK1/2 (selumetinib) inhibitors. 66 In accordance with these studies, we are confident that our approach revealed ODCs with highly selective and synergistic anticancer activity. Nevertheless, clear evidence for the efficacy and safety profile of the combination has to be gained from preclinical and clinical trials. Therefore, we are currently planning validation of the abovedescribed results' in vivo models of RCC where tumour angiogenesis and immune responses can be studied in more detail.
CONCLUSIONS
Optimised drug combinations of four drugs applied at low doses, always containing AZD4547 and pictilisib, have been identified as an effective regimen that is synergistically toxic to RCC cells and non-toxic in non-cancerous cells, both in sunitinib-naive and sunitinib-treated cells. ODC treatment induced apoptosis and outperformed current standard RCC care, sunitinib. Further mechanistic studies and preclinical validation will guide conceivably to clinical evaluation. | 2020-05-22T14:58:16.037Z | 2020-05-22T00:00:00.000 | {
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252209055 | pes2o/s2orc | v3-fos-license | Online Mindfulness-Based Logotherapy Program: A Pilot Study Targeting Depressive Symptoms of Cyberbullied Adolescents During the Covid-19 Pandemic
The outbreak of the Covid-19 pandemic has led to the intensive use of the internet for educational and recreational purposes, leading adolescents to be more exposed to cyberbullying. Empirical studies indicate that cyberbullying has become a pervasive problem among adolescents causing negative consequences like depression. Moreover, the global pandemic lockdown and quarantine measures gave rise to the demand for telepsychology and teletherapy to virtually address the psychological needs of netizens. Thus, utilizing Conklin’s program development model, we created and experimentally tested an online intervention program to target the depressive symptoms experienced by cyberbullied adolescents. We integrated logotherapy and mindfulness to develop an eight-modular, Online Mindfulness-based Logotherapy Program (OnlineMLP). After being validated by experts in various disciplines, we experimentally pilot tested the efficacy of the OnlineMLP in a 3-h session per module in four weeks to a group (N = 10) of cyberbullied adolescents in Tamilnadu, India. The pilot study showed significantly that the OnlineMLP had a positive effect on the participants reducing their depressive symptoms. Furthermore, it confirmed that the online program’s concepts and structure are reliable, feasible, and efficacious for testing a more extensive base of cyberbullied adolescents experiencing depressive symptoms.
Adolescents experience unique opportunities across social media to express their creativities and find people with the same talents and ideology whose interests differ from mainstream culture (Moreno & Kolb, 2012;O'Keeffe et al., 2011;Reid & Weigle, 2014). The Covid-19 pandemic led to an increase in adolescents using digital platforms for their educational and recreational purposes. This unprecedented period and more time spent online pave the ways for bullies to reach the victims at any time in the day and night. Cyberbullying happens via calling and sending offensive and threatening messages, calls, and e-mails, and morphing one's picture with disturbing images, excluding someone from the group, and spreading rumors. These are classified as written, verbal, visual, impersonation, and exclusion (Alvarez-Garcia et al., 2015;Nocentini et al., 2010). L1ght, an organization, conducted a survey (2020) deep dive into millions of websites, teen chat rooms, and gaming platforms and revealed a 70% increase in cyberbullying during these Covid-19 pandemic times. Mkhize and Gopal (2021) surveyed collecting data from Facebook, Twitter, and Instagram posts since the beginning of lockdown and revealed that most had been victims of cyberbullying with the increase of social media usage.
Cyberbullied adolescents experience internalizing problems such as stress, anxiety, depression, anger, suicidal ideation, somatic symptoms, and emotional issues; externalizing problems like conduct problems, drug, and alcohol use; and decreased prosocial behaviors. Similarly, the victims of cyberbullying experience a decrease in self-esteem, life satisfaction, and perceived support (Fisher et al., 2016;Kowalski et al., 2014). In addition, adolescents with severe depressive symptoms showed lower levels of functioning, a lack of meaning in life, and satisfaction in daily activities, role functioning, and social relationships (Kuehner, 2002;Saarijarvi et al., 2002). Cyberbullying brings about significant strain to an adolescent's psychological state that leads to adverse outcomes. The general strain theory (GST) of Robert Agnew describes strain as "events or conditions that individuals dislike," and various forms of strains or stressors increase the likelihood of negative emotions such as anger, frustration, depression, and despair (2006, p.4; 1992). Agnew proposed GST and said that unfavorable conditions that generate strains might pressure individuals into engaging in crime through negative emotions if unaddressed. Agnew presented three significant types of strain (Agnew, 1992): the inability of individuals to achieve their goals, the loss of positively valued stimuli, and the presentation of noxious or negatively valued stimuli. The third strain explains individuals' unexpected exposure to events or conditions such as child abuse, bullying, cyberbullying, hostile relations with parents and teachers, or criminal victimization (Agnew, 1992;Jang & Agnew, 2015). The strain causes dysfunctional beliefs, which also leads to developing cognitive triad: (a) negative view of the self, (b) negative view of the world, (c) negative view of the future (Beck, 1976, p.11). Beck found that such thought happens automatically that the individuals might not even be conscious of their presence or realize that they occur (Sharf, 2016;Sue & Sue, 2008). The cognitive triad has been associated with vulnerability for depression in adolescents (Braet et al., 2015).
A systematic review of school-based cyberbullying prevention and intervention programs that were carried out in 9 countries and 17 studies revealed that programs that address cyberbullying effectively reduce cyberbullying among school-aged youth irrespective of the theory, sampling, or design (Tanrikulu, 2018). The global pandemic with its consequences of lockdown and quarantine measures has led to the emergence and increased demand for telepsychology and teletherapy. In-person or face-to-face therapies and interventions have been pushed to be converted into an online strategy to be executed and administered virtually. After implementing telemental health service in the U.S. Department of Veterans Affairs (VA), patient's hospitalization between 2006 and 2010 was approximately reduced to 25% (Godleski et al. 2012;Caver et al., 2019). In addition, a study conducted among 768 US mental health professionals regarding telepsychology during the Covid-19 pandemic resulted in 39% respondence that used telepsychology before Covid-19, which became 98% during Covid-19 (Sampaio et al., 2021). Furthermore, for the patients with chronic pain during the Covid-19 outbreak, remotely supported (eHealth) service positively helped pain management (Eccleston et al., 2020). Thus, to provide this growing need for telemental health services through online or virtual therapies and intervention programs to provide psychological services remotely and safely amidst the Covid-19 threat, we developed an online intervention program to target the depressive symptoms experienced by adolescents who have been bullied, combining mindfulness therapy and logotherapy.
The mindfulness theory of Shapiro guides us to stand back and witness rather than be immersed in the life story. In perceiving, the "subject" becomes "object." The individual will realize, "this pain is not me," "this depression is not me," and be able to connect moment-to-moment experience and understand what is, instead of a commentary or story about what is. Shapiro and his colleagues defined mindfulness with three axioms: intention (I), intentions as dynamic and evolving, which allows them to change and develop with deepening practice, awareness, and insight; attention (A), paying attention involves observing the operations of one's moment-to-moment, internal and external experience; and attitude (A), the qualities one brings to attention have been referred to as the attitudinal foundations of mindfulness (Shapiro et al., 2006, p. 376). This model gives a shift in perspective, which is termed reperceiving.
On the other hand, logotherapy of Victor Frankl says life has meaning in all circumstances, and people can discern the meaning potential in every passing moment; people have an innate desire and motivation to find purpose in life; everyone has the freedom of attitudinal choice, even in situation of unalterable condition, respectively (Devoe, 2012;Frankl, 2006). Therefore, he emphasized dereflection, meaning attention, which helps to refocus on the proper object (Devoe, 2012;Schulenberg et al., 2008), and also in the treatment instead of hyper-intention (forced intention), Frankl insists on hyper-reflection (excessive attention). Therefore, while practicing reperceiving and dereflection, the adolescent victims of cyberbullying can view self, world, and the future and get relieved from the depressive symptoms.
On this premise, we hypothesized that the Online Mindfulness-Based Logotherapy Program (OnlineMLP) will have a positive effect on the cyberbullied adolescents by reducing their depressive symptoms. Thus, the present study integrated mindfulness therapy and logotherapy in developing the Online Mindfulness-Based Logotherapy Program (OnlineMLP) and pilot tested its efficacy in reducing the depressive symptoms of adolescent victims of cyberbullying.
Design
The present study employed Conklin's (1997) program development model that consists of three phases: (1) planning, (2) design and implementation, and (3) evaluation and validation in constructing the Online Mindfulness-Based Logotherapy Program (OnlineMLP). Using a program development model enhances program relevance and allows for concentrated resources to be focused on the most severe, contemporary needs of many people (Boyle & Mulcahy, n.d.). We designed the OnlineMLP as a group activity and pilot tested it utilizing a true experimental research design, particularly the onegroup pre-test and post-test (Myers et al., 2012), to evaluate the efficacy of our online intervention.
Participants
We selected 10 adolescents (males = 4; females = 6) to participate since about 10 to 30 participants are suggested for a pilot study (Hill, 1998;Isaac & Michael, 1995). The participants studied in different colleges in Tamilnadu, India, ages 18 and 19 years (M = 18.50; SD = 0.527). Participants were randomly selected from a participant pool with the following characteristics: (a) 18 and 19 years old, (b) adolescent boys and girls, (c) had smartphones, (d) understood the English language, (e) experienced cyberbullying victimization as revealed by CYVIC, and (f) had severe depressive symptoms as revealed by BDI-II. All ten participants gave their consent to voluntarily participate in the pilot experiment without compensation or academic credits.
Demographic Information Form (DIF)
The personal data sheet is a researcher-made demographic questionnaire used to obtain the participants' socio-demographic profiles. It provided relevant information and helped in the process of inclusion and exclusion of the participants. The datasheet contained personal and professional information such as age, gender, education status, owning a smartphone or not, and staying at home or relatives' house or hostel. Data collected from the DIF had no identifying features which protected the personal identity of the participants.
Cybervictimization Questionnaire for Adolescents (CYVIC)
CYVIC is a standardized self-reported questionnaire of Alvarez-Garcia et al. (2017) to assess the severity of cyberbullying victimization over the last 3 months (e.g., "I have received calls on my mobile that are not answered, I guess to annoy me."; "Someone has made false complaints about me in some forum, social network, or online game, which have caused me to be expelled."). It comprises 19 items evaluated in a 4-point Likert scale ranging from 1 = never to 4 = always. The higher score indicates the higher degree of cyberbullying victimization.
Beck Depression Inventory-II (BDI-II)
The BDI-II is a self-report 4-point Likert scale with 21 items (e.g., "I feel quite guilty most of the time"; "I do not feel that I look any worse than I used to.") to assess the severity of depressive symptoms. The score is ranging from 0 to 3. While adding up all the responses, a total score of 0-13 is considered minimal range, 14-19 is mild, 20-28 is moderate, and 29-63 is severe (Beck et al., 1996;Cinarbas et al., 2011;Al-Turkait & Ohaeri, 2010;Tusiime et al., 2015; Community-University Partnership for the Study of Children, Youth, & Families, 2011).
Procedure
The UST Graduate School Ethics Review Board approved this research before data collection. Due to the Covid-19 pandemic, we created a google form with the questionnaires and forwarded it via Facebook, WhatsApp, and Messenger to college-going adolescents in Tamilnadu, India. The participants voluntarily completed the google form, which contained the questionnaires, brief description of the study, procedures, benefits, and risks, and gave consent to participate in this study. Of the 612 participants who came within the inclusion criteria, 281 (45.91%) had higher cyberbullying victimization and severe depressive symptoms. We randomly selected ten adolescents for the pilot study from this pool of participants. Following the government regulation during the Covid-19 pandemic, we conducted the OnlineMLP in a 3-h module in 4 weeks via secure version of zoom. Homework was given to practice what they were learning in the sessions. We ran each session with all 10 participants attending all the sessions the entire time. During the program, all participants used laptops, and for every session gave their informed consent to participate and be recorded; they were assured that the recorded sessions are confidential and will be safely secured, adequately deleted, and discarded after completion of the study. On the last day, program evaluation form was given to the participants to get their feedback about the program. In addition, a distress protocol was in place if participants would need them during and after participation. Crisis hotlines (cellphone numbers) were also provided to the 10 participants after completing the online intervention program. The recorded sessions helped us get more information to update this program for the final phase of the program development. All recordings were securely deleted after and participants were informed accordingly.
The development and pilot testing of the Online Mindfulness-based Logotherapy Program (OnlineMLP) had three phases: (1) planning, (2) design and implementation, and (3) evaluation and validation. Phase 1 is planning which entailed identifying program goals, conducting a needs assessment, and setting program priorities. It also involved identifying target audiences, writing program objectives from the extensive study of the available related literature, and analyzing data from surveys, interviews (10), and focus group discussions (10) from the pool of participants. Phase 2 involved designing and implementing the OnlineMLP; selecting and developing themes, delivery methods, and resource materials; and constructing an implementation timeline. The logical flow of the program followed with introducing OnlineMLP and its objectives to the adolescents, educating the participants to understand the interconnection between body, mind, and spirit. This second phase also involved guiding them to notice dysfunctional thoughts, debrief the suffering, engage them to nurture the meaning, establish attitudinal change, and relieve them from depressive symptoms. The third phase, evaluation and validation, involved expert validation and a pilot study to determine the program's efficacy.
Results
The present study was able to develop an online intervention program that integrated logotherapy and mindfulness to target the depressive symptoms experienced by cyberbullied adolescents using Conklin's program development model. A team of seven mental health experts, such as clinical psychologists, counselors, and psychiatrists, evaluated the OnlineMLP. Then, using the standardized evaluation guidelines, which was an adapted form of the tool developed and used by United States Agency for International Development (2016), the experts unanimously graded the program with an overall score of "A," indicating the soundness, relevance, and feasibility of the OnlineMLP. Though all the experts are in complete agreement and gave an overall score of "A," they still gave corrections, modifications, and suggestions to further improve the program. In addition, the inter-rater reliability resulted in a coefficient of 0.709. Thus, the experts' validation assured that the program is reliable and predicts a high chance of bringing about affirmative changes.
Ten adolescent participants were randomly selected for the pilot study to test the feasibility through a one-group pretest and post-test experimental design. In the Appendix shows the schedule of the 3-h module over 4 weeks. And Table 2 shows the pre-and Post-test scores of the participants on depression as measured by BDI-II. Statistical analysis was carried on with the pre-and post-test scores using the "Wilcoxon signed-rank test," and the study results are shown in Table 3. The result confirmed that the OnlineMLP Connecting body, mind, and spirit Identifying the connection between body, mind, and spirit; becoming aware of negative thoughts; and picking one item in each dimension (mind, body, spirit) and working on it throughout the day 3.
Acknowledging thoughts Acknowledge whatever is arising in mind; recognize the tragic triad; using paradoxical intention; practicing self-distancing; guiding to improve self-esteem and self-efficacy; teaching various strategies to create positive vibes 4.
Your suffering is not you To enhance participants, treat themselves with as much kindness as they would when another person is in pain; discuss existential frustration, despair, and loss of hope; make them understand an internal pulling force; help to improve social skills self-acceptance 5.
Explore and express innate power Motivate the participants to scull out the moments that they miraculously solved the problems; encourage, explore the meaning, and express their creativity; guide to reperceive the facts; help to understand that freedom is an innate need 6.
Attitudinal change To encourage the participants to change attitudes in the face of life's difficulties; motivate them to follow "selfless compassion"; encourage them to connect with their inner resources and develop and strengthen a sense of inner calmness; enable the participants to reduce aggressive thinking and building trust 7.
Respond than react Help to practice non-judgmental and let go; encourage to understand and practice a quick exercise that will help bring back positive energy to life; help to practice lovingkindness; allow them to switch from the usual mode of doing to non-doing 8.
Stabilizing the change To enable the participants to feel better about themselves, being accepted and loved; help them be present and be mindful at every moment; prepare the participants for termination is reliable, feasible, and efficacious in targeting depressive symptoms of the cyberbullied adolescents. Table 3 shows the outcome of the statistical analysis using pre-test and post-test mean scores and standard deviation. The results indicated a decrease in depressive symptoms among the adolescent participants after the feasibility test. The level of decreased depression is shown from the pre-test mean and standard deviation score of 44.70 (4.42) to the post-test mean and standard deviation score of 21.00 (5.79). In addition, there is a significant difference in the pre-test and post-test scores of BDI-II (z = − 2.805; p = 0.005).
The ten adolescents who participated in the 4-week-long eight-modular OnlineMLP perceived the experience positively and expressed satisfaction and appreciation for being part of the pilot study. In addition, everybody qualitatively reported improved mindful attention, reperceiving the facts, and decreased depressive feelings. One participant said "Yes, through this program, I have learned a lot to look into the facts as an 'observer' rather than an active participant. It helps me to move on from my negative thoughts." Another participant said "this program helped me cultivate attitudes to find meaning in life and get away from the depression."
Discussion
At present, no online intervention study to address cyberbullying and reduce depressive symptoms has been available. Nonetheless, the Online Mindfulness-based Logotherapy Program (OnlineMLP) development integrated the theories of mindfulness and logotherapy to target the depressive symptoms among the cyberbullied adolescents during the Covid-19 pandemic. The OnlineMLP was pilot tested with ten participants for 4 weeks to assess its feasibility and usability for a larger population. The intervention outcome statistically supported and validated the impact and efficacy of the OnlineMLP as an intervention program. Furthermore, the result shows substantial implications that the mindfulness theory and logotherapy concepts are efficacious and have achieved the program objectives.
Before the pilot test, we carefully examined the research conditions and the demographic profile of the participants to ensure homogeneity of research conditions. This examination revealed that the participants had a high degree of cyberbullying victimization and had severe depressive symptoms. This outcome supported the need for intervention programs like OnlineMLP to address the depressive symptoms of cyberbullied adolescents comprehensively.
The pilot study result is consistent with the evaluation and validation of the seven mental health experts which indicates that the OnlineMLP is scientifically sound and may positively influence change when implemented among adolescents. The adolescents were able to learn to be mindful in each moment, to have positive energy, happiness, and meaning in life. Becoming aware of the connection between body, mind, and spirit has shown changes in seeing the facts as an "observer" rather than an active participant. The adolescents also expressed that reperceiving and dereflection enhanced their capacity to take another person's perspective and facilitate empathy. They were able to identify that freedom is an innate need they all have and want to acquire. These positive changes were reflected in the post-test score and showed a decrease in the depressive symptoms.
Conclusion
We successfully integrated mindfulness therapy and logotherapy in developing the Online Mindfulness-Based Logotherapy Program (OnlineMLP) and tested its efficacy in reducing the depressive symptoms of adolescent victims of cyberbullying through a true experimental research design, specifically the one-group pre-test and post-test. The OnlineMLP had a significant positive effect on the participants and was able to reduce their depressive symptoms. Moreover, our online program's concepts and structure are reliable, feasible, and efficacious for testing a more extensive base of cyberbullied adolescents experiencing depressive symptoms.
Limitations and Future Directions
We designed the OnlineMLP program as a group activity. Yet, it can also be implemented on an individual level. During the pilot test, the participants expressed shyness and uneasiness to share their life events with other adolescent participants. Thus, the OnlineMLP must be tested on a one-on-one engagement held in few sessions. The purpose of OnlineMLP as an intervention program was mainly to address the depressive symptoms caused by cyberbullying victimization during the Covid-19 pandemic. However, we acknowledge that other factors may also contribute to causing depressive symptoms, such as loneliness, pressures from the school or parents, and family issues. Therefore, to maximize the benefits of the OnlineMLP, it may be further tested on a larger base of individuals with extensive planning and among different cultures. The function of gender differences on either variable was not an objective of our study, as such future research could explore and investigate on this.
The OnlineMLP has been pilot tested via a virtual platform and has been proven as an efficacious intervention program to reduce the depressive symptoms among the cyberbullied college-going adolescents in Tamilnadu, India. However, we acknowledge the importance of human contact and recommend that the OnlineMLP be tested in a face-to-face or in-person setup. No matter how hard we try to transfer human communication to be entirely virtual, nothing can replace human contact.
Module
Section Time frame
Declarations
Ethics Approval All procedures performed in the present study that involved human participants were in accordance with the ethical standards of the Ethics Review Committee Board of the Graduate School, University of Santo Tomas.
Consent to Participate
Each participant in the current study gave their informed consent before voluntary participation. In addition, participants were briefed on the nature of the study, and were assured that all data collected will be kept confidential and that participation is voluntary without compensation or academic credits.
Conflict of Interest
The authors declare no competing interests. | 2022-09-14T05:06:52.969Z | 2022-09-10T00:00:00.000 | {
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29707785 | pes2o/s2orc | v3-fos-license | Acute hypocomplementemic post-infectious glomerulonephritis as a complication of sinus-related orbital cellulitis : case report
1 Professor Doutor Colaborador do Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo USP Ribeirão Preto (SP) Brasil. 2 Professor Titular do Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço da Faculdade de Medicina de Ribeirão Preto da USP Ribeirão Preto (SP) Brasil. 3 Médica Assistente do Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço da Faculdade de Medicina de Ribeirão Preto da USP Ribeirão Preto (SP) Brasil. 4 Pós-graduada doutorado pelo Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço da Faculdade de Medicina de Ribeirão Preto da USP Ribeirão Preto (SP) Brasil.
INTRODUCTION
Sinus-related orbital cellulitis is a serious condition that can lead to several intracranial and ophthalmic complications (1) .Hypocomplementemic acute glomerulonephritis (AGN) is an immune disorder that is commonly seen in children with infectious diseases caused by a variety of microorganisms, especially Group A β-hemolytic streptococci (2)(3) .We describe a case of AGN associated with sinus-related orbital cellulitis.
CASE REPORT
A previously healthy 11-year-old boy was evaluated for an acute inflammation in the right orbit.On examination, the right eye was proptotic (Hertel measurements were 22 and 12 mm) with swelling of the upper and lower eyelids.Ocular motility was limited in all gaze directions.Visual acuity was 20/40 of the right eye (OD) and 20/25 of the left (OS).Intraocular pressure was 40 mmHg in OD and 12 mmHg in OS.Orbital computed tomography (CT) disclosed a right inferomedial orbital abscess and ipsilateral pansinusitis (Figures 1 and 2).A cantholysis was performed on the OD during admission.The next day the boy underwent middle turbinate resection and ethmoidal and maxillary sinus drainage under general anesthesia.The orbit was approached by an inferior fornix transconjunctival incision.Abundant pus was aspirated from the orbit and sinuses.The culture grew positive for Group A β-hemolytic streptococci.The patient was treated with parenteral antibiotics (ceftriaxone and clindamycin) and five days after surgery he was discharged with full recovery of eye motility and complete resolution of proptosis.Four days after discharge the child presented with facial edema and signs of distress.A pediatric evaluation disclosed generalized edema and systemic hypertension (200 x 100 mmHg), with no other cardiac findings.Blood count, antinuclear antibody, rheumatoid factor, LE cells and serologic tests were within normal limits.Urinalysis showed hematuria and mild proteinuria.Seric complement component levels C3 and C4 were low.The patient was diagnosed as having acute poststreptococcal glomerulonephritis and was treated with furosemide, nifedipine and salt and fluid restriction.The outcome was favorable and after 12 days of hospitalization the boy was discharged without any signs of renal dysfunction, and with increase of complement levels afterwards.
DISCUSSION
Postinfectious acute glomerulonephritis may be induced by several bacteria including S. aureus, S. epidermidis, meningococcus, atypical mycobacterium, and Corynebacterium (2) .Ho-wever, group A β-hemolytic streptococcus is the most common causative agent implicated, mainly after acute pharyngitis.AGN is not a common complication, with a frequency less than 5% after a streptococcal infection (2,4) .These associations are usually sporadic, notwithstanding they may present in epidemic events in some countries.A progressive renal disease is quite rare and a complete recovery with normal diuresis occurs in more than 90% of treated cases (4) .
The pathogenesis of the disease is linked to the production of specific bacterial antigens which are targeted by the host immune response.Immune complexes are then formed and deposited within the glomeruli, activating the complement cascade and attracting inflammatory cells.The resulting inflammatory reaction provokes proliferation of mesangial and endothelial cells and produces bloodless glomeruli, a decrease in renal filtration and retention of fluid and salt (2)(3) .
The association between sinus-related orbital cellulitis and AGN seems to be quite rare.In a literature search using the Medline database we could not find any report on this association.AGN is usually controlled with supportive measures.However, when the disease is not recognized, serious effects such as congestive heart failure may ensue.The present report highlights the necessity of clinical evaluation of any children with sinus-related orbital cellulitis.Ophthalmologists should be aware that orbital infections can induce renal complications. | 2017-10-21T04:53:18.995Z | 2008-07-01T00:00:00.000 | {
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85182789 | pes2o/s2orc | v3-fos-license | Egg Structures of Anopheles fluminensis and Anopheles shannoni
Eggs of two species belonging to the Arribalzagia Series of the Laticorn Section of Anopheles (Anopheles) collected in Brazil are described from scanning electron micrographs. The An. fluminensis egg is long with shallow floats displaced far dorsally. The narrow deck region is overlain by a frill modified into prominent ridges that are nearly continuous to both ends of the egg. Slightly opened decks at both poles contain an average of four lobed tubercles. Polygonal, plastron-type chorionic cells cover the lateral and dorsal surfaces. The egg of An. shannoni is unique in possessing 22-27 fingerlike filaments that project with regular spacing from each of its massive floats. These filaments and their bases are highly perforated and are believed to trap air and support flotation of the egg with the dorsal surface up, contrary to the usual orientation for anophelines. The eggs are compared with those of related species bearing similar structures, notably An. fluminensis with An. mediopunctatus .s and An. shannoni with An. peryassui .
For many subsequent years anopheline eggs received scant attention until Hinton (1968) recognized the potential of the scanning electron microscope (SEM) for visualizing egg microstructures and, by extension, new morphological characters for species recognition.However, only in the present decade has the SEM been applied extensively to eggs of New World Anopheles for describing structural details (e.g.Linley & Lounibos 1993, 1994), intraspecific variation (Rodriguez et al. 1992), geographic differentiation (Linley et al. 1996), and for separating members of species complexes (Linley et al. 1993) or separate species mistakenly synonymized (Lounibos et al. manuscript in preparation).
The present study describes the ultrastructure of eggs of two anopheline species that had previously been known only at the light microscope level.Anopheles (Anopheles) fluminensis was originally described by Root (1927) from the State of Rio de Janeiro, and its egg was first depicted by Causey et al. (1944) from Amazonian collections of this species.Although it has not been regarded as a vector in malarious regions of Brazil (Cerqueira 1961), mosquitoes identified as An.sp.near fluminensis were incriminated as vectors of human malaria in eastern Peru (Hayes et al. 1987).The first illustration of the egg of An. shannoni Davis, at the time undescribed, was by Bonne and Bonne-Wepster (1925) (as An. mediopunctatus [Theobald]).The highly modified egg of this species was first described by Causey et al. (1944) from collections in the State of Pará, Brazil, the type locality of An. shannoni (Davis 1931).Anopheles shannoni has not been suspected as a vector of human malaria (Deane et al. 1948, Cerqueira 1961), in part because it prefers to bite in the canopy of forested regions (Deane et al. 1953).However, such acrodendrophilic host-seeking behavior makes An. shannoni a possible maintenance vector of simian malaria where this mosquito species and monkeys co-occur in the Amazon region (Lourenço-de-Oliveira & Luz 1996).
Both An. fluminensis and An.shannoni belong to the Arribalzagia Series of the Laticorn Section (Reid & Knight 1961) of the subgenus Anopheles.Wilkerson and Peyton (1990) inferred a monophyletic origin of the Arribalzagia Series based upon wingspot characters.Since eggs of several other species of this Series have been described recently using the SEM (Linley & Lounibos 1994, Linley & Milstrey 1995, Lounibos et al. in preparation), we infer in this paper possible species affinities based on egg structures.
MATERIALS AND METHODS
Female An. fluminensis were collected in January 1995 from human bait at Picinguaba, State of São Paulo, Brazil (23 o 23'S, 44 o 50'W) and female An.shannoni were collected by the same method in July 1994 at Samuel Ecological Station, State of Rondônia, Brazil (9 o 07'S, 63 o 16'W).Blood-fed females, identified with the keys of Consoli and Lourenço-de-Oliveira (1994), were isolated individually in small vials with damp filter paper for oviposition.Laid eggs were allowed 24 hr at 26 o C to embryonate before preservation in alcoholic Bouin's fixative.A few eggs of An. shannoni were immersed in water prior to fixation in order to allow float filaments to unfurl, as accomplished previously for An.peryassui Dyar and Knab (Linley & Lounibos 1994).In preparation for microscopy, eggs were removed from fixative, washed twice in 80% ethanol, then dehydrated in an ethanol series of 5% concentration increments.After critical point drying, eggs were mounted on stubs coated with sticky tape, sputter-coated with gold/palladium, and examined in an Hitachi S-510 SEM.
Electron micrographs of eggs were either scanned and rendered as computer images for measurements with SigmaScan software (Jandel Scientific, San Rafael, CA, U.S.A.), or measurements were made with a digitizing tablet and tabulated with the same software.Except where otherwise noted, measurements of An. fluminensis were made on 10-12 eggs laid by three females, and of An. shannoni on 4-6 eggs from one female.Mean values in the text are followed by ± 1 SE.The descriptive terminology follows that of Harbach and Knight (1980) except for "plastron" which conforms to the usage of Hinton (1968) to describe the network of structures that form a physical gill beneath the water line of anopheline eggs.Voucher specimens of adult females have been deposited in the Department of Entomology, FIOCRUZ.Figs 1-4) Size: egg length 515.4-546.2 µm (mean 530.8 ± 3.3 µm, n=10), width 180.8-203.8µm (mean 187.7 ± 2.2 µm, n=10), length/width ratio 2.68-2.91 (mean 2.83 ± .03,n=10).Color: black.Overall appearance: long and wide across floats, especially anteriorly, but tapering abruptly where floats terminate before anterior and posterior ends (Fig. 1a); egg boat-shaped in lateral view, dorsal surface concave but ventral surface flat (Fig. 1b); floats shallow and displaced unusually far dorsally (Fig. 1b).
Anopheles fluminensis (
Ventral (upper) surface: deck usually hidden by overlapping frills which form ventral ridges for length of the egg except for anterior and posterior poles (Figs 2a,d).When visible, deck deeply recessed between ridges which are deeply grooved in both ventral and lateral views (Figs 4a,d).Both anterior and posterior decks open, containing 3-5 mushroom-like, lobed tubercles (mean anterior 4.0 + 0.2, mean posterior 3.8 ± 0.2, n=12) (Figs 2c,f).Smaller tubercles of both anterior and posterior decks irregular, some star-shaped, and domed with buttressed walls (Figs 3a,c).Tubercles of middle deck, exposed infrequently because of ridge overlap, less densely packed and shorter, some marooned on chorionic islets (Fig. 3b).
Posterior end: blunt in end-on view (Fig. 2e), otherwise similar in conformation to ventral view Dorsal (lower) and lateral surfaces: outer chorionic cells of dorsal plastron tending to form polygons, although some with rounded borders (Fig. 3e), length 24.7-34.7 µm (mean 27.8 ± 1.2 µm, n=10), width 16.9-20.4µm (mean 17.1 ± 0.7 µm, n=10).Interior of cells with tightly packed tubercles like smooth cobblestone pavement (Fig. 3f).Exochorion continuous except for infrequent perforations between tubercles (Fig. 3f).Floats deeply grooved to margin with dorsal surface (Fig. 3d), the grooved sutures undulating near egg midline in lateral view (Fig. 1b).Boundary between float and lateral chorionic cells separated by a narrow strip of tiny, densely packed tubercles (Fig. 4e); high magnification reveals these tubercles to be of varying sizes with smooth domes and buttressed roots, some with fine projections from roots (Fig. 4f).Lateral chorionic cells similar in detail to dorsal counterparts in polygonal shape, boundaries with smooth, raised tubercles, cobblestone-like interior and occasional perforations (Figs 4b-d).
Anterior end, micropyle: anterior end somewhat conical, floats terminating in ventral surface before pole, left and right sides separated by narrow chorion strip (Fig. 6a).Micropylar apparatus located dorsally to an agglomeration of cauliflowerlike tubercles (Fig. 6b).Micropylar collar smooth, slightly scalloped on interior margin (Fig. 6c), radial arms extend from collar approximately halfway into disc area.Micropylar orifice recessed in low mound (Fig. 6c).
Posterior end: no obvious distinctions from anterior end in ventral view, floats terminating dorsomedially before pole (Fig. 6d).End-on view shows cauliflower-like tubercles clustered at pole and distinctive from those forming polygonal cells of dorsal chorion (Fig. 6e).
DISCUSSION
The light microscopic descriptions by Causey et al. (1944) of eggs of An. fluminensis and An.shannoni are accurate insofar as their limited resolution allows.These authors depict the frill of An. fluminensis "surrounding (a) wide, elliptical black area", which indicates that the deck of their specimens was more exposed than those of the current study.For An. shannoni drawn by Causey et al. (1944), the segments of floats near the midline do not give rise to filamentous projections, and the floats are positioned more ventrally than in our specimens.
The egg of An. fluminensis, distinctive for its ventral ridges and dorsally positioned floats, is not likely to be confused with that of any other described anopheline species.Among related species examined with the SEM, the egg of An. mediopunctatus s.s.most resembles that of An. fluminensis.However, the central deck of An. mediopunctatus s.s. is more exposed than in An. fluminensis and its dorsal ridges are coalesced into whorls (Lounibos et al. in preparation).Interestingly, the type locality of both these species is southeastern Brazil.Examinations of eggs of the malaria vector An.sp.near fluminensis from eastern Peru would be valuable for establishing its relationship to An. fluminensis from the type locality.
The filamentous projections of the floats of An. shannoni invite comparisons to the homologous extensions of the floats of An. peryassui (Linley & Lounibos 1994), which species is also placed in the Arribalzagia Series.Filaments of both species are compressed laterally until the newly laid egg touches water and are highly perforated, presumably to entrap air and assist flotation.
However, float filaments differ between An. shannoni and An.peryassui in many structural details.In the former species, these extensions project regularly from the complete length of massive floats.In An. peryassui, filaments are confined to the anterior and posterior ends, and the floats of this related species are much more slender than those of An. shannoni.Further, perforations on the margins and bases of the floats are larger and more abundant in An. peryassui.Additional features distinguish the eggs of the two species: An. shannoni eggs have no frill or crown, which occur at both ends of the An.peryassui egg [although Causey et al. (1944) show some An.peryassui without frills or crowns]; the mean egg length/width ratio is 4.0 for An.peryassui and 2.7 for An.shannoni; the ventral surface is highly concave in An. shannoni but only slightly concave in An. peryassui.
Observations on An. peryassui eggs laid in water led to the conclusion that the natural flotation position of this species is dorsal side up (Linley & Lounibos 1994).In view of the many structural similarities of eggs of the two species, we anticipate that An. shannoni eggs also float in this position, which is unique to these species of Anopheles.The ventral deck of both species, which would be submerged, is covered with porous, plastronlike chorionic cells, usually typical of dorsal surfaces, which entrap air for respiration.The larval habitat of An. shannoni is reported to be filled with leaves, branches and tree trunks (Deane et al. 1948).As conjectured for eggs of An. peryassui which also occur in detritus-laden habitats, the filaments of An. shannoni eggs may support their flotation by attachment to buoyant debris. | 2019-03-22T16:16:49.312Z | 1997-03-01T00:00:00.000 | {
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204032195 | pes2o/s2orc | v3-fos-license | Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans
Abstract Background Inorganic nitrate, abundant in leafy green vegetables and beetroot, is thought to have protective health benefits. Adherence to a Mediterranean diet reduces the incidence and severity of coronary artery disease, whereas supplementation with nitrate can improve submaximal exercise performance. Once ingested, oral commensal bacteria may reduce nitrate to nitrite, which may subsequently be reduced to nitric oxide during conditions of hypoxia and in the presence of “nitrite reductases” such as heme- and molybdenum-containing enzymes. Objective We aimed to explore the putative effects of inorganic nitrate and nitrite on mitochondrial function in skeletal muscle. Methods Mice were subjected to a nitrate/nitrite-depleted diet for 2 wk, then supplemented with sodium nitrate, sodium nitrite, or sodium chloride (1 g/L) in drinking water ad libitum for 7 d before killing. Skeletal muscle mitochondrial function and expression of uncoupling protein (UCP) 3, ADP/ATP carrier protein (AAC) 1 and AAC2, and pyruvate dehydrogenase (PDH) were assessed by respirometry and Western blotting. Studies were also undertaken in human skeletal muscle biopsies from a cohort of coronary artery bypass graft patients treated with either sodium nitrite (30-min infusion of 10 μmol/min) or vehicle [0.9% (wt:vol) saline] 24 h before surgery. Results Neither sodium nitrate nor sodium nitrite supplementation altered mitochondrial coupling efficiency in murine skeletal muscle, and expression of UCP3, AAC1, or AAC2, and PDH phosphorylation status did not differ between the nitrite and saline groups. Similar results were observed in human samples. Conclusions Sodium nitrite failed to improve mitochondrial metabolic efficiency, rendering this mechanism implausible for the purported exercise benefits of dietary nitrate supplementation. This trial was registered at clinicaltrials.gov as NCT04001283.
Introduction
Recent data suggest that there are considerable health benefits (particularly cardiovascular) from diets high in inorganic nitrate (NO − 3 ). The nitrate (NO − 3 )-nitrite (NO − 2 )-nitric oxide (NO) pathway, whereby dietary inorganic nitrate is processed via the entero-salivary circuit to release NO − 2 and NO (1), is attracting considerable interest. Adherence to a Mediterranean-style diet (typically rich in dietary nitrates) is associated with a lower risk of developing coronary artery disease or heart failure (2,3). In healthy individuals, increased dietary nitrate consumption has been shown to reduce the oxygen cost of skeletal muscle work during submaximal exercise, implying increased metabolic efficiency (4)(5)(6)(7)(8); in clinical settings, a regime of dietary nitrate supplementation improved exercise performance in heart failure with preserved ejection fraction (HFpEF) and muscle contractile function in heart failure with reduced ejection fraction (HFrEF) (9,10). However, each of these studies was limited by their small sample size. The recent, comparatively large, INDIE (Inorganic Nitrite Delivery to Improve Exercise Capacity) trial (utilizing inhaled nitrite) showed no benefit on exercise capacity in HFpEF (11). It is unclear whether the smaller studies were false positives, or whether the shorter plasma half-life of the inhaled nitrite approach failed to work because of the pulsatile rather than sustained nature of elevation of nitrite.
The physiological mechanisms underlying these apparent improvements in metabolic efficiency are not well understood. It has been proposed that ingestion of inorganic nitrate can improve mitochondrial coupling efficiency (i.e., the relationship between mitochondrial respiration and ATP generation) via reduced expression of proteins believed to facilitate mitochondrial proton leak [e.g., the ADP/ATP carrier proteins (AACs) and uncoupling proteins (UCPs)] (6). However, recent work has cast doubt on these findings. Whitfield et al. (23) observed a reduction in whole body oxygen consumption in response to beetroot juice with no alteration in mitochondrial coupling efficiency (23). Similarly, Monaco et al. (24) demonstrated improved hemodynamics with no change in cardiac mitochondrial coupling efficiency in response to oral sodium nitrate (24). An alternative mechanism would entail a shift in the Randle cycle [see Hue and Taegtmeyer (25) for a review] away from fatty acids toward glucose utilization with increased coupling of glycolysis to carbohydrate oxidation, which would be accompanied by increased efficiency with respect to oxygen consumption. Previous work has reported that inorganic nitrate is capable of increasing skeletal muscle glucose utilization in the context of type 2 diabetes (26,27).
The aforementioned studies have largely focused on investigating sodium nitrate supplementation, as opposed to sodium nitrite, the putative "active principle" of inorganic nitrate. Given the limitations of entero-salivary conversion of sodium nitrate to sodium nitrite, the following investigation sought to elucidate the molecular mechanisms whereby sodium nitrite may improve metabolic efficiency in skeletal muscle.
Treatment conditions
Male C57/bl6 mice, 3-5 mo old, were obtained from Harlan Laboratory UK Ltd and the University of East Anglia Disease Modelling Unit. After a 2-wk acclimatization period to local vivarium conditions, mice were subjected to a nitrate/nitritedepleted diet for 2 wk. Mice were then administered either sodium chloride, sodium nitrate, or sodium nitrite at a concentration of 1 g/L in drinking water ad libitum for 7 d. All experimental procedures and protocols used in this study were reviewed and approved by the Animal Welfare and Ethical Review Body and were conducted according to the specifications of the United Kingdom Animals (Scientific Procedures) Act, 1986.
Determination of plasma nitrate/nitrite concentrations
Mice were anaesthetized using 5% isoflurane and 4 L/min oxygen, and blood was collected via intracardiac puncture into EDTA-containing anticoagulant tubes containing 100 μL 10 mM N-ethylmaleimide. Plasma was generated by centrifugation at 2000 × g for 10 min at room temperature, then snap-frozen using liquid nitrogen and stored at −80 • C until analysis. Plasma concentrations of nitrate and nitrite were determined by a dedicated HPLC system (ENO-20; Eicom), as previously reported (28).
Mitochondrial isolation
A subsequent cohort of mice (C57/bl6 mice, 3-5 mo old) were killed by cervical dislocation and gastrocnemius muscle samples harvested from both hind limbs were finely dissected in 1 mL cold isolation buffer (100 mM sucrose, 100 mM KCl, 50 mM Tris-HCL, 1 mM KH 2 PO 4 , 0.1 mM EDTA, and 0.2% BSA; pH 7.2) and washed, before incubating with 12 U/g protease (Sigma-Aldrich) in 5 mL isolation buffer for 2 min on ice. The tissue was removed from the protease by brief centrifugation (200 × g for 5 min at 4 • C) and resuspended in 20 mL fresh isolation buffer. The suspension was then transferred into a Dounce homogenizer and homogenized using 10 up-and-down strokes on ice, before centrifugation at 700 × g for 10 min at 4 • C. The supernatants were filtered (70 μm cell strainer) and centrifuged at 8000 × g for 10 min at 4 • C. The pellets were resuspended in cold isolation buffer and centrifuged at 7000 × g for 10 min at 4 • C. Isolated mitochondria were resuspended in 150 μL suspension buffer (225 mM mannitol, 75 mM sucrose, 10 mM Tris, and 0.1 mM EDTA; pH 7.2), assayed for protein content (Pierce BCA Protein Assay Kit; ThermoFisher Scientific), and kept on ice until further use.
Seahorse XF24 analyzer.
The oxygen consumption rate (OCR) of mitochondria isolated from murine skeletal muscle was initially measured using an XF24 analyzer (Seahorse Bioscience). The mitochondrial suspension was deposited onto XF24 cell culture microplates (Seahorse Bioscience) at a concentration of 2.5 μg/50 μL in mitochondrial assay solution (MAS) buffer [70 mM sucrose, 220 mM mannitol, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM HEPES, 1.0 mM EGTA, and 0.2% (wt:vol) fatty acid-free BSA; pH 7.2] containing 10 mM pyruvate/5 mM malate (mitochondrial complex I substrate). The plates were centrifuged at 2000 × g for 20 min at 4 • C. Prewarmed (37 • C) MAS buffer containing complex I substrate was added to the equivalent wells to a final volume of 495 μL. The plate was equilibrated in a carbon dioxide-free incubator for 8 min at 37 • C, then placed in the analyzer. OCR was measured in triplicate at baseline, and after sequential addition of 4 mM ADP, 2.5 μg/μL oligomycin, 4 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 4 μM antimycin A (mitochondrial "stress test") according to the manufacturer's instructions. The OCRs were used to determine maximal mitochondrial respiration (FCCP-induced OCR minus antimycin A OCR), leak (oligomycin-induced OCR minus antimycin A OCR), and the respiratory control ratios (ADP-induced OCR:oligomycin-induced OCR), accordingly.
Oxygen electrode.
For a more detailed analysis of respiratory function, mitochondrial oxygen uptake activity was measured using a Clark-type oxygen electrode (Oxytherm; Hansatech), calibrated following the manufacturer's instructions. Aliquots (0.75 mg) of isolated muscle mitochondria were suspended in 1.5 mL air-saturated MAS buffer at 37 • C with the addition of 10 mM pyruvate/5 mM malate to initiate mitochondrial oxygen uptake activity (at an acquisition rate of 5 Hz, stirring at 27 rpm). For the assessment of key respiratory parameters, OCRs were measured after the sequential addition of 4 mM ADP (to initiate maximal "state 3" respiration), 5 μM oligomycin (to measure the rate associated with proton leak), 7 μM carboxyatractyloside (to determine the contribution of AAC to the proton leak rate), 2 μM FCCP (to give the maximal uncoupled activity), and 2 μg/mL antimycin A (to fully inhibit mitochondrial respiration). Alternatively, 2 sequential additions of 400 nmol ADP (ε 259 = 15,400 M −1 /cm) were used to induce transient state 3 rates for phosphate:oxygen (P:O) ratio determination. The corresponding amount of oxygen consumed during each state 3 burst was estimated as described in Brown and Cooper (29). Importantly, the order in which "treated" and "control" samples were measured in the electrode was alternated each day to avoid any time-dependent effects (e.g., uncoupling) inadvertently being ascribed to one mitochondrial sample type over the other.
Mouse tissue.
Mice were killed by cervical dislocation. Gastrocnemius muscle was harvested, snap-frozen in liquid nitrogen, then stored at −80 • C. Tissue samples were homogenized in lysis buffer (100 mM Tris-HCl, 2 mM Na 3 VO 4 , and 5 mM NaF; pH 7.4) using a mortar and pestle. Homogenate was subject to 3 freeze-thaw cycles using liquid nitrogen, then centrifuged at 14,000 × g for 15 min at 4 • C. Supernatants were retained and stored at −20 • C. Total extracted protein was determined by Bradford assay.
Human tissue.
Participants were recruited into the "Effect of Nitrite on Cardiac Muscle and Blood Vessels in Patients Undergoing Coronary Artery Bypass Grafting Surgery" study (NCT04001283), a randomized, placebo-controlled, double-blind clinical study investigating the potential mechanisms underlying sodium nitrite as a medical therapy (Figure 1). Eligible participants were patients undergoing coronary artery bypass graft (CABG) surgery, with reasons for exclusion being inability to provide informed consent, pregnancy or of child-bearing potential, type 1 diabetes mellitus, New York Heart Association class III-IV heart failure with left ventricular ejection fraction <40%, renal impairment requiring dialysis, and unstable coronary syndrome (occurring within the preceding 2 wk). Study participants were randomly assigned into either the treatment (sodium nitrite 10 μmol/min infused over a period of 30 min) or control [0.9% (wt:vol) saline-infused equivolume over a period of 30 min] arms, with blood samples collected immediately before and after infusion, then 6 h and 24 h postinfusion, and at time of biopsy. Infusion occurred 24 h before scheduled surgery. Skeletal muscle (pectoral muscle), right atrial appendage, and left ventricular biopsies were collected immediately before aortic cross-clamping, snap-frozen using liquid nitrogen, and stored at −80 • C until analysis. The study was approved by the institutional Ethics of Human Research Committee and complied with the Declaration of Helsinki. Written informed consent was obtained in all cases.
Samples were snap-frozen in liquid nitrogen, after which 100-150 μL NP40 cell lysis buffer (ThermoFisher Scientific) containing a Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific) was added. Samples were manually crushed, then homogenized on ice using a Pellet Pestle (Sigma-Aldrich). Samples were then snap-frozen using liquid nitrogen and allowed to thaw on ice, after which they were homogenized for a second time. Samples then underwent a further 2 freezethaw cycles using liquid nitrogen, mixing on a vortex after thawing each time. Samples then underwent centrifugation at 16,700 × g for 10 min at 4 • C; the supernatants were collected and stored at −80 • C. Protein determination was performed using the Bio-Rad DC Assay (Bio-Rad). Samples were loaded into Laemmli buffer at a concentration of 1.5 mg/mL and underwent SDS-PAGE using 10% acrylamide gels under reducing conditions for ∼1 h at 0.08 A, after which they were transferred onto PVDF membrane (GE Healthcare) for ∼1.5 h at 0.38 A. Membranes were subsequently blocked for a minimum of 2 h in either 5% (wt:vol) BSA or skim milk in TBS-Tween, as appropriate. Primary antibody targets were Uncoupling Protein-3 (1:1000, Abcam), Adenine Nucleotide Translocase 1 (1:1000, Abcam), Adenine Nucleotide Translocase Membranes were developed using Pierce ECL western blotting substrate (ThermoFisher Scientific) and images captured using a ChemiDoc-It 2 imager (Ultra-Violet Products Ltd) with VisionWorksLS 8.1.2 software (Ultra-Violet Products Ltd). Images were analyzed using ImageJ and normalized to Vinculin expression.
Pyruvate dehydrogenase activity
The activity of the active form (i.e., dephosphorylated) of pyruvate dehydrogenase (PDH) was determined through acetyl-CoA formation, measured after condensation with radioactive oxaloacetate to form citrate, as previously reported (31,32).
Statistics
All data are expressed as mean ± SD unless stated otherwise. Differences between groups were analyzed by independent t test or ANOVA (as appropriate) for parametric data or Mann-Whitney U test for nonparametric data. Frequency distributions across treatment groups were assessed by χ 2 test. The human studies were powered in order to detect a treatment difference of 20% in expression and phosphorylation status of metabolic proteins (i.e., PDH, UCP3, AAC1, and AAC2), as determined by immunoblotting. Based on an assumed CV of 20%, n = 14 patients per group would be required for 80% confidence at a 2-sided α of 0.05. Similarly, in murine experiments a treatment difference of 15% in leak respiration (oligomycin respiration minus antimycin A respiration) would require n = 8 per group for 80% confidence at a 2-sided α of 0.05. Analyses were performed using GraphPad Prism version 7.01 or IBM SPSS Statistics version 25.0.0.1.
Murine uptake of supplemented sodium nitrate or nitrite
Plasma concentrations of NO − 3 and NO − 2 after 7 d supplementation of mice with sodium nitrate or sodium nitrite are depicted in Figure 2. Supplementation with sodium nitrate resulted in an ∼7-fold increase in plasma NO − 3 concentrations (227 ± 103 μM compared with 33 ± 10 μM, P < 0.0001), with a nonsignificant trend toward increased plasma NO − 2 concentrations (4 ± 3 μM compared with 2 ± 1 μM, P = 0.09). This result may be indicative of a limited entero-salivary circuit for reduction of NO − 3 to NO − 2 (33), because supplementation with sodium nitrite achieved robust increases in both plasma NO − 3 (117 ± 62 μM compared with 41 ± 15 μM, P < 0.05) and plasma NO − 2 (10.7 ± 10.5 μM compared with 1.7 ± 0.8 μM, P < 0.05) concentrations. These results are comparable with what has been reported previously in mice (28), rats (24), and humans (6) in response to sodium nitrate incorporation into the diet.
The effect of sodium nitrate or nitrite supplementation on murine mitochondrial protein expression and phosphorylation status
The mitochondrial proteins AAC (also known as the adenine nucleotide translocases) and UCP have been implicated in the dissipation of mitochondrial membrane potential by facilitating futile proton leak (34,35). Sodium nitrate supplementation has been reported to improve mitochondrial energetic efficiency through decreased expression of AAC and UCP3, in particular (6). However, when investigated we observed no alterations in expression of these proteins in skeletal muscle in response to supplementation with either sodium nitrate or sodium nitrite (Figure 3).
Sodium nitrite supplementation has also been reported to attenuate fatty acid utilization by mitochondria (27). By extension and according to the Randle cycle (25), a reciprocal boost in glucose utilization and PDH complex activity may be expected to occur. Activity through the PDH complex is regulated through phosphorylation of 3 distinct serine residues (serines 232, 293, RCRs were unchanged in response to supplementation with sodium nitrate. (C) Mitochondrial leak was unchanged in response to sodium nitrate. No significant differences were found between treatment and control groups (at P < 0.05; independent t test). See the Methods section for further details. White bars, NaCl; black bars, NaNO 3 . AA, antimycin A; AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; RCR, respiratory control ratio; St2, state 2; St3 * , state 3 (ADP at saturated concentrations). and 300), with decreased phosphorylation of these sites corresponding to increased PDH activity. The effects of sodium nitrate or nitrite supplementation in mice on PDH phosphorylation status in skeletal muscle are summarized in Figure 4. No change in phosphorylation was detected with sodium nitrite supplementation, whereas only a small change was observed with sodium nitrate (Figure 4). These findings correlate with no observable difference in PDH activity in response to nitrite FIGURE 6 Respiratory activity of skeletal muscle mitochondria isolated from mice supplemented with sodium nitrite (n = 5-6 per group) for 7 d, as assessed by Seahorse XF24 analyzer. (A) Skeletal muscle mitochondrial respiration in response to dietary supplementation with sodium nitrite. Nitrite supplementation resulted in decreased oligomycin-induced respiration when compared with control. * Significant differences: * P < 0.05. (B) RCRs were unchanged in response to supplementation with sodium nitrite. (C) Mitochondrial leak was unchanged in response to sodium nitrite. No significant differences were found between treatment and control groups (at P < 0.05; independent t test). See the Methods section for further details. White bars, NaCl; gray bars, NaNO 2 . AA, antimycin A; AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; RCR, respiratory control ratio; St2, state 2; St3 * , state 3 (ADP at saturated concentrations). Figure 1; please see Supplemental Figure 2 for example blots).
The effect of sodium nitrate or nitrite supplementation on murine mitochondrial energetics
After observing limited change in protein expression levels, we investigated the impact of both nitrite and nitrate supplementation on mitochondrial efficiency directly by respiratory analysis of isolated mitochondria. As summarized in Figure 5, sodium nitrate supplementation had no effect on mitochondrial respiration associated with proton leak activity (OCRs in the presence of oligomycin; Figure 5A, C), coupling efficiency (e.g., the respiratory control ratio; Figure 5B), or respiratory activity in any of the conditions tested. In contrast, sodium nitrite supplementation appeared to decrease mitochondrial activity in the presence of oligomycin (Figure 6). However, the respiratory control ratio ( Figure 6B) remained unchanged, suggesting that the apparent decrease was due to a general decrease in respiratory activity in all conditions, and was not specific to proton leak ( Figure 6C). Further detailed analysis at higher resolution using a Clark-type oxygen electrode in follow-up experiments confirmed that neither proton leak activity ( Figure 7A, C), the respiratory control ratio ( Figure 7B), nor P:O ratio estimates of mitochondrial efficiency (ATP generated per oxygen consumed; Figure 7D) had changed in response to sodium nitrite supplementation. In contrast to previous reports (6,14), therefore, these results suggest that sodium nitrate and nitrite do not alter mitochondrial proton leak or coupling efficiency to affect physiology.
Effects of sodium nitrite supplementation on human mitochondrial protein expression
Given our observations with mice, we took the opportunity to investigate the impact of sodium nitrite infusion on expression of mitochondrial UCPs in human skeletal muscle of patients scheduled for CABG surgery (Figure 8). For data on the effect of sodium nitrite infusion on cardiac expression of mitochondrial proteins, please see Supplemental Figures 3 and 4.
The clinical characteristics of this cohort are summarized in Table 1, whereas pharmacokinetics of plasma NO − 3 and NO − 2 in response to infusion are depicted in Figure 9. Infusion of sodium nitrite resulted in an immediate increase in plasma NO − 2 concentrations, whereas plasma NO − 3 concentrations had increased by 6 h postinfusion.
Infusion of sodium nitrite before surgery did not alter the expression of UCP3, AAC1, or AAC2 in human skeletal muscle. Similarly, PDH expression and phosphorylation status remained unchanged. These findings in humans are consistent with our findings in mice, where little or no change in these proteins was observed.
Discussion
Numerous studies have reported on improved exercise performance in healthy participants in response to ingestion of inorganic nitrate (4,5,7,13,14,36). The inclusion of inorganic nitrate into the diet, whether through dietary sources (e.g., beetroot, spinach) or through other therapeutic avenues (e.g., supplements), has been consistently reported to improve muscle performance (8,13,37) and to decrease the oxygen cost of submaximal exercise (4,5,7,14), with no detectable changes in plasma substrate concentrations (7,14,36). These improvements in exercise performance have also been observed in cardiovascular diseases such as stable angina (38), and heart failure with either preserved (9) or reduced (10) ejection fraction.
Based in part on data obtained via near infrared spectroscopy (NIRS), as well as unchanged plasma substrate concentrations, it was postulated that the exercise benefits of inorganic nitrate could be attributed to improvements in mitochondrial efficiency, specifically increased coupling of mitochondrial respiration to ATP generation, through decreased expression of AAC and UCP3 and reduced proton leak activity (6). Given the canonical understanding of the entero-salivary circuit for absorption of exogenous nitrates (39,40), together with the observations regarding NO and mitochondrial respiration (41), these findings provide an attractive mechanism to account for the apparent exercise benefits of inorganic nitrates.
It should be noted that studies using the NIRS technique have shown reduced muscle oxygen consumption after ingestion of inorganic nitrate (4), but this is directly related to alterations in regional blood flow (42,43). This is particularly relevant when considering the known vasodilator properties of nitrite, the putative active component of inorganic nitrate (1,15,(44)(45)(46). Using quantitative fMRI, reductions in muscle oxygen consumption at submaximal exercise with inorganic nitrate were shown to be more prominent in muscles with higher proportions of type 1 fibers, suggesting a mitochondrial, rather than vascular, effect (47). However, previous human studies have observed that oral inorganic nitrate does not alter glucose uptake nor insulin sensitivity (36).
Importantly, our investigation failed to find any alterations in mitochondrial respiration in response to inorganic nitrite, nor were any changes in expression of the proteins AAC1, AAC2, or UCP3 observed in either human or murine skeletal muscle tissue. Indeed, recent work by others has also cast doubt on the idea that inorganic nitrate improves mitochondrial respiratory efficiency: whereas there was a slight reduction in whole body oxygen consumption, Whitfield et al. (23) observed no changes in mitochondrial respiration or UCP content in response to inorganic nitrate (ingested as beetroot juice) (23). Furthermore, we found no substantive changes in the phosphorylation status of the PDH complex, nor any increase in PDH activity. As such, there is now a growing body of evidence to indicate that previously observed exercise benefits in response to inorganic nitrate/nitrite are unlikely due to improvements in mitochondrial oxygen efficiency.
There are some limitations to the present study which need to be acknowledged. Although broadly similar, plasma NO − 3 and NO − 2 concentrations in our murine cohorts were slightly higher than what has been reported previously (28). In addition, we collected no functional data to complement our physiological data. With regards to our human data, plasma NO − 3 and NO − 2 concentrations at the time of biopsy were similar to those at baseline, although this does not preclude the possibility of longlasting effects: indeed, we have recently reported on a persulfidesignaling-based mechanism whereby sodium nitrite may induce long-lasting effects in vascular tissue (48). Lastly, this investigation does not address the possibility of divergent effects between dietary nitrate and dietary nitrite supplementation: dietary nitrate supplementation was observed to reduce whole body oxygen consumption (while leaving the respiratory exchange ratio and indexes of glucose handling unchanged), yet infusion with sodium nitrite failed to demonstrate the same effects (36). These results were replicated to an extent in mice and humans (23,24), where supplementation with nitrate-rich beetroot juice or sodium nitrate solution also reduced whole body oxygen consumption. These latter studies reported similar results to our own in that they did not observe any reductions in mitochondrial leak nor improvements in mitochondrial coupling efficiency. The evidence underlying the vascular effects of inorganic nitrate/nitrite remains convincing, however. Hypoxic potentiation of inorganic nitrite as a vasodilator, and its augmentation in the presence of hemoglobin, have been reported previously (15), and similar work has reported a seeming dependence on myoglobin for the vascular effects of nitrite (49). In a clinical study involving chronic HFrEF patients, nitrite infusion resulted in decreases in pulmonary vascular resistance and right atrial pressure, as well as increased stroke volume, correlating with increased estimated transseptal gradient (defined as pulmonary capillary wedge pressure minus right atrial pressure), with alleviation of diastolic ventricular interaction suggested as a likely mechanism (45). Similar effects have also been observed in response to inhaled nitrite in HFpEF patients (50,51). Elucidation of the mechanisms underlying the vascular effects of nitrite, however, is beyond the scope of the present investigation.
The therapeutic benefits of inorganic nitrate/nitrite are unlikely to be due to direct modulation of skeletal muscle mitochondrial metabolism. These results do not preclude that the underlying benefits of inorganic nitrite may be mediated through vascular effects: indeed, modulation of vascular function may even account for previous reports of altered metabolism in response to inorganic nitrate/nitrite. | 2019-09-17T02:48:04.566Z | 2019-10-10T00:00:00.000 | {
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67755499 | pes2o/s2orc | v3-fos-license | Economic Expansion and Increase in Labout Market Formality : A Poaching Approach ∗
This paper investigates the relationship between economic expansion and the degree of formalization for the Brazilian labour market in the recent period. We present a theoretical framework that attempts to explain this relationship through the dynamics of firms hiring strategies. The main predictions are: the share of formal employment rises as the unemployment rate falls, and that the formal-informal wage gap increases, at least at the beginning of the economic expansion. In the empirical part, we use longitudinal microdata from a Brazilian household survey to check whether these two predictions are confirmed. To a large extent our results corroborate both predictions
INTRODUCTION
Recently, fluctuation in economic activity has stood out in the literature as one of the main forces driving changes in informality rates in the labour market, notably in Less Developed Countries (LDC).The vast empirical evidence in support of this claim (see Perry et al. (2007) and references therein) contrasts with the scarce theoretical efforts proposing specific mechanisms that link labour informality and the business cycle.
The main contribution of the present study is twofold.Firstly, we propose a theoretical framework whose mechanics relies on the dynamics of workers' transitions across unemployment, formal and informal jobs when the economy is expanding.This theoretical framework, which is adapted from the "poaching" version of the job search models recently put forth by Moscarini and Postel-Vinay (2009), offers predictions about the formal/informal composition of employment and the formal/informal wage gap along the expansion of the economy.Secondly, we present an empirical analysis to test whether the predictions made by the proposed theory are consistent with the dynamics of the Brazilian metropolitan labour market between 2003 and 2008.
The proposed theoretical framework focuses on the outflow of workers either from unemployment or from the informal sector into the formal sector.The composition of these flows is influenced by the business cycle in such a way that the share of employment in the formal sector grows during economic expansion.
The proposed framework assumes that unemployed workers tend to have a lower reservation wage than those individuals who hold informal jobs.Therefore, when the unemployment rate is high, formal sector firms can offer a low wage and hire workers who come mainly out of unemployment.As the economy grows, unemployment falls and, little by little, formal sector firms offer higher wages, and begin to poach relatively more workers from informal sector firms, thus increasing the level of labour market formality.To do that, it is assumed that informal sector firms are less productive than formal ones, not being able to adjust wages in a similar fashion.This, in turn, tends to broaden the gap between formal and informal wages along this adjustment period.
In the empirical part of the paper we check whether the business cycle (as measured by the unemployment rate) relates to poaching measures and to the formal/informal wage gap, as predicted by the proposed theory.Specifically, we firstly verify whether a worker newly hired by the formal sector has moved out of unemployment or out of the informal sector, and we check how the worker's previous employment status varies with the aggregate unemployment rate, controlling for his individual observable characteristics. 1 With regard to wages, our aim is to investigate to what extent the gap between the formal and informal sectors varies with unemployment by controlling for workers' observable and unobservable characteristics (as perceived by the analyst).
We consider the recent experience of Brazil a rich "case study" for three reasons.First, the Brazilian economy expanded continuously between 2003 and 2008. 2 Simultaneously, during this recent period, there was a remarkable increase in labour market formality and a sharp decrease in the unemployment rate. 3Second, the data available for Brazil allow for longitudinal links necessary to track the previous state of the workers hired in formal jobs.Finally, and perhaps most importantly, the data distinguish very well workers employed under a legal contract from those under informal arrangements.
Economic Expansion and Increase in Labout Market Formality: A Poaching Approach
The remainder of this paper is organised as follows.Section 2 presents the related literature on informality and business cycles.Section 3 introduces our theoretical framework.The empirical analysis is presented in sections 4 and 5. Section 4 displays some descriptive statistics about the facts we intend to explain, whereas Section 5 shows the estimated parameters that allow testing our hypotheses.The last section concludes.
RELATED LITERATURE
The relationship between informality and the business cycle has already been investigated in LDC, including Brazil.In general, the available studies are based on dualism (or segmentation) theories, dating back to Harris and Todaro (1970).Perhaps the best reference regarding these theories is the framework presented by Fields (1975), which somewhat generalises Harris and Todaro's (1970) theory.There are three remarkable assumptions in their theory: 1. Presence of restrictions (albeit partial) on the flow of workers between informal and formal sectors, 2. Exogenous determination (attributed to institutional aspects) of the wage in the formal sector, and 3.The influence of the business cycle is contemplated by demand shocks restricted to the formal sector.
We have at least two reasons to propose an alternative theory.First, as pointed out in Barros et al. (1993), the dualism theory is not compatible with the fact that the wage gap between the formal and informal sectors is procyclical, a phenomenon that has been previously observed in Brazil in other periods.Second, the imposition of restrictions on the flow of workers between the informal and formal sectors was empirically contested by Bosch et al. (2007), among others.
Therefore, in our model, we seek to relax the three hypotheses above, which are at the core of the dualism theory.
Recently, other authors have also proposed alternative models to the received theory of segmentation in order to assess the relationship between informality and the business cycle.Boeri and Garibaldi (2007) and Bosch and Esteban-Pretel (2009) suggest theoretical frameworks that relax the latter two hypotheses and predict how the level of (in)formality of workers relates to the business cycle.Both are based on extensions of the matching model proposed by Mortensen and Pissarides (1994), to which an informal sector is added.The papers show predictions that are compatible with Brazilian labour market facts.However, the assumption to restrict the flow of workers between formal and informal sectors is present in both papers, being therefore a limitation. 4mpirically, both studies above use data from the Brazilian household survey that is also the source of information for our empirical analysis.Boeri and Garibaldi (2007) describe a positive correlation between unemployment and informality.In addition to corroborating this result, Bosch and Esteban-Pretel (2009) also describe a relationship between the business cycle and the hiring and separation rates in the formal and informal sectors, separately, as well as a relationship between the business cycle and the flow of workers across these two sectors.
It should be pointed out that other factors, specific to the Brazilian experience, have been indicated as possible explanations for the recent decrease in informality observed in Brazil.Among these factors, we highlight: credit expansion for firms (Catao et al., 2009), reduction in the tax burden for small-and medium-sized firms (Corseuil and Moura, 2010), and enforcement of labour laws (Almeida and Carneiro, 2007).
THEORETICAL FRAMEWORK
Our theoretical framework consists of an adaptation of the poaching model developed by Moscarini and Postel-Vinay (2009), which is an extension of the job search model proposed by Burdett and Mortensen (1998).Initially, we show how the model originally proposed by Moscarini and Postel-Vinay works, as well as some predictions of their model.After that, based on some assumptions, we apply the model and its predictions to a labour market with formal and informal jobs.
Suppose that all workers are identical and that firms differ in terms of productivity.Information does not flow perfectly in the labour market, causing firms and workers to engage in a search for the other part.It is not only unemployed individuals who search for a job, but employed ones can search for a better job.As a result, at any time, any worker, unemployed or not, has a fixed probability to get a job offer.The decision to take or not the offer depends on the comparison between the current value of the proposed wage and the worker's reservation wage.The reservation wage is the value that equates the current value of the expected utility flow in the job position the worker is occupying.So, the reservation wage of unemployed workers is expected to be the lowest of all.
On the other end of the labour market, firms produce a homogeneous product using some technology with constant returns to scale.The context of imperfect information generates a monopsony power for firms in the labour market that allows them to offer lower wages than their employees' level of productivity.Therefore, it will always be advantageous for firms to offer vacancies, and firm size will be limited by the difficulty they will have finding workers to fill these positions.
Firms choose wage trajectories to be offered to each and every worker regardless of his level of experience and of his status in the labour market. 5This choice should maximize the current value of the expected profit flow, in compliance with the restriction imposed by a federally mandated minimum wage, as well as with the dynamics of the state variables of this maximization problem. 6Moscarini and Postel-Vinay (2009) show that the wage trajectory to be offered depends on the firm's productivity; consequently, the wage trajectories offered by more productive firms tend to be more advantageous for workers than those offered by less productive firms.7 Also, more productive firms employ more workers than less productive ones.Thus, workers' flow occurs in one direction only: from a less productive firm to a more productive one, that is to say, from a smaller to a bigger firm.
The arguments of Moscarini and Postel-Vinay (2009) can be summarised as follows.Consider an economy that is just finding its way out of a recession and is about to go through a continuous period of economic expansion, with a reduction in unemployment.To begin with, firms prefer to hire workers who are unemployed, given the bountiful supply of workers with such a status, and also considering that it is possible to lure them with relatively low wages.As time moves forward, unemployment tends to decrease and it gets harder to hire workers from this source.Initially, more unemployed individuals find a job in small firms as they are more numerous than their bigger counterparts.Therefore, the total of employees in relatively small firms increases in the first moments of the recovery.However employees in relatively small firms continue to seek jobs in bigger firms.Thus, there are greater chances of a big firm hiring an employee from a small firm as unemployment falls.This flow of employees from small firms into big firms increases the share of workers in big firms in total employment during the remaining moments of economic revival.This is the major prediction explored in this paper.
Another noteworthy prediction refers to the behavior of the wage gap between bigger and smaller firms.With economic expansion, it is optimal for firms to increase both the employment and the wage offers to workers.However, larger firms can increase the wage offers much more than can smaller firms (owing to the productivity differential).So, at first, the wage gap between bigger and smaller firms tends to increase as unemployment decreases.However, this variation in the wage gap can reverse at some point, decreasing as the economy draws near a steady state. 8e explore these predictions using an adaptation that takes into consideration the coexistence of formal and informal sectors in the economy.Note that the highest levels of employment and wage associated with big firms result from the fact that these firms are more productive.This way, all the description outlined for the evolution of employment between big and small firms can be reformulated for more and less productive firms, respectively.If we consider that formal sector firms are more productive than those of the informal sector, the theoretical framework can be adapted straightforwardly.In fact this consideration can be grounded by theoretical works modeling firms' sector choice.Since Rauch (1991) seminal paper it is a standard result that firms above (below) a threshold value for productivity chooses the (in)formal sector.See for instance De Paula and Scheinkman ( 2011) for a more recent contribution providing the same result.Therefore, we propose to classify firms into formal and informal instead of into big and small.9 According to this view, we can observe the increase in the share of formal employment during economic expansion, especially after its initial stage.This growth would be based on an outflow of workers from informal to formal employment.We should also expect the wage gap between the formal and informal sectors to increase during the period of economic expansion.In the remainder of this paper, we check whether the data for the Brazilian metropolitan labour market for the 2003-2008 period confirm these predictions.
DESCRIPTIVE ANALYSIS
We use data from several waves of a monthly household survey that covers the six main Brazilian metropolitan regions.10This survey, known as Pesquisa Mensal do Emprego (PME) and conducted by the Instituto Brasileiro de Geografia e Estatística (IBGE), aims to provide detailed information on participation in the labour market and employment conditions of each respondent, including whether an employment relation complies with the labour legislation or not. 11ME provides longitudinal links since the sample is designed as a rotating panel.A household in the PME sample is typically surveyed for four months in a row, is dropped from the sample for the subsequent eight months, and is reintroduced into the sample one year after the first interview for another set of interviews for four months in a row.Although individuals do not get an identification code, it is possible to identify the household.Therefore, we assume that we can identify individuals when information such as birth date and sex appears with the same values in two consecutive interviews in the same household. 12ur aim in this section is to conduct a preliminary analysis of what happens when the unemployment rate falls, therefore we use all the waves of PME comprised in the period from 2003 to 2008.For this period, we want to check whether: 1.The share of formal workers in the private sector tends to increase; 2. The flow of workers from informal into formal jobs tends to increase relative to the flow of unemployed individuals into formal employment.
As the focus of the theory is on formal sector hirings, in this study, we do not assess how the workers' flows from the formal sector into unemployment and into the informal sector react to the business cycle.
Figure 1 provides evidence about item i), showing the evolution of the unemployment rate and of the share of formal jobs in the private sector (henceforth this indicator will be referred to as formality rate).The raw data seem to be negatively correlated, as predicted by the theory, i.e., there would be an increase in formality in periods of low unemployment.This phenomenon is more evident in the last months of 2004, as shown in the figure.In order to obtain a more accurate indicator of this association, we calculated the correlation coefficient (r) of these series.Not only does the result confirm our suspicion of a negative correlation, but it also indicates a significant magnitude of −0.76.
To analyze claim ii), we use an indicator that captures to what extent formal sector firms hire relatively more or less workers from informal sector vis-à-vis the amount of unemployed workers hired.Thus, we compute the following indicator, which we will call poaching probability: where the numerator represents the number of workers in formal jobs (F J) in month t, hired from informal jobs (IJ) in the previous month (t−1).The denominator includes the flow out of unemployment (U ). 13 The thin lines in Figure 2 show the evolution of this indicator and that of the unemployment rate between 2003 and 2008.In this case, it is much harder to observe a clear correlation between these variables due, in part, to the high volatility of our poaching indicator.In fact, the correlation coefficient between these series is 0.01, showing that, at least in the whole 2003-2008 period, there does not seem to be an association between the unemployment rate and the poaching probability.
The thin lines in Figure 2 show the evolution of this indicator and that of the unemployment rate between 2003 and 2008.In this case, it is much harder to observe a clear correlation between these variables due, in part, to the high volatility of our poaching indicator.In fact, the correlation coefficient between these series is 0.01, showing that, at least in the whole 2003-2008 period, there does not seem to be an association between the unemployment rate and the poaching probability.
However, the lack of correlation in the whole period may be caused by correlations switching signs in different subperiods, which cancel each other out when the whole period is considered.In order to clarify whether movements are uncorrelated at no time between 2003 and 2008, we "smoothed" both series by calculating their respective centered 12-month moving averages, shown by thicker lines in Figure 2.
These smoothed series unveil some information that was not evident in the original series.Specifically, it is possible to observe that the series have a clear negative correlation until the end of 2006 and with the labour legislation.PME provides the information on whether the worker has his card signed by his current employer or not. 13Note that we left out observations of non-adjacent interviews, i.e. those between the fourth and fifth interviews of the household.
Analysis of Transition Probabilities
It is important to underscore that the correlations described in the previous section can result from the use of mechanisms other than those proposed by the theory.For instance, the composition of the group of informal workers and of the group of unemployed workers in relation to productive attributes (schooling, experience) or subject to discrimination (race, gender) can vary to such an extent that the former group becomes more attractive to formal sector firms than the latter.In other words, if informal workers changed their productive traits over time relative to unemployed individuals, it is likely that formal sector firms will hire proportionally more workers from the former group simply because they need a labour force with the productive characteristics more abundantly available among informal workers.
The aim of this section is to test whether the qualitative result of the previous section is still valid when we add the possible influences of individual characteristics in this process.Our basic sample is restricted to workers who have just been hired for a formal sector job, who were either unemployed or worked informally, between January 2004 and December 2008.We used alternative specifications of regression models to test the hypothesis that matters to us.The probability to be estimated can be written as: where Λ(.) denotes the logistic function and y it is equal to 1 if individual i just hired for a formal job in month t was working informally in month (t − 1), and equal to 0 if he was unemployed.The variable u rt denotes the unemployment rate in the metropolitan region r and X it is a control vector we will specify in different ways.The first specification contains only dummy variables for the following individual characteristics: schooling, sex, age, and race.In the second one, we introduce dummy variables to capture the quarterly seasonality and in the third one we include year dummies.The last specification adds a variable to capture fluctuations in reservation wages.To achieve that, we use the per capita labour earnings of the household excluding the earnings of worker i.All specifications contain dummies for metropolitan region.Parameter δ in equation ( 1) provides the most valuable information for our study.It informs us how unemployment in the metropolitan region in which a formal worker lives affects the probability that this worker was previously in an informal job.
Table 1 shows the estimated values for δ and the respective coefficients for the control variables used in the model's specifications.As can be observed, the estimate of our parameter of interest is positive for the first two specifications, but it is negative for the third and fourth ones, when we introduce the year dummies and the variable that controls for reservation wage fluctuations, respectively.Therefore, the assumption that formal sector firms tend to hire relatively more informal workers than unemployed ones when the unemployment rate decreases is only confirmed for the latter two specifications. 14 Our descriptive analysis had indicated that the correlation between the unemployment rate and the poaching probability was not homogenous throughout the analyzed period.Figure 3 shows the estimated value for δ when we consider subsamples of two years with one quarter in between.That is, the first point in each line of Figure 3 correspond to values estimated for the January 2004-December 2005 subsample, the second one refers to the April 2004-March 2006 subsample, and so on and so forth, up to the last point, which has to do with the January 2007-December 2008 subsample.The lines in the figure correspond to the previously outlined specifications.
Figure 3 demonstrates that the estimates of δ have a negative sign for virtually all subperiods and specifications when we consider two-year subperiods. 15This includes the estimates based on the first two specifications, which have a positive sign when the whole period was used in the estimation.Therefore, Figure 3 provides evidence that the hypothesis being tested is confirmed, at least for shorter time periods.
The exercises discussed hitherto leave out some of the worker flows into formal employment, in particular workers who were self-employed or who were inactive.These workers can be added to our analysis if we use broader concepts of informality and of unemployment.Self-employed workers can be included in the group of informal workers, thus constituting a broader group of informally hired workers; on the other hand, inactive workers can be included in the group of those hired out of unemployment, thus constituting a broader group of unemployed workers.Note that, if the hiring dynamics for formal positions does not make a distinction between informal (i.e.employee without a work card) and self-employed workers or between unemployed and inactive individuals, then these extended groups are more suitable for our analysis than the previous groups.
Using these extended groups, we repeat the exercises described in Table 1 and in Figure 3, redefining y it , which is now equal to 1 if individual i just hired for a formal job in month t had an informal job or was a self-employed worker in month (t − 1), and 0 if he was unemployed or inactive.The results of these new exercises are shown in Table 2 and in Figure 4.
As shown in Table 2 and in Figure 4, the estimates of the parameter of interest are negative for all specifications of the model and subperiods analyzed. 16Therefore, the hypothesis that the flow of informal workers into formal employment increases as the unemployment rate decreases is consistently confirmed when extended groups of informal and unemployed workers are used.
The exercises we have just analyzed do not include workers who transit across formal jobs.However, the inclusion of this group can be important for our analysis because some of the formal sector firms hire workers from other formal sector firms.Thus, we redefine y it , which now has three possible values: 1 if individual i just hired for a formal job in month t had another formal job in month (t − 1), 2 if he had an informal job or a self-employed position, and 3 if he was unemployed or inactive.
Given that we have three categories in this exercise, we use the multinomial logit model.The results of this exercise are shown in Tables 3A and 3B, as the multinomial logit model estimates the effect of 14 The unemployment rate in the same month in which the worker takes the formal sector job is likely to be too close to this transition to capture the effect of interest.Thus, we re-estimated the model using unemployment rate (i) lagged one month, (ii) lagged three months and (iii) the mean unemployment rate of the quarter in which the transition took place.
The results, which can be requested from the authors, are qualitatively similar to those shown in Table 1. 15Except for the estimate of specification 2 for the July 2006-June 2008 period, all the remaining estimates of are statistically significant at 1%. 16 All the remaining estimates of δ in Figure 4 are statistically significant at 1%. (1) The dependent variable assumes value 1 if worker transition was from the informal sector (without a work card or self-employed) and 0 if from unemployment or inactivity.
(2) Standard errors in brackets.Significance levels: (*) 10%, (**) 5% and (***) 1 (3)Omitted categories: less than 1 year of schooling and 15 to 17 years old.Specification 1 Specification 2 = Specification 1 + quarterly dummies Specification 3 = Specification 2 + yearly dummies Specification 4 = Specification 3 + income per capita each variable relative to a base category.In our case, we used the unemployed or inactive workers as the base group.Therefore, in Table 3A we report the effect of the respective variable, of each specification, using the odds ratio for "those hired from formal jobs versus those hired from unemployment or inactivity"; while in Table 3B, we report the odds ratio for "those hired from informal jobs versus those hired from unemployment or inactivity".So, we consider Table 3B as the most important for our purpose and is more comparable with the results of previous exercises.The results in this table show coefficients that are always negative for the unemployment rate, indicating that a decrease in unemployment tends to increase the probability of a worker being hired from an informal job rather than from an unemployed status.This result then corroborates our hypothesis.17
Analysis of Wage Gap between Formal and Informal Employment
This subsection tests the prediction of our theoretical framework that the wage gap between the formal and informal sectors increases at least at the beginning of economic expansion.Therefore, let us consider the following regression model specified for panel data: (1) The dependent variable assumes value 1 if worker transition was from the informal sector (without a work card or self-employed) and 0 if from unemployment or inactivity.
(2) Standard errors in brackets.Significance levels: (*) 10%, (**) 5% and (***) 1 (3)Omitted categories: less than 1 year of schooling and 15 to 17 years old.where w it is the actual wage of worker i in month t, X it is a vector of time-varying and time-invariant characteristics of this worker, u rt is the unemployment rate of metropolitan region r in which worker i lives and F it is a dummy variable that assumes value 1 if the worker has a formal job in month t, and 0 otherwise.The term θ i captures the time-invariant characteristics of worker i that are not observed by the analyst and potentially affect his wage (e.g.innate ability).We admit that θ i may be correlated with the model's covariates.The term v it represents a random disturbance with zero mean, possibly heteroskedastic, but uncorrelated with the other covariates.
To estimate this model, we used the first-difference method, which is widely employed in the panel data literature:18 where ∆ denotes the time difference between month t and month (t − 1).Note that, in the transition of worker i from an informal job to a formal one, ∆F it is equal to 1.As before, we test the prediction of interest in two complementary ways.First, we verify whether the value of parameter λ is negative when we estimate the model in equation ( 3) for the whole sample.Then we estimate the model for the two-year subsamples, as they are used in Figures 3 and 4. Table 4 shows the estimates of equation ( 3) for the whole sample.The first pair of columns considers informal workers to be only those without a work card, whereas the second pair includes self-employed workers.The difference between the two columns of each pair is that the second one contains year dummies.
As shown in Table 4, the point estimates of the parameter associated with difference in the interaction F it .urt (i.e., λ) are negative for both informal groups and for both specifications.However, these estimates are only statistically significant when we use the group of workers without a work card.In this regard, the prediction that the wage gap between the formal and informal sectors increases during economic expansion is confirmed, but not in a completely robust fashion. 19(1) The dependent variable is the logarithm of the real wage.
(3) IE stands for informal employee (no legal contract) and IE for self-employed.
(4) The unemployment rate is calculated per metropolitan region's PME.
The estimated parameter of interest for the whole period can be close to zero as the result of variation over time, being negative in part of the sample and positive in the other part, as predicted by the theory.To tackle that possibility, the thin lines in Figure 5 show the estimates of the parameter of interest for the two-year moving windows with one-quarter increments in between, for the two groups of informal workers and for each of the two specifications considered in Table 4.The thick lines in Figure 5 correspond to the linear trends in the estimates presented in the figure.As can be seen, though there is some fluctuation over time, estimates are more negative for the initial subperiods but tend towards positive values as time moves forward.This is in line with the prediction of the theory that posits that 19 The wage model was re-estimated for the two informal groups using the unemployment rate (i) lagged one month, (ii) lagged three months and (iii) the mean unemployment rate for the quarter.
The results (not shown) indicate that the parameter of interest is statistically significant for both groups and specifications, but has a positive sign in some cases.
CONCLUSION
In this study, we sought to check whether a new theory that associates the dynamics of the labour market to business cycle expansions can explain the evolution of formal employment in Less Developed Countries (LDC).The theory, which is based on the poaching model put forward by Moscarini and Postel-Vinay (2009), advocates that the formality rate should go up as unemployment falls as a consequence of some change in the hiring strategy of formal firms.As the economy expands, the new workers in these positions come mainly from informal jobs rather than from the shrinking pool of unemployed workers.The theory also predicts that the wage gap between formal and informal workers increases, at least at the beginning of the economic expansion.
In order to verify the predictions of the theory, we analyzed data for the Brazilian metropolitan labour market between 2003 and 2008, a period of continuous economic expansion in this country.We presented evidence that, to a great extent, confirms both predictions.Thus, we offer an alternative explanation to that of the segmentation theory for the variations observed in formality rates in LDC.
One limitation of the theory is that the job-to-job flow in the labor market occurs only in the direction of low productivity (informal) to high productivity (formal) firms.Though in practice this is the more relevant flow in quantitative terms, the inverse one is also observed.We were able to confirm the main predictions of the theory for the informal-to-formal flow, but it would certainly be interesting to have a more complete model that is also capable to explain the reverse flow as well.
Figure 1 :
Figure 1: Evolution of the share of formal employment and of the unemployment rate (in %)
Figure 2 :
Figure 2: Evolution of the poaching probability and of the unemployment rate (in %)
Figure 3 :
Figure 3: Logit Model Estimates with a Two-Year Moving Window -Informally hired and Unemployed Workers in Month t − 1 who Took a Formal Sector Job in Month t Table 3A: Multinomial Logit Model with self-employed and informally hired workers versus unemployed and
Figure 4 :
Figure 4: Logit Model Estimates with a Two-Year Moving Window -Informally hired and Self-Employed Workers and Unemployed and Inactive Workers in Month t − 1 who Took a Formal Sector Job in Month t Rio de Janeiro v. 66 n. 2 / p. 207-224 Abr-Jun 2012 Economic Expansion and Increase in Labout Market Formality: A Poaching Approach the formal/informal wage gap increases at the initial stages of the economic expansion but tends to decline thereafter.
Figure 5 :
Figure 5: Evolution of the interaction coefficient in wage regressions in first differences
Table 1 :
Logit Model Estimates -Workers Either in Informal Jobs or Unemployed in Month t − 1 Hired for a Formal Job in Month t
Table 2 :
Logit Model Estimates -Informally Hired or Self-Employed and Unemployed or Inactive Workers in Month t − 1 who Were Hired for a Formal Sector Job in Month t
Table 3B :
Multinomial Logit Model with self-employed and informally hired workers versus unemployed and inactive workers
Table 4 :
Wage Regression Estimates in First Differences | 2018-12-27T06:01:03.470Z | 2012-06-13T00:00:00.000 | {
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70752644 | pes2o/s2orc | v3-fos-license | An analysis of human immunodeficiency virus/acquired immune deficiency syndrome-related awareness among masses
Acquired immune deficiency syndrome (AIDS) was first recognized in 1981. It has attained a pandemic status over time and is considered as a global crisis. Majority of the cases are reported from the developing countries and as per a rough estimate, 1 out of 100 sexually active adults worldwide is infected with human immunodeficiency virus (HIV). According to the World Health Organization (WHO) reports, there has been an alarming rise in the incidence of AIDS cases in South-East Asia including Thailand, Indonesia and the Indian sub-continent. The epicenter of the disease, in India, lies in the states of Maharashtra and Tamil Nadu, which collectively constitute about half of all HIV-positive cases (mostly contracted sexually), while the state of Manipur in the North-East accounts for 8% of all cases (mostly among intravenous drug abusers).1
INTRODUCTION
Acquired immune deficiency syndrome (AIDS) was first recognized in 1981. It has attained a pandemic status over time and is considered as a global crisis. Majority of the cases are reported from the developing countries and as per a rough estimate, 1 out of 100 sexually active adults worldwide is infected with human immunodeficiency virus (HIV). According to the World Health Organization (WHO) reports, there has been an alarming rise in the incidence of AIDS cases in South-East Asia including Thailand, Indonesia and the Indian sub-continent. The epicenter of the disease, in India, lies in the states of Maharashtra and Tamil Nadu, which collectively constitute about half of all HIV-positive cases (mostly contracted sexually), while the state of Manipur in the North-East accounts for 8% of all cases (mostly among intravenous drug abusers). 1 HIV is a retrovirus that infects the CD4 cells, i.e., Helper T-cells of the host, thereby leading to a depression in the normal immune response of the body. This decrease in immunity allows various opportunistic microbes to invade the host and cause infections or other complications like Kaposi's sarcoma. 2 Despite many developments in the field of antiretroviral therapy, AIDS still remains invariably lethal. The antiretroviral (ARVs) drugs aim at maximal suppression of virus replication, thereby improving life quality and increasing life expectancy of the infected patients. However, the ARVs cannot cure the patients completely. 3 So, adequate HIV and AIDS related education for young people is considered as an important prevention strategy. Many government/non-government organizations like WHO, Visual AIDS and its "red ribbon" project, National AIDS Control Organization (NACO), Avert, etc., are working actively at national/international levels to create awareness regarding HIV/AIDS. 4-7 Analysis of the awareness of HIV/AIDS among the masses is important for determining the effectiveness of previously existing and current awareness programs, as well as the need for interventions. 8 So, with this background the present study was conducted so as to analyze the awareness about HIV/AIDS among masses and to determine the loopholes in the awareness campaigns currently in action.
Study population
The study was conducted among a total of 80 subjects chosen at random. Of the 80 respondents, 45 were males and 35 were females. Mostly subjects were of North Indian origin and some were from flushing, New York (USA). The distribution of respondents with non-scientific educational background and scientific educational background was even, i.e., 50% of each category. A written consent was obtained from all the participating subjects after explaining to them in detail the aim of the study, extent of their involvement, and the freedom of choice in participation.
Data collection
To analyze the awareness regarding HIV/AIDS among masses, a questionnaire was designed which comprised of questions related to basic and essential knowledge about HIV/AIDS. The questionnaire comprised of 17 yes/no type questions, 3 short descriptive type questions, and 5 multiple choice questions. The questionnaires were distributed among the chosen subjects/population.
Data analysis
The responses of the subjects to each question were analyzed on the basis of sex, educational background, ethnicity/ country of residence, and in general. The responses were analyzed further, so as to determine the perception of masses regarding the modes of HIV transmission.
General awareness
Overall, 95% subjects had heard about HIV, however, only 65% could differentiate between AIDS and HIV. The major source of awareness regarding HIV/AIDS was the counseling done by the teachers (70%). Only 38.75% had heard about NACO. Other sources of information were counseling and discussion with friends, parents, and families. 16.25% believed that AIDS in not fatal and 38.75% believed that ARV drugs can cure AIDS. 25% thought that HIV can spread through mosquito bite and 3% considered it justified to outcast HIV/AIDS patients from jobs or education systems, etc. 91.25% subjects said that they insist the personnel at salons to change the blades of razors and only 13.75% admitted that they share filers, razors, etc., with friends or family members. 81.25% subjects claimed that they check every time whether the doctors use sterilized syringes. However, only 13.75% had got themselves tested for HIV. 18.75% subjects claimed that they asked/received a blood report pertaining to HIV after blood donation, while 58.75% had never donated blood. In response to questions asked to check the specific information, 88.75% knew the full form of HIV and 56.25% were aware regarding its origin from monkeys. 62.50% knew that the count of T-helper cells decreases in AIDS and 30% knew that zidovudine is an ARV drug (Table 1).
Awareness on the basis of sex
Both the sexes had an almost same level of awareness. However, as compared to females only 20% males were counseled for HIV/AIDS by parents/family and 28.89% felt hesitant/awkward when talking about HIV/AIDS.
Awareness on the basis of educational background
It was observed that as compared to the subjects with the scientific educational background, only 90% of the subjects with non-science educational background had heard about HIV. Even subjects with the scientific educational background (62.5%) believed that being HIV-positive means that the person has AIDS. Surprisingly, 17.5% of subjects with scientific background considered mosquito bite as a mode of transmission. 20% of subjects with scientific education believed that AIDS in not fatal and 32.5% believed that ARV drugs can cure AIDS. While in case of subjects with non-scientific education, the percentage was 12.5% and 45%, respectively. 85% subjects with non-scientific education and 97.5% subjects with scientific education mentioned that they insist the personnel at salons to change the blades of razors. 25% of subjects with non-scientific education and 2.5% of subjects with scientific education said that they share filers, razors, etc., with friends or family members. 12.5% of nonscientific background and 65% of the subjects with scientific background claimed that they know something about NACO.
Awareness on the basis of ethnicity
As compared to American subjects, only 94.20% Indian subjects had heard about HIV. Furthermore, it was observed that in case of American subjects counseling done by the teachers, friends, parents, and families played a much significant role in spreading awareness as compared to Indian subjects. As compared to Americans (14.28%), Indians (23.18%) are more hesitant/awkward, when talking about HIV/ AIDS. 100% American subjects claimed that they insist the personnel at salons to change the blades of razors and none of them shared filers, razors, etc., with friends or family members. In comparison with Indian subjects (11.59%), more American subjects (42.85%) had got themselves tested for HIV.
DISCUSSION
Analysis of these questionnaires indicated that American subjects had more awareness regarding HIV/AIDS and they took better/proper precautionary measures as compared to Indian subjects. They were made aware through multiple sources including parents/families while Indian subjects felt hesitant regarding these discussions. Indian subjects lagged in general awareness and even those from scientific background had not much awareness regarding NACO or other awareness/preventive programs. Surprisingly, subjects with scientific background had some misconceptions ( Table 2, Figures 1 and 2) and were lenient regarding precautionary measures.
It was found out that counseling by teachers, friends, parents/family regarding HIV/AIDS in Indians was lesser than that of American subjects. Similarly, it was observed that the awareness related to HIV/AIDS among subjects with non-scientific educational background was less than the subjects with the scientific educational background. On the basis of the present study, it was concluded that the awareness measures running in India are not effective enough as the knowledge of Indian subjects lacked considerably on some basic areas related to HIV/AIDS. So, there is a need to promote more awareness campaigns and to begin the counseling of children/students through parents, families or friends about the disease or the preventive | 2019-03-07T14:06:41.423Z | 2015-01-01T00:00:00.000 | {
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221763119 | pes2o/s2orc | v3-fos-license | Feasibility of Using a Prosthetic-Based Impression Template to Improve the Trueness and Precision of a Complete Arch Digital Impression on Four and Six Implants: An In Vitro Study
Background: Intraoral scanners (IOSs) in implantology represent a viable approach for single teeth or partial arches. However, when used for complete edentulous arches or long-span edentulous areas, it has been demonstrated that there is a need for improvement of IOS-related techniques. Therefore, the aim of this in vitro study was to assess the trueness and precision of a complete arch digital impression on four and six implants taken with or without a customized, prosthetic-based impression template. Materials and Methods: Two experimental models were prepared, representative of a complete edentulous mandible restored with four and six implants with built-in scan abutments. Models were scanned with (test group, TG) or without (control group, CG) the prosthetic-based impression template. Eight scans were taken for each model. The time needed to take impressions, error, trueness, and precision were evaluated. A statistical analysis was performed. Results: In the case of four implants, the time needed for the impression was 128.7 ± 55.3 s in the TG and 81.0 ± 23.5 s in the CG (p = 0.0416). With six scan abutments, the time was 197.5 ± 26.8 and 110.6 ± 25.2 s in the TG and CG, respectively (p = 0.0000). In the TG, no errors were experienced, while in the CG, 13 impressions were retaken due to incorrect stitching processes. In the four-implant impression, the mean angle deviation was 0.252 ± 0.068° (95% CI 0.021–0.115°) in the CG and 0.134 ± 0.053° (95% CI 0.016–0.090°) in the TG. The difference was statistically significant (p = 0.002). In the six-implant impression, the mean angle deviation was 0.373 ± 0.117° (95% CI 0.036–0.198°) in the CG and 0.100 ± 0.029° (95% CI 0.009–0.049°) in the TG (p = 0.000). In the TG, there were no statistically significant differences in the mean angle deviation within the group (p > 0.05), but there were in the CG. A colorimetric analysis showed higher deviations from the original model for the six-implant impression without a prosthetic template. Conclusions: Although all of the impressions exhibited deviation from the original model in the range of clinical acceptability, the prosthetic-based impression template significantly improved the trueness and precision of complete edentulous arches rehabilitated with four or six implants, making the complete arch digital impression more predictable.
Introduction
In the last few years, intraoral scanners (IOSs) have represented a viable approach for the diagnosis, planning, and execution of treatments [1][2][3][4]. One of the major contributions to the rapid spread of digital impressions is the fact that IOSs have been demonstrated to perform within the same range of accuracy as conventional impressions when used for short-span areas (single teeth or partial arches) [2,5]. This allows digital models to reach the high accuracy needed to guarantee the proper fit of dental restorations [6]. Moreover, when compared with analog impressions, digital technologies offer several benefits, such as patient acceptance, time efficiency, direct visualization of the impression, and quick and easy repeatability [2][3][4][5]. Last but not least, IOSs can be used in combination with other digital technologies, such as computer-aided design/computer-aided manufacturing (CAD/CAM) technologies, for chairside production, or in combination with cone beam computed tomography (CBCT), to make computer-guided surgery easier [7][8][9][10]. Nevertheless, when used for complete edentulous arches or long-span edentulous areas, it has been demonstrated that there is a need for improvements of IOS-related techniques, in order to reach the same levels of accuracy achieved with conventional impressions [11][12][13][14][15][16][17][18]. Whilst powdered intraoral scanners showed promising results, they have been withdrawn from the market [19]. Moreover, at the time of writing this article, there were no randomized controlled trials (RCTs) proposing novel techniques or materials to improve the accuracy of implant-based digital impressions for complete edentulous arches.
For the latter, the accuracy is defined with trueness and precision. Trueness can be evaluated by comparing the master model (original geometry) with the digitized impression. Moreover, precision can be obtained by an intragroup comparison of the digitized models [17,18].
In 2017 [19] and 2018 [20], Tallarico et al. published a fully digital workflow to rehabilitate edentulous patients. In order to improve the accuracy of the digital impressions, a novel, prosthetic-based impression template, made by virtual planning, was presented. This prosthetic template was customized by maintaining the original tooth design, but including four windows, to allow the screwing of scan abutments, so that the impression could be matched with the initial planning.
The aim of the present in vitro comparative study was to assess the trueness and precision of complete arch digital impressions on four and six implants taken with or without the prosthetic-based impression template. The null hypothesis of this research was that there are no statistically significant differences between different impression techniques.
Materials and Methods
Two different virtual implant plans of the same, real complete edentulous mandibular arch were performed with four and six implants, respectively (RealGUIDE5, version 5.0, 3DIEMME srl, Cantù, Italy). In the four-implant plan, the implants were placed according to the All-on-4 protocol [21], tilting the distal implants by 30 • (Figure 1). In the six-implant plan, all of the implants were placed according to a pre-established prosthetic setup, straight and parallel between them ( Figure 2). Then, virtual implant positions were exported and two experimental models were designed (Rhino 6, Rhinoceros, McNeel Europe, Barcelona, Spain) and milled in titanium grade 5 (New Ancorvis SRL, Calderara di Reno (BO), Italy). The decision to mill the models in titanium was made in order to create stable and durable models with an opaque, micro abraded surface (no scanning spray required), avoiding a risk of bias. Both models were derived from the same prosthetic setup, simulating a complete denture. The first model was designed by placing the four implants according to the All-on-4 protocol and with built-in scan abutments (Figure 3), while the second model was created by placing six straight implants, with the same in-build scan abutments ( Figure 4). Each scan abutment was designed to be 10 mm in length and 4 mm in diameter. Two prosthetic-based impression templates (prosthetic template) to be used during the complete-arch digitalization were designed (RealGUIDE5) and then prepared for printing (Materialise Magics 24, Materialise, Leuven Belgium). At this point, four ( Figure 5) or six ( Figure 6) windows were created in the prosthetic templates (Materialise Magics 24) to accommodate the scan abutments, ensuring accurate fitting of the template (Figures 7 and 8). The windows were created by subtracting solid shapes from the original STL files, without compromising the stability of the prosthetic template and maintaining at least five teeth that acted as landmarks between scan abutments. To fix the template to the titanium model, three pre-planned screws were used. Finally, the templates were printed using the ProJet MJP 2500 Plus with VisiJet M2R-CL (3D System Inc., Rock Hill, SC, USA).
Materials 2020, 13, x FOR PEER REVIEW 3 of 12 least five teeth that acted as landmarks between scan abutments. To fix the template to the titanium model, three pre-planned screws were used. Finally, the templates were printed using the ProJet MJP 2500 Plus with VisiJet M2R-CL (3D System Inc., Rock Hill, SC, USA). least five teeth that acted as landmarks between scan abutments. To fix the template to the titanium model, three pre-planned screws were used. Finally, the templates were printed using the ProJet MJP 2500 Plus with VisiJet M2R-CL (3D System Inc., Rock Hill, SC, USA). least five teeth that acted as landmarks between scan abutments. To fix the template to the titanium model, three pre-planned screws were used. Finally, the templates were printed using the ProJet MJP 2500 Plus with VisiJet M2R-CL (3D System Inc., Rock Hill, SC, USA). least five teeth that acted as landmarks between scan abutments. To fix the template to the titanium model, three pre-planned screws were used. Finally, the templates were printed using the ProJet MJP 2500 Plus with VisiJet M2R-CL (3D System Inc., Rock Hill, SC, USA). Models with a respective prosthetic template were immobilized using a customized metal base and then manually digitalized using the Medit i500 intraoral scanner (Medit Corp., Seoul, Korea) with level-2 filtering and a 17.0 mm depth, following the manufacturer's guidelines. The right side of each model was digitalized first (Medit Link software version 2.2.2.753, Medit Corp.). An expert operator (MT) started placing the camera on the most right distal scan abutment and then started digitalizing the occlusal surface by pushing the button on the scanner. Following this, the process proceeded from the right to the left side of the arch by rolling the camera to the buccal and lingual areas of the models, until the complete occlusal surface had been digitized. According to the protocol, when reaching the anterior area, zigzag movements between the lingual and buccal areas, centered around the central edge, were performed to extend the anterior digitalized zone, making the remaining areas easier to match. Once the opposite side had been scanned, the lingual and finally buccal sides were digitalized. To move from the occlusal to lingual zone, the tip of the scanner was tilted at around 45 • toward the lingual side and then moved to the opposite side. Once the lingual side was completed, the tip of the scanner was tilted to the buccal site and moved from there to the opposite area. Before processing the impression, the scan data was checked. In the case of incomplete surfaces, the tip of the scanner was positioned in the area to complete the data. Each model (test group, four and six scan abutments) was digitalized eight times. After that, the prosthetic templates were removed by unscrewing the fixing screws, and the models were digitalized individually eight times each, following the same aforementioned protocol. The scanner was calibrated before any impression, according to the manufacturer's instructions. All of the impressions were processed by the software and then exported in Standard Triangle Language (STL) format, in a share folder, using a file hosting service (Dropbox, Inc., San Francisco, CA, USA).
The outcome measures were as follows: The time (seconds) needed to take impressions was automatically calculated using the digital chronometer of the software.
Any error dictating impression retaking, such as distortion, an incorrect stitching process, or failure due to overlap was noted.
Trueness and precision were established by measuring the difference in angle between the original (the truth) and digitalized scan abutment position. The postoperative STL file, derived from the intraoral scan, was geometrically aligned with the original STL file, by automated image registration, using the maximization of mutual information (Optical RevEng4.0, Open Technologies, Rezzato (BS), Italy). The trueness was assessed using GOM Inspect Professional (GOM, Braunschweig, Germany) after aligning the experimental models and the digitalized impressions by using best-fit algorithms. After superimposition, the deviations between selected surfaces were assessed and the qualitative analyses were presented using colorimetric scale measurements. Deviations at tolerance levels from 0.01 to 0.05 mm were analyzed ( Figure 9). The precision was assessed as the angular deviation between the digitalized and original scan abutment position, calculated along the long axis of each scan abutment (Rhino 6) after library alignment (Exocad Plovdiv, Exocad GmbH, company, Darmstadt, Germany). An expert biomedical engineer performed all of the measurements (RA) (Figures 10 and 11). Statistical analyses were performed using NUMBERS, version 10.0 (6748) (Apple Inc., Cupertino, CA, USA) and online calculators [22][23][24][25]. Mean values, standard deviations (SD), and the 95% confidence interval (CI) were calculated. Comparisons between groups for continuous outcomes (time and accuracy) were made by paired tests, in order to detect any changes in the impression accuracy. One-way analysis of variance (ANOVA) was conducted to determine the effect of the scan abutment position and angulation on the overall accuracy. Differences in the proportion of errors during the impression (dichotomous outcomes) were compared between groups using the 2 × 2 Fisher exact test. Impression was the statistical unit. The statistical significance was set at 0.05. Based on the authors' knowledge, there are no similar studies in the scientific literature. Therefore, an a Statistical analyses were performed using NUMBERS, version 10.0 (6748) (Apple Inc., Cupertino, CA, USA) and online calculators [22][23][24][25]. Mean values, standard deviations (SD), and the 95% confidence interval (CI) were calculated. Comparisons between groups for continuous outcomes (time and accuracy) were made by paired tests, in order to detect any changes in the impression accuracy. One-way analysis of variance (ANOVA) was conducted to determine the effect of the scan abutment position and angulation on the overall accuracy. Differences in the proportion of errors during the impression (dichotomous outcomes) were compared between groups using the 2 × 2 Fisher exact test. Impression was the statistical unit. The statistical significance was set at 0.05. Based on the authors' knowledge, there are no similar studies in the scientific literature. Therefore, an a priori sample size calculation was not performed. We decided to scan each model eight times, according to or better than previous in vitro studies comparing IOSs [3,4]. A post hoc analysis of continuous variabilities (mean angular deviation between groups) was performed by calculating the effect size (Cohen's d) and giving the means of each group, number of samples (n = 16), and alpha value (0.05).
Results
The time needed to take the impressions with four scan abutments was 128.7 ± 55.3 and 81.0 ± 23.5 s in the test and control group, respectively. The differences were statistically significant (p = 0.0416). The time needed to take the impressions with six scan abutments was 197.5 ± 26.8 and 110.6 ± 25.2 s in the test and control group, respectively. The differences were statistically significant (p = 0.0000). In the test group, no errors were experienced during impression taking, while in the control groups, 13 impressions were retaken (11 impressions with six scan abutments and two impressions with four scan abutments) due to incorrect stitching processes ( Figure 12). The differences were statistically significant when six scan abutments were digitalized (p = 0.008), but not when four scan abutments were digitalized (p = 0.447).
In the test group, there were no statistically significant differences in the mean angle deviation within groups (four scan abutments, p = 0.391 and six scan abutments, p = 0.372). In the control group, there were statistically significant differences in the mean angle deviation within groups. In the case of four digitalized abutments, a higher angle deviation was found in the last left scan abutment (position 34, 0.510 ± 0.191°, p = 0.00005). In the case of six digitalized abutments, a higher angle deviation was found in the first scan abutment (position 46, 0.616 ± 0.306°; p = 0.00766).
Post hoc power analysis demonstrated power in a range of 94.9 to 100% in the case of four and six implants, respectively. The mean angular deviation values between groups are summarized in Table 1. In the test group, there were no statistically significant differences in the mean angle deviation within groups (four scan abutments, p = 0.391 and six scan abutments, p = 0.372). In the control group, there were statistically significant differences in the mean angle deviation within groups. In the case of four digitalized abutments, a higher angle deviation was found in the last left scan abutment (position 34, 0.510 ± 0.191 • , p = 0.00005). In the case of six digitalized abutments, a higher angle deviation was found in the first scan abutment (position 46, 0.616 ± 0.306 • ; p = 0.00766).
Post hoc power analysis demonstrated power in a range of 94.9 to 100% in the case of four and six implants, respectively. The mean angular deviation values between groups are summarized in Table 1. Colored representation showing the smallest deviation using the surgical template in the four-implant model. Nevertheless, similar deviations when observed without surgical templates for the same four-implant model. Deviations of six-implant models were larger. However, the worst results were observed without the surgical templates.
Discussion
This in vitro study was designed to provide preliminary data on whether it would be more advisable to use the proposed prosthetic template when a digital impression was taken to fabricate an implant-supported complete arch restoration. The results of the present study demonstrated that the prosthetic-based impression template significantly improved the trueness and precision of complete edentulous arches rehabilitated with four or six implants. Hence, the null hypothesis of this research was rejected in favor of the alternative hypothesis of differences.
A precise impression still remains as one of the most important steps for fabricating dental-tooth and implant-supported restorations with an adequate fit, avoiding risks of mechanical and biological complications [13,14,26]. Digital impressions have been suggested as a valid alternative to conventional impressions for partial arch rehabilitation [2], while complete-arch impressions still remain a challenge when IOS devices are used [13][14][15][16]. Intraoral scanning systems are not without technology-related errors. Most of the drawbacks in complete-arch impressions could be due to a lack of fixed references. It has been suggested that the longer the scanning field, the more stitching processes with possible errors are presented [5].
To reduce this possible drawback, in the present study, the proposed prosthetic template, derived from the original tooth-setup by duplicating the complete denture by using an IOS or desktop scanner, was used. The main benefit of the prosthetic template is its ability to provide fixed references between scan abutments, in order to improve the readability of the IOS, even in complex scenarios, making digital impressions for complete-arches more predictable. A second benefit is that it allows matching between the implant positions and the original prosthetic setup. This allows transferring the match between the prosthetic volume (esthetic and function) of the setup and the final implant position to be transferred. The vertical dimension of occlusion and centric relation are also transferred.
To the best of the authors' knowledge, no previous study has compared the accuracy of a complete arch digital impression on four and six implants with and without a prosthetic template.
Data from the present study demonstrate that the overall accuracy of digital impressions is statistically significantly higher when a prosthetic template is used. The mean angular deviation experienced when using the prosthetic template was 0.100 and 0.134 • with six and four implants, respectively. These values correspond to a linear deviation of about 88 and 119 µm. Previous clinical studies showed that the threshold for a clinically acceptable fit implant-supported fixed prosthesis is within a range of 59-200 µm [27][28][29][30][31]. On the other hand, when the surgical templates were not used, the present research found a mean angular deviation of 0.252 and 0.373 • with four and six implants, respectively. These values correspond to a linear deviation of about 224 and 331 µm that appear to be in disagreement with what has been previously reported. The aforementioned findings suggest that the complete arch digital impression still remains a challenge and efforts must be performed to increase the accuracy. To date, it is the authors' opinion that a try-in with an aluminum framework is mandatory before the manufacturing of the definitive prosthesis. Nevertheless, data from the present study demonstrated that when using the prosthetic template, the mean accuracy was statistically significantly higher. This makes the digital impressions taken with the prosthetic template more predictable. In fact, even if the overall time needed to take the impressions was lower in the control group, 13 impressions were retaken due to incorrect stitching processes, making the impressions without the prosthetic template more time consuming. In the present study, the time needed to properly set the prosthetic template was not calculated because it was fixed to the model for all of the impressions. In a real practice, the prosthetic template can be fixed using the same anchor pins planned for the surgical implant placement or can be fixed in occlusion with a flowable resin composite, extending the time needed to take the impression.
The present study failed to find any statistically significant differences in the mean angle deviation within groups when the prosthetic template was used. The same results were not experienced when the prosthetic template was not used, with a higher angle deviation in the last (distal) abutments, in both four-and six-implant impressions. This means that when using the prosthetic template, the accuracy of each scan abutment is predictable regarding the implant position and angulation. This could be helpful for reducing the learning curve, making the impression not operator-dependent.
In 2017 [19] and 2018 [20], Tallarico et al. published a customized prosthetic template with the purpose of improving the accuracy of the digital impressions for implant-supported complete-arch restorations. After that, some case reports describing similar concepts were proposed. In 2019, Venezia et al. [32] presented the evolution of the previously published BARI technique, which allowed the digital transfer of the maxillary-mandibular relationship, from the complete denture to the implant-supported 3D-printed hybrid prosthesis. For the definitive impressions, prosthetic stents derived from the original plan were used. At the beginning of 2020, Ahmed et al. [33] published a digital scanning and maxillomandibular relationship workflow for maxillary complete-arch implant-supported restoration. Even in this case, a custom scanning device was used during the complete-arch intraoral scan. However, the present research is the only study evaluating the trueness and precision of the prosthetic template.
The prosthetic template can be fabricated starting from the original prosthetic setup used to virtually plan the implants or duplicating the existing complete denture of the patient [34][35][36][37]. The prosthetic template includes a certain number of windows to allow screwing of the scan abutments, as well as teeth surfaces that act as reference points to improve the accuracy of IOS and later match the digitalized impression with the initial plan [37]. There are some potential limitations for the use of the prosthetic template, such us the fixation method of the template, as well as its costs. When used in combination with a guided surgery, the prosthetic template can be designed with the same anchor pins used to stabilize the surgical guide [29,30]. This allows the prosthetic template to be stabilized during impression taking. On the other hand, when used after surgery, the prosthetic template can be stabilized in occlusion, fixing the scan abutments to the prosthetic template, avoiding possible negative effects due to template movements during scanning, and leading to the recording of an accurate impression that can be taken chairside or extra-orally. All of these methods have been published in previous clinical case reports [19,20], showing promising results. Regarding the overall costs, looking at the whole scenario, the prosthetic template allows the overall treatment time to be reduced, avoiding vertical dimension and occlusion recording. Considering this, the overall cost could even be reduced.
The main limitation of this research was the in vitro nature of the study design. The in vitro evaluation may not have fully simulated the clinical practice environment or condition [38,39], such us the natural gingiva. However, it is highly likely that the benefits of the prosthetic template can even exceed the positive results of the present study. The accuracy of the IOS required as many reference points as possible. Moreover, it can be affected by the presence of saliva, such as movement of the soft-tissue area. The challenge of taking digital impressions of edentulous mandibular arches remains in the elasticity of gingiva. To partially overcome this drawback, artificial gingiva could be used. Nevertheless, in the present study, artificial gingiva was not used in order to avoid possible bias due to its possible shift or detachment. The prosthetic template can also be used extra-orally after fixing the scan abutment on its surfaces. Of course, the results of the present study require confirmation through further clinical studies, even if the clinical report suggests encouraging results.
Conclusions
Although all of the impressions showed a mean deviation from the original model in the range of clinical acceptability, the prosthetic-based impression template significantly improved the trueness and precision of complete edentulous arches rehabilitated with four or six implants. Due to the limitations of in vitro studies, further in vitro studies are needed to confirm these preliminary results. Funding: This study was partially supported by a grant of the University of Sassari for publication costs (I.A.L. FAR2019). The New Ancorvis SRL donates models and templates. | 2020-08-13T10:07:26.045Z | 2020-08-01T00:00:00.000 | {
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248866175 | pes2o/s2orc | v3-fos-license | The Pyroptosis-Related Signature Predicts Diagnosis and Indicates Immune Characteristic in Major Depressive Disorder
Pyroptosis is recently identified as an inflammatory form of programmed cell death. However, the roles of pyroptosis-related genes (PS genes) in major depressive disorder (MDD) remain unclear. This study developed a novel diagnostic model for MDD based on PS genes and explored the pathological mechanisms associated with pyroptosis. First, we obtained 23 PS genes that were differentially expressed between healthy controls and MDD cases from GSE98793 dataset. There were obvious variation in immune cell infiltration profiles and immune-related pathway enrichment between healthy controls and MDD cases. Then, a novel diagnostic model consisting of eight PS genes (GPER1, GZMA, HMGB1, IL1RN, NLRC4, NLRP3, UTS2, and CAPN1) for MDD was constructed by random forest (RF) and least absolute shrinkage and selection operator (LASSO) analyses. ROC analysis revealed that our model has good diagnostic performance, AUC = 0.795 (95% CI 0.721–0.868). Subsequently, the consensus clustering method based on 23 differentially expressed PS genes was constructed to divide all MDD cases into two distinct pyroptosis subtypes (cluster A and B) with different immune and biological characteristics. Principal component analysis (PCA) algorithm was performed to calculate the pyroptosis scores (“PS-scores”) for each sample to quantify the pyroptosis regulation subtypes. The MDD patients in cluster B had higher “PS-scores” than those in cluster A. Furthermore, we also found that MDD patients in cluster B showed lower expression levels of 11 interferon (IFN)-α isoforms. In conclusion, pyroptosis may play an important role in MDD and can provide new insights into the diagnosis and underlying mechanisms of MDD.
INTRODUCTION
Pyroptosis, a programmed cell death mode closely related to the inflammatory response, plays an important role in a variety of physiological processes and disease progression (Broz et al., 2020). The characteristics of pyroptosis include activation of caspase-1, 4, 5, and 11; formation of cell membrane pores mediated by gasdermin protein; cell swelling and rapid rupture; and release of intracellular inflammatory factors (Shi et al., 2017). Therefore, inflammatory vesicles, gasdermin protein, and pro-inflammatory cytokines are key factors involved in pyroptosis. The expressions and functions of these core regulatory components influence pyroptosis progression. Further study of these regulatory components may help to clarify the role of pyroptosis in disease pathogenesis (Ahechu et al., 2018).
Major depressive disorder (MDD) is a serious neuropsychiatric disorder and a leading cause of suicide (Lépine and Briley, 2011). The incidence of depression is increasing annually to rank third among global disease burdens (Malhi and Mann, 2018). The pathogenesis of depression is complex, and inflammation is one of the main pathogenic factors (Troubat et al., 2021). Inflammation results from abnormal immune system activation. The imbalance of immune cells in the body can lead to illness, including mental illness such as MDD. Patients with depression show dysregulation of the innate and adaptive immune systems; for example, monocyte activation, decreased T-cell number and/or activity, and increased production of pro-inflammatory cytokines (Beurel et al., 2020). Excessive inflammation caused by pyroptosis and the release of various inflammatory factors after cell rupture may aggravate the disease development by forming an inflammatory immune microenvironment .
Comprehensive analysis of pyroptosis characteristics alteration in depression may be a key strategy for diagnosis and physiopathologic mechanism exploration of depression. Due to technical limitations, previous studies were limited to one or two key factors of pyroptosis. However, disease occurrence and progression involve a series of factors that form a highly synergistic network. Nowadays, the developments of highthroughput genomics technology and bioinformatics analysis have helped researchers to study genes expression profiles at the genomic level, generated new ideas for the interpretation of genomic results, and provided an ideal resource for the comprehensive analysis of pyroptosis and immune regulation in MDD (Gururajan et al., 2016;Ferrúa et al., 2019;Takahashi et al., 2019). In this study, we first established a novel diagnostic model by eight pyroptosis-related genes (PS genes) for MDD based on the GSE98793 dataset from the Gene Expression Omnibus (GEO) database. We found that MDD patients could obtain a good clinical benefit based on this model. Secondly, we explored the role of pyroptosis in physiopathologic mechanism of MDD. According to PS genes, data from patients with depression were clustered and two MDD subtypes were identified. We observed different immune properties and biological functions of these two subtypes. In all, our present study indicated that pyroptosis plays an important role in depression occurrence and progression, which may guide depression diagnosis, treatment and intervention plans.
Data Acquisition and Processing
GSE98793 dataset, the expression profile of whole blood samples, was downloaded from the Gene Expression Omnibus (GEO) database. This dataset totally included 128 MDD cases (64 with anxiety symptoms and 64 without) and 64 healthy controls. 64 MDD cases without anxiety symptoms and 64 healthy controls were included in our analysis. All the participants of GSE98793 were from the GlaxoSmithKline-High-Throughput Diseasespecific target Identification Program (GSK-HiTDiP) study. The MDD patients were evaluated by the semi-structured Schedule for Clinical Assessment in Neuropsychiatry (SCAN) (Wing et al., 1990), which was administered by trained staff. Furthermore, patients had a diagnosis of recurrent MDD (at least two episodes of depression satisfying DSM-IV or ICD10 criteria) were included as well. The exclusion criteria were as follows: if they had experienced mood incongruent psychotic symptoms, a lifetime history of intravenous drug use or diagnosis of drug dependency, depression secondary to alcohol or substance abuse or depression as clear consequence of medical illnesses or use of medications. Patients with co-morbid anxiety disorders, with the exception of obsessive compulsive and post traumatic stress disorders, were included. Patients with diagnosis of schizophrenia, schizoaffective disorders and other axis I disorders were excluded from the study (Leday et al., 2018). The detail information of participants included in the present study was showed in Table 1. GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array) was detection platforms for GSE98793. Gene symbols were used to annotate the downloaded gene probes, eliminate probes without matching, and retain any gene probes with multiple matching.
Screening for Pyroptosis-Related Differentially Expressed Genes "Limma" package (R Foundation for Statistical Computing) was used for gene differential expression analysis with the processed gene expression matrix (Diboun et al., 2006). Before the bioinformatic analysis, all the samples were tested in two batches, and batch information could be extracted from phenotypic data. Thus, removeBatchEffect from the limma package was used to remove the batch effect (Ritchie et al., 2015). We conducted the gene differential expression analysis with set threshold: |log 2 (FC)|>0.1 and Benjamini-Hochbergadjusted p < 0.05. Finally, we identified 2,216 DEGs in GSE98793. A total of 184 PS genes were obtained by inputting
Screening MDD-Specific Genes and Constructing Diagnostic Model for MDD
We applied random forest (RF) and least absolute shrinkage and selection operator (LASSO) to establish a diagnostic model for MDD. First, we used RF to screen candidate MDD-specific genes from pyroptosis-related DEGs. RF is a general technique for the training and prediction of samples based on the classification tree. The number of decision trees (ntree) and the value of MTRY in this study were 300 and 4, respectively. The RF was performed by R package "randomforest" (Kursa, 2014). Subsequently, to reduce the number of genes in the model and to solve the multicollinearity problem in regression analysis, we used LASSO logistic regression to screen feature genes and then construct a diagnostic model for MDD. The "glmnet" package was applied for LASSO algorithm (Friedman et al., 2010). Finally, a receiver operating characteristic (ROC) curve was created to investigate whether the built model could effectively predict MDD.
Internal and External Validation for Diagnostic Model 10-fold cross-validation as internal validation method was performed to confirm the predictive performance of our diagnostic model for MDD (Martinez et al., 2011). We chosed 10-fold cross-validation because 10-fold cross-validation techniques could test all data in the dataset and produce stable predictive accuracy. Therefore, the 10-fold cross-validation method with 2000 iterations of resampling was used for internal validatIon. GSE76826 was used as an external validation dataset to examine the universality and reliability of the diagnostic model. GSE76826 dataset (expression profiling by array) included 20 MDD patients and 12 healthy controls (Miyata et al., 2016).
In addition to this, we examined the effectiveness of this diagnostic model in other mental illness as well. The GSE38484 dataset was used to analyze the diagnostic model. This dataset, based on the microarray platform of the Illumina HumanHT-12 V3.0 expression beadchip (GPL6947), included 96 healthy controls and 106 schizophrenia patients (Van Eijk et al., 2015).
The same model and the same coefficient were conducted for GSE76826 and GSE38484. The characteristic information of participants in GSE76826 and GSE38484 datasets were exhibited in Tables 2, 3.
Diagnostic Markers Verified by Postmortem Brain Tissue Samples
GSE53987 was based on the platform of the Affymetrix Human Genome U133 Plus 2.0 Array (Lanz et al., 2019). There were 17 subjects with MDD and 18 healthy controls in this dataset, and three brain regions (hippocampus, prefrontal cortex, and striatum) were included. We used the GSE53987 dataset to verify the diagnostic model. The characteristic information of participants in GSE53987 dataset were showed in Table 4.
Consensus Clustering of 23 PS Genes by Partitioning Around Medoids
Consensus clustering is an algorithm used to identify subgroup members and verify subgroups based on resampling. We performed consensus clustering with PAM mehod (Wilkerson and Hayes, 2010) to identify distinct pyroptosis regulation clusters according to the expression profiles of 23 PS genes. PCA was then used to further validate different regulation clusters.
Immune Cell Infiltration Estimation by ssGSEA
Single-sample gene set enrichment analysis (ssGSEA) was used to quantify the relative abundance of 28 immune cell types related to immune response. In ssGSEA, the relative abundance of each immune cell was expressed as an enrichment score that was normalized to a uniform distribution of 0-1. A deconvolution approach CIBERSORT (http://cibersort.stanford.edu/) was used to evaluate the relative abundances of 22 distinct leukocyte subsets with gene expression profiles in the blood samples.
Gene Set Variation Analysis and Gene Ontology Annotation
We utilized GSVA analysis by "GSVA" package (Hänzelmann et al., 2013) to explore the differentiation in biological processes between different pyroptosis regulation clusters. The well-defined biological signatures were derived from the Hallmark geneset (MSigDB database v7.1) (Denny et al., 2018). The GO annotation for different clusters was performed using the R package "clusterProfiler" (Kursa, 2014) with a false discovery rate (FDR) cutoff of <0.01.
Identification of DEGs in Distinct Pyroptosis Regulation Subtypes
The consensus clustering algorithm classified MDD patients into two distinct pyroptosis regulation subtypes. We next identified DEGs between two different clusters using the "limma" package. Specifically, gene expression data were normalized using "voom" function and then inputted to the "lmFit" and "eBayes" functions to calculate the differential expressed statistics. The selection criteria were an adjusted P of <0.01 and |FC| of >1.0.
Construction of the Pyroptosis Score
To quantitatively analyze the pyroptosis subtypes, PCA was used to quantify the pyroptosis level of individual patients. First, PCA was used to distinguish pyroptosis subtypes. Then, the formula was performed to measure the pyroptosis scores (PS) as following: PS score = PC1 i , where PC1 represents principal component 1 and i represents DEG expression.
Study Design
The framework and workflow are described as following. First, the characteristic information and gene expression profiles of MDD cases and healthy controls in GSE98793, GSE76826, and GSE53987 were obtained from the GEO database (https://www.st-va.ncbi.nlm. nih.gov/gds/?term=). Using GSE98793 dataset, we developed a novel diagnostic model for MDD based on PS genes by machine learning methods (RF and LASSO). GSE76826 and GSE53987 datasets were used as external validation and postmortem brain tissue samples valiadtion. Furthermore, GSE38484 dataset (96 healthy controls and 106 schizophrenia patients) was used to examine whether this diagnostic model was unique for MDD. Then, immune cell infiltration profiles and immune-related pathway enrichment were compared between healthy controls and MDD cases as well.
To discover the connections between PS genes and MDD subtypes, MDD cases were divided into two subtypes (A and B clusters) by consensus clustering analysis according to the PS genes expression profiles. The particular immune and biological characteristics of these two clusters were observed.
We developed a pyroptosis-related signature score, the "PSscore," to quantify the pyroptosis phenotype subtype.
Interactions among the 23 pyroptosis-related DEGs were observed, and each gene was mapped to the STRING database to show their interaction relationships (interaction minimum>0.9, highest confidence) and visualize in Cytoscape.
The correlation analysis showed that CD14 was significantly positively correlated with NLRP3, while CD14 was significantly negatively correlated with HMGB1 in all healthy controls and MDD samples (Figure 2). These results indicated that expression imbalances of PS genes played important roles in the occurrence and development of MDD.
A Diagnostic Model for MDD Constituting of PS Genes
Firstly, the RF was used to screen MDD-specific genes that optimally differentiated MDD cases from healthy controls. When the number of decision trees reached 300, the error changes of the three kinds gradually decrease ( Figure 3A). The 23 PS genes identified by MeanDecreaseAccuracy and MeanDecreaseGini were showed in Figure 3B. The top 10 of these-UTS2, NLRC4, GZMA, GPER1, IL1RN, CAPN1, NLRP3, HMGB1, ATF6, and ADORA3-were candidate genes. Secondly, to establish an MDD-specific diagnostic model, LASSO regression was conducted based on the above 10 genes and basic phenotype information (age and sex), we contained eight genes (GPER1, GZMA, HMGB1, IL1RN, NLRC4, NLRP3, UTS2, CAPN1) according to lambda.1se ( Figures 3C,D).
Internal and External Validation of Diagnostic Model Performance
The internal validation of our diagnostic model was performed by 10-fold cross-validation (n = 2000). The model demonstrated good discrimination (bias-corrected AUC = 0.774, 95% CI = 0.679-0.864) ( Figure 4A). The external analysis using independent dataset GSE76826 revealed a good performance of our diagnostic model for stratifying MDD patients (AUC = 0.891, 95% CI = 0.657-0.941) ( Figure 4B). Furthermore, the accuracy of our diagnostic model was validated using brain tissue GSE53987 dataset. The AUC was 0.86 (95% CI = 0.669-0.933), which also indicated good performance ( Figure 4C).
In order to observe the effectiveness of this diagnostic model in other mental illness, the analysis of schizophrenia GSE38484 dataset was performed. The AUC of GSE38484 dataset was 0.593 (95% CI = 0.507-0.686) ( Figure 4D), suggesting that this diagnostic model was not effective and accurate for schizophrenia. We speculated that our diagnostic model constituting of eight PS genes was more appropriate for MDD.
Immune Cell Infiltration Profile and Immune-Related Pathway Enrichment Between Healthy Controls and MDD Cases
Many studies have indicated that MDD is accompanied by immune dysregulation, pyroptosis is closely related to immune response as well, thus we want to further explore the relationship between pyroptosis and immune response in MDD. The differences in immune cell infiltration profile and immune-related pathway enrichment between healthy controls and MDD cases were quantified.
Firstly, ssGSEA was used to calculate the relative abundance of immune cells in each sample. Eight of the immune cell types discovered significant changes in depression samples, with activated dendritic cells, immature dendritic cells, monocytes, and neutrophils showing upregulation and activated B cells, activated CD4 T-cells, effector memory CD8 T-cells, and immature B cells showing downregulation ( Figure 5A). These results suggested significantly altered immune cell profiles in MDD cases comparing with healthy controls ( Figure 5B). BIRC3 exhibited the most obvious positive correlation with activated CD4 cells, with low scores in MDD ( Figures 5C-E). Meanwhile, HMGB1 showed a significant negative correlation with monocyte. The score for HMGB1 was lower in MDD, while monocytes showed the opposite phenomenon ( Figures 5F-H).
Similarly, the enrichment fractions of immune-related pathways were also calculated by ssGSEA ( Figure 6A). We observed differences in the antimicrobial pathway, B cell receptor (BCR) signaling pathway, chemokines, cytokines, TCR signaling pathway, and transforming growth factor (TGF)-β family member receptors between healthy controls and MDD cases ( Figure 6B). CAPN1 was positively correlated with antimicrobials ( Figures 6C-E), while FOXO3 was negatively correlated with the TCR signaling pathway (Figures 6F-H). We speculated that these changes immune cells and immune-related pathways played important roles in occurrence and development of MDD.
PCA analysis indicated these two clusters differentiated significantly ( Figure 7D). The heatmap and boxplot showed the expression differences of 23 PS genes between two clusters,
FIGURE 5 | Correlation between pyroptosis-related genes and immune infiltrating cells (A). Differences in the abundance of immune infiltrating cells between healthy control and MDD samples (B). Correlation between pyroptosis-related genes and immune infiltrating cells (C-E).
The most positive correlation between immune infiltrating and pyroptosis-related genes (F-H). The most negative correlation between immune infiltrating and pyroptosis-related genes. Adjusted p-values were showed as: ns, not significant; *p < 0.05; **p < 0.01.
Distinct Immune and Biological Characteristics Between Two Clusters
To explore the immune characteristics between these two clusters, we measured the immune cells enrichment fractions, immune pathway activity, and human leukocyte antigen (HLA) gene expression profiles. The two clusters revealed completely different immune characteristics: for example, memory B cells and cytokines were mainly enriched in cluster A, while HLA genes mostly concentrated in cluster B (Figures 8A-C).
In addition to immune characteristics, we further explored the biological functions. We analyzed the enrichment scores of the Hallmark pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the two clusters by GSVA, which revealed enrichment of pathways in cluster A, including fatty acid metabolism and neuroactive receptor interaction ( Figures 8D,E).
Generation of "PS-Scores" and Functional Annotation
To further explore the pathological mechanisms of MDD related to pyroptosis, we developed a pyroptosis-related signature score, the "PS-score," including the phenotyperelated genes to quantify the pyroptosis regulation pattern of each MDD sample. The "PS-scores" for two distinct subtypes were calculated. The "PS-scores" of cluster B was significantly higher than that of cluster A ( Figure 9A). Figure 9B exhibited that the relationships between gender, pyroptosis clusters, and "PS-scores".
IFN-α is currently suggested to be an important link in the pathogenesis of MDD. Clinical studies have shown significantly higher serum IFN levels in depression patients than those of normal people. After 12 weeks of antidepressant treatment, IFN levels were significantly lower than those before treatment (Shelton et al., 2011). Thus, we explored the relationship between two subtypes and expression profiles of IFN-α isoforms. Figure 9C indicated that the expression profiles of IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17, and IFN-α21 were all lower in cluster B, although the difference was not significant for IFN-α17.
DISCUSSION
Major depression is a debilitating mental illness and a leading cause of suicide (Ting et al., 2020). Pyroptosis is originally identified as a key mechanism in fighting infection, and a growing number of research suggests its role in the development of several diseases. However, the role of pyroptosis in MDD remains unclear. Our present study first constructed a diagnosis model for depression based on PS genes and then explored the role of pyroptosis in depression.
Currently, there is no clear boundary between normal and depressive behavioral manifestations (Wakefield et al., 2007); thus, the diagnosis of depression is subjective and difficult to implement (Pan et al., 2018). Previous studies have reported several diagnostic biomarkers for depression. Leday, the original author of the GSE98793 dataset, reported an AUC of 0.71 for 165 gene combinations (Leday et al., 2018). In addition, in two independent sample sets of patients with depression, Papakostas et al. (2013) reported high diagnostic performance and sensitivity and specificity >80% for nine biomarkers (alpha1 antitrypsin, apolipoprotein CIII, brain-derived neurotrophic factor, cortisol, epidermal growth factor, myeloperoxidase, prolactin, resistin, and soluble tumor necrosis factor-alpha receptor type II). We previously constructed a diagnostic model with the signature of four autophagy-related genes used GSE98793 dataset and autophagy gene set as well. The AUC of autophagy-related diagnostic model was 0.779 (He et al., 2021). In the present study, we explored a molecular diagnostic model for MDD based on PS genes and observed whether this model had higher efficiency. We firstly identified 23 differentially expressed PS genes between MDD cases and healthy controls. Further analysis using RF in machine learning identified UTS2, NLRC4, GZMA, GPER1, IL1RN, CAPN1, NLRP3, HMGB1, ATF6, and ADORA3 as key PS genes affecting MDD classification. Then, the LASSO logistic regression finally screened feature genes and develop a diagnostic model based on eight genes (GPER1, GZMA, HMGB1, IL1RN, NLRC4, NLRP3, UTS2, CAPN1). Our present diagnostic model showed the AUC was 0.795 (95% CI: 0.721-0.868) for this model, indicating the high performance for differentiating MDD cases from healthy controls.
The activation level of NLRP3 inflammasome was increased in multi-pathway induced depressed mouse models. Furthermore, the specific inflammasome inhibitor VX-765 blocked NLRP3 activation in the hippocampus and improved depression-like behavior in chronically unpredictable stressed mice. Clinical investigation reported increased NLRP3 mRNA and protein levels in peripheral blood mononuclear cells of MDD patients compared with healthy controls (Kaufmann et al., 2017). HMGB1 can be highly expressed in the cerebral cortex (Lian et al., 2017) and hippocampus (Liu et al., 2019) of CUMS mice. It is reported that stress can induce depression-like behaviours through the HMGB1/TLR4/NF-κB signalling pathway in the hippocampus (Liu et al., 2019) and PFC (Xu et al., 2020). G protein-coupled receptor 30 (GPR30), also known as G protein-coupled estrogen receptor 1 (GPER1), levels in MDD patients were significantly higher than that in the healthy controls (Findikli et al., 2017). GPER stimulates IL-1β secretion via JNK and p38 MAPK signaling pathways, and then induce pyroptosis (Deng et al., 2020). IL1RN (the gene encoding IL-1RA) encodes a classical signal peptide that secretes cytokines through the endoplasmic reticulum and Golgi apparatus (Lennard, 2017). The NLRP3 and caspase-1 affect the release of IL-1RA, which has a broad effect on the inflammatory response and pyroptosis in bladder epithelial cells (Lindblad et al., 2019). A meta-analysis reported significantly higher levels of cytokine receptor antagonists (IL-1RA) in MDD patients compared to those in normal controls (Goldsmith et al., 2016). However, no associations between the other six candidate genes and depression have been reported. We believe that our present results will provide a direction for future research on early diagnosis of depression based on pyroptosis.
A growing number of studies have reported the associations between pro-inflammatory cytokines and emotional, cognitive and behavioral changes, many of which are associated with depression. For example, higher levels of pro-inflammatory cytokines such as interleukin-6 and C-reactive protein have been discovered in MDD patients (Howren et al., 2009). Poor response to antidepressant treatment is obviously associated with elevated levels of proinflammatory cytokines (Hiles et al., 2012). Traditional antidepressants, such as fluoxetine and imipramine, had no obvious effects on more than 40% of MDD patients. Surprisingly, the use of anti-inflammatory drugs in conjunction with antipsychotics can relieve a range of psychotic symptoms (Berk et al., 2020). Therefore, we next observed the immune status of MDD patients and healthy controls in GSE98793 dataset. We found that activated dendritic cells, immature dendritic cells, monocytes, and neutrophils were higher, and activated B cells, activated CD4 T-cells, effector memory CD8 T-cells, and immature B cells were lower in MDD patients compared with healthy controls, with similar to Pfau' (Pfau et al., 2018) and Kronfol's studies (Kronfol, 2002). The up-regulation profiles of dendritic cells, monocytes, and neutrophils illustrate infection and inflammation occur and progress. Our results indicated MDD patients might present immune imbalance and inflammatory status. Pyroptosis is a type of programmed cell death that is closely related to the inflammatory response. In the process of pyroptosis, cells form various vesicles, with pores 10-20 nm in diameter appearing on the cell membrane after gasdermin shear. It is reported that the release of many inflammatory factors leads to cascade amplification of cellular inflammatory responses (Frank and Vince, 2019), and pyroptosis is closely related to the immune response. We also discovered PS genes were closely associated with the changes of immune cells and immune-related pathway in GSE98793 dataset. Furthermore, our molecular diagnostic model based on pyroptosis showed a good performance. It was attractive for us to explore the immune-related mechanisms and search effective therapy based on pyroptosis can target a specific group of depression patients. So, we divided the MDD patients in GSE98793 into two subtypes according to pyroptosis, and found the immune characteristics and "PS scores" of subtypes were different. The "PS score" of cluster B was significantly higher than that of cluster A, and patients in cluster B presented lower profiles of B cells memory, dendritic cells, eosinophil, cytokines receptor pathway, and IFN-α family, which suggesting higher "PS score" might indicate lower levels of inflammatory condition. In addition, HLA genes play an important role in immune response and immune therapy. Attractively, most HLA genes were up-regulated in cluster B, indicating that patients in cluster B might be more sensitive to immune therapy. Previous studies identified different pyroptosisrelated subtypes for gastric cancer and melanoma, providing a new method for the prognosis and survival of tumor patients and promoting the development of personalized therapy (Liu et al.,FIGURE 9 | Differences in PS-scores and IFN-α isoforms between two pyroptosis-related clusters (A). Differences in PS-scores between cluster A and cluster B (B). The relationships between clusters, gender, and "PS-scores" (C). Differential expression levels of IFN-α isoforms between cluster A and cluster B. Adjusted p-values were showed as: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
Frontiers in Pharmacology | www.frontiersin.org May 2022 | Volume 13 | Article 848939 2021). According to our results, deep exploration of immune regulation in depression could help us understand and develop accurate and effective anti-inflammatory therapy for MDD patients. However, unfortunately, the clinical information of MDD patients is very limited, so we only conducted a partial study to observe the phenomenon, and further study is needed. Few studies have assessed the role of pyroptosis in depression. This preliminary study explored the diagnostic values of PS genes in depression, and systematically analyzed the relationships between PS genes and the immune response in depression, providing theoretical support for future research. However, our study has some limitations. First, the total sample size was relatively small (MDD: n = 64; Normal: n = 64). Secondly, this study was based on bioinformatics analysis; thus many of the results were theoretical. However, as experiments are the only standard for verifying the results, their accuracy requires verification without experimental methods. Third, due to the lack of abundant clinical data, we cannot determine the specific role of these PS genes in depression, which warrants further study.
In summary, our study provided a molecular model for MDD diagnosis. We additionally revealed the pyroptosis was closely related with immune imbalance in MDD. Comprehensive analysis of the pyroptosis pattern of MDD will improve our understanding of the internal mechanism of the MDD immune regulatory network and inform the development of more effective treatment methods.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article
AUTHOR CONTRIBUTIONS
WG and SH designed the study. ZD analyzed the data and drafted the manuscript. JL analyzed the data. | 2022-05-19T13:34:12.932Z | 2022-05-19T00:00:00.000 | {
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259874797 | pes2o/s2orc | v3-fos-license | Relation between Maximum Oral Muscle Pressure and Dentoalveolar Characteristics in Patients with Cleft Lip and/or Palate: A Prospective Comparative Study
Background: Perioral muscle function, which influences maxillofacial growth and tooth position, can be affected in patients with oral clefts due to their inherent anatomical characteristics and the multiple surgical corrections performed. This research aims to (1) compare the maximum oral muscle pressure of subjects with and without isolated cleft palate (CP) or unilateral cleft lip and palate (UCLP), (2) investigate its influence on their dentoalveolar characteristics, and (3) investigate the influence of functional habits on the maximum oral muscle pressure in patients with and without cleft. Material and methods: Subjects with and without CP and UCLP seeking treatment at the Department of Orthodontics of University Hospitals Leuven between January 2021 and August 2022 were invited to participate. The Iowa Oral Performance Instrument (IOPI) was used to measure their maximum tongue, lip, and cheek pressure. An imbalance score was calculated to express the relationship between tongue and lip pressure. Upper and lower intercanine (ICD) and intermolar distance (IMD) were measured on 3D digital dental casts, and the presence of functional habits was reported by the patients. The data were analyzed with multivariable linear models, correcting for age and gender. Results: 44 subjects with CP or UCLP (mean age: 12.00 years) and 104 non-affected patients (mean age: 11.13 years) were included. No significant differences in maximum oral muscle pressure or imbalance score were detected between controls and clefts or between cleft types. Significantly smaller upper ICDs and larger upper and lower IMDs were found in patients with clefts. A significant difference between controls and clefts was found in the relationship between oral muscle pressure and transversal jaw width. In cleft patients, the higher the maximum tongue pressure, the wider the upper and lower IMD, the higher the lip pressure, the smaller the upper and lower ICD and IMD, and the higher the imbalance score, the larger the upper and lower IMD and lower ICD. An imbalance favoring the tongue was found in cleft patients. The influence of functional habits on the maximum oral muscle pressure was not statistically different between clefts and controls. Conclusion: Patients with CP or UCLP did not present reduced maximum oral muscle pressure compared with patients without a cleft. In cleft patients, tongue pressure was consistently greater than lip pressure, and those who presented a larger maxillary width presented systematically higher imbalance scores (favoring the tongue) than those with narrow maxillae. Therefore, the influence of slow maxillary expansion on maximum oral muscle pressure in cleft patients should not be underestimated.
Introduction
An appropriate lip and tongue function is indispensable for speech and non-verbal oral functions such as swallowing. Large discrepancies in the activity of perioral muscles such as the orbicularis oris, buccinator, or tongue can affect oral aesthetics and ultimately contribute to a deterioration of the patient's oral health-related quality of life (OHRQoL; Table 1 includes a description of abbreviations and acronyms used) [1]. The balance between intra-and extra-oral muscles is believed to influence maxillofacial growth and the position of the teeth [2]. Lip and tongue function affects the form of the dental arch [3] and has been linked with occlusal traits such as overjet and incisor inclination [4][5][6], the growth of the cranial base [4], and vertical skeletal facial patterns [5,6]. In addition, functional asymmetries of masticatory muscles can affect postural equilibrium by disturbing the neck and facial muscle balance [7]. Therefore, alterations in muscle strength and activity may result in dental and/or skeletal disharmony and discrepancies in facial morphology.
In patients with cleft lip and/or palate, perioral muscle function could be restricted due to the cleft affecting the anatomy of some of these muscles. Additionally, the multiple surgical corrections performed on these patients in order to attempt reconstruction of the oral muscles could influence both their aesthetics and function [7].
Nevertheless, there is limited research regarding oral muscle function in patients with cleft lip and/or palate. The scarce evidence reports a reduced range of upper lip movement [8] and the presence of compensatory movements of the lower lip and chin in patients with clefts [9], as well as increased upper lip function and compression of the lips during swallowing [10]. Normal lip and tongue function in 25 patients with UCLP aged 6.7 to 18.2 years has also been reported [11]. However, the literature focuses almost exclusively on unilateral cleft lip and palate [9,[12][13][14], includes very small control groups of unaffected patients [13,14], and research regarding tongue and cheek pressure is scarce [9,12]. Additionally, the balance between tongue and lip function has been barely investigated [5]. In patients with anatomical muscle conditions such as cleft lip and/or palate, it could be of added value to not only look at tongue and lip function separately but also at how they both work in relation to each other.
Therefore, this research aims to (1) compare the maximum tongue, lip, and cheek muscle pressure and the imbalance of intra and extraoral muscles of subjects with and without CP or UCLP; (2) investigate the influence of the maximum oral muscle pressure and muscle imbalance on the dentoalveolar characteristics; and (3) examine the influence of functional habits on the maximum oral muscle pressure of both groups.
Materials and Methods
The protocol of this prospective comparative study was defined prior to the start and approved by the Ethics Committee of University Hospitals Leuven and KU Leuven University, Belgium, with registration number s64829. Informed consent was obtained from all the participating subjects and their parents/guardians before enrollment in the study.
Subject Recruitment
In the cleft group, subjects with CP and UCLP, under the age of 18 and of selfreported European descent, seeking treatment at the Department of Oral Health Sciences-Orthodontics of University Hospitals Leuven between January 2021 and August 2022 were invited to participate. Subjects presenting only submucosal clefts, only clefts of the lip or the alveolar bone, bilateral cleft lip and palate, the Robin sequence, and have a confirmed diagnosis of a syndromic disorder or pathogenic mutations were excluded.
All the included cleft patients were operated on by the same two surgeons at the same hospital with the following surgical protocol: In patients with UCLP, lip closure was performed at the age of 3 months using a modified Millard's technique. In patients with CP and UCLP, soft palate closure took place at 12 months-old by using a modified Veau-Wardill-Kilner push-back technique with supraperiosteal dissection and hard palate closure between the ages of 5 and 7 years in order to minimize maxillary growth restriction due to scar tissue by using the modified von Langenbeck procedure. It is important to remark that cleft patients undergo orthodontic maxillary expansion on two occasions: before the hard palate closure and before the secondary alveolar bone graft, normally planned around the age of 9 to facilitate the eruption of the maxillary canines.
In the control group, subjects without oral clefts, muscular diseases, or syndromes, aged under 18 years and of self-reported European descent, seeking treatment at the same department between October 2021 and November 2021, were invited to participate. This control group, composed at random over two months, represents the 'average' orthodontic patient.
Assessment of Dentoalveolar Data
Dental alginate impressions, a wax bite registration, and intra-and extraoral photographs were taken from all patients as part of the standard orthodontic diagnosis since all included subjects needed orthodontic treatment. The dental impressions and the wax bite registration were then digitalized, and the 3D digital dental casts were analyzed using the software DigiModel ® (OrthoProof ® version 3.46.2, Nieuwegein, The Netherlands). The following parameters were measured on the casts: intercanine and intermolar distance (ICD and IMD, as described by Van de Velde et al. [15]), overjet, overbite, and the presence of a crossbite. The sagittal occlusion of canines and first molars was noted by using an ordinal system where 0 was defined as neutro-occlusion, -1.00 as a full premolar width mesio-occlusion, and +1.00 as a full premolar width disto-occlusion. Intervals of 0.25 were used to express all intermediate occlusions. The measurements were taken before the start of comprehensive orthodontic treatment (including functional or fixed appliances) for all patients.
Assessment of Functional Habits
The following functional habits were clinically evaluated: the patient's swallowing pattern (mature or infantile), the position of the tongue at rest (physiological, interdental, or caudal), and the competence of the lips (competent or not). Additionally, the following parafunctions were verbally asked about and, if possible, clinically checked: nail biting, pen chewing, thumb sucking, and bruxism.
Assessment of Oral Muscle Pressure
Perioral muscle pressure was measured with the Iowa oral performance instrument (IOPI) [16,17], which is a widely used, validated tool. It is easy to use in children, and it keeps the diagnostic procedure as short as possible. It measures the amount of pressure a certain muscle exerts by means of an air-filled bulb. Anterior tongue pressure was measured by placing the bulb on the midline of the tongue and instructing the patient to push it against the palate at the height of the anterior rugae as hard as possible. Lip pressure was measured by positioning the air-filled bulb between two wooden blades. These were then placed between the lips at the region of the incisors, and the patient was asked to close the lips as hard as possible. Right and left cheek pressure were measured by placing the bulb between the cheek and the canine-premolar region and then asking the patient to squeeze as hard as possible. All measurements lasted up to 10 s and were repeated three times with a resting period of 30 s between each trial. The maximum values of the tongue, lip, right, and left cheek pressure were recorded. The measurements were taken by five senior orthodontic residents, trained and calibrated in advance. The measurements were taken before the start of comprehensive orthodontic treatment (including functional or fixed appliances) for all patients. The intra-observer reliability of the myofunctional outcome parameters (maximum tongue, lip, and cheek pressure) was calculated by using the intra-class correlation coefficient (ICC) for each of the 5 observers.
An imbalance score, previously described in the literature [5], was used to represent the imbalance between lip and tongue pressure. The score was calculated as follows: (maximum tongue pressure − maximum lip pressure) × 100 maximum tongue pressure + maximum lip pressure .
Statistical Analysis
The first aim of this research was to compare the maximum tongue, lip, and cheek pressure of subjects with and without cleft lip and/or palate. For this, multivariable linear models were used, modeling the muscle pressure as the response variable and the type of patient (cleft or non-cleft) as the explanatory variable of interest. Age and gender were included as covariates to account for possible confounding.
For the comparison of the transversal width (ICD and IMD) in the upper and lower jaws of subjects with and without clefts, a series of t-tests were used for pairwise comparison.
To investigate the influence of oral muscle pressure on dentoalveolar characteristics in patients with and without cleft (second aim), multivariable linear models were also used, and again, age and gender were included as covariates to account for possible confounding. The dentoalveolar characteristics were divided into two big groups: transversal width (ICM and IMD) and occlusal parameters (occlusion on the left and right sides, overjet, overbite, and crossbite), with these dentoalveolar characteristics as the response variable. The type of patient (cleft or non-cleft), muscle pressure, and the interaction between both terms were included in the explanatory model. A similar analysis was performed, defining the three patient groups as control, CP, or UCLP.
The third aim was to investigate the influence of functional habits on oral muscle pressure in patients with and without cleft palates. Multivariable linear models were also used, and age and gender were included as covariates to account for possible confounding. The regression analyses modeled muscle pressure as the response variable, type of patient (cleft or non-cleft), functional habits (dichotomous variable), and the interaction between both terms in the explanatory model.
Analyses were performed with SAS software (version 9.4 of the SAS System for Windows). A probability level of <0.05 was considered significant.
Results
Forty-four cleft patients were included (13 girls and 31 boys, mean age 12.00 years old, range 7.06-16.57 years old). 16 and 28 subjects presented CP and UCLP, respectively. Of the CP patients, 7 presented a cleft of the soft palate only. From the UCLP subjects, 17 presented the cleft on the right side, and 11 presented Simonart's band. Three of the UCLP patients had undergone presurgical nasoalveolar molding (PNAM), and one UCLP patient had used lip straps before the surgical lip closure.
The control group consisted of 104 patients (53 girls and 51 boys, mean age 11.13 years old, range 7.17-13.97 years old). Table 2 presents the demographic information of the included sample. Gender distribution was significantly different between the cleft and non-cleft groups (p = 0.019, Fisher's Exact test), which was expected since isolated CP is more common in women while UCLP occurs more frequently in men [18]. The mean age of the cleft group was nearly 1 year older than the non-affected group (p = 0.026, Mann-Whitney U test). Since literature shows that oral strength differs between males and females and that it increases with age in healthy subjects [19], age and gender were included as covariates in the statistical model to account for possible confounding. Figure ?? shows the strength of the association between myofunctional outcomes and age, quantified by Pearson correlation coefficients and visualized by means of scatter plots. The only association that could be demonstrated was an increase with age in maximum tongue pressure in the control group. The distribution of the occlusion in both the cleft and control groups is shown in Figure 2. The fact that control patients also present malocclusion was a conscious choice since comparing patients with cleft lip and/or palate to an 'ideal' group without malocclusion will be more likely to show differences. Most patients presented a Class II malocclusion (66. 35 The distribution of the occlusion in both the cleft and control groups is shown in Figure 2. The fact that control patients also present malocclusion was a conscious choice since comparing patients with cleft lip and/or palate to an 'ideal' group without malocclusion will be more likely to show differences. Most patients presented a Class II malocclusion (66. 35 Of all 148 included participants, one subject failed to have the lip function properly measured, which was considered a missing value. The mean intra-observer reliability (ICC) of the myofunctional outcome parameters (maximum tongue, lip, and cheek pressure) of the 5 observers was 0.803. Table 2 also shows the mean values of muscle pressure and imbalance score in each group. Table 3 shows the statistical significance and the mean differences in muscle pressure and imbalance scores between the groups. Positive mean differences correspond with higher values for the first group compared to the second one, and vice versa. No significant differences in maximum oral muscle pressure or imbalance score were detected between controls and clefts, nor between cleft types. Of all 148 included participants, one subject failed to have the lip function properly measured, which was considered a missing value. The mean intra-observer reliability (ICC) of the myofunctional outcome parameters (maximum tongue, lip, and cheek pressure) of the 5 observers was 0.803. Table 2 also shows the mean values of muscle pressure and imbalance score in each group. Table 3 shows the statistical significance and the mean differences in muscle pressure and imbalance scores between the groups. Positive mean differences correspond with higher values for the first group compared to the second one, and vice versa. No significant differences in maximum oral muscle pressure or imbalance score were detected between controls and clefts, nor between cleft types. Table 4 reports the values of maxillary width per group (intercanine and intermolar distances). Results show significantly smaller upper ICDs and larger upper and lower IMDs in patients with clefts compared to controls, and larger lower ICDs and upper IMDs in patients with CP compared to controls. The upper ICD was smaller and the lower IMD was larger in patients with UCLP compared to the control group. No significant differences in ICD or IMD were found between patients with CP and UCLP. Table 3. Mean differences in muscle pressure between the groups, as well as the statistical significance. Positive mean differences correspond with higher values for the first group compared to the second one, and vice versa. Age and gender were included as covariates to account for possible confounding. Table 5 reports whether there is a relation between jaw width (ICD and IMD) and perioral muscle pressure (tongue, lip, and cheek pressure and imbalance score) within and between the different groups. Positive slope values refer to positive relations (higher muscle pressure values for larger width values). A significant relationship was found between maximum anterior tongue pressure and jaw width in cleft patients. The higher the maximum tongue pressure, the wider the upper and lower IMD in patients with clefts compared to controls. The same relationship was found for CP patients compared to controls. For UCLP patients, the wider the upper and lower ICDs. Additionally, the higher the tongue pressure, the wider the upper ICD in patients with UCLP compared to patients with CP. Lip pressure was also significantly related to jaw width. The higher the lip pressure, the smaller the upper and lower ICD and IMD in patients with cleft and CP (compared to controls). In patients with UCLP, the effect of lip pressure on the upper ICD and lower IMD was not statistically significant. Regarding the imbalance score, the higher it was in cleft patients, the larger the upper and lower IMD and lower ICD. In patients with CP, the higher the imbalance score, the larger the upper and lower IMD. Furthermore, in UCLP patients, the higher the imbalance score, the larger the upper and lower IMD and ICD. An imbalance favoring the tongue was observed in patients with CP and UCLP. A significant relationship was found between cheek pressure and transversal width. The higher the right cheek pressure in patients with CP and UCLP and the higher the left cheek pressure in patients with UCLP, the smaller the upper ICD. The higher the right cheek pressure, the smaller the lower IMD in patients with CP compared to patients with UCLP. Table 6 shows the relation between muscle pressure and the remaining occlusal characteristics (sagittal molar occlusion, overjet, overbite, and crossbite) within and between the groups. Results for ordinal outcomes are presented as slopes indicating the change in dental outcome for a 1 unit increase in perioral muscle pressure. Positive slope values refer to positive relations (higher muscle pressure values for larger occlusal values). Results for cross-bite as a binary outcome are presented as odds ratios (OR), indicating the change in odds for cross-bite for a 1 unit increase in perioral muscle pressure. OR > 1 refers to positive relations (higher muscle pressure values for higher odds of a crossbite). Age and gender were included as covariates to account for possible confounding.
Muscle
Statistically significant differences were found between the control and cleft groups regarding maximum tongue pressure on the sagittal (right and left) occlusion, whereby a larger tongue pressure led to a tendency towards mesio-occlusion in patients with cleft compared to controls. The effect of maximum lip pressure was significantly different between controls versus cleft, controls versus CP, and controls versus UCLP for the right sagittal occlusion, whereby a larger lip pressure led to a tendency towards disto-occlusion in the cleft group. On the left side, the same effect was seen in patients with UCLP compared to controls. This can be explained by the increased occurrence of the unilateral variant on the right side. Significant differences were also observed between clefts and controls regarding the effect of the imbalance score on right occlusion: the higher the imbalance score in cleft patients (more tongue than lip pressure), the greater the tendency to mesio-occlusion. Additionally, the higher the imbalance score, the greater the tendency towards mesio-occlusion on the right and left sides in patients with UCLP compared to controls. A significant difference was seen between controls and CP and between CP and UCLP regarding overbite: the higher the imbalance score, the more tendency there is towards an open bite in CP. Significant differences were found between controls and UCLP regarding the effect of maximum right and left cheek pressure on overjet: the higher the cheek pressure, the more negative the overjet (and thus, a greater tendency towards reverse overjet). A significant difference was seen between controls and cleft patients regarding right cheek pressure: the higher the right cheek pressure in cleft patients, the higher the chance of a cross bite.
Finally, the influence of the observed functional habits on the maximum oral muscle pressure was statistically different between cleft and controls ( Table 7), but this significance presented a very wide range of 95% CI and was not considered valuable. Within the control group, infantile swallowing and the interdental position of the tongue at rest significantly affected maximum tongue pressure. The caudal and interdental positions of the tongue at rest also significantly affected the imbalance score. Table 4. Intercanine and intermolar distance of the included subjects, as well as the statistical significance (pairwise comparison between the four groups) according to a series of t-tests. A probability level of <0.05 was considered significant (* p < 0.05).
Maxilla Mandible
Control ( Abbreviations: 13g-23g: distance between the most lingual and cervical points of the permanent maxillary canines if present; 13c-23c: distance between the cusps of the permanent maxillary canines if present; 16g-26g: distance between the most lingual and cervical points of the permanent maxillary first molar if present; 16f-26f: distance between the central fossae connected to the vestibular groove of the permanent maxillary first molar, if present; 16MB-26MB: distance between mesio-buccal cusps of the permanent maxillary first molar, if present; 16DB-26DB: distance between disto-buccal cusps of the permanent maxillary first molar, if present; 33g-43g: distance between the most lingual and cervical points of the permanent mandibular canines if present; 33c-43c: distance between the cusps of the permanent mandibular canines, if present; 36g-46g: distance between the most lingual and cervical points of the permanent mandibular first molar, if present; 36f-46f: distance between the central fossae connected to the vestibular groove of the permanent mandibular first molar, if present; 36MB-46MB: distance between the mesio-buccal cusps of the permanent mandibular first molar, if present; 36DB-46DB: distance between the disto-buccal cusps of the permanent mandibular first molar, if present. Table 5. Relation between jaw width (ICD and IMD) and perioral muscle pressure (tongue, lip, and cheek pressure and imbalance score). Within-group results are presented as slopes indicating the change in dental outcome for a 1 unit increase in perioral muscle pressure. Positive slope values refer to positive relations (higher muscle pressure values for larger width values). Age and gender were included as covariates to account for possible confounding. p-values for differences between slopes between groups are presented in the last 4 columns. A probability level of <0.05 was considered significant (* p < 0.05). Table 6. Relation between oral muscle pressure (tongue, lip, and cheek pressure and imbalance score) and the remaining occlusal characteristics. Within-group results for ordinal outcomes are presented as slopes indicating the change in dental outcome for a 1 unit increase in perioral muscle pressure. Positive slope values refer to positive relations (higher muscle pressure values for larger occlusal values). Within-group results for crossbite as the binary outcome are presented as odds ratios (OR) indicating the change in odds for crossbite for a 1 unit increase in perioral muscle pressure. OR > 1 refer to positive relations (higher muscle pressure values for higher odds of crossbite). Age and gender were included as covariates to account for possible confounding. p-values for differences between slopes or odds ratios between groups are presented in the last 4 columns. A probability level of <0.05 was considered significant (* p < 0.05). Table 7. Relation between oral muscle pressure (tongue, lip, and cheek pressure and imbalance score) and functional habits within and between the different groups, as well as the statistical significance. Results are presented as mean differences and 95% confidence intervals. Age and gender were included as covariates to account for possible confounding. A probability level of <0.05 was considered significant (* p < 0.05).
Discussion
This prospective study measures the maximum pressure of the lips, tongue, and cheeks in 44 subjects with CP and UCLP, comparing it with that of a non-affected control group consisting of 104 subjects, which is a very large control group in comparison to similar studies [9,[12][13][14]. The data was collected before the start of comprehensive orthodontic treatment. Results show that there is no significant difference in maximum tongue, lip, or cheek pressure or imbalance score between subjects with and without clefts. Our findings are similar to those from the study of Van Lierde et al. [11], which compares the oral muscle pressure of 25 patients with UCLP with that of 25 control patients, also using the IOPI and reporting normal lip and tongue pressure. However, a comparison between patients with CP and UCLP has never been made in the literature before. Our results show no significant differences in maximum oral muscle pressure between patients with CP and UCLP. This suggests that the involvement of the lip and alveolar bone in patients with UCLP does not necessarily affect maximum oral muscle pressure and that cleft repair surgery performed at a young age ensures a good functional recovery of the muscles.
Important to note is that 11 of the 28 included UCLP patients (39%) had a Simonart's band on the cleft side, which according to the literature has a minor positive effect on the facial pattern, reduces the need for the secondary nose and lip repair [20], and requires less traumatic lip and palate repair [21]. Furthermore, three of the UCLP patients had undergone PNAM, which improves nasal asymmetry [22], prevents the positioning of the tongue between the palatal shelves [23], molds the protruded premaxilla, and reduces the size of the cleft lip, alveolus, and palate [22]. One UCLP patient had used presurgical lip straps to facilitate lip closure [23]. These presurgical techniques could influence our outcome since they aim to reduce the width of the defect and the lip tension.
It is relevant to take into consideration that all cleft patients included in this study were operated on by the same two surgeons at the same hospital, following the same surgical protocol. However, between 2012 and 2013, a new surgical protocol for lip and palate repair was gradually implemented by the Cleft Team of University Hospitals Leuven. In the new surgical protocol, the hard palate is closed using a vomer flap at the time of lip repair. The repair of the soft palate still takes place at 10-12 months old, but no longer by supra-periosteal push-back; radical intravelar veloplasty as described by Sommerlad [24] is used instead. In this new protocol, cleft patients undergo an orthodontic expansion only before the secondary alveolar bone graft. Research is ongoing to compare both surgical protocols and examine if the new surgical techniques cause less scar tissue and have a positive influence on oral muscle pressure in cleft patients.
The present study also explores the influence of the maximum oral muscle pressure on jaw width, measured by ICD and IMD. Significantly smaller upper ICDs and larger upper and lower IMDs were found in cleft patients compared with controls. It is important to remark that 19 of the 44 included cleft patients (43,18%) (5 with CP and 14 with UCLP) had previously undergone interceptive orthodontic treatment with removable expansion plates, while only 8 of the 104 control patients (7.69%) had undergone interceptive expansion of the maxilla, which can influence the outcome. Early orthodontic treatment in cleft patients often overexpands the upper jaw, having more effect at the height of the molars than the canines [25], since the canines are positioned closer to the cleft and thus also to the scar tissue. However, it is practically impossible to recruit a sample of cleft patients that did not undergo early maxillary expansion since the lack of growth in the maxillary suture and the presence of scar tissue on the palate lead to maxillary growth impairment [26]. Early orthodontic expansion is then performed in preparation for the closure of the hard palate and the secondary alveolar bone graft. The fact that the lower intermolar distance was larger in patients with clefts in comparison to patients without clefts may be due to a lower position of the tongue in patients with clefts [27].
Furthermore, since we measured jaw width with 32 dental landmarks depending on the dentition stage, we can also make statements regarding tooth position. For example, it can be concluded that the higher the tongue pressure, the more the permanent molars are inclined buccally in patients with CP. This is also true for the canines and molars in the upper jaw in UCLP patients.
The functional harmony and balance of the perioral muscles are critical not only in craniofacial growth and development but also in the position and inclination of the teeth. According to the equilibrium theory, teeth receive intraoral and extraoral muscle forces [28]. A lack of balance in these muscle pressures would affect the occlusion and, in turn, craniofacial growth. However, occlusal forces may also need to be taken into account, as well as the presence of functional habits [4,[28][29][30][31]. In this study, a significant difference in tongue and lip pressure on the transversal width was found between clefts and controls. This significant relationship means that the intra-oral muscles increase the transversal width, and the extra-oral muscles would cause a decrease in the transversal width since the pressure comes from the outside towards the teeth. In patients with a cleft, the higher the imbalance score, the higher the transversal width, meaning that intra-oral forces are more prominent than extra-oral forces. In other words, in cleft patients, there is an imbalance favoring the tongue. Literature has described that the forces from the tongue are consistently greater than those from the lips in non-affected patients [4,[28][29][30][31]. The results of this study suggest that this phenomenon could be extended to the cleft population.
However, perioral muscle pressure at rest is considered to be more crucial than that exerted during function [4,32], since forces at rest are exerted for much longer periods of time [17]. Several authors have attempted to measure oral muscle pressure at rest with various devices, but this was found to be very challenging [33,34]. Measurements during functional activities such as swallowing are also demanding because the procedure of swallowing itself is very complex, consists of different steps [33], and depends on parameters such as the type of device used [35], the type of bolus [36], the amount of occlusal units [35], etc. Maximum tongue pressure is a simpler measurement to take, is less dependent on other external factors, can be compared between studies and/or patients, is correlated with the swallowing function [35], and has been used as a diagnostic feature to detect muscular diseases [37,38].
This study also investigates the potential relationship between oral muscle pressure and sagittal occlusion, overjet, overbite, and cross bite in patients with and without clefts. The effect of the tongue on sagittal occlusion was significantly different between patients with and without cleft: in cleft patients, a greater tongue pressure led to more mesioocclusion. Patients with cleft palate often present a skeletal and dental class III relationship due to the underdevelopment of the maxilla [39], caused by the lack of transversal growth in the midpalatal suture and the presence of scar tissue on the palate [25]. Pereira et al. [27] stated that patients with class III malocclusion exhibit recessed tongue positioning on the floor of the mouth. Our results confirm these findings and suggest that this effect will be amplified with higher maximum tongue pressure. However, according to Wen-hua et al. [2], children with class III have fewer perioral forces, while Lambrechts et al. [40] found no significant relationship between class III and lip and tongue forces in non-affected patients. It would be very interesting for future research to compare the oral muscle pressure of class III patients with and without cleft palate, which has not yet been performed in the literature.
The effect of the maximum lip pressure on the occlusion was significantly different between the controls and clefts: a greater lip pressure led to more disto-occlusion on the right side in the total cleft group, the CP group, and the UCLP group compared to the control group. The same effect was seen on the left side in the UCLP group compared to the controls. This can be explained by the increased occurrence of the unilateral variant on the right side. The findings of the cleft group could be due to compensatory movements of the lower lip, a more prominent lower lip, or a higher stomion position. The literature reports both a reduced range of upper lip movement [13] and compensatory movement of the lower lip [9] in cleft patients. Since the IOPI does not discriminate between upper and lower lip pressure, these aspects could not be explored in our study.
The present research found a significant difference between the cleft and control groups regarding the right cheek pressure: the higher the right cheek pressure, the greater the chance of a cross bite being present. This could be explained by the presence of a cleft on the right side in 17 of the 28 UCLP patients. Literature is controversial on this topic. While Kecik et al. [41] also found higher masseter activity at the cross bite side in patients without cleft, Szyszka-Sommerfeld et al. [42] found lower masticatory muscle activity in cleft patients. However, the literature does agree with the fact that orthodontic correction of a cross bite is essential to restore the balance of the oral muscles [43].
The present study also looked into the relationship between maximum oral muscle pressure and functional habits in control and cleft patients. The influence of the swallowing pattern, resting position of lips and tongue, and several parafunctions on the maximum oral muscle pressure was not statistically different between the groups.
In addition, it would also have been useful to know whether the patients had undergone speech therapy since this could influence the myofunctional results and can be seen as a limitation. Furthermore, the presence of scar tissue at the level of the lips and the palate, as well as the shape of the palate, could also play a role in the measurement of tral muscle pressure. These aspects were also not investigated in this study.
Finally, the results of this study may have some clinical implications regarding early orthodontic expansion in patients with CP and UCLP. Maxillary expansion in patients with clefts is essential as it contributes to speech development, masticatory function, tooth eruption, and stabilization of the dental arches. However, maxillary expansion has been reported to relapse in 80% of patients with cleft [44,45], since they are especially susceptible to occlusal instability due to congenitally missing teeth, a lack of bone in the cleft site [45,46], and the presence of surgical scar tissue. For these reasons, some studies recommend compensatory overexpansion [47] or prolonging the retention period for 2-3 years [48]. However, our results suggest that cleft patients with a larger maxillary width present a higher imbalance score favoring the tongue than those with a narrower maxilla. Since almost half of the included cleft patients had previous interceptive orthodontic treatment with removable expansion plates, we can conclude that maxillary orthodontic expansion has an important influence on the maximum oral muscle pressure. For this reason, attention should be paid to the expansion protocols, for instance, by optimizing the design of the appliance, the amount of expansion, and the retention phase. It could also be an option to revise the retention protocols for patients with cleft palates. Hawley retainers could be additionally equipped with a transversal screw or extra springs, which could be activated in case of relapse of the upper transversal width. Retainers could be supplemented by myofunctional trainers in order to improve the oral muscle function of cleft patients and prevent relapse after orthodontic expansion, keeping the palate as wide as possible. Speech therapy could also be integrated before, during, and after orthodontic treatment [49]. However, the motivation and burden of care for the patient have to be taken into account.
Conclusions
Patients with cleft lip and/or palate did not present reduced maximum oral muscle pressure compared with patients without this condition. An imbalance favoring the tongue (the tongue pressure is greater than the lip pressure) was found in both patients with and without clefts. Patients with CP and UCLP with a larger transversal maxillary width presented systematically higher imbalance scores (favoring the tongue) than those with narrow maxillae. Therefore, the influence of slow maxillary expansion on maximum oral muscle pressure in cleft patients should not be underestimated. It is important for cleft teams to take these myofunctional aspects into consideration in the treatment of patients with cleft lip and/or palate, especially regarding orthodontic treatment planning.
Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the medical ethical committees of University Hospitals Leuven and KU Leuven with registration number s64829.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author. | 2023-07-15T15:47:27.192Z | 2023-07-01T00:00:00.000 | {
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237875929 | pes2o/s2orc | v3-fos-license | MPC Based Coordinated Active and Reactive Power Control Strategy of DFIG Wind Farm with Distributed ESSs
: The ESS is considered as an effective tool for enhancing the flexibility and controllability of a wind farm, and the optimal control scheme of a wind farm with distributed ESSs is vital to the stable operation of wind power generation. In this paper, a coordinated active and reactive power control strategy based on model predictive control (MPC) is proposed for doubly fed induction generator (DFIG)-based wind farm (WF) with distributed energy storage systems (ESSs). The proposed control scheme coordinates the active and reactive power output among DFIG wind turbines (WTs), grid-side converters (GSCs), and distributed ESSs inside the WF, and the aim is to decrease fatigue loads of WTs, make the WT terminal voltage inside the extent practicable, and take the WF economic operation into consideration. Moreover, the best reactive power references of DFIG stator and GSC are produced independently based on their dynamics. At last, the control scheme generates optimal power references for all ESS to make the SOC of each ESS converge to their average state. With the distributed ESSs, the WF controller regulates the WTs inside WF more flexibly. A WF composed of 10 DFIG WTs was utilized to verify the control performance of the proposed coordinated active and reactive power control strategy.
Introduction
In order to deal with the environmental issue and depletion of fossil fuels, wind power has been developing rapidly over the world [1][2][3]. The increasing installed capacity of wind farms (WFs) brings challenges to power system operation and control [4]. WFs are required to operate as conventional power plants to preserve safety of the power system with high wind power penetration rate. To solve these challenges, the WFs have to use reasonable control strategy to meet grid operating demands from the transmission system operators (TSOs) [5]. The DFIG-based wind turbine (WT) has been broadly applied for in advanced WFs benefit from excellent controllability and little converter capacity. By means of power electronic converters, the reactive power of DFIG-based WF could be separated adjusted and the power system reactive power support could be offered [6].
In the conventional active power dispatch strategy of WFs, the power reference is proportionally distributed to individual WTs on the basis of either WTs available power or actual output power. Without considering the operating condition of each WT, the fatigue load increases which results in reduction of WT lifetime [7,8]. For minimizing fatigue loads, in [9], a MPC-based active power control strategy for WFs was proposed to decrease the WTs fatigue loads.
Due to the energy storage system (ESS) has good performance of adaptable charging/discharging, it can be used as an useful tool to improve the adaptability and controllability of WF. For a WF with a centralized ESS [10,11], the requirements for electronic devices of centralized ESS will be rapidly increasing with WF size increasing. If the centralized ESS fails, it will result in security operation problems for the wind farm. For a WF with distributed ESSs, each WT inside the WF is equipped with a small capacity ESS on the DC side of the WT converter. One ESS failure will not have a big impact on the WF operation. WT with distributed ESS has motivated a great number of studies [12][13][14]. In [13], a constant power control strategy was presented for a DFIG WT WF, at the same time, ESS is installed on every DFIG. The scheme enabled the WF to effectively regulate active power of the grid. Reference [14] has proposed a model and control framework to attain coordinated run for grid connected DFIG-based WT and ESS.
For the purpose of the DFIG-based WF safe operation, voltage performance also should be addressed. Every DFIG-based WT inside DFIG-based WFs is prepared with power electronic converter. The DFIG-based WF can utilize the capabilities of the electronic converters to regulate the bus voltages inside the WF. A few control modes involve voltage, reactive power at the point of connection (POC) and power factor have been designated in a lot of grid codes [15]. Voltage control mode usually exhibit excellent execution of transmission systems [16,17]. In [18], A optimized voltage control strategy of offshore WFs cluster was proposed, the pilot bus voltage is controlled under steady-state operation by assigning reactive power references to every WT according to their accessible reactive power margin. An ideal reactive power dispatch strategy was presented in [19] to keep WF electrical losses at a minimum, containing transmission cables losses, WT transformers, and the wind energy generation systems. In [20], a hierarchical automatic voltage control was presented to keep every WT terminal voltage inside the attainable extend.
As mentioned above, there are some currently studies researches have made incredible contributions to the WFs voltage/reactive power control. But most of references described the dynamic model of WT as a first-order lag function, and did not consider the real WT generator and converter dynamic model [21][22][23]. Therefore, an MPC-based active and reactive power integrated control strategy for DFIG WF with distributed ESSs is proposed in this paper, which has two stages. The control objectives of the first stage are controlling the DFIGs inside the WF to follow the active power reference issued by TSO, decrease fatigue loads through minimizing the change of WT thrust force and shaft torque, meanwhile generate a ESSs active power reference. Under the second stage, the controller offer optimized reactive power references of DFIG stator and grid-side converter to adjust the voltage of each bus in the reasonable range, and reduce the WF network losses. Moreover, controller generates the active power references for ESSs to keep its SOCs close to a common state and consider the WF economic operation.
The most important contribution of this paper is the MPC based coordinated active and reactive power control strategy for distributed large-scale WFs with ESSs. The ideal reactive power references of DFIG stator and GSC are generated separately to keep bus voltage deviation and network power losses at a minimum. With the distributed ESSs, the WF controller regulates WTs more flexibly. The WTs and ESSs active and reactive power output could be best distributed to promote the optimal operation of WF. Compared with conventional reactive power control for DFIGs WF, reactive power references are generated separately on the basis of stator and GSC dynamics to achieve a better performance.
The paper includes the following parts. Section 2 introduces a brief overview of the coordinated active and reactive power control structure. Section 3 introduces the prediction model for RSC, GSC, ESS and WT in detail. Section 4 presents the MPC based mathematical formulation of coordinated power control scheme is described. Section V introduces and discusses the simulation results, and gives conclusions. This article takes DFIG-based WF with distributed ESS as the research object, different from the traditional control method that control WT active power independently to reduce fatigue load or control WT reactive power independently to stabilize WT terminal voltage, the proposed control method, the proposed control method can coordinate active and reactive power control at the same time, reduce WT fatigue load and stabilize WT voltage at the same time, and reduce the WF network losses. meanwhile, tracking the dispatch command and maintaining the state-of-charge (SOC) of ESSs within a specified range.
Configuration of DFIG Wind Farm
A representative topology of a WF is shown in Figure 1. It is linked to a outside AC grid by transmission cable. The HV/MV on-load tap changer (OLTC) is located between point of connection (POC) in HV side and collection point in MV side. Several feeders are connected to the MV collection point, and each feeder is connected to series WTs. reduce the WF network losses. meanwhile, tracking the dispatch command and maintaining the state-of-charge (SOC) of ESSs within a specified range.
Configuration of DFIG Wind Farm
A representative topology of a WF is shown in Figure 1. It is linked to a outside AC grid by transmission cable. The HV/MV on-load tap changer (OLTC) is located between point of connection (POC) in HV side and collection point in MV side. Several feeders are connected to the MV collection point, and each feeder is connected to series WTs.
Concept of Coordinated Power Control Scheme
The structure of the presented active and reactive power coordinated control strategy is shown in Figure 2. The WF total active power reference is set by TSO and send to the WF controller. The MPC-based WF controller implements a two-stage optimization scheme.
Configuration of a wind farm.
Concept of Coordinated Power Control Scheme
The structure of the presented active and reactive power coordinated control strategy is shown in Figure 2. The WF total active power reference is set by TSO and send to the WF controller. The MPC-based WF controller implements a two-stage optimization scheme.
Configuration of DFIG Wind Farm
A representative topology of a WF is shown in Figure 1. It is linked to a outside AC grid by transmission cable. The HV/MV on-load tap changer (OLTC) is located between point of connection (POC) in HV side and collection point in MV side. Several feeders are connected to the MV collection point, and each feeder is connected to series WTs.
Concept of Coordinated Power Control Scheme
The structure of the presented active and reactive power coordinated control strategy is shown in Figure 2. The WF total active power reference is set by TSO and send to the WF controller. The MPC-based WF controller implements a two-stage optimization scheme. , , Figure 2. Structure of control scheme. The first stage controls all DFIGs inside the WF to follow the active power reference issued by TSO, and decreasing WTs fatigue loads, brought by the changes of WTs shaft torque and thrust force. And the total active power reference value of ESSs and the best active power reference of every DFIG WT are obtained by the controller.
In the second stage control, controller make the ESSs follow its power reference from the first stage control, while keep its SOCs to an average state. Meanwhile controller regulates the reactive power output of DFIG stator and GSC to minimize bus voltage deviation and decrease network power losses of WF, and consider the influence of active power outputs of WTs and ESSs. Figure 3 shows the structure of the DFIG WT equipped with an ESS. DFIG stator joins to the AC grid directly. DFIG rotor is linked to the AC network by a back-to-back PWM converter, including a rotor side converter (RSC), a GSC and an ESS.ESS contains an energy storage unit (ESU) and a DC/DC converter. The ESU is linked to a DC bus by a DC/DC converter to adjust charge/discharge power of ESU to track the active power reference from the WF controller. The first stage controls all DFIGs inside the WF to follow the active power referen issued by TSO, and decreasing WTs fatigue loads, brought by the changes of WTs sh torque and thrust force. And the total active power reference value of ESSs and the b active power reference of every DFIG WT are obtained by the controller.
WF Model
In the second stage control, controller make the ESSs follow its power reference fro the first stage control, while keep its SOCs to an average state. Meanwhile controller re ulates the reactive power output of DFIG stator and GSC to minimize bus voltage dev tion and decrease network power losses of WF, and consider the influence of active pow outputs of WTs and ESSs. Figure 3 shows the structure of the DFIG WT equipped with an ESS. DFIG stator joi to the AC grid directly. DFIG rotor is linked to the AC network by a back-to-back PW converter, including a rotor side converter (RSC), a GSC and an ESS.ESS contains an e ergy storage unit (ESU) and a DC/DC converter. The ESU is linked to a DC bus by DC/DC converter to adjust charge/discharge power of ESU to track the active power r erence from the WF controller.
RSC Model
The RSC is mainly used to control active and reactive power output of DFIG. R could decouple active and reactive power by adjusting rotor current in a synchronou rotating reference frame oriented by the stator flux. Figure 4 shows the RSC control stru ture.
DFIG active power output is: stator active power output is the first item in function (1), and GSC active power outp is the second item. DFIG reactive power is DFIG stator reactive power QS. QS is controlled through a justing DFIG rotor d-axis current idr, its calculation formula can be derived and express as:
RSC Model
The RSC is mainly used to control active and reactive power output of DFIG. RSC could decouple active and reactive power by adjusting rotor current in a synchronously rotating reference frame oriented by the stator flux. Figure 4 shows the RSC control structure. where ψS is the stator flux, Lm is the mutual inductance, ωS is the supply angular speed, LS is DFIG stator inductance. Uqr is q-axis rotor voltage, iqr is rotor q-axis current. DFIG active power output is: stator active power output is the first item in function (1), and GSC active power output is the second item. DFIG reactive power is DFIG stator reactive power Q S . Q S is controlled through adjusting DFIG rotor d-axis current i dr , its calculation formula can be derived and expressed as: where ψ S is the stator flux, L m is the mutual inductance, ω S is the supply angular speed, L S is DFIG stator inductance. U qr is q-axis rotor voltage, i qr is rotor q-axis current. Normally, The time constant of power outer loop is much smaller than that of the current inner loop The first-order lag function could describe the dynamic behavior of the current loop. Hence, the model of RSC active power and reactive power could be simplified re which is shown in Figures 5 and 6 spectively. Where k P r_P , k Q r_P , k P r_i , and k Q r_i are the proportional gain and integral gain of PI controllers, respectively. T ir is the time constant of current loop. T fr denote filter time constant. Superscript '*'indicates reference value. where ψS is the stator flux, Lm is the mutual inductance, ωS is the supply angular speed, LS is DFIG stator inductance. Uqr is q-axis rotor voltage, iqr is rotor q-axis current. Define ∆i qr , ∆i dr , ∆P g , ∆Q S , ∆P ref g , ∆P int , ∆Q ref S , and ∆Q S int are the incremental values between its current value and the initial value at operation point, respectively. Where P int is the error integral of P ref g and P g , Q S int is the integral of error between Q ref S and Q S . Then, RSC active power incremental state space model is as follows, The state space matrix form is, where The RSC reactive power incremental state space model is derived as follows, The state space matrix form is, where
GSC Model
The DC bus voltage is adjusted by GSC, which offering reactive power support for WF AC grid at the same time. GSC can decouple DC voltage and reactive power control by adjusting current in a synchronously rotating reference frame oriented by the grid voltage [24]. Figure 7 shows the GSC control structure. GSC reactive power in the grid-voltage-oriented synchronously rotating reference frame can be modeled as, where Um is the grid phase voltage amplitude. The same as the RSC, the GSC model is shown in Figure 8, k GSC reactive power in the grid-voltage-oriented synchronously rotating reference frame can be modeled as, where U m is the grid phase voltage amplitude. The same as the RSC, the GSC model is shown in Figure 8, k Q g_P , k Q g_i is the proportional gain and integral of PI controller, T ig denote current loop time constant, T fg denote GSC filter time constant. Superscript '*'indicates reference value.
where Um is the grid phase voltage amplitude. The same as the RSC, the GSC model is shown in Figure 8, k Q g_P , k Q g_i is the proportional gain and integral of PI controller, Tig denote current loop time constant, Tfg denote GSC filter time constant. Superscript '*'indicates reference value. Define the ∆i q , ∆Q C , ∆Q ref C , and ∆Q C int are the incremental values between its current value and the initial value at operation point, respectively, and Q C int is the integral of error between Q ref C and Q C . Superscript '*'indicates reference value. GSC incremental state space model is derived as follows, The state space matrix form can be get, where
ESS Model
The calculation formula of ESS stored energy C ESS is, where C ESS,0 denotes the initial energy, and P ESS denotes the charge/discharge power of ESS.
To enhance the DC side current response speed, a double closed loop control structure diagram of DC/DC converter, as shown in Figure 9. Figure 10 shows the ESS simplified block diagram. Where k P d_P and k P d_i denotes the proportional gain and integral of the PI controller. U ESS denotes ESU voltage. T id denotes the current loop time constant, T fd denotes ESS filter time constant. Superscript '*' indicates reference value.
where CESS,0 denotes the initial energy, and PESS denotes the charge/discharge power of ESS.
To enhance the DC side current response speed, a double closed loop control structure diagram of DC/DC converter, as shown in Figure 9. Figure 10 shows the ESS simplified block diagram. Where k P d_P and k P d_i denotes the proportional gain and integral of the PI controller. UESS denotes ESU voltage. Tid denotes the current loop time constant, Tfd denotes ESS filter time constant. Superscript '*'indicates reference value. where CESS,0 denotes the initial energy, and PESS denotes the charge/discharge power of ESS.
To enhance the DC side current response speed, a double closed loop control structure diagram of DC/DC converter, as shown in Figure 9. Figure 10 shows the ESS simplified block diagram. Where k P d_P and k P d_i denotes the proportional gain and integral of the PI controller. UESS denotes ESU voltage. Tid denotes the current loop time constant, Tfd denotes ESS filter time constant. Superscript '*'indicates reference value.
The state space expression of the ESS is, where
WT Model
The mechanical system model of the WT can be obtained according to the analysis in [25].
Let K RSC = L m ψ S ω S T fr L S + 3U qr 2T fr L S , and combine the RSC active power model and the WT model, the incremental state-space matrix of DFIG active power dynamic model is, where ∆x W = ∆P g , ∆P int , ∆i qr , ∆β ref , ∆β, ∆ω g , ∆ω f T , where θ denotes pitch angle, β = K C θ, K C = K 0 + 2K 1 θ, where K 0 and K 1 are constants, T θ denotes time constant of pitch servo function, T a denotes aerodynamic torque, and T g denotes generator torque, K p and K i denote the proportional gain and integral gain of pitch controller, ω f is filtered speed at which the generator speed ω g is filtered by the low-pass filter, T f is filter time constant, η g denotes gear box ratio, J r denotes rotor mass, J g denotes generator mass, J t = J r + η 2 g J g is the equivalent mass, µ g is the generator efficiency; T S denotes shaft torque, F t denotes thrust force, K βT , K ωT , K βF and K ωF are the coefficients obtained the Taylor expansion of T S and F t at the operating point, and subscript 0 is used to denote the initial value in control period.
Whole System Model
The continuous state-space matrix of a WF consist of N T WTs can be get as follows, where The discrete state space matrix is,
MPC Based Coordinated Active and Reactive Power Control for Wind Farm with ESSs
The DFIG-based WF requires the WF controller consider the coordinated control of the all the energy sources inside the WF. All WTs, RSCs, GSCs, and VSCs within the WF are optimally coordinated to reduce fatigue loads, keep the each bus voltage in a reasonable range and decrease network power losses. In order to decouple active and reactive power optimal control and keep fatigue loads at a minimum, the control strategy is divided into two steps. every DFIG active power reference and a total ESSs active power reference are produced by the controller in the first stage, which aims to follow the dispatch command and reduce the fatigue loads by minimizing variations in TS and Ft of WTs. The controller coordinates control the reactive power of DFIG stator and GSC inside this WF in the second stage, aims to keep the all buses voltage of WF in a feasible range, and reduces WF network power losses. Moreover, the WF controller regulates ESSs charge/discharge power to follow the ESSs active power reference obtained in the first step and keeps every ESS SOC value converging to average.
Objective Function
The WF controller reduces fatigue loads of WTs. In addition, in order to enable the ESS to operate stably for a long time, the CESS of ESS is controlled to keep at the intermediate level. Then the objective function can beget as follows, where N P denotes MPC predictive steps, and C total ESS,mid denotes intermediate level of total ESS capacity. λ C , λ T , λ F are the weighting coefficients for the total ESSs energy management, variations of T S and F t , separately.
Constraints
The active power constraint of the WF is, The constraint of a WT is as follows, The constraint of total ESSs is as follows,
Objective Function
The first objective is voltage regulation, which aims to keep the each MV bus voltage and WT terminal bus voltage in a reasonable range [26]. Therefore, the objective function about voltage regulation is, Keep the total network power losses at a minimum is the second objective, which is, where P pre LOSS is the predictive value of network power losses, where sensitivity coefficients regarding power injections in (34), (35), In addition, (37) can be obtained by using the analytical method in [26].
The third objective is to ensure the SOC of each ESS converging to an average value, which is, According to (33), (36), (38), the objective function of the second step is, where λ V , λ L , λ S are the weighting coefficients for Obj V , Obj L , Obj S , respectively.
Constraints
For a distributed ESS, its active power reference constraint is as follow, where Q min S,i /Q min C,i and Q max S,i /Q max C,i are the minimum and maximum Var capacity of the DFIG stator and GSC, separately.
The entire charge/discharge active power output should be set equal to the total ESS active power reference obtained by the first stage as follow,
Test System
A WF composed of 10 × 5 MW DFIG WTs with distributed ESSs is used to verify presented control strategy in the section. The WF modeling that considers the effects of turbulence and wakes is created by SimWindFarm, a toolbox for dynamic WF modeling, simulation and control. The control period T C , prediction horizon T P and the prediction sampling period ∆T P are set as 1 s, 5 s, and 0.1 s, separately. For the purpose of verify the effectiveness of the presented control scheme, the simulation results are contrast with the one without ESSs and reactive power control.
Control Performance
In the MATLAB/Simulink, the simulation time is set as 550 s. The total WF available active power and TSO dispatch demand are shown in Figure 11. During t = 0-280 s, the WF available power fluctuates between 38-50 MW. At t = 280 s, the total available active power begin increasing and reaches the maximum available power 50 MW at 300 s. The available active power gradually reduces after t = 480 s. Figure 12, thanks to the control effect of ESSs on WF active power adjustment, Ft variation with ESSs is smaller than that without ESSs. In Figure 13, during t = 0-340 s, the fluctuations of TS with ESSs is much less than that without ESSs. After t = 340 s, wind farm is operating in the maximum power point tracking (MPPT) mode, the changes in TS with ESSs is also less than that without ESSs. The fluctuations of thrust force are also less than that without ESSs. The control strategy proposed in this paper can effectively decrease the fatigue loads of DFIG WT by minimizing the changes of Ft and TS, then improve the security of the WF. Figure 11. WF total available and reference active power. Figures 12 and 13 show the thrust force and shaft torque changes of WT4. In Figure 12, thanks to the control effect of ESSs on WF active power adjustment, F t variation with ESSs is smaller than that without ESSs. In Figure 13, during t = 0-340 s, the fluctuations of T S with ESSs is much less than that without ESSs. After t = 340 s, wind farm is operating in the maximum power point tracking (MPPT) mode, the changes in T S with ESSs is also less than that without ESSs. The fluctuations of thrust force are also less than that without ESSs. The control strategy proposed in this paper can effectively decrease the fatigue loads of DFIG WT by minimizing the changes of F t and T S , then improve the security of the WF.
The First Stage Control Performance
12, thanks to the control effect of ESSs on WF active power adjustment, Ft variation with ESSs is smaller than that without ESSs. In Figure 13, during t = 0-340 s, the fluctuations of TS with ESSs is much less than that without ESSs. After t = 340 s, wind farm is operating in the maximum power point tracking (MPPT) mode, the changes in TS with ESSs is also less than that without ESSs. The fluctuations of thrust force are also less than that without ESSs. The control strategy proposed in this paper can effectively decrease the fatigue loads of DFIG WT by minimizing the changes of Ft and TS, then improve the security of the WF. Figures 12 and 13 show the thrust force and shaft torque changes of WT4. In Figure 12, thanks to the control effect of ESSs on WF active power adjustment, Ft variation with ESSs is smaller than that without ESSs. In Figure 13, during t = 0-340 s, the fluctuations of TS with ESSs is much less than that without ESSs. After t = 340 s, wind farm is operating in the maximum power point tracking (MPPT) mode, the changes in TS with ESSs is also less than that without ESSs. The fluctuations of thrust force are also less than that without ESSs. The control strategy proposed in this paper can effectively decrease the fatigue loads of DFIG WT by minimizing the changes of Ft and TS, then improve the security of the WF.
Simulation Analysis of the Second Stage
The WT 4 terminal voltage is shown in Figure 14. With the reactive power control, the voltage could be kept within the reasonable range effectively. The voltage deviation from nominal value with the reactive power control is significantly smaller than that without this control, which guarantee the WF secure operation.
Simulation Analysis of the Second Stage
The WT 4 terminal voltage is shown in Figure 14. With the reactive power control, the voltage could be kept within the reasonable range effectively. The voltage deviation from nominal value with the reactive power control is significantly smaller than that without this control, which guarantee the WF secure operation. Figures 15 and 16 show the active power and reactive power output of WT 4, respectively. During t = 0-300 s, the WT 4 stator and GSC absorb reactive power separately to regulate the WF voltages. After t = 300 s, due to the increasing active power output, the reactive power absorbed by stator is decreasing. At this time, the GSC supply reactive power. Due to GSC reactive power output limits, the WT 4 terminal voltage is increased, but it is still within the feasible operating range. During t = 0-300 s, the WT 4 stator and GSC absorb reactive power separately to regulate the WF voltages. After t = 300 s, due to the increasing active power output, the reactive power absorbed by stator is decreasing. At this time, the GSC supply reactive power. Due to GSC reactive power output limits, the WT 4 terminal voltage is increased, but it is still within the feasible operating range. The WF total network power losses is shown in Figure 17. The network power losses with reactive power control is smaller than that without reactive power control. The WF total network power losses is shown in Figure 17. The network power losses with reactive power control is smaller than that without reactive power control. The WF total network power losses is shown in Figure 17. The network power losses with reactive power control is smaller than that without reactive power control. Figure 17. Network power losses of wind farm. Figure 18 shows the SOC of each ESS. All the ESSs SOC are kept to the average value. From t = 0-350 s, the SOC fluctuates around 0.5, which means the proposed scheme manage the ESSs to keep the SOCs at the medium level. From t = 350-500 s, the WF output rated active power. The ESSs also needs output a spot of active power to decrease the WTs Figure 18 shows the SOC of each ESS. All the ESSs SOC are kept to the average value. From t = 0-350 s, the SOC fluctuates around 0.5, which means the proposed scheme manage the ESSs to keep the SOCs at the medium level. From t = 350-500 s, the WF output rated active power. The ESSs also needs output a spot of active power to decrease the WTs fatigue loads. The SOCs are decreased slightly, but they are kept closing to the medium level. After 500 s, the available power gradually decreases, and the dispatch command remain unchanged. The available power is less than the dispatch command. Therefore, all ESSs discharge power to track the dispatch command, causing SOCs decrease gradually. Figure 19 shows the WF total active power output that equipped with distributed ESSs. With the ESSs, the WF total active power output could accurately follow the active power dispatch command from the TSO. Figure 19. Active power output of wind farm.
Conclusions
In this paper, an MPC based coordinated active and reactive power control strategy is proposed for DFIG wind farm with distributed ESSs, which coordinates the active and reactive power output among DFIG WTs, GSC and distributed ESSs inside wind farm to track the dispatch command the TSO set, reduce the fatigue loads of the WTs, and manage the distributed ESSs. Moreover, the control scheme can provide the best reactive power references for DFIG stator and GSC separately to regulate all the bus voltages and network power losses while track the dispatch command from TSO. Case studies show the control strategy could effectively decrease the fatigue loads, regulate the voltage in the feasible range, decrease the WF network power losses and ensure the SOC of each ESS converging to an average value. The presented coordinated active and reactive power control strategy is suitable for real-time control of large-scale wind farm. Figure 19 shows the WF total active power output that equipped with distributed ESSs. With the ESSs, the WF total active power output could accurately follow the active power dispatch command from the TSO. Figure 19 shows the WF total active power output that equipped with distributed ESSs. With the ESSs, the WF total active power output could accurately follow the active power dispatch command from the TSO. Figure 19. Active power output of wind farm.
Conclusions
In this paper, an MPC based coordinated active and reactive power control strategy is proposed for DFIG wind farm with distributed ESSs, which coordinates the active and reactive power output among DFIG WTs, GSC and distributed ESSs inside wind farm to track the dispatch command the TSO set, reduce the fatigue loads of the WTs, and manage the distributed ESSs. Moreover, the control scheme can provide the best reactive power references for DFIG stator and GSC separately to regulate all the bus voltages and network power losses while track the dispatch command from TSO. Case studies show the control strategy could effectively decrease the fatigue loads, regulate the voltage in the feasible range, decrease the WF network power losses and ensure the SOC of each ESS converging to an average value. The presented coordinated active and reactive power control strategy is suitable for real-time control of large-scale wind farm.
Conclusions
In this paper, an MPC based coordinated active and reactive power control strategy is proposed for DFIG wind farm with distributed ESSs, which coordinates the active and reactive power output among DFIG WTs, GSC and distributed ESSs inside wind farm to track the dispatch command the TSO set, reduce the fatigue loads of the WTs, and manage the distributed ESSs. Moreover, the control scheme can provide the best reactive power references for DFIG stator and GSC separately to regulate all the bus voltages and network power losses while track the dispatch command from TSO. Case studies show the control strategy could effectively decrease the fatigue loads, regulate the voltage in the feasible range, decrease the WF network power losses and ensure the SOC of each ESS converging to an average value. The presented coordinated active and reactive power control strategy is suitable for real-time control of large-scale wind farm. Data Availability Statement: The study did not report any data.
Conflicts of Interest:
The authors declare no conflict of interest. | 2021-09-01T15:05:42.036Z | 2021-06-29T00:00:00.000 | {
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264073342 | pes2o/s2orc | v3-fos-license | Differential Evolution Particle Swarm Optimization for Phase-Sensitivity Enhancement of Surface Plasmon Resonance Gas Sensor Based on MXene and Blue Phosphorene/Transition Metal Dichalcogenide Hybrid Structure
A differential evolution particle swarm optimization (DEPSO) is presented for the design of a high-phase-sensitivity surface plasmon resonance (SPR) gas sensor. The gas sensor is based on a bilayer metal film with a hybrid structure of blue phosphorene (BlueP)/transition metal dichalcogenides (TMDCs) and MXene. Initially, a Ag-BlueP/TMDCs-Ag-MXene heterostructure is designed, and its performance is compared with that of the conventional layer-by-layer method and particle swarm optimization (PSO). The results indicate that optimizing the thickness of the layers in the gas sensor promotes phase sensitivity. Specifically, the phase sensitivity of the DEPSO is significantly higher than that of the PSO and the conventional method, while maintaining a lower reflectivity. The maximum phase sensitivity achieved is 1.866 × 106 deg/RIU with three layers of BlueP/WS2 and a monolayer of MXene. The distribution of the electric field is also illustrated, demonstrating that the optimized configuration allows for better detection of various gases. Due to its highly sensitive characteristics, the proposed design method based on the DEPSO can be applied to SPR gas sensors for environmental monitoring.
Introduction
Surface plasmons (SPs) comprise a special mode of electromagnetic field that exists at the dielectric-metal interface [1,2].As the resonance absorption peak, resonance angle, and resonance wavelength generated when the surface plasmon resonance (SPR) occurs are closely related to the variation in the refractive index in the sensing medium, sensors find widespread applications in fields such as cell analysis, metamaterial absorbers, and gas detection [3][4][5][6][7][8].The utilization of SPR in gas detection has garnered significant attention due to its label-free and real-time characteristics [9,10].However, most SPR sensors rely on measuring the intensity change in the reflected beam, which is susceptible to environmental influences and exhibits a low sensitivity to changes in the refractive index of small molecules [11,12].
In order to promote the sensitivity and resolution of SPR sensors, phase-sensitivity configuration has been proposed in several studies in the literature [13][14][15], based on the fact that the phase of the TM-polarized component of the incident light wave undergoes drastic changes, while the phase of the TE-polarized component remains essentially unchanged [16].The conventional phase-modulated SPR sensor, which employs the Kretschmann prism coupler, typically utilizes monolayer gold (Au) [17], silver (Ag) [18], aluminum (Al) [19], or copper (Cu) [20] film as the plasmonic metal.Among these metallic Sensors 2023, 23, 8401 2 of 16 materials, Ag is widely favored due to its strong resonance peak resulting from the characteristics of the bulk plasma frequency and low D-electron band [21,22].However, the inherent absorption of the metal layer broadens the response, and the Ag film is vulnerable to oxidation at ambient temperatures, which negatively affects the phase-sensitivity of the SPR sensor.To mitigate these issues, researchers have proposed the use of two-dimensional (2D) materials.These materials offer a high photoelectric performance, promoting light absorption and providing an improved biological compatibility.
Blue phosphene (BlueP) is a promising 2D material produced by Zhu and Tomanek, following the experimental discovery of black phosphorus [23].The lattice arrangement of phosphorus atoms in BlueP is located in the lower curved honeycomb of its lattice [24].BlueP demonstrates thermal stability comparable to black phosphorus, and also features a band gap of 2ev, making it highly suitable for application in SPR sensors [25].Additionally, due to the hexagonal crystal structure shared by the BlueP and TMDC monolayers, it is possible to construct a van der Waals (vdw) hybrid structure of BlueP/TMDCs [26].This vdw heterostructure exhibits novel optoelectronic characteristics previously unseen in 2D materials.BlueP-based SPR phase sensors have been utilized in practical biosensing due to their superior sensitivity compared to traditional monolayer biosensors [27][28][29].Liao successfully developed an SPR sensor using ITO and BlueP/MoS 2 , achieving a maximum phase sensitivity of ~3.600 × 10 6 deg/RIU [30].Furthermore, Li proposed an SPR biosensor based on BlueP-graphene, which achieved the highest phase sensitivity of 1.473 × 10 5 deg/RIU [31].
MXene consists of metal carbide and metal nitride materials with a 2D layered structure [32].It is exfoliated via the MAX phase and has a universal chemical formula M n+1 AX n (n = 1, 2, or 3), where M represents early transition metals, A usually represents chemical elements of the third or fourth main group, and X represents nitrogen or carbon [33][34][35].Due to its large surface-to-volume ratio and metal level conductivity, MXene significantly enhances the detection limit of small molecules for SPR sensors modified with MXene.However, the sensors discussed in the aforementioned literature employ a conventional method (CM) based on layer-by-layer optimization.As the number of SPR sensor layers increases, this method becomes time-consuming and makes it challenging to determine the optimal thickness of all the layers simultaneously.
An intelligent optimization algorithm is developed to design a multi-layer SPR sensor for high sensitivity and resolution.The structure and performance of SPR sensors are optimized using the particle swarm optimization (PSO) algorithm.Sun proposed a SPR biosensor structure with the PSO algorithm and analyzed its sensing performance under four different modulation modes (wavelength, angle, phase, intensity) [36].The results demonstrate that the optimization structure based on the PSO algorithm outperforms the experimental structure.Additionally, Amoosoltani used the PSO algorithm to optimize the thickness of the metal layer in an SPR gas sensor [37].The simulation results show a significant improvement in the Q-factor and FWHM using this method.However, the PSO algorithm is prone to parameter selection and multi-objective parameter optimization, leading to a faster convergence in the early stage and slower convergence in the later stage, and a higher likelihood of being trapped in the local optima [38].To address these limitations, the differential evolution (DE) algorithm introduces mutation and crossover concepts into the particle position update, enabling the algorithm to escape local optima and effectively handle a large number of design parameters.
The DEPSO algorithm is used in this article to design a high-phase-sensitivity SPR gas sensor based on the Ag-BlueP/TMDCs-Ag-MXene hybrid structure for gas detection.The DEPSO algorithm uses an objective function with a restraint condition of minimum reflectivity.By optimizing the layer thickness in the Ag-BlueP/TMDCs-Ag-MXene modified structure, the phase sensitivity of the SPR gas sensor can be increased while maintaining a low reflectivity, making it more suitable for gas detection.The performance of the optimized structure is then evaluated via detecting different gases.Finally, the ultrasensi-tive properties of the optimized structure are further analyzed via illustrating the electric field distribution.
Theoretical Modeling and Design Consideration
The schematic diagram of the phase-sensitivity SPR gas-detection setup is shown in Figure 1.The proposed SPR gas sensor consists of a BK7 prism (n BK7 = 1.5151); a Ag film with RI is n Ag = 0.0803 + 4.2347i based on the Drude-Lorentz model as the plasmonic metal.Table 1 shows the RIs and monolayer thicknesses of the BlueP/TMDCs and MXene at the wavelength of 633 nm.The sensing medium is air, and its RI is 1.To provide a basis for the numerical simulation method, Figure 1 shows the feasibility of the proposed method in the experiment.Firstly, the He-Ne laser passes through a laser beam expander and polarizer to obtain 45 • linear polarization for the P-wave and S-wave.Then, the light is incident upon the SPR gas sensor at angle θ, which is controlled by the 3-axis rotation stage.At last, it enters the Mach-Zehnder interference to obtain the interference patten and calculate the actual phase sensitivity of the SPR gas sensor.
Sensors 2023, 23, x FOR PEER REVIEW 3 of 17 fied structure, the phase sensitivity of the SPR gas sensor can be increased while maintaining a low reflectivity, making it more suitable for gas detection.The performance of the optimized structure is then evaluated via detecting different gases.Finally, the ultrasensitive properties of the optimized structure are further analyzed via illustrating the electric field distribution.
Theoretical Modeling and Design Consideration
The schematic diagram of the phase-sensitivity SPR gas-detection setup is shown in Figure 1.The proposed SPR gas sensor consists of a BK7 prism ( Table 1 shows the RIs and monolayer thicknesses of the BlueP/TMDCs and MXene at the wavelength of 633 nm.The sensing medium is air, and its RI is 1.To provide a basis for the numerical simulation method, Figure 1 shows the feasibility of the proposed method in the experiment.Firstly, the He-Ne laser passes through a laser beam expander and polarizer to obtain 45° linear polarization for the P-wave and S-wave.Then, the light is incident upon the SPR gas sensor at angle θ , which is controlled by the 3-axis rotation stage.At last, it enters the Mach-Zehnder interference to obtain the interference patten and calculate the actual phase sensitivity of the SPR gas sensor.For the sake of validating the performance of the sensor, the transfer matrix method (TMM) is used to compute the total reflection coefficient r of p-polarized and s-polarized light, as follows: The characteristic matrix of the N-layer structure is given by: For the sake of validating the performance of the sensor, the transfer matrix method (TMM) is used to compute the total reflection coefficient r of p-polarized and s-polarized light, as follows: Sensors 2023, 23, 8401 4 of 16 The characteristic matrix of the N-layer structure is given by: In these equations, λ represent the wavelength of the incident light, and q k is the optical admittances.d m and ε m represent the thickness and dielectric constant of each film layer, respectively.From the above equation, the reflectance R p is R p = r p 2 , and the phase of the p-polarized and s-polarized light can be expressed as: Furthermore, the phase difference (φ d ) between the p-polarized (φ p ) and s-polarized (φ s ) light can be described as: Therefore, the phase sensitivity of the sensor is given by: S = ∆φ d ∆n bio (7) where ∆φ d is the differential phase corresponding to changes in the refractive index of gas.
In this work, a high-phase-sensitivity SPR sensor based on a Ag-BlueP/TMDCs-Ag-MXene hybrid structure is designed and numerically investigated using MATLAB software (R2021a), based on differential evolution particle swarm optimization and the transfer matrix method with the Fresnel equation for the detection of various gases.
Differential Evolution Particle Swarm Optimization
The particle swarm optimization (PSO) is a group-based intelligence optimization algorithm that simulates the foraging behavior of birds.It achieves this by imagining each bird as a particle and representing each possible solution as a particle in the population [39].Each particle has its own velocity and position.The updating of the particle position and velocity is mainly achieved via comparing itself with the surrounding particles and the current optimal value of the population.Due to these characteristics, it can be seen that the PSO is suitable for optimizing the solution of multi-dimensional problems.However, the PSO has the problem of a fast convergence speed in the early stage.Additionally, the information exchange between each particle is unidirectional, which causes the population to lack diversity in the later stages of the algorithm and easily fall into the local optimum.
To address this issue, this paper designs a differential evolution particle swarm optimization algorithm.The idea is to introduce mutation and crossover strategies from the differential evolution algorithm into the process of particle updates.This integration aims to maintain the diversity of the particle swarm in the later stage of the search, thereby improving the global optimization performance of the algorithm.
The specific steps of designing the phase-sensitivity SPR gas sensor based on the DEPSO are as follows.At first, the population positions and velocities need to be randomly initialized, and then the positions and velocities of the particles are updated after a comparison.Afterwards, we perform mutation operations on the updated population to generate temporary intermediate individuals: where the randomly selected serial numbers r 1 , r 2 , r 3 , and r 4 are different from each other, and F denotes the scaling factor, which determines the degree of variation.Similar to the crossover operator in genetic algorithms, in order to enhance the population diversity, the crossover operation in the differential algorithm can be represented as: where CR represents the crossover probability, and j rand is a random integer between [1, 2, . .., D].
Finally, new populations are generated through the above mutation and crossover operation, and the new populations are evaluated and the global optimal solution is updated until the termination condition is satisfied.The pseudocode of the DEPSO can be described as below (Algorithm 1): %Update the velocity and position of the particle (7) If func(x_pso ij ) > func(p ij ) then p ij = x_pso ij (8) End If func(x_pso ij ) > func(p gj ) then p gj = x_pso ij (9) End % Mutation and Crossover operation End % Update globe values (16) If max(func(p g )) > max(func(x i )) then (17) [fmax,r]= max(func(p g )); bestx=x_pso(r,:);() (18) else [fmax,r]= max(func(x i )); bestx=x i (r,:); ( 19) The DEPSO is used to optimize the thickness of the proposed structure to simultaneously obtain a high phase sensitivity and low reflectivity at the resonance angle.Therefore, the objective function is defined as: where S is the phase sensitivity, and R min represents the minimum reflectivity.The aim is to find the maximum value of the objective function in the search region.Apart from that, the process of finding the maximum phase sensitivity is also the process required to minimize the value of R min ; if the R min is larger than 0.01, then the solution will be discarded.
Results and Discussion
In order to verify the effectiveness of the proposed method, the conventional method and particle swarm optimization (PSO) are used to optimize and verify the same heterostructure.First of all, in the conventional method, the thickness of the first layer(d 1 ) and the third layer(d 3 ) of the Ag film are randomly set to 20 nm, the RI of the gas is changed to 0.0001, and the phase sensitivity with respect to different SPR sensor structures at the same change in RI are obtained.As shown in Figure 2 In addition, to better illustrate the improvement in the phase sensitivity of the SPR sensor through the addition of the BlueP/TMDCs and MXene layer, the influence of the number of layers of BlueP/TMDCs and MXene on the phase sensitivity is shown in Figure 3.It is clear from the figure that the phase sensitivity is related to the number of BlueP/TMDCs and MXene layers, with the increase in layers, the phase sensitivity is not It can be concluded from the above analysis that the sensor structure with the BlueP/TMDCS and MXene layer is feasible in improving the phase sensitivity.However, the number of BlueP/TMDCs and MXene layers cannot be arbitrarily determined.Therefore, it is necessary to use an algorithm to simultaneously optimize the thickness of each layer of the SPR sensor.A Ag-BlueP/TMDCs-Ag-MXene hybrid-structure-based SPR gas sensor is designed via the PSO and the DEPSO to verify the effectiveness of the method.In Equations ( 1)-( 8), it is evident that the value of the objective function is related to the thickness of Ag d1 and d2, the thickness of BlueP/TMDCs N1-N4, and the number of layers in MXene L1.Thus, It can be concluded from the above analysis that the sensor structure with the BlueP/ TMDCS and MXene layer is feasible in improving the phase sensitivity.However, the number of BlueP/TMDCs and MXene layers cannot be arbitrarily determined.Therefore, it is necessary to use an algorithm to simultaneously optimize the thickness of each layer of the SPR sensor.
A Ag-BlueP/TMDCs-Ag-MXene hybrid-structure-based SPR gas sensor is designed via the PSO and the DEPSO to verify the effectiveness of the method.In Equations ( 1)-( 8), it is evident that the value of the objective function is related to the thickness of Ag d 1 and d 2 , the thickness of BlueP/TMDCs N 1 -N 4 , and the number of layers in MXene L 1 .Thus, there are seven design parameters, which are as follows: x 2 , x 3 , x 4 , x 5 , x 6 , x 7 ] T .The search range for each variable is set as 0 < d 1 , d 2 < 50 nm, 0 < N 1 . . .N 4 , L 1 ≤ 10 layers.At 633 nm, silver film thicknesses (d 1 , d 2 ) less than 50 nm usually have a good sensing performance [16,18] and, for N 1 . . .N 4 , L 1 > 10 layers, the resonant dip vanishes.Before invoking the algorithm, the initialization variables are listed in Table 2, all of which are set based on experience to enable the algorithm to perform optimally [40].
After 100 iterations, the change in the optimized layer thickness of the Ag-BlueP/ TMDCs-Ag-MXene structure-based SPR sensor via the PSO is shown in Figure 4.The corresponding structural parameters, including the minimum reflectivity at the resonance angle and the phase sensitivity, are presented in Table 3. From Figure 4 and the relevant data in Table 3, it can be seen that as the number of iterations increases, the thickness of the optimized SPR biosensor oscillates first and then converges to a stable value.For Figure 4a and Table 2, the optimized Ag-BlueP/MoS 2 -Ag-MXene heterostructure reaches the highest phase sensitivity of 1.824 × 10 6 deg/RIU, the minimum reflectivity of 2.360 × 10 −4 , when the thickness of the first and third Ag are 19.064nm and 22.244 nm, and the BlueP/MoS 2 and MXene are bilayer and monolayer, respectively.For the Ag-BlueP/MoSe 2 -Ag-MXene heterostructure, when the BlueP/MoSe 2 and MXene are both monolayer, the thickness of the first and third Ag layer are 16.310 nm and 25.367 nm, the highest phase sensitivity is 1.808 × 10 6 deg/RIU, and the minimum reflectivity is 5.289 × 10 −5 .In Figure 4c, the optimized Ag-BlueP/WS 2 -Ag-MXene hybrid structure achieves the highest phase sensitivity of 1.821 × 10 6 deg/RIU, the minimum reflectivity of 1.271 × 10 −4 , when the thickness of first and third Ag layer are 16.310 nm and 25.367 nm, and the layer of both the BlueP/WS 2 and graphene is a monolayer.Finally, the SPR sensor of the Ag-BlueP/WSe 2 -Ag-MXene heterostructure is optimized when the BlueP/WSe 2 and MXene are monolayer, the thickness of first and third Ag layer are 21.502 nm and 20.142 nm, the highest phase sensitivity can achieve 1.816 × 10 6 deg/RIU, and the minimum reflectivity is 1.395 × 10 −4 .The above results demonstrate that, by optimizing the thickness of each layer, the phasesensitivity to RI change can be promoted in the SPR gas sensor.Figure 5 presents the schematic of the implementation of the DEPSO algorithm in conjunction with TMM.Initially, a population of 100 configurations is generated, and the phase difference and minimum reflectivity for each configuration are computed.Subsequently, the configurations are sorted based on their phase sensitivity, given that the minimum reflectance is less than 0.01.Following this, the position and velocity of the particles are updated, and mutation and crossover operations are performed to generate the offspring population.Furthermore, TMM calculates the phase sensitivity of the offspring population and compares the OF value with the current optimal solution to determine the configuration to be transferred to the next generation.Ultimately, the entire process is repeated until the termination condition is achieved, leading to the identification of the best solution.
Figure 6 and Table 4 gives the change in the optimized layer thickness of the Ag-BlueP/TMDCs-Ag-MXene structure-based SPR sensor via the DEPSO.Under the premise of ensuring a minimum reflectivity of less than 0.01, it can be seen that, compared with the PSO and the conventional method, the phase sensitivity based on the DEPSO is greatly improved, and the number of iterations of the algorithm to reach the stable optimal value is significantly reduced.For the BlueP/MoS 2 structure, for the highest phase sensitivity of 1.833 × 10 6 deg/RIU, the minimum reflectivity is 1.824 × 10 −4 , when the thickness of the first and third Ag film are 18.401 nm and 22.609 nm, the BlueP/MoS 2 is three layers, and the MXene is one layer.Subsequently, the optimized Ag-BlueP/MoSe 2 -Ag-MXene structure via the DEPSO is shown as Figure 6b.The highest phase sensitivity is 1.811 × 10 6 deg/RIU, the minimum reflectivity of 3.597 × 10 −5 , when the thickness of the first and third Ag are 17.668 nm and 22.948 nm, the BlueP/MoSe 2 is four layers, and the MXene is one layer.Then, when the BlueP/WS 2 is three layers, the MXene is one layer, and the thickness of the first and third Ag layers are 18.513 nm and 22.712 nm, the maximum phase sensitivity can reach 1.866 × 10 6 deg/RIU for the Ag-BlueP/WS 2 -Ag-MXene hybrid structure.Finally, for the SPR gas sensor of the Ag-BlueP/WSe 2 -Ag-MXene heterostructure, when the BlueP/WSe 2 is bilayer and the MXene is monolayer, and the thicknesses of the first and third Ag films are 17.226 nm and 24.236 nm, the maximum phase sensitivity can reach 1.821 × 10 6 deg/RIU, and the minimum reflectivity is 1.283 × 10 −4 .In comparing Figure 5 with Figure 6, and Table 3 with Table 4, it can be seen that the DEPSO algorithm requires fewer iterations to reach an optimal solution, which shows better convergence characteristics.Moreover, the DEPSO algorithm exhibits a superior global search ability, which enables it to jump out of the local optimal value and more fully utilize the high electron concentration and mobility of BlueP/TMDCs and MXene, thus enhancing the performance of SPR sensors, making them suitable for detecting mixed gases.At the same time, as the number of SPR sensor layers increases, the DEPSO algorithm can find the optimal thickness of all layers simultaneously and save time, which has the advantage of working with a large number of design parameters.Figure 5 presents the schematic of the implementation of the DEPSO algorithm in conjunction with TMM.Initially, a population of 100 configurations is generated, and the phase difference and minimum reflectivity for each configuration are computed.Subsequently, the configurations are sorted based on their phase sensitivity, given that the min- 4 gives the change in the optimized layer thickness of the Ag−BlueP/TMDCs−Ag−MXene structure-based SPR sensor via the DEPSO.Under the premise of ensuring a minimum reflectivity of less than 0.01, it can be seen that, compared with the PSO and the conventional method, the phase sensitivity based on the DEPSO is greatly improved, and the number of iterations of the algorithm to reach the stable optimal value is significantly reduced.For the BlueP/MoS2 structure, for the highest phase sensitivity of 1.833 × 10 6 deg/RIU, the minimum reflectivity is 1.824 × 10− 4 , when the thickness of the first and third Ag film are 18.401 nm and 22.609 nm, the BlueP/MoS2 is three layers, and the MXene is one layer.Subsequently, the optimized Ag−BlueP/MoSe2−Ag−MXene structure via the DEPSO is shown as Figure 6b.The highest phase sensitivity is 1.811 × 10 6 deg/RIU, the minimum reflectivity of 3.597 × 10− 5 , when the thickness of the first and third Ag are 17.668 nm and 22.948 nm, the BlueP/MoSe2 is four layers, and the MXene is one layer.Then, when the BlueP/WS2 is three layers, the MXene is one layer, and the thickness of the first and third Ag layers are 18.513 nm and 22.712 nm, the maximum phase sensitivity can reach 1.866 × 10 6 deg/RIU for the Ag−BlueP/WS2−Ag−MXene hybrid structure.Finally, for the SPR gas sensor of the Ag−BlueP/WSe2−Ag−MXene heterostructure, when the BlueP/WSe2 is bilayer and the MXene is monolayer, and the thicknesses of the first and third Ag films are 17.226 nm and 24.236 nm, the maximum phase sensitivity can reach 1.821 × 10 6 deg/RIU, and the minimum reflectivity is 1.283 × 10− 4 .In comparing Figure 5 with Figure 6, and Table 3 with Table 4, it can be seen that the DEPSO algorithm requires fewer iterations to reach an optimal solution, which shows better convergence characteristics.Moreover, the DEPSO algorithm exhibits a superior global search ability, which enables it to jump out of the local optimal value and more fully utilize the high electron concentration and mobility of BlueP/TMDCs and MXene, thus enhancing the performance At the same time, the objective function curves of the Ag-BlueP/TMDCs-Ag-MXene hybrid structure of the SPR gas sensor via the PSO and the DEPSO are shown in Figure 7.It is observed that, with increasing iterations, the value of the objective function of the DEPSO increases much more rapidly than the PSO, and the higher value of OF has been stable for about 30 times when the value of the objective function of the PSO has been stable for about 65 times.Based on the above analysis, it can be seen that the DEPSO algorithm not only effectively improves the phase sensitivity of the SPR gas sensor, but also reduces the number of iterations.Therefore, the DEPSO has a high efficiency and accuracy in optimizing the multi-layer sensor structure.
To further illustrate the performance of the optimized sensor, the electric field distribution has been investigated in Figure 8. From (a) to (d), one can see that, after coating the BlueP/TMDCs and MXene onto the surface of the traditional SPR sensor, there is a great increase in the electric field intensity, which means a stronger excitement of SPs.Furthermore, the electric field intensity shows tremendous changes at the MXene-sensingmedium interface when the RI of the air changes from 1 to 1.001, which illustrates that a small change in the sensing medium will result in dramatic variation in the surface wave characteristics.At the same time, the objective function curves of the Ag-BlueP/TMDCs-Ag-MXene hybrid structure of the SPR gas sensor via the PSO and the DEPSO are shown in Figure 7.It is observed that, with increasing iterations, the value of the objective function of the DEPSO increases much more rapidly than the PSO, and the higher value of OF has been stable for about 30 times when the value of the objective function of the PSO has been In addition, Figure 9 shows that the visible wavelength range has been utilized to study the effect of changes in phase difference on sensitivity.From the figure, it can be observed that the highest phase sensitivity can be obtained around 633 nm but, in other visible regions, the proposed sensing structure in this paper also achieves a high phase sensitivity.This indicates that the proposed sensor structure can be used not only to detect gases at 633 nm, but also to detect gases at different wavelengths.
stable for about 65 times.Based on the above analysis, it can be seen that the DEPSO algorithm not only effectively improves the phase sensitivity of the SPR gas sensor, but also reduces the number of iterations.Therefore, the DEPSO has a high efficiency and accuracy in optimizing the multi-layer sensor structure.To further illustrate the performance of the optimized sensor, the electric field distribution has been investigated in Figure 8. From (a) to (d), one can see that, after coating the BlueP/TMDCs and MXene onto the surface of the traditional SPR sensor, there is a great increase in the electric field intensity, which means a stronger excitement of SPs.Furthermore, the electric field intensity shows tremendous changes at the MXene-sensing-medium interface when the RI of the air changes from 1 to 1.001, which illustrates that a small change in the sensing medium will result in dramatic variation in the surface wave characteristics.Moreover, different gases are used to benchmark the adaptivity of the proposed sensor at 633 nm.From Table 5, it can see that the sensor structures optimized in this paper had a high phase sensitivity when detecting different gases, which indicates that the BlueP/TMDCs and MXene are suitable for SPR gas sensor due to their unique characteristics.In addition, Figure 9 shows that the visible wavelength range has been utilized to study the effect of changes in phase difference on sensitivity.From the figure, it can be observed that the highest phase sensitivity can be obtained around 633 nm but, in other visible regions, the proposed sensing structure in this paper also achieves a high phase sensitivity.This indicates that the proposed sensor structure can be used not only to detect gases at 633 nm, but also to detect gases at different wavelengths.Moreover, different gases are used to benchmark the adaptivity of the proposed sensor at 633 nm.From Table 5, it can see that the sensor structures optimized in this paper had a high phase sensitivity when detecting different gases, which indicates that the BlueP/TMDCs and MXene are suitable for SPR gas sensor due to their unique characteristics.
Conclusions
A novel method for designing SPR gas sensors with a high phase sensitivity using bilayer metal film, BlueP/TMDCs, and MXene is proposed in this work.It has been proven that the DEPSO method, with restrained conditions to simultaneously optimize the thickness of each layer of the SPR gas sensor, exhibits a better efficiency and accuracy compared
Conclusions
A novel method for designing SPR gas sensors with a high phase sensitivity using bilayer metal film, BlueP/TMDCs, and MXene is proposed in this work.It has been proven that the DEPSO method, with restrained conditions to simultaneously optimize the thickness of each layer of the SPR gas sensor, exhibits a better efficiency and accuracy compared to the conventional and PSO methods.The results indicate that the sensor with the Ag-BlueP/TMDCs-Ag-MXene structure, where BlueP/WS 2 has three layers and MXene is monolayer, achieves the maximum phase sensitivity of 1.833 × 10 6 deg/RIU, 1.811 × 10 6 deg/RIU, 1.866 × 10 6 deg/RIU, and 1.821 × 10 6 deg/RIU.Furthermore, the ultrasensitive properties are demonstrated by the distribution of the electric field and the detection of different gases, which support the suitability of the proposed structure for gas-sensing applications.
Drude-Lorentz model as the plasmonic metal.
Figure 1 .
Figure 1.Schematic diagrams of the proposed phase-sensitivity SPR gas-detection setup.
Figure 1 .
Figure 1.Schematic diagrams of the proposed phase-sensitivity SPR gas-detection setup.
Population N, dimension D, iteration T, Scaling factor F, Leaning factor C, hybrid probability (2) Randomly initialize particle position x_pso, velcolity v, mutation operator w, selection operator u, P i and P g of particles (3) Cycle (4) For i = 1:N (5)For j = 1:D
17 Figure 2 .
Figure 2. The phase sensitivity with respect to the different SPR sensor structures at the same change in RI.
Figure 2 .
Figure 2. The phase sensitivity with respect to the different SPR sensor structures at the same change in RI.In addition, to better illustrate the improvement in the phase sensitivity of the SPR sensor through the addition of the BlueP/TMDCs and MXene layer, the influence of the
Figure 3 .
Figure 3.The phase sensitivity with (a) the number of BlueP/TMDCs layers under the condition of monolayer Mxene; (b) the number of Mxene layers under the condition of optimized BlueP/TMDCs.
Figure 3 .
Figure 3.The phase sensitivity with (a) the number of BlueP/TMDCs layers under the condition of monolayer Mxene; (b) the number of Mxene layers under the condition of optimized BlueP/TMDCs.
Figure 5 .
Figure 5. Schematic of the implementation of the DEPSO alongside TMM.
Figure 6
Figure 6 and Table4gives the change in the optimized layer thickness of the Ag−BlueP/TMDCs−Ag−MXene structure-based SPR sensor via the DEPSO.Under the premise of ensuring a minimum reflectivity of less than 0.01, it can be seen that, compared with the PSO and the conventional method, the phase sensitivity based on the DEPSO is greatly improved, and the number of iterations of the algorithm to reach the stable optimal value is significantly reduced.For the BlueP/MoS2 structure, for the highest phase sensitivity of 1.833 × 10 6 deg/RIU, the minimum reflectivity is 1.824 × 10− 4 , when the thickness of the first and third Ag film are 18.401 nm and 22.609 nm, the BlueP/MoS2 is three layers, and the MXene is one layer.Subsequently, the optimized Ag−BlueP/MoSe2−Ag−MXene structure via the DEPSO is shown as Figure6b.The highest phase sensitivity is 1.811 × 10 6 deg/RIU, the minimum reflectivity of 3.597 × 10− 5 , when the thickness of the first and third Ag are 17.668 nm and 22.948 nm, the BlueP/MoSe2 is four layers, and the MXene is one layer.Then, when the BlueP/WS2 is three layers, the MXene is one layer, and the thickness of the first and third Ag layers are 18.513 nm and 22.712 nm, the maximum phase sensitivity can reach 1.866 × 10 6 deg/RIU for the Ag−BlueP/WS2−Ag−MXene hybrid structure.Finally, for the SPR gas sensor of the Ag−BlueP/WSe2−Ag−MXene heterostructure, when the BlueP/WSe2 is bilayer and the MXene is monolayer, and the thicknesses of the first and third Ag films are 17.226 nm and 24.236 nm, the maximum phase sensitivity can reach 1.821 × 10 6 deg/RIU, and the minimum reflectivity is 1.283 × 10− 4 .In comparing Figure5with Figure6, and Table3with Table4, it can be seen that the DEPSO algorithm requires fewer iterations to reach an optimal solution, which shows better convergence characteristics.Moreover, the DEPSO algorithm exhibits a superior global search ability, which enables it to jump out of the local optimal value and more fully utilize the high electron concentration and mobility of BlueP/TMDCs and MXene, thus enhancing the performance
Figure 5 .
Figure 5. Schematic of the implementation of the DEPSO alongside TMM.
Table 2 .
The setting of the parameters for the algorithms.
Table 3 .
The performance parameters of the optimized Ag-BlueP/TMDCs-Ag-MXene structure via the PSO.
Table 4 .
The performance parameters of the optimized Ag-BlueP/TMDCs-Ag-MXene structure via the DEPSO.
Table 4 .
The performance parameters of the optimized Ag-BlueP/TMDCs-Ag-MXene structure via the DEPSO.
Table 5 .
The performance parameters of the Ag-BlueP/WS 2 -Ag-MXene structure for various gases.
Table 5 .
The performance parameters of the Ag-BlueP/WS2-Ag-MXene structure for various gases. | 2023-10-14T15:19:52.998Z | 2023-10-01T00:00:00.000 | {
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265196525 | pes2o/s2orc | v3-fos-license | EU trade liberalisation, sectoral coalitions and development: insights from Moldova and Georgia
ABSTRACT This paper contributes to the literature on EU trade policy by introducing insights from the political economy literature on development to the study of how EU trade liberalisation affects development in trading partners. Drawing on a comparison of EU trade liberalisation with Moldova and Georgia, we argue that the type of coalition between public and private actors in partner countries’ top export sectors determines which firms benefit from better market access to the EU, as indicated by their ability to increase their exports. We show that liberalised trade with the EU tends to contribute to achieving the EU’s declared objective of inclusive development if the presence of inclusionary development coalitions ensures that a broad range of firms is enabled to increase their export capacities through a mechanism that we call ‘inclusive empowerment’. Otherwise, trade liberalisation contributes to exclusive development, benefitting big, mostly foreign firms, or, at worse, consolidating rent-seeking practices.
Introduction
The question of how the EU -through its trade policy -affects development in its trading partners has received increasing scholarly attention in recent years.Scholars have examined the extent to which EU trade policy is prioritising EU market interests over its normative ambitions by revealing the conditions under which EU free-trade agreements include non-commercial, political objectives (Meissner and McKenzie 2019;Orbie, Alcazar, and Sioen 2022;Saltnes 2021).Others have looked into the macro-level economic effects of EU trade liberalisation in terms of trade flows, economic growth or (un-)employment rates (see, for example, Nekhay, Delgado, and Cardenete 2021;Tröster et al. 2020) or examined how EU trade agreements reduce the local 'policy space' for development (Langan and Price 2015).Still others have investigated how domestic factors in the EU trading partners influence how EU trade policy affects local development.Along these lines, Langan (2015) analysed how corrupt power networks in less developed economies consolidate their position thanks to financial assistance provided by the EU as part of its trade agreements.Further, Potjomkina (2021) investigated how domestic power relations shape the extent to which different local stakeholders are included in trade negotiations with the EU and how this affects local development agendas.
This paper contributes to scholarship on the link between EU trade policy and development by introducing insights from the political economy literature on development to the study of how the EU affects its trading partners through trade liberalisation (Bollen, De Ville, and Orbie 2016).Drawing on authors who reject a depoliticised view of development, we start from the assumption that embarking on (or changing) developmental pathways requires coalitions between public and private actors that are ready to seize opportunities for development, including those arising from trade liberalisation (Doner and Schneider 2016;Evans 1989).In other words, state agencies, research institutes, marketing agencies and business associations need to coordinate and exchange knowledge with firms in order to ensure that the latter are able to benefit from trade liberalisation.For example, to increase their export capacity, firms need to have the technology and knowledge to comply with the prevailing product and process standards in highvalue markets (Stiglitz and Charlton 2006).To facilitate this, the state needs to be autonomous from status quo-oriented groups since the political connections of firms may result in misallocations of trade benefits, e.g.via preferential access to state aid or monopoly profits (Atkin et al. 2022).
Against this background we focus on EU trade liberalisation with Moldova and Georgia and examine what kinds of sectoral coalitions between public and private actors have evolved in these countries' top export sectors to the EU and how these coalitions determine which firms benefit from EU trade liberalisation in these sectors, as indicated by their ability to increase their exports.We take a closer look at the composition of exports to the EU to identify top export sectors, determine the dominant exporters in these sectors and their possible political connections, and analyse the sectoral coalitions in which these firms are embedded.We argue that particular types of sectoral coalitions, which we conceptualise as inclusionary and exclusionary development coalitions, as well as rent-seeking coalitions, affect which firms benefit from EU trade liberalisation, thereby setting export sectors on pathways towards exclusive or inclusive development or helping to consolidate rent-seeking behaviour.What is more, we reveal the different mechanisms that link particular types of sectoral coalitions to specific developmental pathways induced by trade liberalisation.
Our paper reveals that the type of sectoral coalition between public and private actors is an important factor in who reaps the benefits of EU trade liberalisation.We therefore add to existing studies that analysed how domestic power relations in the EU's trading partners shape the effects of EU trade policy on local development (Langan and Price 2015;Potjomkina 2021).While these studies focused on the dynamics underlying the negotiation of EU trade agreements, our study emphasises the effects of EU trade liberalisation and how they are shaped by vested interests in the trading partners.Our argument is also politically relevant, since inclusive development, which creates benefits for a broad range of economic actors, including small and medium-sized enterprises (SMEs), is at the programmatic heart of the EU's trade and development policy (European Commission 2017).Having a better understanding of the conditions under which trade liberalisation helps to bring about inclusive development can contribute to making the EU's respective toolbox more effective.The paper proceeds as follows: in the next section we discuss the link between trade liberalisation, the type of sectoral coalitions in top export sectors and sectoral developmental outcomes, and conceptualise key variables.We also justify our case selection and discuss our methods.Sections three and four zoom in on the cases of Moldova and Georgia.The conclusion summarises our theoretical and empirical contribution to the literature on EU trade policy and discusses the implications of our findings for advancing an EU trade policy that can live up to its normative ambition of promoting inclusive development in its partner countries.
Theorising the link between trade liberalisation, sectoral coalitions and development
Development is a broad concept.The notion that some countries are poor, while others are rich is, however, implicit in the term 'development', suggesting that an important dimension of development is economic growth, which is -among others achieved through higher productivity and integration in transnational markets and value chains (Bruszt and McDermott 2014;Gereffi, Humphrey, and Sturgeon 2005;Szirmai 2015).Over the past decades, additional dimensions of development have entered the debate.They range from a decline in income equality, to the reduction of poverty, the provision of public goods like health or education, democratisation and good governance, and the notion that economic growth must be generated in a sustainable manner (for an excellent overview of this debate, cf.Szirmai 2015).
In this paper, we apply a narrow definition of development and conceive it as a process creating welfare gains.More precisely, we are interested in how some firms in exporting sectors manage to benefit from trade liberalisation by increasing their export shares and getting access to transnational value chains.The existing literature defines three possible effects of trade liberalisation on firms in export sectors: In inclusive development, better market access creates welfare gains that are distributed evenly across firms of all sizes in export sectors.This is the case if an increasing number of firms, including domestic SMEs, can take advantage of market opening by managing to increase their competitiveness and get access to transnational value chains (Acemoglu and Robinson 2012).But trade liberalisation may also create losers, as it may increase the vulnerability of local industries, or even marginalise or exclude a large share of firms in exporting sectors (Stiglitz and Charlton 2006).This is mainly because SMEs, in particular, often find it difficult to align with transnational regulatory norms and to export and become part of transnational value chains (Gereffi, Humphrey, and Sturgeon 2005). 1 By contrast, big business tends to benefit most from trade liberalisation, as larger companies find it easier to access new markets and value chains due to economies of scale and lower opportunity costs (Baccini 2019;Chase 2003).Therefore, trade liberalisation can also promote exclusive development, which is mainly beneficial for a narrow group of economic actors, mostly big, powerful and often foreign firms (Evans 1989, 563).Even worse, in many developing economies exporting firms with political connections may use their privileged access to trade for seeking and obtaining rents defined as an 'income which is higher than the minimum which an individual or firm would have accepted given alternative opportunities' (Khan and Jomo 2000, 5).Hence, trade liberalisation might even help to consolidate rentseeking behaviour, imposing welfare losses on society (Adserà and Boix 2002;Hirschman 1968).
Economists tend to explain uneven distributions of trade benefits across exporting firms or sectors with reference to differences in factor endowments or levels of intraindustry trade (for excellent overviews cf.Martin 2015;Peterson and Thies 2015).In a contribution to this debate, proponents of New New Trade Theory stress that different levels of productivity explain the uneven distribution of trade benefits.Put differently, increased trade forces low productivity firms out of the market, while high productivity firms can reap the benefits of better market access (Bernard, Jensen, and Schott 2006;Fernandes 2007).Such approaches help us to understand why different firms and sectors gain more from open markets than others in terms of value added.Yet, such a depoliticised view neglects political economy dynamics that may affect why in some export sectors, a broad range of firms, including SMEs, are able to meet export requirements, while in others, predominantly large (mostly) foreign firms or firms with strong political connections are able to benefit from new export opportunities.While we do not want to deny the validity of economic approaches when it comes to explaining the uneven distribution of trade benefits, we wish to enrich the debate by looking at the process through the lens of political economy.
When studying the link between development and trade liberalisation, political economists suggest that the type of coalition between public and private actors affects whether and how opportunities for development are used in a particular sector.Drawing on these insights, we identify three types of sectoral coalitions that are likely to mediate the effect of trade liberalisation on development.These coalitions differ as regards the range of public and private actors who participate in them and how these actors interact when it comes to enabling firms to reap the benefits of trade liberalisation: First, export sectors can be dominated by an inclusionary developmental coalition characterised by the involvement of a broad range of actors in entrepreneurial activities.Such coalitions include different state authorities, firms of different sizes, business associations, research institutes and/or marketing firms (Acemoglu and Robinson 2012;Doner and Schneider 2016;Langbein, Gazizullin, and Naumenko 2021).Inclusionary development coalitions help firms to pool resources, obtain intimate knowledge about sectoral needs with regard to skills or technological know-how, and identify market niches (Gereffi, Humphrey, and Sturgeon 2005).We argue that inclusionary development coalitions are likely to set exporting sectors on a pathway towards inclusive development through a mechanism that we call inclusive empowerment, because such coalitions ensure that a broad range of firms (domestic and/or foreign; small, medium and large firms) manages to access transnational markets.Accordingly, we take an increasing number of firms that are able to export after trade liberalisation -especially SMEs or companies without political connections to ruling elites -as an indicator for inclusive development.
Second, exclusionary development coalitions are formed between state actors and a narrow group of economic actors.Big and powerful, mostly foreign, firms often seek to capitalise on peripheral economies' convenient location and cheap labour.However, they rarely engage in technology or know-how transfer that would help their subsidiaries to achieve a better position in transnational value chains.The state's role is limited to accommodating the needs of strong economic actors and implementing investment-friendly policies rather than nurturing the capacities of domestically owned business for market integration and upgrading (Nölke and Vliegenthart 2009;Pavlínek 2017).We suggest that trade liberalisation is more likely to contribute to exclusive development if export sectors are dominated by exclusionary development coalitions.The latter focus on enabling a narrow group of actors to increase exports as tariff rates decline through a mechanism that we call exclusive empowerment.If only a narrow group of large, mostly foreign firms without political connections to ruling elites manages to increase their export volumes following the reduction of tariff rates, we take this as an indicator for exclusive development.
Third, when ties between state and non-state actors are based on clientelism and corruption, the coalition becomes rent-seeking.In this case, state officials provide business actors with monopoly profits or subsidies to secure their loyalty (Khan and Jomo 2000).Applied to the dynamics underlying trade liberalisation, exporting firms with political connections to the ruling elite are able to reap most of the benefits of trade liberalisation thanks to preferential treatment.In turn, these firms often use their rents to finance the ruling elites and their political parties, thereby reinforcing their political connection to the latter (Hirschman 1968).The dominance of rent-seeking coalitions in top export sectors therefore increases the likelihood that trade liberalisation will help to consolidate rent-seeking behaviour through a mechanism that we call clientelist empowerment.
Case selection
In order to test whether our theoretical assumptions hold for the case of EU trade liberalisation, we focus on trade relations between the EU and two Eastern neighbours, Moldova and Georgia, as part of the EU's European Neighbourhood Policy (ENP).This is the most likely case for reinforcing tendencies towards inclusive development because the EU combines trade liberalisation with relatively far-reaching assistance to enable local firms to benefit from market access (Bossuyt et al. 2017).This assistance has been stepped up since the EU established Deep and Comprehensive Free Trade Areas (DCFTAs) with Moldova and Georgia as part of their Association Agreements in 2014. 2 If we find that EU trade liberalisation is only likely to contribute to inclusive development under certain conditions even in the context of two associated Eastern partners, we can assume that the EU faces similar limitations in other contexts.
Further, our focus on Moldova and Georgia keeps a couple of potential country-level explanatory variables for different development effects of EU trade liberalisation constant: For most of the time in the period under scrutiny (2004-2020), both countries were classified as lower middle-income economies (LM) 3 and were hardly integrated in transnational markets and value chains (EBRD 2020).Further, Moldova and Georgia received similar amounts of EU assistance under the ENP (Wolczuk and Zeruolis 2018).With regard to corruption, which is often seen as an obstacle to development (cf., for example, Cieślik and Goczek 2018;Mauro 1996), Georgia has scored better than Moldova (and many other LMs) in Transparency International's Corruption Index since 2009. 4This is due to the effective fight against petty corruption.High-level corruption based on connections between political and economic elites, however, remained widespread in both Moldova and Georgia (Transparency International Georgia 2023).
In Moldova, oligarchic groups controlled the country for their own benefit during the period under scrutiny (Marandici 2021).Up to 2016, state institutions were embedded in the networks of two dominant oligarch politicians: Vladimir Filat (the leader of the Liberal-Democratic Party, PLDM) and Vladimir Plahotniuc (a billionaire who de facto controlled the Democratic Party, PDM).After 2016, Plahotniuc's network monopolised political power (Marandici 2021).In the run-up to the 2019 parliamentary elections, Moldova's heterogeneous opposition groups united to oust Plahotniuc, who escaped to the US to avoid prosecution.Despite the temporary unification of the opposition, rent-seeking practices by no means disappeared.They simply lost their centralised character (Interviews 1 and 2).While the 2020 presidential and 2021 parliamentary elections brought a reform-oriented and pro-European leadership to power for whom the fight against corruption is a key priority, it remains to be seen whether it will be successful in undermining rent-seeking practices (Baltag and Burmester 2021).
Georgia's broader political economy context is similar; here too, oligarchic networks control access to economic and political resources, despite improvements to Georgia's governance capacity after the 2006 Rose Revolution.Back then, the fight against petty corruption was impressive.However, property rights violations and high-level corruption remained widespread (Ademmer, Langbein, and Börzel 2019).In 2012, billionaire Bidzina Ivanishvili, who united various opposition parties in the coalition Georgian Dream (GD), won the elections and managed to concentrate power in his hands.He no longer held an official position after 2013, but had a stint as GD chairperson in the run-up to the 2018 elections before resigning from all duties.Ivanishvili's wealth is estimated at 6 billion USD, mostly built on Russian assets.He owns a huge network of companies and uses control over the judiciary and other state institutions to protect his and his allies' power and wealth (Gherasimov 2019).
While a number of country-level factors for different development effects of EU trade liberalisation are constant in our case selection, we create variation on the mediating variable, i.e. the type of sectoral coalitions between public and private actors in top export sectors.More precisely, we selected two countries that differed in the structure of their exports to the EU in 2004 -two years before trade liberalisation gathered pace (Tables 1 and 3).Unlike in Moldova, the major share of Georgia's exports to the EU come from resource sectors.The latter produce more opportunities for rent-seeking (Torvik 2002), particularly in the context of weak institutions and governance quality (Chambers and Munemo 2018).We therefore expect to detect more rent-seeking coalitions in Georgia's top export sectors.We can also assume that trade liberalisation with the EU is more likely to set Moldova's export sectors on a pathway to exclusive or even inclusive development, while in Georgia we expect to mainly detect the consolidation of rent-seeking practices.
Last but not least, for reasons of data availability we decided to focus on the top five export sectors in each country.Since they account for more than 80-90% of exports to the EU at each point in time under scrutiny, our analysis reveals the dominant trend regarding the effects of EU trade liberalisation on development in Moldova and Georgia.
Methods
Parts of our empirical research for this study rely on statistical data from Eurostat, the Moldovan National Bureau of Statistics and the National Statistics Office of Georgia.Based on this data, we measure the extent to which sectors benefit from trade liberalisation on the basis of changes in their shares in total exports to the EU from 2004 to 2020.To account for the price effect, we also look at changes in the total volume of sectoral exports to the EU.We then analyse the structure of those exports to assess the development effects of trade liberalisation, because liberalised trade leads to an expansion of foreign trade (Helleiner 1994).An important caveat that remains is that we cannot precisely establish to what extent larger exports to the EU are due to better terms of trade or result from a loss of export opportunities for other markets (esp.Russia and other post-Soviet countries).That said, the opportunity for Moldovan and Georgian exports to re-orient towards the EU also depends on greater accessibility of the EU market.
It turned out to be more difficult than initially assumed to access data on the share of SMEs vis-à-vis larger firms in total exports to the EU from most of the top Georgian or Moldovan export sectors.That is because neither the EU nor national statistical offices systematically collect export performance statistics by sector and size class (i.e.number of employees and value added).Further, ownership structures of economic sectors in Moldova and Georgia and rent-seeking practices at the level of particular sectors or firms are an extremely under-researched topic in the scholarly literature.Therefore, we relied on other publicly available primary and secondary sources.For example, in the case of Georgia, we got information about top export companies from the National Statistics Office of Georgia.For each company that consistently featured in the category of top exporters, we then accessed company registration dates and registration statements through the website of the public registry (reestri.gov.ge) in order to reveal possible connections to oligarchic groups and/or leading figures in ruling political parties.Further, the website companyinfo.gecreated by Transparency International Georgia was used to trace (changes in) company owners and managers.On this basis, we were able to identify power networks and corporate relationships across different companies and to track down business interests and possible rent-seeking behaviour.
We used articles and reports published by media outlets, non-governmental organisations or sectoral business associations as an additional resource for both case studies.In most cases, especially with regard to the disclosure of a firm's ties to oligarch structures or their involvement in rent-seeking practices, we were able to secure at least two sources for an empirical observation.
Interviews played only a minor role in the data collection since only few experts appeared to have intimate knowledge about the relationship between public and private actors in top export sectors.That said, expert interviews with two key decision-makers from Moldova, who initiated and implemented the parliamentary inquiry into rentseeking practices in the post-2014 period, provided further validation of the data collected through the study of media articles and reports.Another interview with a representative of Georgia's exporters' association gave us more insights into the share of SMEs in exports to the EU and obstacles to SME development.
Moldova's top export sectors to the EU: on a pathway towards inclusive and exclusive development
Trade liberalisation between Moldova and the EU started to gather pace in 2006 when Moldova became a beneficiary of the special incentive arrangement for Sustainable Development and Good Governance (GSP+).Hence, we start our analysis prior to GSP+ in 2004 by looking at the top five export sectors at each point in time under scrutiny.Table 1 suggests that between 2004 and 2020 the significance of individual sectors for exports to the EU changed with increasing liberalisation.Moldova's agrifood sector is the biggest winner of EU trade liberalisation.Machinery and textiles rank second and third, respectively.These sectors were able to significantly increase their export volumes to the EU.By contrast, export shares and volumes of metals, transport vehicles and footwear have declined since 2004, suggesting that these sectors did not benefit to the same extent from EU trade liberalisation.
To understand the effects of liberalised trade with the EU on the type of development characterising Moldova's top export sectors, we will take a closer look at changes in key market players and their ownership structures over time and examine the type of sectoral coalitions that exist between public and private actors.Our analysis shows that with regard to Moldova's top five export sectors, trade liberalisation with the EU has offered more opportunities for exclusive and even inclusive development than for consolidating rent-seeking practices.
Metals and vegetable products: clear links to oligarchic networks
Two of Moldova's top export sectors to the EU are clearly embedded in a rent-seeking coalition between key exporting companies and the ruling political elites: metals and vegetable products.
The key player in Moldova's metals sector, which accounted for 9.5% of total exports to the EU in 2020 (Table 1), is the state-owned Metalferos (European Business Association 2020).The company is an important source for rent-seeking, as it is used to distort economic operations with scrap metal.Recently leaked documents from Moldova's General Prosecutor's Office revealed that before the scrap metal market was liberalised in the second half of 2019, oligarchic networks operated a scheme that allowed them to buy scrap metal cheaply by artificially dumping the prices on the local market.They then sold it through proxy companies at a higher price to Metalferos, which used to hold the monopoly for the export of scrap metal thanks to a regulation dating back to 2002. 5 As a result, Metalferos became the major exporter of metals to the EU and has thus far been able to maintain this position (EBA 2020).According to media reports, Metalferos' revenues were used to finance the PDM when the party was in power between 2015 and 2019 and controlled by oligarch Vladimir Plahotniuc (Necşuţu 2021;Ziarul de Gardă 2021).Further, a recent report revealed a long list of discriminatory measures taken by Moldovan state authorities against Metalferos' competitors to undermine their entrepreneurial activities despite the formal de-monopolization of the scrap metal market in 2019 (EBA 2020).This shows that rent-seeking practices are still widespread even if Plahotniuc left Moldova in summer 2019 to avoid prosecution (Esanu, Calmus, and Mosneag 2020).
Another leading export sector dominated by rent-seeking coalitions between monopolists and the Moldovan state is vegetable products (nuts, oil seeds and vegetable oil and grain), which accounted for about 21% of total exports to the EU in 2020 (Table 2).These coalitions promoted state aid policies that allowed key market players in this sector to access financial resources on a preferential basis.
For example, one of the biggest Moldovan exporters of walnuts, Monicol SRL, owned by Dumitru Vicol, has steadily increased its share on the local walnut market since 2014 thanks to generous state aid. 6At about the same time, Vicol was able to build close personal connections to the oligarchic network of Vladimir Plahotniuc, then the unofficial leader and sponsor of the leading political party PDM (Agora.md 2019a;Gulca and Necşuţu 2019).
Similar trends can be seen in the production of oilseeds and vegetable oils, which increased their export shares between 2007 and 2020 (Table 2).About 90% of Moldova's processing capacities for sunflower oil belong to FFA Trans Oil LTD (hereinafter TransOil) (Agrobiznes.md 2018).It controls not only companies in the oil production field, such as Floarea Soarelui SA, but also important infrastructure like silos and transportation routes to Moldova's only port -Giurgiulesti International Free Port.Despite changing ownership structures involving different Russian individuals, TransOil managed to maintain its close relationship with Plahotniuc (Gulca and Necşuţu 2016).With Plahotniuc's backing, Moldovan state authorities facilitated the construction of more transportation capacities for TransOil via rail and air, securing the company's monopoly of the local grain business (Gulca and Necşuţu 2016;JurnalTV 2018).TransOil used its control over storage and transportation to dump local grain prices.As a result, it was able to consolidate its position as a leading exporter to the EU in this sector and make massive profits with grain exports.Similar developments shaped the dynamics on the local oilseed market (Gulca and Necşuţu 2016).
Our analysis shows that at least up until 2020, trade liberalisation helped to consolidate rent-seeking practices in the metals and vegetables sectors, since the dominant exporters used their close political connections to the ruling elites to get preferential treatment, allowing them to reap most of the benefits of better market access to the EU.In exchange, they supported the ruling parties.As mentioned earlier, since 2019 pro-European political forces have tried to dismantle monopolies (Agora.md 2019b;Ziarul National 2019).Recently adopted anti-monopoly legislation under Sandu's leadership aims at weakening monopolies, at least in Moldova's metal and nuts industries (Government of the Republic of Moldova 2019; see also Infotag 2019).The jury is still out as to whether these changes will suffice to eradicate rent-seeking practices (Marandici 2021).
Machinery: towards exclusive development?
Moldova's machinery sector is a clear winner of trade liberalisation with the EU.Since 2011, it has witnessed impressive growth rates in export volumes to the EU (from 2% in 2007 to 26% in 2020).Increasing exports went hand in hand with the emergence of an exclusionary development coalition between foreign investors and Moldovan state authorities.Local authorities made sure that foreign investors in Moldova's machinery sector were shielded from corruption and non-transparent business practices and could execute their entrepreneurial activities smoothly, especially in the seven free economic zones that the Moldovan government has established over the last 19 years, starting in 2001 (Ministry of Economy and Infrastructure 2014).Particularly since the DCFTA with the EU was provisionally enforced in 2014, the sector has witnessed an inflow of large foreign component producers. 7They established assembly and production platforms for low value-added automotive parts and components in Moldova.Engagement in research and development (R&D) activities is, however, limited, although some local experts are optimistic that more value-added activities are likely to develop in the future (Shehadi 2019).In exclusionary development coalitions, the state's role is generally limited to accommodating the needs of foreign investors rather than nurturing backward linkages with local component producers.That said, the achievements of the exclusionary development coalition in Moldova's machinery sector are clear: statements by top managers of foreign investors in the sector suggest that the state's investment-friendly policies were key to their decision to open subsidiaries in Moldova (InvestMoldova 2022).As a result, machinery exports to the EU more than doubled between 2013 and 2020 (Table 1), and characteristic of exclusive development, these exports are driven by foreign investors, mainly from European countries (InvestMoldova 2022).
Moldova's textile and wine industry: towards inclusive development?
As for Moldova's textile industry, its share in the country's total exports to the EU decreased from 23% in 2004 to 12% in 2020, while the export volume more than doubled during the same period (Table 1).The ownership structure of Moldova's textile industry is diverse: apart from a few large companies, the majority of producers are SMEs (UNECE 2021).There is a remarkable level of inclusionary sectoral organisation here.Most textile companies are members of the Light Industry Employers Association (APIUS).Together with Moldovan government agencies, the Technical University of Moldova, and with the support of USAID, APIUS initiated several public-private partnerships (PPPs) from 2012 onwards to boost competitiveness and promote the integration of Moldova's textile industry into European value chains.Moldova's State Agency on Intellectual Property (AGEPI) actively supported the project in particular, by training participating companies in the use of intellectual property (IP) systems to protect their brands and designs.According to industry representatives, the joint promotional campaign 'From the Heart -Brands of Moldova', which started in 2012, contributed to changing perceptions of Moldovan brands by both foreign investors and local consumers (Apostol and Vieru 2016).Further, in 2015, the Technical University of Moldova established the ZIPHouse Center of Excellence and Acceleration in Design Technologies.The project serves as a platform for innovation and entrepreneurial development in the industry, targeting in particular young designers, start-ups and SMEs (Kosta 2023;Vogler 2017).In terms of exports to the EU, the sector is on a pathway to inclusive development.Micro and small businesses tend to focus on the domestic market, but in the past ten to fifteen years an increasing number of mainly locally owned medium to large businesses has emerged in the sector.Sectoral studies suggest that these firms benefitted from better market access to the EU thanks to the aforementioned horizontal networks with universities, business associations and state authorities, which facilitated the transfer of know-how about how to produce competitive products for the EU market and access the respective value chains.They started to manufacture for export and occasionally even managed to export their own brands to surrounding countries (Kosta 2023;Vogler 2017).
Moldova's wine sector occupies a tiny place in total exports to the EU, accounting for about 2% to 3% of the country's total exports to the European market.An inclusionary development coalition helped local wine producers to attract new markets in the EU.To begin with, the National Vine and Wine Office (NVWO) started to enhance transparency and quality assurance by introducing an electronic wine register in 2013 with the help of the Czech Development Agency and USAID.Western donors also helped smaller companies to form associations like the Association of Small Wine Producers of Moldova, which today controls about one-third of the vineyards (Gheorgita and Crucerescu 2018).The fact that hundreds of wineries and dozens of producers and exporters organised themselves into sectoral business associations not only prevented the establishment of monopolies and the development of rent-seeking schemes.It also helped to overcome information asymmetries regarding EU funding criteria and export certificates (Business Intelligence Service 2020).According to a recent study, the Moldovan wine sector is dominated by micro, small and mediumsized wineries.Only about 6% of wineries can be classified as large.Considering that about two-thirds of Moldovan wine is exported, of which nearly 40% is exported to the EU, it is safe to say that trade liberalisation benefits not only large producers but also SMEs (Business Intelligence Service 2020).This is indicative of inclusive development in Moldova's wine industry.
Summing up, we showed that quite a few of Moldova's top export sectors to the EU have recently seen the emergence of exclusionary and inclusionary development coalitions.An exclusionary development coalition in Moldova's machinery sector mainly served the interests of larger foreign firms.Through a mechanism that we call 'exclusive empowerment', the Moldovan state implemented investment-friendly policies targeted at big foreign machinery producers, but neglected to nurture a local component industry and create corresponding backward linkages.By contrast, inclusionary development coalitions in Moldova's wine and textile industries enabled a broader range of firms to reap the rewards of trade liberalisation through a mechanism that we accordingly call 'inclusive empowerment'.As a result, Moldova's trade liberalisation with the EU has so far created more opportunities for exclusive (machinery) and even inclusive development (textiles and wine) than for consolidating rent-seeking practices.In 2020, machinery, textiles and wine accounted for around 41% of Moldova's exports to the EU.
Still, as of 2020, almost one-third (31%) of Moldova's total exports to the EU came from sectors (metals and vegetables) that are controlled by rent-seeking coalitions between key sectoral players and state authorities.Through a mechanism which can best be described as 'clientelist empowerment', these coalitions ensured that firms with political connections enjoyed preferential treatment, allowing them to reap most of the benefits of better market access to the EU.While the importance of metal exports to the EU as a source of revenue for Moldova's oligarchic networks is shrinking, Moldova's vegetable production sector has seen growing exports to the EU.
Georgia's top export sectors to the EU -towards the consolidation of rentseeking practices
As in the case study of Moldova, we begin our analysis of the effects of Georgia's liberalised trade with the EU by looking at how the structure of the country's exports to the EU has changed between 2004 and 2020.The significance of individual sectors started to change after trade liberalisation gathered pace as a result of GSP+ 8 (Table 3), the only exception being minerals, which maintained its top position.Without a doubt, agrifood (mainly nuts, mineral waters and wine, according to Geostat data) was the sector that received the greatest boost from EU trade liberalisation.Chemicals and textiles were able to increase their export share in both percentage and volume.Metals, stone and glass lost in significance, while machinery lacked sustainable growth perspectives.
Our analysis shows that where Georgia's top five export sectors are concerned, trade liberalisation with the EU has offered more opportunities for consolidating rent-seeking practices than for exclusive or inclusive development.
Minerals and metals: close links to oligarchic networks
Minerals (in particular copper ore) were Georgia's key export commodity to the EU between 2004 and 2020.Rich Metals Group (RMG) Copper was the key market player during this time period.According to Georgia's National Agency of Public Registry, 9 RMG Copper has been owned by Mining Investments LLC, which is controlled by Russian billionaire Dmitriy Troitskiy, since 2019.Together with other Russian businessmen, Troitskiy had already controlled RMG B.V., the previous owner of RMG Copper.The Georgian NGO Green Alternative documented close links between RMG and Bidzina Ivanishvili's networks (Green Alternative 2021), pointing to the existence of a rentseeking coalition in this sector (see also Kupatadze 2018).A case in point is the decision taken by the Georgian Ministry of Culture in 2013 to revoke the status of the world's oldest gold mines in Sakdrisi as a protected historical site.As a result, RMG was allowed to start mining operations there (Kupatadze 2018).Furthermore, government authorities turned a blind eye to RMG's environmentally and socially harmful mining operations (Swann-Quinn 2019).These decisions helped to consolidate the concentration of mine ownership within RMG.In terms of exports, RMG Copper benefitted from its central position in Georgia's mining business.According to Geostat data, the company was among the top 50 exporters every year from 2004 to 2019 and is Georgia's most important exporter of copper ore to the EU.RMG uses parts of its revenues to maintain its close political connections to the ruling elite.Individuals directly or indirectly connected with RMG reportedly donated more than 800 thousand GEL (approx.192,000 euros) to the ruling GD party, which is close to Ivanishvili, in the run-up to the 2020 parliamentary elections (IDFI 2020).Trade liberalisation with the EU therefore contributes to consolidating rentseeking practices in Georgia's mining industry.
The metals sector is also closely linked to Georgia's ruling elites.Its significance for exports to the EU declined, however in particular, after the DCFTA entered into force (Table 3).According to the company website, 10 Georgian Manganese LLC (GM) is Georgia's leading producer of ferroalloys and manganese ore.From 2006 to 2013, 75% of GM's shares belonged to Privat Group, one of the most closed and non-transparent Ukrainian holdings.During this time, its founders Igor Kolomoisky and Henadiy Boholyubov were among the five richest people in Ukraine (Pleines 2019).In 2013, Privat Group transferred GM to the US-American Holding Georgia American Alloys, an offshore company assumed to be registered in Luxembourg (Gujaraidze 2021).
GM has a dubious track record and is said to be closely linked to the GD party, which is de facto controlled by oligarch Bidzina Ivanishvili (Gujaraidze 2021;Mtivlishvili 2021).There are a few indicators for the existence of a rent-seeking coalition in Georgia's metals sector, which helped GM maintain its dominant market position.To begin with, GM has faced repeated accusations of exploitative labour practices and pollution: the company was fined on a few occasions up to 2017 for environmental damages and tax evasion.The payment of these fines ensured that GM was discharged from liability for environmental damages (Gujaraidze 2021;Kavanagh 2017).Such deals first became legal in 2012 11 and used to be considered a prominent rent-seeking activity in Georgia, since the fines were assumed to fill the pockets of corrupt state officials (Gujaraidze 2013).Further, in 2017, Nikoloz Chikovani, a protégé of the GD Party, was appointed special manager at GM. Until today, GM still operates unsafe mine quarries and continues to damage the environment with GD's backing (Gujaraidze 2021; Business and Human 12 Rights Resource Center, 2021).In terms of exports, GM was among the top 50 exporters every year from 2004 to 2019 according to Geostat data and is Georgia's most important exporter of copper ore to the EU.Trade liberalisation with the EU has therefore brought benefits to a company heavily involved in rent-seeking practices to avoid criminal prosecution. 13
Agrifood and textiles: between exclusive and inclusive development
Georgia's agrifood and textile industries made up roughly 25% of the country's exports to the EU in 2020.The ownership structures of these sectors are more diverse than in the case of minerals: a few larger foreign and domestic companies dominate, with an increasing number of SMEs.
Thus far, exclusionary development coalitions have emerged in Georgia's textile sector and in nut production, setting these industries on pathways towards exclusive development.In textiles, an exclusionary development coalition between state authorities and a few international brands and larger local companies have empowered the latter.More precisely, tariff eliminations in the context of GSP+ and the DCFTA, along with other favourable government decisions, helped to boost foreign direct investment in Georgia's textile industry from 2006 onwards.The government's decision to create special economic zones and the launch of the 'Produce in Georgia' programme in 2014 certainly played an important role in attracting investors with reduced tax rates, competitive labour costs, good transportation links, and a more favourable investment environment (Invest in Georgia 2016; OECD 2020).Indeed, Georgia's textile and apparel industry witnessed a considerable increase in production (up 30% since 2008) and export volumes (up 24% in the period from 2009 to 2015).But the government's tendency to give investors and larger local textile producers a free rein, especially when it comes to labour standards, comes at a high social cost for the local workforce (Chkareuli 2020).Georgia has become an attractive low-cost production base for some leading world-class brands like Adidas and Moncler.These international brands work closely with the local apparel industry, which comprises around 200 enterprises, but seldom engage in any technological transfer or upgrading.
Similar developments characterise Georgia's nut production sector.As increasing export volumes suggest, the two large companies that dominate Georgia's nut exports to the EU -Dioskuria XXI and Westnut -have benefitted from better market access.Russian citizen Nona Kharebava owns Dioskuri XXI and Westnut is owned by Georgian businessmen and UK-based companies.Westnut receives its raw materials from approximately 2,000 farmers.However, most small Georgian nut producers cannot reap the full benefits of free trade because they export their product through intermediary companies from Turkey or EU countries that take on the burden of regulatory and export procedures.While this approach helps small producers to increase their sales, the prices paid to them by the intermediaries are much lower than the sale prices on the EU market (EastFruit 2021; Europe for Georgia 2016).
As for Georgia's wine industry, two large companies, Tbilvino and Telavi Wine Cellar, dominate the market.Leading company figures seem to enjoy some political connections to the ruling GD Party, as indicated by several donations to the party in 2017 and 2020 (Transparency International Georgia 2021).Unlike the minerals and metals sectors, Georgia's wine industry is, however, not controlled by a rent-seeking coalition.SMEs operating in the wine industry are on the rise and make up a higher market share than in the nuts and textile industries (Interview 3).Most of the 300 wine companies exporting between 2017 and 2020 were SMEs, and the emergence of an inclusionary development coalition between government agencies, business associations and a broad range of firms has helped set the sector on an inclusive path.The National Wine Agency (founded in 2014) and the Georgian Wine Association (founded in 2010) played an important role in diversifying the industry's ownership structures.They helped local wine producers to overcome information asymmetries, initiated certification procedures for export companies, and fostered participation at international fairs, which increased brand awareness of Georgian wines (The Financial 2016;Vinoge 2015).EU twinning initiatives facilitated exchange between Georgian authorities and their EU counterparts (EU4Business 2016).Nonetheless, it should be remembered that Georgia's wine sector only accounts for 3.5% of total exports to the EU.
All in all, the above analysis shows that more than 60% of Georgia's exported goods are produced in sectors where rent-seeking coalitions dominate.These rent-seeking coalitions engage in clientelist empowerment, as they give preferential treatment to leading exporters with political connections to Ivanishvili's oligarch network, thereby enabling them to reap most of the benefits from trade liberalisation in exchange for political loyalty.Only a minor share of Georgia's exports to the EU are produced in sectors dominated by exclusionary or inclusionary development coalitions, which empower firms without political connections to benefit from better access to the EU market.Georgia's trade liberalisation with the EU has thus so far consolidated the power position of rent-seeking elites rather than supporting exclusive -let alone inclusive -developmental pathways.
Conclusion
This study makes a theoretical and empirical contribution to the literature on EU trade policy.On a theoretical level, we introduce insights from the political economy literature on development to the study of how the EU -through trade liberalisation -affects development in its trading partners.In so doing, we reveal an additional yet under-researched factor affecting who benefits from increased trade liberalisation with the EU: the type of sectoral coalition between public and private actors in top export sectors.Our analysis shows that particular types of sectoral coalitions, which we conceptualise as inclusionary and exclusionary development coalitions, as well as rent-seeking coalitions, mediate the effect of increasing EU trade liberalisation on development, as they set sectors on pathways towards exclusive or inclusive development or help to consolidate rent-seeking practices.Relatedly -and here we also add to the literature on the link between trade liberalisation and development more generallyour analysis reveals three different mechanisms by which the type of sectoral coalition mediates the effects of trade liberalisation on export sector development by enabling different groups of firms to reap the benefits of trade liberalisation.We call these mechanisms inclusive empowerment (of a broad range of firms including SMEs), exclusive empowerment (of a narrow group of -mostly big and foreign -firms) and clientelist empowerment (of politically connected firms).
Our findings are based on a comparative study of EU trade liberalisation with Moldova and Georgia.We show that despite being a most likely case for reinforcing inclusive development, EU trade liberalisation with the two Eastern neighbours does not necessarily contribute to this type of development.In fact, it may also contribute to exclusive development or even to the consolidation of rent-seeking practices, depending on the type of coalition between public and private sectors present in top export sectors.Further, our focus on Moldova and Georgia allowed us to establish variation when it came to the assumed presence of rent-seeking coalitions in top export sectors.A similar causal relationship between trade liberalisation, the type of sectoral coalition characterising top export sectors and the type of sectoral development is therefore likely to be found in EU trade relations with other partners which have similar country-level characteristics to Moldova and Georgia, but receive even less assistance with capitalising on better access to the EU market.Here we think especially of most countries in Northern Africa with which the EU is cooperating through its partnership with the Southern Neighbourhood.
Regarding our empirical contribution to the literature on EU trade policy, to our best knowledge this study is the first to examine the type of sectoral coalitions present in Moldova's and Georgia's top export sectors and how they shape the effect of EU trade liberalisation on sectoral development.Our analysis thus helps us to understand under what conditions the EU can achieve its declared objective to promote inclusive development in its neighbourhood, which is seen as a precondition for social cohesion and, ultimately, political stability (European Commission and EEAS 2020).More precisely, we show that trade liberalisation with the EU has offered more opportunities for exclusive and even inclusive development in Moldova than in Georgia.By contrast, the majority of Georgia's exports to the EU comes from resource sectors like minerals and metals where the key market players are embedded in a rent-seeking coalition closely connected to the ruling oligarchic network under the leadership of billionaire Bidzina Ivanishvili.
Our findings suggest an important avenue for future research: While it is well established that natural resources produce more opportunities for rent-seeking (Torvik 2002), in particular in the context of weak institutions and governance quality (Chambers and Munemo 2018), more work is needed to systematically examine the factors behind the emergence of exclusionary or inclusionary development coalitions in non-resource sectors.According to the existing literature, the way the EU ties trade liberalisation to the provision of assistance to enable a broad group of domestic economic actors to reap the benefits of trade liberalisation may be key in this respect (Bruszt and Langbein 2020).
Notwithstanding this, our findings already hold important implications for the conditions under which the EU can live up to its normative ambition of promoting inclusive development through trade liberalisation.First, when implementing policies and programmes aimed at facilitating access to the EU market for firms from less developed trading partners, the EU would be well-advised to take into account the ownership structure of top export sectors and the involvement of key owners in rent-seeking practices.Doing so may help the EU to avoid the risk that trade liberalisation and related funding to facilitate market access will help to stabilise the power position of rent-seeking elites.
Second, our analysis suggests that the emergence of inclusionary development coalitions is closely linked to the provision of assistance targeted at strengthening sectoral organisation, e.g. through promoting the establishment of business associations, and/or bringing different public and private actors together.To improve local conditions for inclusive development, the EU could therefore make sure that trade liberalisation goes hand in hand with assistance programmes that put greater emphasis on fostering cooperation between a diverse set of private and public actors in promising sectors where firms not linked to oligarchic networks are important market players.Such a strategy will help to broaden access to trade for a wider group of economic actors, thereby laying the foundation for more inclusive development in the EU's Eastern neighbours and beyond.(around EUR 193,000) in subventions from 2014 to 2018 (Gulca and Necşuţu, 2019).In 2017, Monicol exported 95% of its production to the EU (https://www.eumonitor.nl/9353000/1/j9vvik7m1c3gyxp/vkj6g5k5ypzs?ctx= vhyzn0ozwmz1 7.These companies include, among others, DRÄXLMAIER Group, Gebauer & Griller, Fujikura Automotive MLD.Their local subsidiaries operate mainly in the free economic zone ZEL 'Bălţi' (cf.http://zelb.md).8. Compared to Moldova, Georgia was a frontrunner in terms of trade liberalisation with the EU.
List of interviews
As early as 2006, 90% of Georgia's trade turnover with the EU was at zero-tariff due to a decision of the Georgian government to scrap most tariffs on EU imports.The EU for its part extended the tariff preferences to Georgia under a special incentive arrangement rewarding development and good governance (GSP+) in 2006.When the DCFTA between the EU and Georgia was provisionally enforced in 2014, its immediate impact on bilateral trade was therefore limited (Economic Policy Research Center, 2014).9. https://napr.gov.ge/10. https://www.gm.ge/en/ 11.The Law on Making Amendments to Some Legislative Acts of Georgia (Source: https://www.matsne.gov.ge,registration code: 360000000.05.001.016640)12. Miscellaneous comprises commodities such as furniture, bedding and mattress supports.13.The data exclude Transnistria, cf:https://ec.europa.eu/eurostat/cache/metadata/Annexes/enpe_esms_an1.pdf.It is worth mentioning, however, that Transnistrian companies seem to take advantage of trade liberalisation with the EU.Cf.Gumene (2019).
Interview 1 :
Dumitru Alaiba, Member of Parliament of the Republic of Moldova, representing the Action and Solidarity Party (PAS) parliamentary group, 20 January 2021, online.Interview 2: Igor Munteanu, researcher in politics and governance and lecturer on public policy at the Academy of Economic Studies in Chisinau, former Member of Parliament of the Republic of Moldova and former Ambassador of Moldova to the US, 25 January 2021, online.Interview 3: Giorgi Gudabandze, Export Development Association of Georgia, 22 January 2021, online.Notes 1. Certainly, trade liberalisation may also create losers, if local producers cannot compete with imports (Stiglitz and Charlton 2006).The focus of this paper is, however, on the effect of trade liberalisation on exporting sectors and firms.2. The DCFTAs were provisionally applied in 2014.The Association Agreements entered into force in 2016.3. Georgia was classified as an upper middle-income country (UM) in 2015.In 2018 the country was again classified as an UM.Moldova has only been classified as an UM since 2020 according to the World Bank (cf.https://datacatalogfiles.worldbank.org/ddh-published/0037712/DR0090754/OGHIST.xlsx, accessed 4 May 2023).4. Cf. https://www.transparency.org/en/cpi/2022 5.According to Regulation Nr. 1284/2002 of 02.10.2002'On the conduct of the competition for obtaining the waste marketing license and scrap ferrous and non-ferrous metals', Metalferos was the only company to receive the license, see also: Munteanu et al. (2020); Rață (2017).6.According to the Moldovan Agency for Intervention and Payments in Agriculture (AIPA), Monicol SRL received a total amount of MDL 4.1 million
Table 1 .
Moldova's top five export sectors to the EU (share in total exports to the EU in %/share in total export volume to the EU in mln €).
Table 2 .
The top five Moldovan agri-food products exported to the EU (%).
Table 3 .
Georgia's key sectors with the highest shares in exports to the EU (%). | 2023-11-15T17:29:19.709Z | 2023-11-08T00:00:00.000 | {
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237701608 | pes2o/s2orc | v3-fos-license | Identification of the sex chromosome system in a sand fly species, Lutzomyia longipalpis s.l
Abstract In many animal species, sex determination is accomplished by heterogamety i.e., one of the sexes produces two types of gametes, which upon fertilization will direct the development toward males or females. Both male (“XY”) and female (“ZW”) heterogamety are known to occur and can be easily distinguished when the sex-chromosomes are morphologically different. However, this approach fails in cases of homomorphic sex chromosomes, such as the sand fly Lutzomyia longipalpis s.l. (Psychodidae, Diptera), which is the main vector of visceral leishmaniosis in Brazil. In order to identify the heterogametic sex in L. longipalpis s.l., we did a whole-genome sequencing of males and females separately and used the “Y chromosome Genome Scan” (YGS) method to find sex-specific sequences. Our results, which were confirmed by PCR, show that L. longipalpis s.l. has XY system. The YGS method can be especially useful in situations in which no morphological difference is observed in the sex-chromosomes or when fresh specimens are not readily available.
Introduction
Sex determination is a major biological phenomenon with important practical and theoretical consequences. In many animal species, sex determination is accomplished by genes in specialized chromosomes, called sex-chromosomes (White 1973;Bull 1983;Rice 1996;Charlesworth 2006;Bachtrog et al. 2011;Blackmon et al. 2017). Sex chromosomes are often heteromorphic and are called XY system when males are the heterogametic sex and ZW when females have the heterogametic chromosome (Bull 1983;Blackmon et al. 2017). Most Diptera have heteromorphic sex chromosome systems (mainly XY) (Kaiser and Bachtrog 2010;Ashman et al. 2014;Vicoso and Bachtrog 2015;Blackmon et al. 2017) but other systems occur like in sand flies (Psychodidae; Diptera) where the sex chromosomes are homomorphic (Kreutzer et al. 1987(Kreutzer et al. , 1988. The commonness of heteromorphic XY systems in Diptera might suggest that this is the ancestral state, but actually there were numerous replacements of both the X and the Y chromosomes (Vicoso and Bachtrog 2015;Dupim et al. 2018). The search for sex determination systems and sex determination genes in Diptera and insects in general is an active area of research, and an important phenomena remain to be elucidated (Hall et al. 2015;Krzywinska et al. 2016;Sharma et al. 2017).
The standard theory of the evolution of a new sex chromosome invokes the acquisition of a sex-determining locus by an ordinary autosome. Sexually antagonistic selection then favors the suppression of recombination around the associated sexdetermining region, which may extend to the whole chromosome (Charlesworth 2017). The lack of recombination will cause an increase in genomic divergence between the homologs with and without the sex-determining locus, and the chromosome associated with the heterogametic sex gradually degenerates. This process will ultimately lead to the formation of morphologically different (heteromorphic) sex chromosomes, making it easy to tell them apart. XY systems originated when the sex-determining locus determines maleness ("M factor"), whereas ZW systems originated from a female determining gene factor ("F factor") (Charlesworth 1991;Kaiser and Bachtrog 2010;Wright et al. 2016;Furman et al. 2020). The degree of differentiation is a continuum across species from full homology (which is expected in very young systems) to nearly full differentiation (e.g., mammals, birds); it is assumed to correlate with the time of divergence, but there are numerous exceptions (Charlesworth and Charlesworth 2005;Palmer et al. 2019;Furman et al. 2020).
Sex chromosome systems are generally stable, but transitions across groups are more common than previously thought (Carvalho and Clark 2005;Kaiser and Bachtrog 2010;Bachtrog et al. 2011;Ashman et al. 2014;Blackmon and Demuth 2015;Vicoso and Bachtrog 2015;Abbott et al. 2017;Blackmon et al. 2017;Dupim et al. 2018;Jeffries et al. 2018;Pennell et al. 2018). The classical approach to determine the sex chromosome system (XY vs ZW) is to observe the chromosomes during meiosis or mitosis in males and females. However, when there are no morphological differences between the sex chromosomes (homomorphy), cytogenetics cannot determine which system (XY or ZW) is present in a given organism. This is the case of most sand fly species studied so far, from both the Phlebotomus and Lutzomyia genera, (Kreutzer et al. 1987(Kreutzer et al. , 1988. The exceptions are Phlebotomus perniciosus and Lutzomyia shannoni, in which heteromorphic chromosomes were observed (Kreutzer et al. 1987;Jiménez et al. 2001). In both cases, the authors considered the heteromorphic pair as an XY pair, however, neither work was able to determine the sex of the larvae before dissection (Kreutzer et al. 1987;Jiménez et al. 2001), and hence these species may have as well a ZW system. As mentioned, despite the fact that most Diptera have an XY system, ZW species occur (Kaiser and Bachtrog 2010;Ashman et al. 2014;Vicoso and Bachtrog 2015;Blackmon et al. 2017). Therefore, the sex determination system of sand flies remains an open question.
Lutzomyia longipalpis s.l. (2N ¼ 8) (Kreutzer et al. 1987) is the main vector of visceral leishmaniosis in Brazil. It actually is a complex of cryptic species (Souza et al. 2008(Souza et al. , 2017Araki et al. 2009Araki et al. , 2013Vigoder et al. 2015), and identifying sex-specific sequences in L. longipalpis s.l. may provide important data about the evolutionary history of this group. It may also provide important tools for vector control strategies, especially in regard to genetic manipulation (Achee et al. 2019).
Recent bioinformatics methods have been used to identify the sex chromosome system, however, most rely on producing many genetic lines through crossing and/or expensive sequencing of DNA and/or RNA from many individuals (Tomaszkiewicz et al. 2017;Palmer et al. 2019). In order to identify the heterogametic sex, we adapted the "Y chromosome Genome Scan" (YGS) method developed by Carvalho and Clark (2013). This method uses separate sequencing from male and female DNA, followed by de novo assembly, and computational identification of sex-specific sequences. YGS was originally used to detect Y chromosome sequences in organisms with a known XY system. Since we do not know the sex-determination system of L. longipalpis s.l., we ran the YGS pipeline twice, assuming either a XY or a ZW system. The rational is that only one of these runs will detect sex-specific sequences; searching for a W in a XY system or searching for a Y in a ZW system should fail to retrieve any sequences.
Sand flies and DNA extraction
Sand flies were obtained from Dr. Fernando Genta (Laborató rio de Fisiologia de Insetos Vetores, FIOCRUZ, RJ). The colony was founded over 30 years ago from flies collected at the locality of Jacobina (Bahia State, Brazil; 11 11' S, 40 32' W) with multiple reintroductions from the same population throughout the years. Larvae at L4 stadium were collected from rearing cups and individually put in eppendorf vials for pupation, to ensure that all emerging adults were virgins. Emerged adults were sexed and pooled into groups of 50 males and 50 females and preserved in À20 C ethanol. DNA extraction was performed with the Puregene DNA kit (Qiagen) following the manufacturer's recommendations.
DNA sequencing and assembly
A draft genome of L. longipalpis s.l. available in VectorBase (vectorbase.org/) was obtained from male individuals from Jacobina (LlonJ1_VB). However, for our analysis, we needed sequences from males and females separately and hence we did them as follows. Illumina DNA sequencing (100 bp paired-end; fragment size 470 bp) was performed by Macrogen (South Korea), with a HiSeq 2000 machine. A total of 4.4 Gb of data was produced for each sex. The raw coverage was $ 20Â fold for each sex, as estimated by GenomeScope (Vurture et al. 2017). Illumina reads were trimmed using TrimGalore-0.5.0 (https://www.bioinformatics.bab raham.ac.uk/projects/trim_galore/) and de novo assembled with SPADES v. 3.11.1 (Bankevich et al. 2012) using reads from both sexes. Scaffolds smaller than 200 bp were removed. Contaminants were detected with the Blobtools pipeline (Laetsch and Blaxter 2017), and scaffolds with hits with Bacteria in both BlastX and BlastN were also removed. The completeness of the assembly was evaluated using BUSCO v3 (Simão et al. 2015) using a dataset of 2799 conserved genes that are present in almost all available Diptera genomes (database name: diptera_odb9).
YGS method
The YGS method was developed by Carvalho and Clark (2013) to computationally identify sex-specific sequences. Note that these sequences should be present only in the heterogametic sex (males in XY systems, or females in ZW), and hence YGS can be used to identify the sex determination system. Briefly, the YGS method starts with a genome assembly using sequences from the heterogametic sex or from both sexes, in order to have all chromosomes represented. Next, the assembled genome is decomposed into k-mers (we used 16 bp k-mers) and the repetitive kmers (i.e., those occurring more than once in the assembled genome) are identified. This step aims to avoid the interference of repetitive sequences such as transposable elements, which can be shared between, say, a Y chromosome and an autosome. The unassembled trimmed reads from the homogametic sex were also decomposed into k-mers. Finally, each scaffold is individually decomposed into k-mers, and the repetitive ones (identified as described above) are removed; this result in a set of single-copy kmers, which was compared with the set of k-mers present in the reads of the homogametic sex. Scaffolds with a high percentage of unmatched k-mers are likely to belong to the Y (or W) chromosome, which can be experimentally confirmed with PCR for details Carvalho and Clark (2013). Because we did not know which sex of L. longipalpis s.l. is heterogametic (and hence has the sex-specific sequences) we performed the YGS twice, using the same mixed sex assembly and either males' or females' reads for comparison. Note that scaffolds coming from the autosomes and from the X or Z are expected to have a very low (or zero) proportion of unmatched k-mers. when compared with reads from males or from females.
On the other hand, scaffolds that came from the Y or W chromosome will have a high proportion of unmatched k-mers when compared against the reads of the homogametic sex (females in the case of Y, males in the case of W), but not when compared against the heterogametic sex. Hence, as shown in Figure 1, the sex-determination system is revealed by examining at the pattern of the distribution of unmatched sequences.
If the sex-determination system is male heterogamety (i.e.,a XY system), the proportion of unmatched k-mers should have a bimodal distribution when we use the reads from females, and a unimodal distribution when we use the reads from the males (Figure 1, left panels); the opposite pattern is should occur with female heterogamety (a ZW system; Figure 1, right panels).
YGS in a ZW system
The YGS method had been only used in organisms with a XY system (Carvalho and Clark 2013;Dupim et al. 2018). As a proof of principle that it can work on an ZW system we used reads of males and females of Tephritis californica (Diptera: Tephritidae) from Vicoso and Bachtrog (2015) (SRR1738208 and SRR1738207, respectively). All the procedures used in the L. longipalpis s.l. analysis was used with T. californica, starting with the de novo assembly with SPAdes (see above).
Data availability
The raw reads and de novo genome assembly of L. longipalpis s.l. is available in Bioproject PRJNA699015. Supplementary material is available at figshare: https://doi.org/10.25387/g3.14794824.
Results
As a preliminary assessment of the quality of our genome assembly, we compared it with the one from VectorBase. Our de novo assembly of L. longipalpis s.l. has 39,920 scaffolds with a total of 148,058,403 bp and a N50 of 14,820 (Table 1). The VectorBase genome, in contrast, has only 11,532 "scaffolds," a total size of 154,229,266, and N50 of 85,244. Hence, the VectorBase assembly is more contiguous, probably due to the use of long-range matepair libraries. However, BUSCO indicated that our assembly is more complete than VectorBase's both in terms of complete genes (82 vs 77%, respectively) and of missing genes (4.9 vs 16.6%, respectively; Table 2).
The analysis of contaminants was done using the Blobtools pipeline (Simão et al. 2015). We performed both BlastN and BlastX to assign scaffolds to their likely origin (Arthropod, Bacteria, and so on). Scaffolds that had hits for bacteria, protozoa, and/or virus in both BlastN and BlastX were deemed as contaminants and removed. Note that this is a quite stringent criteria (e.g., Figure 1 Identification of the sex-determining system using the YGS method. A genome assembled with both sexes was compared to male and female short reads, and the percentage of unmatched sequence of each scaffold is calculated. Blue: scaffolds which are present in both sexes (autosomes, and X or Z chromosomes); red: scaffolds that are present only in the heterogametic sex (Y or W chromosome). Note that the pattern of the distributions reveals the sex-determining system. We represented both peaks with the same height (i.e., containing the same number of scaffolds), but in real cases they can be very different, pending on the relative amount of sequence on Y or W chromosomes, and their fragmentation in the assembly [see contaminant scaffolds containing only noncoding regions will not be flagged); we adopted it to avoid discarding real L. longipalpis s.l. sequences. Six hundred and fifty-seven scaffolds (amounting to 215,133 bp) were deemed as contaminants and were removed resulting in a final assembly of 147,843,270 bp. Figure 2 shows the result obtained with Blobtools using BlastN.
Identification of sex-specific sequences
In order to identify the sex chromosome system of L. longipalpis s.l. we ran the YGS method twice on the same mixed-sex assembly. In the first run, we compared the assembly with female reads (a procedure which we will refer to as XY genome scan, or "XY-GS," aiming to detect Y-linked contigs), and in the second run, we compared it to male reads ("ZW-GS," aiming to detect W-linked contigs) (Supplementary File S1). In both cases, we removed from the analysis contigs that have low quality (6163 contigs, totaling 2,279,222 bp), defined as having more than 10% of their k-mers absent from the male and female reads. These spurious k-mers came from assembly errors. Figure 3 shows the result of the two YGS runs: the distribution is clearly bimodal in the XY-GS analysis ( Figure 3A) and unimodal in the ZW-GS ( Figure 3B). This strongly suggests that L. longipalpis s.l. has a XY sex determination system, which we experimentally confirmed as follows. To avoid the possible interference of contaminants (e.g., a sex-specific bacteria), we first sought among the 1539 scaffolds those that have BlastX hit against an Arthropod protein; we found 35 scaffolds. We then picked at random six candidates to test with PCR using male and female DNA to confirm sex linkage (Supplementary Table S1). All six candidates were amplified only in males (Figure 4) confirming that L. longipalpis s.l. has a XY/XX sex determination system. In total, 1539 scaffolds (3.8% of the total) with 1,212,666 bp (0.7%) altogether had more than 70% unmatched k-mers (XY-GS), and are suspect of being Y-linked in L. longipalpis s.l. This amount of sequence is comparable with other species (e.g., D. virilis, Supplementary Table S2). These values should be seen as rough and probably sub-estimates: Y and W chromosomes frequently have a lot of repetitive sequences which fail to assemble.
Although there is no sign of a W-linked sequences such as a bimodal distribution when using male reads, we also looked at the scaffolds in the right tail of Figure 3B. There are 223 scaffolds with more than 70% unmatched k-mers (ZW-GS), containing 161,858 bp. Seven of them had a BlastX hit with an Arthropod using our Blobtools database, but when we manually checked them using NCBI BlastX, they are repetitive sequences such as transposable elements. Repetitive sequences are prone to misassemblies that can generate spurious YGS signals (Carvalho and Clark 2013).
Given that we have identified a set of Y-linked scaffolds, we performed a preliminary search for a sex determining gene. We looked at scaffolds with more that 70% unmatched k-mers (XY-GS) with BlastX hit with Arthropod as they would be more likely to contain a sex determining gene. The candidates from the 35 scaffolds were manually annotated. Upon this closer analysis, they were found to be either spurious hits, transposons, or had early stop codons or only represented a small part of the gene sequence, suggesting (in the latter two cases) that they are pseudogenes. It is important to keep in mind that our preliminary analysis would not be able to find the sex determining gene if it is noncoding RNA or a fast-evolving protein, as is the case in several other insects (Kiuchi et al. 2014;Hall et al. 2015). The identification of the Muller element from the candidates with a D. melanogaster ortholog did not show any clear pattern that would had allowed us to determine the Muller element that corresponds to the sex chromosome in L. longipalpis s.l. (Supplementary Table S2).
Discussion
The sex chromosome system is an important trait as it will influence many aspects of genome evolution. Identifying the system (XY/XX or ZW/ZZ) is difficult when there are no morphological differences between the sex chromosomes, or when live samples are not available. Here, we applied the YGS method (Carvalho and Clark 2013) to identify the sex chromosome system (XY or ZW) in the sand fly L. longipalpis s.l., which is a challenging case due to its homomorphic sex chromosomes. The computational results clearly indicated a XY system (Figure 3), which was experimentally confirmed by PCR (Figure 4). Hence YGS is a reliable tool for the identification of the sex determination system. This conclusion is strengthened by the finding that the known ZW species T. californica produced the inverse pattern when compared with L. longipalpis, confirming that the YGS method can detect a ZW system (Supplementary Figure S1). Readers interested in applying the approach we describe may benefit from the practical guidance we presented in Supplementary File S2.
The primary purpose of the present work was to identify the sex-determination system, but we also did a preliminary (and unsuccessful) attempt of identification of the sex-determination gene by looking at protein-coding genes is the candidate Y-linked scaffolds. This failure is quite expected: Identifying the sexdetermining gene by sequence similarity is a difficult task since it varies among Diptera families, and is unknown in many of them ( Saccone et al. 2011;Salz 2011;Petrella et al. 2019). None of the candidate protein-coding genes that were identified on the Y seem good candidates for the sex-determining gene, as they either had a premature stop codon or were otherwise incomplete genes. The L. longipalpis s.l. genome seems to be somewhat difficult to assemble, as judged by the fragmentation of our assembly and by the number of missing genes in the VectorBase assembly (Table 2). Hence, it is possible that some of the incomplete genes we found in the Y-linked contigs represent full-length genes, which were not fully assembled, or that the gene was lost altogether during the assembly. Our scan for candidates of a sexdetermining gene was only preliminary and would not be able to identify poorly conserved or novel genes, as occurs in some mosquitoes and medfly (Hall et al. 2015;Krzywinska et al. 2016;Meccariello et al. 2019), if there are multiple copies of the gene and/or a similar paralog or if it might be located in an autosome as it can happen in Musca domestica (Hamm et al. 2014;Sharma et al. 2017), or even noncoding RNA genes as in Bombyx mori (Katsuma et al. 2018). Another possible explanation for the lack of candidates is that, although L. longipalpis s.l. has XY system, it might not have a M-factor, as happens in Drosophila (Salz 2011). Further work will be needed to uncover the sex determination pathway in sandfly. This effort would greatly benefit from a more contiguous assembly.
Nonetheless, besides the identification of the sexchromosome system, our analysis provides the first description of male-specific markers in L. longipalpis s.l. Considering that L. longipalpis s.l. is a species complex (Araki et al. 2009;Vigoder et al. 2015;Souza et al. 2017) the use of such markers may offer information about the evolution of the L. longipalpis complex, such the role of sex-chromosomes as barriers to gene flow (e.g., Fontaine et al. 2015). Also, the identification of male-specific genetic markers may provide an important tool for the development of insect control strategies such as allowing the production of male-only lines for SIT programs (Meza et al. 2014;Achee et al. 2019).
Other techniques have been used to identify the sex chromosome system; however, they require RNA sequencing or RAD sequencing (Jeffries et al. 2018;Meisel et al. 2020). The YGS method was originally designed for species with heteromorphic sexchromosomes (Carvalho and Clark 2013), and our results show that it also works in a system with homomorphic sexchromosomes. The expanded use of the YGS method (Carvalho and Clark 2013) to identify the sex chromosome system needs no more than separate Illumina DNA sequencing of males and females. It is a simple and reliable method for identification of the heterogametic sex (and of sequences from the Y or W chromosomes), and it is especially useful in species with homomorphic sex chromosomes or when fresh specimens are required for cytogenetic studies are not readily available. | 2021-09-01T15:08:15.446Z | 2021-06-26T00:00:00.000 | {
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251732600 | pes2o/s2orc | v3-fos-license | Chorea‑acanthocytosis: 3 New Families with Novel Genetic and Metabolic Findings
Annals of Indian Academy of Neurology ¦ Volume 24 ¦ Issue 3 ¦ May-June 2021 452 GBS associated with rickettsial infections is very rare. An association between an infection with Rickettsia conori and GBS was reported in a French study in 1968.[9] Additionally, GBS with Rocky Mountain spotted fever was reported in 1996.[10] Although its pathogenesis has not been completely determined, GBS may be induced by molecular mimicry, toxins, or immune dysregulation.[2] Interestingly, in patients with scrub typhus, both humoral and cellular immunities are activated and play a role in the clearance of O. tsutsugamushi.[5] In our case, mild pleocytosis (10 cells/mm3) was detected. However, CSF antibodies to O. tsutsugamushi and other viral markers (antibodies for EBV, CMV, Hepatitis A, B, C, E, and HIV) were not detected. In general, CSF findings in GBS show albuminocytologic dissociations. In other words, a few or no lymphocytes exist.
Chorea-acanthocytosis: 3 New Families with Novel Genetic and Metabolic Findings
Sir, Neuroacanthocytosis syndromes are a group of rare heterogeneous neurological disorders characterized by the presence of acanthocytes in peripheral blood smear and basal ganglia degeneration. [1] Chorea-acanthocytosis (ChAc), one of the core neuroacanthocytosis syndromes, is inherited by autosomal recessive transmission and is caused by mutations of the Vacuolar Protein Sorting 13 homolog A (VPS13A) gene on chromosome 9q21, which encodes for a protein called "chorein". [2,3] In this case series of four genetically diagnosed patients from three families of various ethnic origins, we describe two novel mutations of VPS13A and highlight elevation of plasma ammonia and lactate as possible disease markers.
subject 1
A 32-year-old man from South India, born of second-degree consanguineous parentage, presented with history of poorly controlled generalised seizures (both complex partial and generalised tonic clonic) for 10 years. He was tried on multiple anti-epileptic medications (phenytoin, levetiracetam, carbamazepine and lacosamide). He presented to us 10 years later with abnormal orofacial movements for the past 2-3 years. On examination, his cognition and extra-ocular movements were normal. He had orofacial dyskinesia that was more prominent while eating than at rest, along with tongue dystonia.
subject 3
A 27-year-old woman from eastern India, born of non-consanguineous parentage, presented with a 4-year history of anxiety and depression, for which she was under psychiatric care. She later developed generalized abnormal movements and gait disturbances over next 2-3 years. On examination, her cognition was normal, ocular movements were full, vertical saccades were absent. She had orofacial dyskinesia with tongue dystonia, prominent while eating (feeding dystonia).
There was a generalized chorea, multifocal motor tics and a bizarre gait pattern with a combination of axial spasms and chorea ("rubber person" gait). Deep tendon reflexes were absent throughout. The sensorimotor and cerebellar examination was otherwise normal. Her investigations are summarized in Table 1. In view of the clinical pattern and positive peripheral blood smear, she underwent whole exome sequence analysis which identified a previously reported homozygous nonsense variation (Lys1037Ter) in exon 29 of the VPS13A gene (NM_033305.3:c. 3109A >T; GRCh38. p13). The substitution results in an early stop codon leading to premature truncation of mRNA translation at codon 1037. [4] The variant was classified as pathogenic based on ACMG guidelines.
Her 25-year-old younger brother, who carried the same mutation, had a two-year history of declining academic [5][6][7] Movement disorders are observed in around two-thirds of patients at initial presentation; hyperkinetic disorders (chorea, dystonia, tics, myoclonus) often appear at an early stage, while Parkinsonism occurs in more advanced disease. [1] As this is a very rare disorder, low clinical suspicion can result in significant diagnostic delay, as was observed in our second subject.
The persistent CPK elevation during the inter-ictal period can suggest the diagnosis, but prior to the development of a movement disorder, may suggest a mitochondrial or other neuromuscular disorder, potentially leading to an unwarranted muscle biopsy. [5][6][7][8] Acanthocytes can be positive in 5-50% patients, [1] and if present, can narrow the diagnosis. Western blot indicating the absence of chorein is supportive in diagnosis. [9] Genetic analysis is considered as gold standard and should be undertaken in presence of strong clinical suspicion. [1] VPS13A is localised on chromosome 9q21 and has 73 exons. The transcript variant A (exons 1-68, 70-73) is the main expressed form and encodes a 3174 amino acid protein "chorein". The absence/or alteration of this protein results in disease. [1] There are around 84 pathogenic and 9 likely pathogenic variants described so far. [10] These mutations are dispersed throughout the gene without any particular hotspot. [1] The mechanism by which absent chorein results in basal ganglia neurodegeneration, and the cause of acanthocytes, is not known at present. [1] Chorein is expressed in several cellular organelles and has diverse functions. It is a membrane protein, localised at membrane contact sites including nucleus-vacuole, endoplasmic reticulum (ER)-vacuole and endosome-mitochondria contact sites. It is involved in the transport of membrane bound proteins between trans-Golgi network and prevacuolar compartment and from endosome to vacuole. VPS13A gene mutations can result in impaired autophagic degradation, defective protein homeostasis, delayed endocytic and phagocytic processing, and abnormal calcium homeostasis. The actin polymerization defects, altered protein phosphorylation and red cell membrane properties are postulated to result in acanthocyte formation. Impairment of mitochondrial functions such as fusion and mitophagy could predispose to neurodegeneration. Gene mutations also affect lipid droplet mobility within the cell organelles, leading to their accumulation within neurons and glia, and thus possibly causing neurodegeneration. [11][12][13] Two novel mutations were observed in our study. One subject presented initially with seizures, while movement disorders were observed in the second patient. The literature does not support a phenotype-genotype correlation, and significant heterogeneity has been reported within families. [1,5] However, similar to our first subject, there are case reports of patient with seizures as a prominent feature having certain mutations such as c2343 deletions and truncating mutation c.4326 T > A (p. Tyr1442*) in VPS13A. [6,7] Further research on possible phenotype-genotype relations may enhance our understanding of the disease.
We observed elevated fasting serum ammonia levels (in 2/3 of cases; venous sample), which have not been previously reported, nor systematically studied. These subjects had a normal liver function and secondary factors like hepatoxic drugs were absent This assumes significance as hepatic clearance play a major role in metabolism of these substances through the urea cycle and gluconeogenesis, respectively. Patients with ChAc often have hepatomegaly and altered liver function, although this is not usually clinically significant. [1] In addition, lactate was elevated in the two patients in whom it was measured. Muscle biopsy results are either normal or show non-specific neurogenic and myopathic atrophy, [1] while ischemic lactate test [7] and mitochondrial enzymes in muscle were normal. [5][6][7] The other source of excess lactate could be increased haemolysis, which has been observed in ChAc patients. [1] These findings require further validation in a large number of patients, and may have the potential to serve as an additional disease biomarkers.
In conclusion, ChAc is a rare disorder with varied clinical presentations. The rarity of the condition and low clinical suspicion can lead to a diagnostic delay. Elevation of serum ammonia and lactate levels in the absence of overt liver and muscle disease is intriguing and requires further validation.
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.
Transient Tunnel Vision as Initial Presentation of Anti-MOG Antibody Positive Optic Neuritis
To the Editor, Myelin oligodendrocyte glycoprotein (MOG) is an essential component of the oligodendrocytes and is expressed in the myelin of the central nervous system (CNS). Optic neuritis (ON) and acute disseminated encephalomyelitis are inflammatory demyelinating disorders that been associated with anti-MOG antibodies. [1] We describe transient monocular tunnel vision, as a novel clinical presentation of anti-MOG antibody associated ON.
A 21-year-old nursing student presented with complaints of painful ocular movements since 2 weeks, followed by transient episodes of tunnel vision involving the left eye. She first noticed tunneling of her vision in the left eye during eye movement and noted that it was associated with peri-ocular pain. She noticed that her field of vision in the left eye had reduced to one third her normal and was able to see central objects such as faces and read text from her mobile phone, but was unable to see peripherally. The tunnel vision was paroxysmal, lasting about 20-30 minutes, resolving spontaneously but recurring every few hours. At this point, there were no symptoms in the right eye. Since 4 days after the onset of transient tunnel vision, she also developed blurring of vision, which was more prominent in the right eye. She also reported decreased brightness and impaired color vision in both eyes. On ocular examination her visual acuity in right eye was 6/18 and left eye was 6/9 and there was relative afferent pupil defect in the right eye. Fundus examination showed bilateral disc edema. The visual filed appeared normal on confrontation and there was loss of color vision in both eyes. Clinical localization was to the anterior visual pathway. Provisional diagnosis of an atypical optic neuritis was considered. MRI Brain with orbits showed thickening and enhancement of the optic nerves and nerve sheath (right >left) without any other white matter lesions in the brain [ Figure 1]. Visual evoked potentials showed bilateral anterior optic pathway dysfunction. CSF analysis was normal (cells-3 glucose-53 mg/dl protien-32 mg/dl). Serum angiotensin converting enzyme (ACE) levels were in normal range. ESR, CRP, and antinuclear cytoplasmic antibodies was negative, connective tissue work up was negative. She was found to be negative for Aquaporin-4 antibodies but positive for serum myelin oligodendrocyte glycoprotein (MOG) antibody. She was given pulse therapy of Inj. Methylprednisolone pulse therapy 1 gm for 5 days, following which her visual symptoms improved dramatically | 2021-05-11T00:07:31.971Z | 2021-03-27T00:00:00.000 | {
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139339329 | pes2o/s2orc | v3-fos-license | THULIUM LASER VAPORIZATION VERSUS VAPOENUCLEATION ( WITHOUT MORCELLATION ) TECHNIQUE FOR BPH : DO WE HAVE A WINNER ?
Background and Objective The thulium laser surgery is a relatively new approach in which a wavelength of approximately 2 μm is emitted in continuous-wave mode, thus enabling the precise incision of tissue by using a wavelength that matches the water absorption peak of 1.92 μm in tissue. However, no published multinational study or other evidence definitively declares the superiority of thulium vaporization (ThuVAP) over thulium vapoenucleation (ThuVEP) without morcellator for better management of bothersome benign prostatic hyperplasia. The present study aims to evaluate the efficacy of vaporization and vapoenucleation (without a morcellator) in thulium laser prostatectomy for the treatment of benign prostatic hyperplasia. Methods A retrospective analysis of 82 patients who underwent thulium laser prostatectomy between February 2017 and January 2018 with ThuVAP and ThuVEP techniques was done and outcome measures analyzed were International Prostate Symptom Score (IPSS), quality-of-life score (QoL), maximum flow rate (Q max), post-void residual (PVRU), total operating time, laser time and resected tissue weight. Results No significant differences were noted between ThuVAP and ThuVEP in terms of post-operative prostate volume (22.4 vs. 21.7 mL) and post-operative prostate specific antigen (PSA) (2.54 vs. 1.85 ng/mL). Nonetheless, there were differences between the groups in total lasing time (56.5 vs. 44.8 min, p = 0.001) and total operative time (88.5 vs. 71.5 min, p= 0.001). There was also a significant difference in IPSS, QoL score, Q max, and PVRU at 6 weeks, 3 months, 6 months and 9 months after surgery. J Endolum Endourol Vol 2(1):e24-e36; February 15, 2019. This article is distributed under the terms of the Creative Commons Attribution-Non Commercial 4.0 International License. ©2019 Sarma et al. Thulium Laser Vaporization versus Vapoenucleation (Without Morcellation) Technique for BPH: Do We Have A Winner? e25 Conclusion ThuVEP provides a superior reduction of prostate volume and better short-term clinical outcomes than ThuVAP in the treatment of patients with benign prostatic hyperplasia. Furthermore, ThuVEP results in significantly lower urethral discomfort rates with significant improvement in IPSS as compared to ThuVAP which is maintained over time. The hospital stay and catheter indwelling time are shorter in the ThuVEP group and the complications associated with it appear to be lower than ThuVAP group.
The past decade witnessed an advent of minimally invasive laser treatment for effective management of benign prostatic hyperplasia (BPH) and substitute transuretheral resection of the prostate by attaining a similar surgical outcome with decreasing morbidity.Despite the fact that traditional TURP is both effective and efficient, it is plagued with significant morbidities with 2-8% transfusion rate. 1,2Not very long ago, Yu and colleagues had analyzed practice patterns and stated that, traditional TURP accounted for 39% of surgical interventions for bothersome BPH while laser procedures accounted for a whopping 57%. 3 The high-power thulium laser was first introduced in 2005 for the treatment of BPH. 4 Since then, the technique of thulium prostatectomy has gained significance with ThuVEP (Thulium laser vapoenucleation) showing encouraging clinical results using a similar technique comparable with holmium laser enucleation of the prostate (HoLEP). 5The wavelength of the thulium (2013 nm) laser is very close to the peak for absorption in water (1940 nm), and this high-density energy is delivered in a continuous wave.The energy transforms to more efficient vaporization with shallower depth (0.2 mm) of penetration in tissue. 6which results in a smaller zone of thermal damage, which decreases post-operative dysuria that is commonly associated with transurethral laser prostate procedures. 7The continuous wave mode is more suitable for hemostasis and coagulation of tissue, whereas the pulsed mode for is more suited for lithotripsy. 8The past researches using the 70-150 W thulium laser systems had demonstrated good short-term efficacy with low morbidity and few complications.This paper reviews the current evidence of safety and efficacy to evaluate the efficacy of vaporization, and vapoenucleation (without a morcellator) in thulium laser prostatectomy for the treatment of bothersome lower urinary tract symptoms due to BPH.
METHODS
In the present retrospective study, 82 patients with bothersome LUTS due to BPH who underwent thulium laser surgery [ThuVAP (n=38) and ThuVEP (n=44)] between March 2017 and January 2018 were analyzed to evaluate the comparative efficacy and safety profile between the 2 given methods after obtaining institutional ethics committee clearance.Inclusion criteria consisted of age ≥50 years, acute urinary retention with failed catheter free trial, symptomatic BPH with a maximal flow rate (Q max) of <15 mL/s, and International Prostate Symptom Score (IPSS) ≥ 8. Exclusion criteria were IPSS ≤ 7, Q max ≥15 mL/s, known case of adenocarcinoma, urodynamically confirmed neurogenic bladder and previous endoscopic surgery for BPH.Patients were thoroughly evaluated for all comorbidities preoperatively and at 6 weeks, and at 3, 6, and 9 months after surgery, using IPSS score, quality-of-life (QoL) score, Q max and post-void residual urine (PVRU).All intraoperative measures such as total operating time, laser-activation time, and estimated blood loss were noted for all the cases.The post-operative prostate volume was measured both pre-surgery and again at 3 months after the procedure to assess the vaporized prostate volume with the help of transrectal USG (TRUS).The operative time was calculated from insertion of the resectoscope into the urinary bladder to the perurethral catheterization at the end of the procedure.The lasing time was calculated when the laser was in use.Complications were graded according to the modified Clavien -Dindo-Dindo classification system.Because of inherent heterogeneous baseline characteristics among patients undergoing ThuVAP and ThuVEP, we used 1:1 propensity score matched analyses to adjust for these differences.A total of 64 patients who underwent ThuVAP (n = 32) or ThuVEP (n = 32) were analyzed.All the procedures were carried out by a single surgeon.
SURGICAL METHODS
Thulium laser prostatectomy was performed with the use of Quanta Cyber TM Thulium laser (Quanta System, Oloma, Italy).All patients were in lithotomy position and spinal or general anesthesia was achieved.We used a conventional 24 Fr resectoscope in both the procedures.A 550 μm end firing fiber was used to transmit laser energy to the prostate with an energy setting of 90 W for ThuVAP and 70 W for ThuVEP.For ThuVAP, we carried out the procedure with an "insideout" vaporization of the prostatic adenoma starting at verumontanum.The surgeon vaporized the adenoma arbitrarily which was not dependent on the exact surgical capsule between the adenoma and the peripheral zone. 9(9).(10).All the continuous pre and perioperative variables were analyzed using the independent t-test.The categorical data were analyzed using the χ2-test.The correlation was established by using the Pearson's R coefficient.Propensity scores were obtained by multivariate logistic regression, based on age, prostate size and IPSS.All statistical analyses were carried out using SPSS software (version 21), and statistical significance was assumed at p< 0.05.
RESULTS
All baseline characteristics are listed in Table 1 and 1:1 propensity matched variables in Table 2 with their mean ± SD values.None of the pre-operative variable had any significant differences between the two groups (Table 1 and 2).When considering both the groups for the intraoperative variables, it was found that when compared with ThuVEP, ThuVAP required longer operation time (88.5 vs. 71.5 min, p = 0.001), lasing time (56.5 vs. 44.8minutes, p = 0.001), energy used (305.1 kJ vs. 211.28kJ, p = .001)and post-operative serum PSA level (2.54 ng/mL vs 1.85 ng/mL).Four constant parameters such as IPSS, FIG. 1 The Cyber TM 150 Watt thulium laser machine used in our study.QoL, Q max, and PVRU were constantly monitored at 6 weeks, 3 months, 6 months, and 9 months.The IPSS score was significantly different between the 2 groups at 6 weeks and maintained at 6 months and 9 months (Table 4, Figure 2).The drop in IPSS from the pre-operative period to 9 months was 56.86% for the ThuVAP group and 71.16 % for the ThuVEP group and was significantly different between the 2 groups (p=.012).The QoL parameter was similarly analyzed at similar follow-up periods as mentioned earlier with significant difference observed at 6 weeks, 3 months and maintained at 9 months (Table 4, Figure 3).The increase in QoL from the pre-operative period to 9 months was 28.05% for the ThuVAP group and 40.28% for the ThuVEP group and was significantly different between the 2 groups (p=.032).The Q max was assessed and a significant difference was observed at 6 weeks, 3 months and maintained at 9 months (Table 4, Figure 3, Figure 4).The increase in Q max from the pre-operative period to 9 months was a staggering 94.23% for the ThuVAP group and 102.28% for the ThuVEP group (p=.150).Finally, PVRU was assessed at similar time periods and a significant difference was observed at 6 weeks and 3 months (Table 4, Figure 5).The decrease in PVRU from the pre-operative period to 9 months was 73.94% for the ThuVAP group and 78% for the ThuVEP group (p=.350).Most patients in each group were catheterized for 2 or 3 days, and if placed, the catheter was removed on the third postoperative day.When the per urethral catheter (PUC) time was analyzed, it was found that that the mean time for ThuVAP group was 3.3 days while it was 2.5 days for the ThuVEP group (p=.001).Similarly the mean hospital stay was 4.5 days in the ThuVAP and 3.6 days in the ThuVEP group (p=.007).The Table 5 lists the complication according to the modified Clavien -Dindo classification system.The majority of the patients went through grade I-II complication (57.8%).According to the 2004 Clavien -Dindo classification system, there were 22 (34.38%)cases with grade I complications consisting of clot retention, transient incontinence, and bladder mucosal injury.Only one patient in the ThuVEP group required a blood transfusion.Within the 6 weeks after surgery, a significant difference in urethral discomfort (dysuria) was noted, with 14 patients (43.75%) complaining of dysuria in the ThuVAP group versus 4 patients (12.5%) in the ThuVEP group.Most of the dysuria was of persisting nature and remained bothersome till 6 months (28.13%, n=9) post-surgery in the ThuVAP group.Delayed bleeding was observed in 3 of 32 patients (9.38%) in the ThuVAP group, and none in the ThuVEP group and these patients required transurethral coagulation under general anesthesia.In the ThuVAP group, there were 2 patients with urethral strictures and bladder neck stenosis, resulting in voiding difficulty at 6 months after surgery, and these patients required optical internal urethrotomy bladder neck incision.All 3 patients of grade 4 complications recovered after a brief stay in ICU with intense monitoring and hemodialysis.
DISCUSSION
The existing laser platforms can be successfully used in various BPH surgery techniques.The scrutiny of their physical properties (target chromophore, wavelength, power and emitting mode) has enabled us to understand their mode of action such as vaporization, vaporesection, vapoenucleation and enucleation on prostatic adenomatous tissue and their possible clinical application.The thulium laser has efficient and even cutting combined with thorough hemostasis which is required for surgical safety in an unmatched manner. 11,12 (11) (12).Since the properties of water remain static until water attains boiling point, the effect of the thulium laser remains constant throughout the surgery. 13 (13).In the present analysis, the mean IPSS had improved threefold and fourfold in ThuVAP and ThuVEP group respectively post-surgery at 9 months.Similarly, the QoL parameter improved nearly 1.5 times in both the groups after surgery.In addition, the Q max had increased approximately twofold, and the PVRU volume had decreased by nearly 75% in both groups compared with the baseline values.It was observed that there was an immediate improvement in IPSS at 6 weeks after surgery which was maintained at 9 months and there was a significant difference between the two groups.This might be attributed to the higher post-operative irritative symptoms and urethral discomfort caused by the longer lasing time of the prostate adenoma in the ThuVAP group.Our analysis suggested that the number of patients who complained of urethral discomfort increased disproportionately in the ThuVAP group when compared to the ThuVEP group.It was hypothesized that in vaporization, the adenoma becomes rugged leading to application of high laser energy to the surface of the irregular adenoma to smoothen it for a longer period of time.This might be a possible aggravator for increased irritative symptoms and urethral discomfort immediately after surgery.In addition, an inside-out approach to vaporization might have led to the longer lasing time in travelling to the correct plane.In the present analysis it was proved beyond any shadow that ThuVAP required longer operation and laser times compared with ThuVEP.ThuVEP showed a significantly higher vaporization potential and prostate volume reduction rate after surgery (see Table 3).There was a meaningful difference of lasing time (56.5 minutes vs. 44.8minutes) between the two groups which might have been the sole cause of increased irritative symptoms and urethral discomfort.We could remove more prostatic adenoma with the shorter surgical time in the ThuVEP group.
When the post-operative parameters were analyzed between the 2 groups (ThuVAP vs. ThuVEP), it was seen that the vaporized volume (59.05 gm vs. 73.65 gm, p= .001),prostate volume reduction (78.06% vs. 72.49%,p=.001), and PSA reduction (62.24% vs. 46.80%,p=.001) were significantly different between the two groups (see Table 3).Bach et al. reported a mean prostate volume reduction of 86% and a PSA reduction of 88% after thulium laser vapoenucleation using a 120 W Tm:YAG laser with a routine power output of 90 Watt. 14 (14).The present analysis failed to reach those reported percentages of prostate volume reduction and PSA decrease.Nevertheless, The present study was compared to the previous available literature in terms of efficacy and safety profile with 2 different arms compared with different observational studies as no past study has solely compared thulium vaporization with vapoenucleation (Table 6).
6][17] The ThuVAP group had 3 cases of acute urinary retention (AUR) post PUC removal while the ThuVEP had 4 such cases.All these cases were recatheterized again and PUC removed after 48 to 72 hours after monitoring clinical progress.Reich and team 20 specified a rate of AUR of 5.8% for TURP and bleeding necessitating transfusion of 2.9%. 18 (18).In our series, 40.63% (ThuVAP =12, ThuVEP = 2) of the patients remained on anticholinergic treatment, complaining of irritative LUTS 9 months after procedure.We adjudge these irritative symptoms post ThuVAP to the higher energy used and longer urethral time but at the same time the possible role of the bladder in irritative LUTS in men must be considered and rules out.It is estimated that approximately 35% of patients presenting with obstructive LUTS may report concomitant irritative symptoms. 19 (19).In addition, up to 1/3 of patients undergoing prostatectomy may present with urgency symptoms up to 11 months after surgery. 20(20).A recent study described the appearance of urge urinary incontinence not associated with detrusor hyperactivity in 50% of patients who underwent vaporization with 120 Watt LBO, which remitted in the subsequent 3 to 12 months. 25 (25).
The present analysis showed no statistically significant difference in any other complications except transient SUI and dysuria in both arms.There was a single bladder mucosal injury in the ThuVEP group developed in the process of vaporizing that enucleated portion of the prostate intravesically.Three patients required transurethral coagulation under general anesthesia because of hemorrhage or delayed bleeding.In the present analysis patients were requested to discontinue anticoagulant and antiplatelet therapies 5-7 days before surgery after taking cardiology consultation.The data of the patients who were still taking anticoagulants due to unavoidable cardiac circumstances were excluded from the propensity matching analysis.Stress urinary incontinence was found in 4 (12.5 %) patient in the ThuVEP group, and 1 (3.13%) in the ThuVAP group with significant difference between them (p=.012).Though this was a transient condition, this was attributable to the loss of hammock like support to the external urethral sphincter.The similar incidence of urinary incontinence, compared with HoLEP could be as a result of less injury to the external sphincter or a smaller portion of the prostate adenoma removed. 22,23t was observed in our analysis that with the use of ThuVAP, the urethral discomfort rate did not decrease over time (3 to 6 months) irrespective of the surgeons' experience.The sexual function was preserved in both the groups (6 to 9 months) based on the IIEF -5 questionnaire confirming the fact that ThuLEP was mostly safe from the grievous complication of erectile dysfunction.
The present analysis was not without any limitations.First, ours was not a randomized prospective study and some selection bias might have crept in despite of best efforts in terms of complication recordings.
We tried to have telephonic conversation whenever the data was insufficient or misguiding.Second, there was a learning curve initially for the surgeon's surgical technique which got refined and tuned with time but these factors were not part of our analysis, making it impossible to evaluate their impact.Thirdly, the current follow-up period might not be long enough to draw parallel on long term post-operative efficiency between the 2 study arms and their and complications.Nonetheless, efficacy and feasibility could be demonstrated by the aforementioned results, further follow-up will be required to verify improvement in urinary symptoms and the need of redo surgery if any.
CONCLUSION
This is the first effort to our knowledge to evaluate the efficacy and safety of two different methods in thulium laser prostatectomy though retrospective in nature.Our key observations were that ThuVEP at 70 W has several advantages over ThuVAP at 90-Watt power.The lasing time was shorter with ThuVEP than with ThuVAP leading to better post-operative urethral comfort profile and lower IPSS in terms of irritative symptoms.The mean prostate volume reduction rate, mean vaporized volume, along with mean PSA drop percentage was significantly better with ThuVEP than with ThuVAP group.The hospital stay and catheter indwelling time are shorter in the ThuVEP group and the complications associated with it appear to be lower than ThuVAP group.The future of thulium laser surgery has just knocked on the endless opportunities of BPH management and there is a strong conviction that thulium would sooner or later replace TURP as the gold standard measure to deal with big prostate glands.Further RCTs with both arms of ThuVAP and ThuVEP will pave the way for validation of our results and new insights in surgical techniques.
TABLE 3
Perioperative and Post-operative Post Propensity Score Matched Patients Data According to Surgical Methods PSA = prostate specific antigen; ThuVAP = thulium vaporization; ThuVEP = thulium vapoenucleation.
TABLE 4
Follow-Up Functional Outcome; IPSS score, QoL, Q Max, PVRU at Showed Time Periods
TABLE 6 .
Thulium Non-comparative Observation Studies in Comparison to Present Study J Endolum Endourol Vol 2(1):e24-e36; February 15, 2019.This article is distributed under the terms of the Creative Commons Attribution-Non Commercial 4.0 International License.©2019 Sarma et al. | 2019-04-30T13:09:09.184Z | 2019-02-15T00:00:00.000 | {
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259818870 | pes2o/s2orc | v3-fos-license | Protocol and Methods: Role of Levothyroxine on the Progression of Chronic Kidney Disease in Subclinical Hypothyroid Populations (LP-CKD) – A Multicenter Randomized Controlled Trial
Introduction: Subclinical hypothyroidism (SCH) is highly prevalent and associated with chronic kidney disease (CKD). However, it is still unanswered whether the restoration of euthyroid status in these patients will be beneficial in retarding a decline in glomerular filtration rate in early CKD patients. We aim to evaluate the efficacy of levothyroxine therapy versus placebo in slowing estimated glomerular filtration rate (eGFR) decline among CKD patients (stage 2–4) with SCH. Methods: This study will be a multicentric, double-blind, randomized, parallel-group, placebo-controlled study. A total of 500 CKD patients, 250 patients in the treatment group and 250 patients in the placebo group, will be randomized. The randomization between the treatment arm and placebo arm will be performed as per the computer-generated random number table in a 1:1 ratio. The sample size was calculated based on the assumed reduction in eGFR after 1-year follow-up in the treatment and placebo groups of 10% and 25%, respectively, at a minimum two-sided 99% confidence interval and 90% power of the study and considering 20% loss on follow-up. Each patient will be followed every 3 months for at least 1 year after randomization. Individuals completing 1-year follow-up visits will be considered for analysis. The baseline and follow-up data will be compared between the treatment and placebo groups. The study will evaluate the efficacy and safety of levothyroxine therapy versus placebo in slowing eGFR decline among CKD patients (stage 2-4) with SCH. The primary endpoint will be the end of follow-up of the patients, reduction of eGFR by ≥50% from a baseline of that patient, or development of ESKD or death of the patients. The secondary endpoint will be any cardiovascular event or arrhythmia after the institution of the drug.
Introduction
[3] The kidney plays an important role in the metabolism, degradation, and excretion of thyroid hormones.CKD affects thyroid function in several ways, including low circulating thyroid hormone levels, altered peripheral hormone metabolism, insufficient binding to carrier proteins, reduced tissue thyroid hormone content, and altered iodine storage in the thyroid gland. [4,5]CKD is associated with a higher prevalence of overt and subclinical hypothyroidism (SCH). [6,7]Experimental studies revealed that deficiency of 3 Osmania Medical College, Osmania General Hospital, Hyderabad, Telangana, 4 Post Graduate Medical Education and Research, Kolkata, West Bengal, India thyroid hormone lowers blood pressure, [8] increases vascular resistance, [9] and reduces renal sodium reabsorption, [10,11] which leads to volume contraction and reduced renal blood flow. [12]These events ultimately lead to a decline in the glomerular filtration rate (GFR).
Protocol and Methods: Role of Levothyroxine on the Progression of Chronic Kidney Disease in Subclinical Hypothyroid Populations (LP-CKD) -A Multicenter Randomized Controlled Trial
Lo et al. [6] showed that the decline in GFR and CKD is associated with hypothyroidism with a higher prevalence of subclinical hypothyroidism up to the tune of 56%, which increases with a decline in GFR. [6]while another study revealed an 18% prevalence among the non-dialysis CKD population. [7][18][19] CKD patients are at higher risk of cardiovascular complications. [20]A few observational studies showed clear associations between SCH and an increased risk for heart failure, coronary heart disease, and mortality. [21,22]25] Two studies from the same group showed that thyroid hormone replacement attenuates the decline in GFR in patients with SCH and CKD. [23,24]However, no association between thyroid hormone status and a decline in renal function was observed in a population-based study of elderly patients. [25]One recent study has shown that low thyroid function is not associated with a deterioration of renal function and has emphasized that the cross-sectional association may be explained by renal dysfunction causing thyroid hormone alterations.On the other hand, hyperthyroidism is known to accelerate the deterioration of renal function. [14,26]Levothyroxine (LVT) has a narrow therapeutic index and may pose the risk of iatrogenic thyrotoxicosis.LVT may also lead to increased protein catabolism and arrhythmia. [5,12]However, a very smaller dose of LVT (25-50 mcg/d) is required to treat SCH, and regular monitoring may obviate the side effects.
The multiple observational studies notwithstanding, there is a lack of randomized controlled trial (RCT) data on the effect of the progression of CKD with thyroid hormone replacement therapy for SCH in CKD patients.We plan to conduct a Randomized controlled trial (RCT) to look for the impact of LVT therapy, with the primary objective of finding its effect on the progression of CKD and the secondary objective of determining its effects on cardiovascular disease and mortality in SCH populations of CKD.
Materials and Methods
Study design: This study will be a multicentric, double-blind, randomized, parallel-group, placebo-controlled study.All patients of CKD (stages 2-4) with SCH visiting the outpatient clinics will be screened for evidence of SCH.Informed written consent will be obtained from each patient.All those patients who fulfill the criteria for the study will be allocated into two groups as per the computer-generated random number table in a 1:1 ratio.Patient enrollment will be stratified according to the CKD stage, aiming for at least 30% in each stage (stage 2, 3, and 4) of CKD.The total study duration will be 3 years, and patients will be recruited over 1½ years of the study.Each patient will be followed every 3 months for 1 year after randomization.
Participating centers: Department of Nephrology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, is the principal coordinating center, with five participating centers across India [Figure 1].SGPGIMS has all accountability regarding randomization, operational, financial, and quality control aspects of the study.Investigators from all the centers will be supervising trial progress, data collection, data analysis, and publication of study results.Every year, the progress report will be submitted to the Indian Council of Medical Research (ICMR), New Delhi.The present trial has received funding from the ICMR, New India (5/4/7-8/2019-NCD-II).
Study subjects: They include stage 2, 3, and 4 CKD patients with SCH, as per the inclusion and exclusion criteria given in Table 1.The randomized patients will be either receiving LVT or a matching placebo.
Table 1: Inclusion and exclusion criteria of the LPCKD study
Inclusion criteria 1.
Any degree of albuminuria 3.
Stable renal function for 3 months Exclusion criteria 1.
Patients already on thyroid hormone replacement therapy 4.
Unstable renal course or acute kidney injury in the last 3 months 5.
History of recurrent UTI 6.
Obstructive uropathy or structural disease of kidney or renal tract 7.
History of analgesics abuse, alternative medication, and nephrotoxic agents in the past 3 months 8.
Patients on chronic or intermittent use of NSAIDs for the nature of diseases such as rheumatoid arthritis and so on 9.
H/O coronary artery disease, arrhythmia, structural heart disease, and congestive cardiac failure in the last 6 months 10.
Patients with acute and chronic liver disease 11.Malignancy 12.
Pregnancy and lactation CKD=chronic kidney disease, eGFR=estimated glomerular filtration rate, GFR=glomerular filtration rate, LPCKD=role of levothyroxine on the progression of chronic kidney disease in subclinical hypothyroid populations, NSAID=nonsteroidal anti-inflammatory drug, UTI=urinary tract infection hormone (TSH) concentration (4.0-10 µIU/ml) and a normal serum T4/free T4 (fT4) and T3 concentration.The tests will be performed two times within 4 weeks to confirm the diagnosis.
Adverse cardiac events
They are defined as new-onset acute coronary syndrome (ACS) and arrhythmia.Coronary artery disease is defined as a history of myocardial infarction or angina, angiographic evidence showing coronary artery disease, history of angioplasty, coronary artery bypass grafts, and any other investigations showing evidence of coronary artery disease.Arrhythmia will be labelled based on the history of palpitation and confirmation from an electrocardiogram (ECG).
Sample size calculation:
With the assumed reduction in eGFR after 1 year follow-up in the treatment and placebo groups of 10% and 25%, respectively, at a minimum two-sided 99% confidence interval and 90% power of the study, the calculated sample size in each of the two groups came out to be 189.After considering the loss of follow-up of 20%, the minimum required targeted sample size in each of the two groups was 227.Thus, we will randomize 250 patients in the treatment group and 250 patients in the placebo group.The sample size has been estimated using software G*power version 3.1.9.2 (Dusseldorf, Germany).2. Patient screening will be done by the primary clinician from the outpatient clinic.Patients will be screened to satisfy the inclusion and exclusion criteria.The initial evaluation will include detailed clinical history, measurement of height, weight, body mass index (BMI), pulse rate, blood pressure, T3, T4/fT4, TSH, anti-Thyroid peroxidase (anti-TPO) antibody, serum creatinine, complete blood count, and lipid profile.Patients will be randomly assigned to the study drug LVT and placebo.Standard therapy for CKD will be continued in both groups.Both groups will continue antihypertensive medications, including angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), and other conservative treatments for CKD as before the study, unless there is a specific contraindication.Drugs and placebo will be continued throughout the study period.
Description of intervention:
Patients will be randomly assigned to the study drug LVT and placebo.Allocation concealment will be done using sealed, sequentially numbered, opaque envelopes.They will be consecutively numbered, and medicine will be given out according to the number allocated to the participant.The investigator will be blinded to the allotment as the procedure will be carried out by a third person, a clinical coordinator, and a senior research fellow employed for this purpose.Patients in both groups will receive either LVT or placebo tablets once daily before breakfast.Drugs (LVT or placebo) will be delivered to the patient by hand on a visit to the center, which will be centrally regulated.Both LVT tablets and placebo will be provided as white tablets identical in appearance, taste, and packing.LVT tablets will be supplemented as 25 µg only.The patients will be initially treated with l-thyroxine 25 µg/d or a placebo.After the start of the thyroid hormone supplement, serum TSH concentration will be remeasured every 12 weeks.If the level of TSH remains above the normal reference range, the dose of the trial drug (LVT 25 µg or placebo) will be increased by one tablet (25 mcg) in the next visit after 3 months.Thyroid hormones T3, T4, and TSH levels will be measured at each visit.The dose will be adjusted until the patient's serum TSH concentration is reduced to the normal reference range.
Apart from LVT, other renoprotective drugs such as ACEI, ARB, febuxostat, and other medicines for the conservative treatment will be continued in both arms as per clinicians' decision and standard indications.
Outcome parameters
The study will evaluate the efficacy and safety of levothyroxine therapy versus placebo in slowing eGFR decline among CKD patients (stage 2-4) with SCH.The primary endpoint will reduction of eGFR by ≥50% from a baseline of that patient, or development of ESKD or death of the patient.The secondary endpoint will be any cardiovascular event or arrhythmia after the institution of the drug.
Follow-up and compliance monitoring: Patients will be evaluated at each visit for a detailed history, physical examination, assessment of any adverse events or endpoints, serum creatinine levels, complete blood count, lipid profile, T3, fT4/T4, and TSH, and any other tests as needed by the treating clinician.History will be taken from the patient by the treating physician, intending to inquire about a drug-related adverse event (as mentioned above), nonsteroidal anti-inflammatory drug (NSAID) abuse, and use of alternative medication.Compliance will be assessed at each visit by history taking and pill counting.On follow-up, if any patient converted into clean hypothyroid with TSH >10, the patient will be terminated from the study and LVT will be started.
Collection, storage, and testing of samples: At baseline and at each visit, the sample will be collected for complete blood count, blood urea nitrogen(BUN), serum creatinine, T3, fT4/T4, TSH, and lipid profile (total cholesterol, low-density lipoprotein [LDL], high-density lipoprotein [HDL], and triglyceride).BUN, serum creatinine, T3, fT4/T4, TSH, and lipid profile will be done at individual centers if the same laboratory facilities are available.Otherwise, they will be done in the principal investigator's central lab identified for this purpose.Blood will be centrifuged; serum will be separated and will be stored at −80°C until final analyses will be carried out if required.Serum T3, fT4/T4, and TSH levels will be analyzed by chemiluminescence assay on a Roche Cobas E601 analyzer.Serum anti-TPO concentration will be analyzed by chemiluminescence assay on a Siemens Immulite 1000 analyzer.The reference ranges of T3, fT4/T4, TSH, and anti-TPO antibodies will be 1.2-2.1 nmol/l, 12-22 pmol/l, 0.4-4.0µIU/ml, and <30 kIU/l, respectively.
Discontinuation of patients from the study: Patients who will develop adverse cardiac event or acute kidney injury or will need kidney replacement therapy (KRT) for a reason other than the natural course of the disease or who are lost to follow-up during the study period will be discontinued from the study.Any patient who will withdraw consent for the study will also be discontinued.The CO-PI of the respective centre will decide about the KRT and other clinical adverse events.
Ethical review:
The study protocol has been approved by the Institutional Ethics Committee, SGPGIMS, Lucknow, India (IEC Code: 2019-52-EMP-108).The study is also registered in the Clinical Trials Registry-India (REF/2019/02/024293).Each participating center has obtained ethics clearance from the ethics committee of the respective institute.The study protocol and procedures are concordant with the principles of the Declaration of Helsinki.Informed consent will be taken from all patients before screening and inclusion in the study at all centers.
Data collection and statistical analysis plan: Data collection will be done through the specific website developed for this study purpose.The LPCKD study's website has been created with the domain name https://lpckd.com/.Separate login and passwords have been created for each center included in the study.The website includes registration, screening, randomization, and follow-up sections.The demographic, biochemical, medications, and events columns are given in the data entry section, and the data can be entered at each patient visit.The consent and prescription can also be attached separately.A specific user login name and password will be created for each participating center.Individuals completing 1-year follow-up visits will be considered for analysis.The baseline and follow-up data will be compared between the LVT and placebo groups.The normality of the continuous variables will be examined using the Kolmogorov-Smirnov test.Data will be presented as mean ± standard deviation for normally distributed variables and median (interquartile range) for non-normally distributed variables.Two-way repeated measures analysis of variance (ANOVA) will be used to test the changes in mean values on follow-up and their association with the study groups (treatment/ control).Bonferroni corrections will be used for multiple comparisons.McNemar test will be used to determine the differences in dichotomous dependent variables between two related groups.Cochran's Q test will be used to test the changes in binary outcomes at different time points of follow-up in the case of more than three related variables.A P value <0.05 will be considered statistically significant.Statistical Package for Social Sciences version 20 (SPSS 20; IBM, Chicago, IL, USA) will be used for statistical analyses.
Discussion
The uremic milieu with inflammation and metabolic acidosis in CKD affects the functioning of the hypothalamic-pituitarythyroid axis. [5]The peripheral conversion of T4 to T3 and its protein binding is reduced.Isolated low T3 level is the most common finding, followed by SCH. [26]It has been observed that as the GFR declines, the prevalence of primary hypothyroid, particularly the subclinical form, increases. [7][34] A linear trend between TSH elevation and CKD was observed in a case-control analysis. [35]The schematic diagram of complications and alterations in CKD with SCH is shown in Figure 3.
However, a few studies did not reveal the same association. [25]Attempts to replace thyroid hormones may lead to increased muscle catabolism, and both overt hypothyroidism and SCH may lead to an increased risk of cardiovascular disease and death in CKD. [36,37][40] One study [39] in elderly SCH and two retrospective studies [23,24] showed a reduction in decline in GFR in CKD patients with SCH.Biondi et al. [41] recommended LVT treatment for patients with SCH and TSH levels of 10 mU/l or higher, as well as those with mild hypothyroid symptoms.Recently, a retrospective database analysis on LVT treatment in CKD and SCH patients showed an absence of the overall benefit in eGFR; however, LVT use shortens the length of hospital stay. [42]There is possibly no association between low free thyroxine levels and CKD progression. [43]The progression of CKD was higher in the presence of proteinuria, with no effect on thyroxine levels.The present study is a large double-blind, placebo-controlled multicentric RCT.The design of the study is such that the study includes all early CKD patients This is the first large multicentric, well-powered study to resolve the issue of treating or not SCH in CKD patients.
Conclusion
SCH is prevalent in CKD patients.The association of SCH with the decline in GFR has been studied.However, whether achieving euthyroid status with LVT replacement will improve the GFR of the patients is unknown.This will be the first RCT to address the treatment of SCH in CKD patients.
Financial support and sponsorship
The study is funded by the Indian Council of Medical Research, New Delhi, India (5/4/7-8/2019-NCD-II).
Figure 1 :
Figure 1: Participating centres in the study across India
Figure 2 :
Figure 2: CONSORT flow diagram of the study | 2023-07-12T16:34:10.518Z | 2023-06-05T00:00:00.000 | {
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119231101 | pes2o/s2orc | v3-fos-license | Non-reciprocal coherent dynamics of a single spin under closed-contour interaction
Three-level quantum systems have formed a cornerstone of quantum optics since the discovery of coherent population trapping (CPT) and electromagnetically induced transparency. Key to these phenomena is quantum interference, which arises if two of the three available transitions are coherently driven at well-controlled amplitudes and phases. The additional coherent driving of the third available transition would form a closed-contour interaction (CCI) from which fundamentally new phenomena would emerge, including phase-controlled CPT and one atom interferometry. However, due to the difficulty in experimentally realising a fully coherent CCI, such aspects of three-level systems remain unexplored as of now. Here, we exploit recently developed methods for coherent driving of single Nitrogen-Vacancy (NV) electronic spins to implement highly coherent CCI driving. Our experiments reveal phase-controlled, single spin quantum interference fringes, reminiscent of electron dynamics on a triangular lattice, with the driving field phases playing the role of a synthetic magnetic flux. We find that for suitable values of this phase, CCI driving leads to efficient coherence protection of the NV spin, yielding a nearly two orders of magnitude improvement of the coherence time, even for moderate drive strengths<~1MHz. Our results establish CCI driving as a novel paradigm in coherent control of few-level systems that offers attractive perspectives for applications in quantum sensing or quantum information processing.
The well-established approaches for addressing threelevel systems rely on simultaneous, coherent driving of two dipole-allowed transitions in the system and lead to applications ranging from light-storage [10] and atomic clock frequency standards [11,12], to coherent quantum control [13][14][15]. A closing of the interaction contour in the three-level system by additional, simultaneous driving of the third available transition (Fig. 1a) would be of fundamental and practical interest [5][6][7][8]16], but is severely complicated by selection rules that prevent this closing in most experimental systems. Indeed, for symmetry reasons, only two of the three available transitions can be dipole-allowed for the same type of driving field. The use of a combination of electric and magnetic dipole transitions has been proposed as a remedy for this fundamental limitation [17][18][19][20]. However, all reported systems allowing for such driving suffer from fast dephasing compared to the rate of coherent manipulation, which prevented the experimental observation of quantum coherent CCI dynamics thus far.
In this work, we exploit the unique properties of the electronic spin of the Nitrogen-Vacancy (NV) centre in diamond to overcome these limitations and observe coherent CCI and the associated, non-reciprocal dynamics in a single three-level quantum system. Exquisite control of the NV centre spin degrees of freedom and innovative ways to coherently drive the NV spin allow us to implement CCI and establish the strong influence of the driving field phase on coherent spin dynamics. A surprising outcome is the realisation that for appropriate phases of CCI driving, the scheme allows for a significant enhancement of the NV's inhomogenous dephasing time, which we extend by nearly two orders of magnitude compared to the undriven case.
The negatively charged NV centre, a substitutional ni-trogen atom next to a vacancy in the diamond lattice, forms an S = 1 spin system (Fig. 1a) in its orbital ground state. Conveniently, the NV spin can be initialised using optical spin pumping under green laser excitation and optically read out by virtue of its spin-dependent fluorescence [21]. The NV's spin sub-levels are |0 and |±1 , where |m s are the eigenstates of the spin operatorŜ z along the NV's symmetry axis z (i.e.Ŝ z |m s = m s |m s ).
In the absence of symmetry breaking fields, the electronic spin states |±1 are degenerate and shifted from |0 by a zero-field splitting D 0 = 2.87 GHz. Applying a static magnetic field B NV along z splits |±1 by ∆ Z = 2γ NV B NV , with γ NV = 2.8 MHz/G, and leads to the formation of the three-level "∇-system" we study in this work (Fig. 1a) [22]. Although each of the states |m s has additional nuclear degrees of freedom due to hyperfine coupling to the NV's 14 N nuclear spin, we restrict ourselves to the hyperfine sub-space with nuclear spin quantum number m I = +1 [23], while other states remain out of resonance with our driving fields and do not contribute to CCI dynamics.
To implement and study CCI dynamics, we employ coherent driving of the NV spin using a combination of time-varying magnetic and strain fields. Specifically, we use the well established method of coherent driving of the |0 ↔ |±1 transitions with microwave magnetic fields [24]. In addition, we utilise a time-varying strain field to drive the |−1 ↔ |+1 transition -a recently developed method for efficient, coherent driving of this magnetic dipole-forbidden transition, which is difficult to address otherwise [23,25]. Considering the combined action of these three driving fields of amplitudes (Rabi frequencies) Ω i and frequencies ω i (Fig. 1a), the dynamics of the NV spin in an appropriate rotating frame [26] Closed contour interaction (CCI) scheme and experimental setup. a) Schematic representation of the three-level CCI system studied here. Levels |−1 , |0 and |+1 are the S = 1 ground state spin levels of a negatively charged diamond Nitrogen-Vacancy (NV) centre. All three possible spin-transitions are coherently driven, either by microwave magnetic fields (for |∆ms| = 1; purple arrows) or by strain (|∆ms| = 2; red arrow). The fields have frequency ωi, amplitude (Rabi frequency) Ωi, and phase φi (i ∈ {1, 2, 3}). b) Spin dynamics under CCI are investigated using a confocal microscope for optical initialisation and readout of the NV spin. Driving fields for microwave-and strain-driving are applied through a microwave antenna and piezo-excitation using appropriate generators (see SOM), which are mutually phaselocked to control the global interaction phase Φ = φ1+φ3−φ2.
are described by the Hamiltonian
if the three-photon resonance ω 1 + ω 3 = ω 2 is fulfilled ( is the reduced Planck constant). HamiltonianĤ 0 is expressed in the basis {|−1 , |0 , |+1 } and δ 1 (2) represent the detunings of the microwave driving fields from the |0 ↔ | (+) − 1 spin transition. Importantly, and in stark contrast to the usual case of coherent driving of multi-level systems, the resulting spin dynamics are strongly dependent on the phases φ i (i ∈ {1, 2, 3}) of the driving fields, through the gauge-invariant, global In the following, we will examine the case of resonant, symmetric driving, for which δ 1 = δ 2 = 0 and Ω i = Ω, ∀i. In this case,Ĥ 0 can be readily diagonalised with resulting dressed eigenstates and eigenenergies with k ∈ {−1, 0, 1} and ϕ 0 = 2π/3. To experimentally observe CCI dynamics and generate the required, time-varying strain field, we place a single NV centre in a mechanical resonator of eigenfrequency ω 3 /2π = 9.2075 MHz, which we resonantly drive using a nearby piezo-electric transducer [23]. The me-chanical Rabi frequency Ω 3 is controlled by the amplitude of the piezo excitation. To achieve resonant strain driving, i.e. ω 3 /2π = ∆ Z , we apply a static magnetic field B NV along the NV axis. The two microwave magnetic fields used to address the |0 ↔ |±1 transitions at frequencies ω 1,2 = 2πD 0 ± ω 3 /2 are delivered to the NV centre using a homebuilt near-field microwave antenna, (see SOM [26] for information on phase control and microwave field generation). Finally, a confocal microscope is used for optical initialisation and readout of the NV spin (Fig. 1b).
We study the NV spin dynamics under closed-contour driving by measuring the time evolution of the NV spin population for different values of Φ, using the experimental sequence shown Fig. 2a (inset). For each value of Φ, a green laser pulse initialises the NV spin in |ψ (τ = 0) := |0 = (|Ψ −1 + |Ψ 0 + |Ψ 1 ) / √ 3, after which we let the system evolve under the influence of the three driving fields for a variable evolution time τ . Finally, we apply a green laser pulse to read out the final population in |0 , The resulting data (Fig. 2a), here for Ω/2π = 500 kHz, show oscillations of P |0 in time, with a marked π-periodic dependence of the population dynamics on Φ.
To obtain a complete picture of the resulting spindynamics, we additionally monitor the populations P |±1 of spin-states |±1 for Φ = 0 and ±π/2 (Fig. 2b) by applying a microwave π-pulse resonant with the |0 → |+1 or |0 → |−1 transition at the end of the evolution time τ (Ω probe , dashed box in the inset of Fig. 2a). The resulting spin dynamics show that at Φ = ±π/2 the spin exhibits time-inversion symmetry breaking circulation (Fig. 2b, right) of population between the three states |0 , |+1 and |−1 [9], with a period T ±π/2 = 4π/ √ 3Ω. In perfect analogy to chiral currents of electrons hopping on a plaquette with three sites, threaded by a synthetic magnetic flux Φ, this circulation of population demonstrates the tunable synthetic gauge field [9] created by our CCI driving scheme. Conversely, for Φ = 0 the spin level population oscillates between |0 and an equal superposition of |±1 in a "V-shaped" trajectory (see Fig. 2, middle) at a period T 0 = 4π/3Ω. This shortening of T 0 compared to T ±π/2 is consistent with the different trajectories (Fig. 2b, right) the spin populations undergo. To further support that HamiltonianĤ 0 provides an accurate description of our system, we calculate the population dynamics and find excellent agreement with data (see SOM [26] for details of the simulation and comparison with experiment).
In addition to the spin dynamics under CCI, our experiment also allows us to directly access the eigenenergies E k of the driven three-level system (see Eq. (3) and black lines in Fig. 3a). After initialisation into |0 = (|Ψ −1 + |Ψ 0 + |Ψ 1 ) / √ 3, each component |Ψ k acquires a dynamical phase E k τ / , which governs the time evolution of the NV spin. The population P |0 (τ ) there- Closed-contour driving of the NV spin leads to periodic evolution of P |0 due to quantum interference in the NV ground state. Period and decay times of the interference pattern depend strongly on Φ. b) Linecuts of P |ms (τ ) (with ms ∈ {−1, 0, +1}) for Φ = π/2, 0 and −π/2 (top, middle and bottom panel, respectively). At phases Φ = ±π/2, population is shuffled clockwise or counterclockwise while for Φ = 0 it alternates between |0 and an equal superposition of |±1 (with some addmixture of |0 ).
fore shows spectral components at frequencies ∆ m,n = (E m − E n ) /h with m = n ∈ {−1, 0, 1} ( Fig. 3a and b). A Fourier transformation of P |0 (τ ) (Fig. 3c) thus reveals ∆ m,n and thereby the eigenenergies of the driven NV spin, which for most values of Φ are in excellent agreement with the predictions based onĤ 0 (colored lines in Fig. 3c). Around Φ = 0 and ±π, we find anti-crossings instead of the expected frequency crossings in the spectrum; an observation we assign to environmental fluctuations and slow drifts. Indeed, the resulting, non-resonant or asymmetric drive lifts the degeneracies of the dressed states and explains our observation ( Fig. 3a and b). Taking these effects into account, we conducted numerical modelling of our experiment and found good qualitative agreement with our observed spectra (see SOM).
The effect of environmental fluctuations is already visible in the phase dependent interference patterns in Fig. 2a, where the resulting quantum-beats decay fastest for phase-values close to Φ = 0 and ±π -an indication that at these phase values, the dressed states |Ψ k are most vulnerable to environmental fluctuations, but protected from them at other values of Φ. Fig. 4a shows linecuts taken at Φ = 0 (top panel) and Φ = −π/4 (bottom panel), which evidence a dramatic change of the dressed state coherence time from T dec −1,0 = (8.5 ± 1.9) µs at Φ = 0 to T dec To understand the phase-dependance of the dressedstate dephasing times, we conducted extensive numerical modelling together with perturbative, analytical calculations of T dec m,n (Φ) (see SOM). Our second order perturbative calculations account for magnetic field fluctuations with Ornstein-Uhlenbeck statistics, together with a random field that was held static over each experimental run. The result (dashed lines in Fig. 4b) reveals that for each of the values Φ ≈ ±π/4 and ±3π/4, two dressed states exist whose energies show the same perturbative response to magnetic field fluctuations and thus form a coherence-protected subspace in the dressed state manifold, in which T dec m,n (Φ) approaches the spin relaxation time. We assign the significantly reduced, measured value T dec −1,0 (−π/4) ≈ 105 µs to driving field fluctuationsa hypothesis which we could quantitatively support with our numerical modelling (see SOM). Our data also show that the four local maxima of T dec m,n vary significantly in magnitude. We attribute this variation to slow experimental drifts of the zero field splitting parameter D 0 due to temperature variations [27] FIG. 3. Spectrum of the driven NV spin under closedcontour driving. a) Calculated eigenenergies E k of the driven spin for Ω/2π = 500 MHz, as a function of Φ for detuning δ1,2 = 0 (black lines) and δ1,2/2π = ±50 kHz (dotted lines). b) Transition frequencies |∆m,n| as a function of Φ for δ1,2 = 0 (blue, orange and red lines) and δ1,2/2π = ±50 kHz (dotted lines). c) Discrete Fourier transform of the data shown in Fig. 2a, as a function of Φ. The spectral components observed agree well with the calculated values of |∆m,n|; discrepancies around Φ = 0, ±π arise from environmental magnetic field fluctuations (see text). The observed Fourier amplitude (contrast) is inversely proportional to linewidth and therefore gives an indication of the decay time for each spectral component.
these drifts into account in our model yields excellent agreement between simulation and experiment for realistic temperature variations of ±1.3 K (see SOM). Our results establish the driving field phase under CCI driving as a novel control parameter for coherent manipulation and dynamical decoupling of single spins. They indicate that further experimental improvements would readily yield coherence protected dressed states with inhomogeneous dephasing times approaching the T 1 -limit. Such dressed states have recently been establishes as powerful resources for quantum sensing of GHz fields [28,29]. The efficient tunability and coherence protection we demonstrate here for dressed states offer highly interesting avenues for enhanced sensitivities and phase-tuning of the sensing-frequencies for such sensing schemes. In addition, our results yield an attractive platform to test, and ultimately implement proposed schemes for phase dependent CPT [5][6][7], since the NV centre would readily allow for promoting CCI to the optical domain, where established optical Λ-transitions [30] Spin-oscillations under closed-contour driving for Φ = 0 and Φ = −π/4, revealing strongly phase-dependent decay times T dec m,n (Φ). A fit of exponentially damped harmonics (see text) yields T dec −1,0 (Φ = 0) = 8.5 ± 1.9 µs, and T dec −1,0 (Φ = −π/4) = 124.8 ± 28.3 µs for the most long-lived spectral components. b) Systematic measurement of decay times as a function of Φ, showing minima of T dec m,n (Φ) at Φ/π = ±1, 0 and pronounced maxima at Φ ≈ ±nπ/4, n ∈ 1, 3. Dashed lines are the results of a second-order perturbative calculation of T dec m,n (Φ) (see text). Note that data in a) and b) originate from separate measurement runs and therefore result in slight differences in decay times.
be combined with strain driving in a phase-coherent way. Lastly, we note the strong analogy between the nonreciprocal spin dynamics under CCI driving we demonstrated and recent realisations of synthetic gauge fields in optomechanical systems [31]. Pursuing this analogy using ensembles of NV centres with engineered dissipation offers interesting avenues for realising on-chip, nonreciprocal microwave elements, such as microwave circulators or directional amplifiers [31].
We thank A. Retzker, N. Aharon, A. Nunnenkamp and H. Ribeiro for fruitful discussions and valuable input. We gratefully acknowledge financial support through the NCCR QSIT, a competence centre funded by the Swiss NSF, through the Swiss Nanoscience Institute, by the EU FP7 project DIADEMS (grant #611143) and through SNF Project Grant 169321.
SUPPLEMENTARY INFORMATION FOR "NON-RECIPROCAL COHERENT DYNAMICS OF A SINGLE SPIN UNDER CLOSED-CONTOUR INTERACTION"
In the first part of the supplementary material, we give more background about the Hamiltonian describing our system, including some discussion of the population dynamics for different values of Φ. We then simulate the effects of noise on the population dynamics and compare the results, including the phase-dependent dressed state coherence times, to the experimental data. In the second part, we discuss the perturbation terms for dressed state energies to first and second order in magnetic field noise along the NV axis. We find an analytical expression for the induced energy fluctuations as a function of Φ, which we compare to the Φ-dependence of the coherence times. Finally, in the third and last part we give additional experimental details on how we generate and control the microwave fields.
Closed-contour Hamiltonian and time evolution
In this section we derive the Hamiltonian (1) of the main text. The NV's S=1 ground state, driven by two MW fields and one strain field, is described by the Hamiltonian written in the {| − 1 , |0 , | + 1 } basis and expressed in the lab frame. Ω i , ω i , and φ i with i = {1, 2, 3} denote driving field amplitudes, frequencies, and phases, respectively. ω |i with i = 0, ±1 are the energies of the three spin sublevels.
In the main text, we study the time evolution of the population P |0 (τ ) in spin sublevel |0 . In the corresponding experiments we initialize the NV in |0 with a green laser pulse. The following abrupt onset of the three driving fields creates the closed-contour interaction, and we express as a linear combination of the system's eigenstates |Ψ k . After an evolution time τ the system is in the state whereÛ = e −iĤ0τ / is the unitary time evolution operator. The final green laser pulse yields a measurement of the resulting population in |0 and we determine therefore oscillates due to the presence of three frequency components ∆ i,j that correspond to the differences of eigenenergies E k . At Φ = 0, ±π , and spin population oscillates between |0 and a state composed of an equal superposition of | ± 1 with a small admixture of |0 at a period T 0,±π = 4π/3Ω (see Fig. 2a of the main text). Eq. (14) also demonstrates that P 0,±π |0 (τ ) = 0 for any time τ . At Φ = ±π/2, however, and P ±π/2 |0 (τ ) is maximized every T ±π/2 = 4π/ √ 3Ω. To extract the frequency components and decay times from the data, we fit our measured values of P |0 (τ ) with a sum of three exponentially decaying sinusoids: Doing this for each value of Φ yields the phase-dependence of Rabi decay times T dec i,j (see Fig. S3).
Analysis of environmental noise sources
Overview of existing noise sources The observed spin dynamics under closed-contour interaction and the dependence of Rabi decay times T dec i,j on driving phase Φ are significantly affected by the presence of several noise sources in our experiment. Most importantly, our measurements are influenced by fluctuations in [23]. c) Fluctuations of the zero-field splitting D0 for different measurement times for a single NV.
• the zero-field splitting D 0 , caused by variations in temperature or environmental strain or electric fields.
Other noise sources, for example frequency noise of the driving fields, are neglected in the following as we could not find any experimental evidence for their relevance.
To characterize the existing fluctuations in driving field amplitudes and zero-field splitting we performed longtime measurements, using the NV centre as a probe (Fig. S1). Low frequency drifts in Ω MW were analyzed by performing Rabi oscillation measurements on a single hyperfine transition. Similarly, slow fluctuations in Ω m and D 0 were investigated via the amplitude of strain-induced Autler-Townes splittings and ESR transition frequencies, respectively (see Barfuss et al. [23]). We found relative driving amplitude fluctuations of σ MW /Ω MW = 0.5 % and σ m /Ω m = 0.7 % within a bandwidth of ≈ 2 mHz, with σ being the corresponding standard deviations, assuming Gaussian distributions. Additionally, these fluctuations are accompanied by even slower amplitude drifts of δ Ωm /Ω m ≈ ±1 % and δ ΩMW /Ω MW ≈ ±1.5 % for strain and MW driving, respectively, which happen on timescales of several hours. In contrast, changes in the zero-field splitting δ D0 /2π are characterized solely by slow drifts, with drift amplitudes varying between a few and several tens of kHz. The data presented in the main text was taken on an NV with coherence time T * 2 = (2.1 ± 0.1) µs, which we determined through Ramsey interference. Magnetic noise is thus characterized by a Gaussian distribution with width σ T * 2 /2π = 1/ √ 2πT * 2 = 107 kHz [33] and is the strongest noise source in our experimental setting.
Modeling existing noise sources
To simulate the influence of noise on the observed spin dynamics we average over N avg solutions of the timedependent Schrödinger equation where each solution is obtained for a differentĤ 0 . In particular, we set Ω 1,2 = Ω + δ ΩMW (18a) where Ω/2π = 500 kHz denotes the applied Rabi frequency and δ ΩMW and δ Ωm describe Gaussian fluctuations, taken from a normal distribution with zero mean and standard deviations σ MW /2π = 2.5 kHz and σ m /2π = 3.5 kHz, as determined above, respectively. Magnetic and zero-field splitting fluctuations are included by setting inĤ 0 . Variations δ D0 in D 0 appear as simultaneous shifts of detunings δ 1,2 while magnetic fluctuations δ T * 2 induce an opposite change. δ D0 can in principle be modeled by a random walk approach, but for our simulations presented in the following we usually set it manually. δ T * 2 is taken from a normal distribution with zero mean and standard deviation σ T * 2 /2π = 107 kHz (see above). When solving the time-dependent Schrödinger equation, we update δ T * 2 , δ ΩMW and δ Ωm every step along Φ, i.e. N Φ times per complete phase sweep. In contrast, δ D0 is changed at a much smaller rate, because the zero-field splitting changes on timescales of several hours (see Fig. S5 to justify this approach). Please refer to the provided plots of δ D0 (Φ) (see Fig. S4). The presented procedure is repeated N avg times and averaging over all solutions yields mean values for |Ψ(τ ) and E k with k = 0, ±1. Note that this approach limits us to low frequency fluctuations with a bandwidth of 1/τ max ≈ 15 kHz, as the experimental environment is kept constant as long as the global phase Φ remains unchanged (τ max ≈ 60 µs the maximum evolution time for which |Ψ(τ ) is calculated). The excellent agreement between simulations and experiment (see Sec. ) justify our approach.
Comparing simulation and experiment
In Fig. S2 and Fig. S3, we compare experimental data presented in the main text and our modeled results, which we obtained as described above (note that no fluctuations in the zero-field splitting D 0 are included unless stated otherwise). In the time domain (Fig. S2), we observe excellent agreement between experiment and model. Yet a few differences do exist. First, the measured interference pattern of P |0 (τ ) in Fig. S2a is characterized by a slightly lower oscillation contrast compared to its simulated counterpart in Fig. S2d. We assign this difference to the limited signalto-noise ratio of our experimental data due to finite integration time. Also, slow fluctuations in driving fields and zero-field splitting have been neglected in our simulation. Second, the experimentally obtained P |±1 (τ ) in Fig. S2b are characterized by slightly smaller oscillation amplitudes than predicted by our simulations. The simplified noise environment in our model is partly responsible for this discrepancy, as is the fact that the strain field is still applied evolution time τ (µs) Closed-contour spin dynamics controlled by global phase Φ -a comparison between experiment (panels a+b) and our model (panels c+d). In general, we observe excellent agreement between experimental data and simulations in the time domain. Furthermore, the experimentally obtained P |±1 (τ ) in panel b are characterized by smaller oscillation amplitudes than predicted by our simulations. The disagreement is caused the fact that the strain field is still applied during the weak microwave swap-pulse used to readout the spin populations P |±1 . We were unable to quickly switch off the strain field because the finite Q factors of our mechanical resonators leads to a long response time. Finally, we want to point out that, due to the finite sampling rate of Φ, the linecuts for Φ = 0 in panel b have not been taken exactly at Φ = 0, but slightly offset at Φ ≈ +2 • , causing the observed small mismatch in oscillation frequencies, weaker damping, and the non-degeneracy of P |±1 .
during the weak microwave swap-pulse applied to read out spin populations P |±1 . We are unable to quickly switch off the strain field due to the non-zero Q factors of our resonators, which leads to a long mechanical response time. Finally, we want to point out that the linecuts for Φ = 0 in Fig. S2b have not been taken exactly at Φ = 0, but slightly offset at Φ ≈ +2 • , due to finite sampling rate of Φ. As a consequence, P |±1 are not degenerate.
Regarding the precession frequencies of the driven NV spin (Fig. S3a+c), we again find remarkable agreement between experiment and model. However, our data in Fig. S3a feature avoided crossings of varying gap sizes (∼ 20 kHz at Φ = 0; ∼ 100 kHz at Φ = ±π). In the simulated precession frequency spectrum (Fig. S3c), however, all gaps are of equal size. As we will see later (see Fig. S4), we assign this mismatch to slow fluctuations in the zero-field splitting D 0 . In contrast to the excellent agreement in Fig. S3a+c, the Rabi decay time spectra in Fig. S3b+d different. The modeled phase dependence of T dec features four, equally long living frequency components with maximum decay times of T dec ≈ 105 µs at Φ = ±π/4, ±3π/4. In our experimental data, T dec is strongly asymmetric. Again, this difference is caused by slow fluctuations, most likely in the zero-field spitting D 0 .
To investigate the influence of zero-field splitting variations, represented by detunings δD 0 in our model (see Eq. (19)), on spin precession frequency spectra and Rabi decay time T dec , we repeated our simulations with the same noise environment, but non-zero detunings δD 0 , and extracted precession frequencies ∆ i,j and Rabi decay times T dec as described above. The results are shown in Fig. S4 and indicate the following: • A fixed δD 0 /2π = −100 kHz (Fig. S4a) increases avoided crossings and induces a strong asymmetry in T dec (Φ).
• Decreasing D 0 , i.e. setting δD 0 /2π = +100 kHz, also increases avoided crossings in the spin precession frequency spectrum. Inverting the polarity of the detuning, however, also inverts its effect on T dec (Φ). Now, the ∆ ±1,0 components decay with T dec ±1,0 ≈ 120 µs and the frequency components ∆ +1,−1 disappear on timescales T dec +1,−1 ≈ 65 µs, the time-dependent Schrödinger equation, we can qualitatively reproduce the experimentally determined phase dependence of T dec very well (compare Fig. S4c and Fig. S3b). The employed variations in zero-field splitting D 0 of δD 0 /2π ≈ ±100 kHz are in good agreement with the experimentally determined fluctuations (see Fig. S1c), and are most likely caused by environmental temperature fluctuations [27]. Note that we did not directly measure how D 0 actually evolved during our experiment, but rather infer the temporal variation of D 0 so as to find the best agreement between data and simulation. We attribute the remaining mismatch between experiment and theory to our lack of knowledge about the precise noise environment, especially slow fluctuations in driving field amplitudes and zero-field splitting, during the measurement. The results from Fig. S4c should not be considered as fits, but rather a demonstration that including noise and drifts allows us to accurately reproduce our experimental observations through simulations.
Our simulations indicate that slow fluctuations are responsible for the observed asymmetry in T dec with respect to Φ. We can support this statement by further analysis of our experimental data. Typically, P |0 (τ, Φ) is obtained by averaging several complete phase sweeps over a total measurement duration of approximately 240 hours. In Fig. S5 we plot P |0 (τ ) (left), spin precession frequencies ∆ i,j (middle) and the root-mean square (RMS) of P |0 (τ ) for τ ∈ [50, 60] µs (right) versus Φ for four of these phase sweeps (note that the data set used here is not the one from the main text, but was recorded under similar conditions). The root-mean square of P |0 (τ ) serves as a measure for the remaining contrast of spin precession and therefore corresponds to the decay time T dec (extracting T dec by fitting was not possible with these data due to limited signal-to-noise ratio). which could not be extracted by fitting due to the limited signal-to-noise ratio of the experimental data. Whenever the RMS of P |0 (τ ) is large, the decay time is long. One can see that the individual RMS spectra show different behaviors with respect to Φ, i.e. the observable maxima differ in amplitude and slightly in position. This behavior is very similar to the influence of fluctuations in zero-field splitting D 0 (see discussion above) and thus confirms the presence of slow experimental fluctuations.
Overview
In addition to numerically modeling the effects of noise, we also used perturbative techniques to derive an analytical expression for the global phase dependence of the coherence times, to better understand how to improve coherence times further. We consider a total HamiltonianĤ =Ĥ 0 +Ĥ (τ ) consisting of the unperturbed Hamiltonian of Eq. 8 and a time dependent perturbation. Under symmetric resonant driving, in the rotating frame, where δ 1,2 could be due to, e.g., a small error in the frequency of the driving fields or a detuning due to the drift of the ZFS. As discussed in the previous section and as often noted in the literature [33], the NV coherence time T * 2 is often limited by magnetic field noise. We will therefore consider a perturbation Hamiltonian corresponding to magnetic field noise given byĤ = g NV µ B B z (τ )Ŝ z , where g NV = 2.0028 is the NV g-factor [35,36], µ B is the Bohr magneton, B z (τ ) is a fluctuating magnetic field along the NV axis, andŜ z is the spin-1 operator along the NV axis.
To find the effect of the perturbations, we calculate the corrections to the propagatorÛ in the interaction picture [34]: where the perturbation Hamiltonian in the interaction picture is given bŷ Using this expression for the perturbation Hamiltonian, we will calculate the transition probabilities and phase fluctuations caused by the perturbation.
Transition Probabilities Between Eigenstates
The first order corrections primarily affect the populations and are related to the transition rates between eigenstates.
If we prepare our system in one of the driven eigenstates |Ψ i , the first order transition probability to eigenstate |Ψ j can be written succinctly as [34] | Ψ j |Û (1) ) /h is the frequency difference between the initial and final states, S noise (∆ i,j (Φ)) is the noise power spectral density at the frequency ∆ i,j (Φ), and δ i,j is the Kronecker delta. Due to the dependence of ∆ i,j on the global phase Φ, the spectral region to which the spin is sensitive can be tuned by changing the global phase. With the driving strength of Ω/2π = 500 kHz we used in our experiments, this corresponds to a frequency range of ∆ min = 0 to ∆ max = 800 kHz. Stronger driving strengths will lead to larger dynamic ranges. This suggests we can use the dressed states to develop novel, Φ-dependent relaxometry techniques.
Effect of Noise on Coherence Times
If we prepare the system in one of the dressed states |Ψ i , the phase ϕ i accrued by |Ψ i to second order in the interaction picture under the influence of the perturbationĤ is given by [34] tan (ϕ i ) = Im{ Ψ i |Û (1) where we have used the fact that for our system Ψ i |Û (1) I (τ )|Ψ i = 0, i.e. there is no first-order contribution to the phase in this case. If the perturbation is small, we can also simplify the denominator as Re{1 + Ψ i |Û (2) I |Ψ i } ≈ 1 and use the approximation tan (ϕ i ) ≈ ϕ i . Putting these approximations all together, we arrive at a simple expression for the additional phase due to the magnetic field noise: There are several integral terms involved in calculating ϕ i , but most of them average approximately to zero. Neglecting these terms and treating the magnetic field as an Ornstein-Uhlenbeck process B O−U , we find that the phase correction for |Ψ −1 , for example, is given by dτ is the mean square value of B O−U during the evolution time τ and τ c is the noise correlation time [34]. Note that we have explicitly indicated the Φ-dependence of the eigenenergies, to indicate that the amount of phase acquired depends on Φ. Expressions for ϕ +1 and ϕ 0 can be found by permuting the indices. Notice that ϕ −1 = 0 at Φ = −π/2, while ϕ +1 = 0 at Φ = +π/2 (see Fig. S6), so that at certain values of Φ, |Ψ +1 and |Ψ −1 are unaffected to second order by the presence of magnetic field noise along the NV axis.
The approximate expression in Eq. 29 accurately predicts the asymmetric shape around the maxima, though the positions of the maxima are slightly off. Also note that the analytic expressions in Eq. 29 diverge for certain values of Φ. In addition to the relatively quickly changing magnetic field B O−U , however, we must also account for very slowly changing magnetic fields, which are constant during a single measurement or evolution time but can change between subsequent ones; these constant fields have the effect of changing the values of δ 1 , δ 2 in the unperturbed Hamiltonian Eq. 20, with the condition We include the effect of slowly varying fields by integrating over the possible dephasing rates as a function of the induced detuning, weighted by the probability of that detuning: where Γ dec −1,+1 (Φ, δ B ) is the dephasing rate in Eq. 28 (where we now treat the eigenergies E k as functions of δ B ) and P (δ B ) is the probability distribution of the detunings caused by the slowly varying fields. For P (δ B ), we use a normal distribution with mean µ = 0 and variance σ 2 = (107 kHz) 2 , corresponding to T * 2 = 2.1 µs. Performing this integral removes the divergence and leads to a maximum decoherence time on the order of 1 ms. Other discrepancies between Eq. 29 and the data are likely due to the contributions of other noise sources (such as fluctuations in the zero field splitting or noise in the strength of the driving fields), which will also have the effect of reducing the maximum achievable coherence time. Even so, the simple expression in Eq. 29 captures the functional form and scale of the coherence times, simply by entering realistic values for the parameters.
Our perturbative calculations show that the improved coherence times are due to the fact that for certain values of Φ two eigenstates experience the same amount of phase acquisition, such that their relative phase is unchanged by the Note that the rates of phase acquisition for the |Φ−1 and |Ψ+1 states, for example, are closest at Φ ≈ ±0.75π. Since their rates of phase acquisition are similar, a two-level system spanned by these two states has a minimal dephasing rate as this value of Φ, leading to longer coherence times at Φ ≈ ±0.75π. (b) The values of T dec i,j extracted from the experimental data, as a function of Φ, plotted alongside our analytical expressions.
presence of the magnetic noise. Furthermore, Eq. 29 shows the intuitively obvious result that stronger driving leads to improved coherence times, since stronger driving leads to larger energy splittings. Finally, the similarity between the data and the calculations confirms that the bulk of the noise in our system is due to fluctuating magnetic fields projected along the NV axis. Thus, removing excess spins from our sample would prolong coherence times and change the Φ-dependence of the coherence times, as other sources of noise come to dominate.
CREATION OF PHASE-LOCKED DRIVING FIELDS
We create the two MW tones used to drive the |0 ↔ | ± 1 transitions by frequency modulating a carrier signal S c at frequency ω c and amplitude A c with two time-dependent modulation signals S mod,I (t) = A mod sin (ω mod t + φ mod ) (31) and S mod,Q (t) = A mod sin (ω mod t) S7. Creation of driving fields for closed-contour interaction. The two MW tones with frequencies ω1,2 = ωc ± ω mod are created through frequency modulating a carrier signal of frequency ωc at frequency ω mod = ω3/2. Phase-locking of the three driving fields is achieved via pulsed output synchronization and locking of MW source, IQ and piezo function generators to the same 10 MHz reference signal.
of equal, but constant amplitudes A mod . These signals are mixed to the carrier signal as I and Q modulation inputs, and we obtain the output signal S out (t) = S mod,I (t)Re{S c } + S mod,Q (t)Im{S c } = A c A mod sin φ mod − π/2 2 · cos (ω c + ω mod ) t + π/2 + φ mod 2 which consists of two MW tones separated by 2ω mod and symmetrically located around ω c . The relative phase φ mod of the two modulation signals allows for modifying the relative amplitudes of the two MW tones. This is usually necessary to establish the condition Ω 1 = Ω 2 , as our MW antenna does not deliver a fully linearly polarized MW field to the NV centre.
As demonstrated earlier, we can only define a time-independent global phase Φ if the closed-contour condition ω 1 + ω 3 = ω 2 is fulfilled. To ensure that this is always the case we choose ω c /2π = D 0 and ω mod = ω 3 /2 with ω 3 being the eigenfrequency of our mechanical resonator. The global phase Φ = φ 1 + φ 3 − φ 2 becomes Φ = φ 3 − (φ mod + π) (34) under such conditions and for 0 ≤ φ mod < π/2. The global phase Φ can therefore be controlled by changing the individual phase φ 3 of the sinusoidal signal that drives the mechanical actuation of our diamond resonator.
To create the phase-locked driving fields experimentally, we connect the MW generator (Stanford Research Systems, SRS384), the function generator driving the piezo for mechanical actuation (Keysight, 33522A) and the function generator that supplies the IQ modulation signals (Keysight, 33622B) to the same 10 MHz reference signal. To set the global phase to a reproducible value, the output of the piezo function generator is triggered via a software command. Upon receiving the software trigger, it emits another trigger pulse which starts the output of our IQ modulation function generator (Fig. S7). During our experiment mechanical actuation of our resonator is always active and MW pulses are created by employing a MW switch (MiniCircuits, ZASWA-2-50DR+) with a rise-time of 2 ns, controlled via digital pulses from our fast pulse generator card. | 2018-02-13T19:11:57.000Z | 2018-02-13T00:00:00.000 | {
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14614848 | pes2o/s2orc | v3-fos-license | Food niche overlap among neotropical frugivorous bats in Costa Rica
: Food habits of 15 species of frugivorous bats were studied at La Selva Biological Station, Costa Rica. Eight hundred and fifty-four (854) fecal samples and 169 samples from fruit parts and seeds discarded by bats beneath feeding roosts were analyzed. During eight months of study, 47 fruit species consumed by bats were identified. Five plant genera ( Cecropia, Ficus, Piper, Solanum , and Vismia ) constituted 85% of all plants found in fecal samples. Feeding niche breadth differed significantly among the six most common species of frugivorous bats ( Artibeus jamaicensis, Carollia sowelli, C. castanea, C. perspicillata, Dermanura sp ., and Glossophaga commissarisi) . All species, except for Dermanura sp., showed a diet dominated by one or two plant species. This suggests a pattern of resource partitioning at a generic level, in which Carollia consumed mainly Piper , Artibeus consumed Ficus and Cecropia , and Glossophaga consumed Vismia . Cluster analysis revealed higher values of food niche overlap in congeneric species than among species of different genera. Results show that if food is a limiting factor, mechanisms other than trophic selection must reduce interspecific interference or competition for food in this frugivorous bat guild. Rev. Biol. Trop. 55 (1): 301-313. Epub 2007
In tropical environments, nearly 80% of plants produce fruits dispersed by vertebrates (Frankie et al. 1974, Janzen 1978, Opler et al. 1980, Charles-Dominique 1991, Ganesh and Davidar 2001. In some tropical areas, over 90% of pioneer plants produce fruits eaten by bats and birds (Charles-Dominique 1986). Plants dispersed by bats produce green fruits externally located on the plant upon maturing to favor their removal by flying animals (Charles-Dominique 1991). Bats are considered instrumental in regeneration of forests on abandoned mining areas (Parrotta et al. 1997), islands (Shanahan et al. 2001), and agricultural areas (Galindo 1998, Medellin and Gaona 1999, Galindo et al. 2000. Bats are also affected by extensive alteration of insectivorous and nectarivorous bats. Frugivory plays an important role in determining bat community structure in the tropics (Heithaus et al. 1975). Competition for food resources is thought to be a key element in bat community structure (McNab 1971, Fleming et al. 1972, Sosa and Soriano 1993, Muñoz-Romo et al. 2005. Fleming (1986a) proposed that mutualistic relations between bats and plants are very important in determining structure of bat communities. However, empirical data are needed to support or refute ideas proposed by the above-mentioned authors. The objective of this study is to evaluate trophic assemblages of frugivorous bats by studying their food habits at La Selva Biological Station, Costa Rica (LSBS).
MATERIALS AND METHODS
Study area: LSBS is administered by the Organization for Tropical Studies (OTS) and is located approximately 2 km east of the town of Puerto Viejo de Sarapiqui, Heredia Province, Costa Rica (10 o 26' N, 83 o 59' W). LSBS, is 1536 ha in size and located at the base of the Central Volcanic Mountain Range with an elevation varying between 35 and 137 m.a.s.l. (McDade and Hartshorn 1994). LSBS contains a mosaic of forest types in successional states within the tropical humid forest and premontane rain forest life zones (Hartshorn and Hammel 1994). Annual precipitation is 3962 mm with the greatest amount (over 400 mm/mo) falling between June and July and November and December while the least falls between February and April. Average monthly temperature is 25.8 o C with little monthly variation (Sanford et al. 1994).
Sample sites: using aerial photographs, topographic maps and field visits, four sites were selected which provided the greatest variability in secondary forest types. The vegetation types were: a) early successional pastures, b) young secondary forest, c) abandoned plantations, and d) mature secondary forest (Hartshorn and Hammel 1994).
Captures: Between January and August 1995, each site was sampled on a monthly basis for bat species and numbers. Each sampling event consisted of four consecutive capture nights between 17:30 and 24:00 hours using four to six mistnets, each measuring 18.6 x 2.7 m and with a 4 cm 2 mesh (Avinet Inc., Dryden, New York 13053-1103). Each bat specimen captured was identified, measured, weighed, sexed, and reproductive condition and age class were determined. Age class was determined for each bat by considering fur condition and color, corporal mass and level of fusion between metacarpal epiphyses.
Food habits: diet was studied by collecting feces from mistnetted individuals and feces and fruit parts found under resting bat tents and feeding perches (Thomas 1988). Individuals captured in mistnets were placed in a clean cloth bag for up to two hours to obtain fecal samples and then liberated. Each fecal sample was stored in an individual waxed paper envelope and the following information was taken: identity number, species, capture site and date. Also, piles of pulp and seed parts regurgitated during bat feeding (Handley et al. 1991) were found at resting or sleeping perches in hollow trees, underneath foliage and in leaf-tents used by bats of tribe Stenodermatini y (Timm 1987). To collect fruit and seed samples, a plastic screen supported by four stakes (so it did not touch the ground) was placed in the afternoon under each perch and checked the following morning. All fruit and seed samples were placed in waxed paper envelopes, and the following data were taken: date, perch location, perch type and bat species (when possible). Samples were left drying at room temperature inside the envelopes.
Reference collection of fruits and seeds:
during the study period, a reference collection was made of fruits and seeds found in the sampled forest types. Monthly collections of fruiting plants potentially eaten by bats were placed in the herbarium at LSBS. Fruit and seed samples preserved in 70% ethanol formed the reference fruit and seed collection. Plant species were identified by botanists and a reference collection deposited in the herbarium at the Instituto Nacional de Biodiversidad (INBio). A stereoscope was used to compare fruit and seed parts found in bat feces to reference collections. In difficult cases, taxonomists identified plant species. Insects were included in the analysis. Presence or absence of insects and plant species was noted for each sample. A sample was defined as presence of one or more seeds of a determined plant or insect parts in each fecal sample.
Data analysis:
The program NICHE (Krebs 1989) was used to calculate food niche breadth and overlap indices. To estimate food niche breadth, Levin´s standardized measurement was used (Colwell and Futuyma 1971). Pianka´s (1973) symmetric equation was used to estimate food niche overlap index. This index varies between 0 (no overlap) to 1 (total overlap). Indices were recalculated using the jackknife method that estimated 95% confidence intervals (Sokal andRohlf 1981, Krebs 1989). The jackknife index of food niche breadth was compared for the six most frequently captured bat species, with a Kruskal-Wallis test (Sokal and Rohlf 1981). A Kolmogorov-Smirnov test (Sokal and Rohlf 1981) was used to determine if differential consumption existed between principal plant genera in a bat species diet. For the tests, the statistical package STATGRAPHICS PLUS was used (Statistical Graphics Corporation 1994). Using feeding niche overlap indices, a trophic overlap matrix was made. Based on this matrix, a cluster analysis was performed (Jaksic and Medel 1987). The results of this analysis were graphed as a phenogram of trophic niche overlap comparing the nine most commonly captured frugivorous bat species. For cluster analysis, non-weighted arithmetic means employing SYSTAT for Windows were used (Systat Incorporate 1992).
RESULTS
Captures: during eight months between January and August 1995, 1 426 bats were captured at the four sample sites. Of total captures, 1 316 individuals (92%) were of 15 frugivorous bat species. At each sample site, monthly sampling effort was 144 net hours (SD = 15.3 net hours) for a total effort of 1 152 net hours with bat captures. An average 1.14 bats were captured per net hour.
Food habits: the captured 15 species of frugivorous bats produced 854 fecal samples (64.8%). One hundred and sixty-nine (169) fecal samples and food parts were found under perches (Table 1). About 92% of fecal samples came from the six bat species (Artibeus jamaicensis, Carollia sowelli, C. castanea, C. perspicillata, Dermanura sp. and Glossophaga commissarisi) captured most frequently at LSBS. Fecal samples from the remaining nine bat species were rare or infrequent (Table 1).
During the study, fruit or seeds of 47 identified plant species were consumed by one or more of the 15 frugivorous bat species in LSBS. Forty-two plant species were identified in bat feces (Table 2) and five additional plant species were found in feces or fruits ejected under tents and feeding stations (Dipteryx panamensis, Hernandia stenura, Quararibea parvifolia, Symphonia globulifera and Spondias radlkoferi). Seeds of four shrub species (Piper sancti-felicis, P. auritum, P. multiplinervium, Vismia panamensis) and a cecropia (Cecropia obtusifolia) made up over 50% of the samples ( Table 2). The genus Piper was most commonly found (55%), while Vismia was a distant second (8.5%).
Fruit and seed species richness consumed by frugivorous bats was greater during the dry season than the wet season (Table 3). However the proportion of samples declined during the dry season. Total numbers of samples of the most common plant genera in the feces (Ficus, Piper, Solanum, and Vismia) for the six most commonly captured bat species were significantly different (Kolmogorov-Smirnov D = 1.73, p = 0.0049).
April and May were the months of greatest reproductive activity for lactating or pregnant females. These events were associated with an increase in consumption of two food items (Fig. 2). A significant positive correlation was found between the number of female reproductive events and number of insects consumed (R 2 = 0.821, p < 0.05) and Vismia panamensis, an understory shrub (R 2 = 0.892, p < 0.05).
Other food items showed no significant relationship between reproductive events and consumption rate.
DISCUSSION
Bat food niches: at least 18 frugivorous bat species coexist at LSBS. This is 28% of LSBS's reported bat species (Howell and Burch 1974, Gardner 1977, Levey et al. 1994, Timm 1994. McNab (1971) stated that two sympatric species coexist in equilibrium only if sufficient differences exist between their ecological niches. Food is a parameter in the bat ecological niche that can be used to test McNab's (1971) hypothesis (Fleming et al.1972). In this study, frugivorous bat seed and fruit consumption patterns for 47 fruit species differed among bat species. Food habit indices differed significantly among the most common frugivorous bat species at LSBS and significant differences were found in fruit consumption patterns between bats at the generic level (Table 2). Frugivorous bats in LSBS most frequently consumed common plant taxon (genus or species) and supplemented their diet with lessfrequently consumed species. Seeds from the genus Piper appeared six times more frequently in feces than Vismia, the next most common plant genus. Only four of the 15 bat species studied did not consume Piper fruits, indicating its importance in frugivorous bat diets in LSBS.
According to Fleming (1988), bats favor seeds and fruits from plant species fruiting year-round, because they offer a predictable food source. Bats complement this diet with other less stable food types, which vary spatially and temporally. In LSBS, Artibeus consumed principally Ficus and Cecropia, Carollia consumed Piper, and G. commissarisi consumed Vismia. These plant genera in LSBS produce fruit all year (Hammel 1986a, Hammel 1986b, Greig 1993a, Greig 1993b. We conclude that (Krebs 1989 diets. However, our study (and others) found that congeneric bat species have similar diets (Tamsitt, 1967, Fleming et al. 1972, Marinho-Filho 1991. Therefore, high trophic niche overlap values between congeneric species suggests that the potential exists for competition or that species coexist in apparent equilibrium and other niche dimensions are involved in resource partitioning. Gorchov et al. (1995) found little overlap in diet between 31 and 29 species of frugivorous bats and birds, while Muller and Reis (1993) found partitioning of in LSBS, Artibeus, Carollia, and Glossophaga behave as proposed by Fleming (1988).
Food niche overlap: the six most abundant frugivorous bat species in LSBS showed greatest niche overlap between congeners; the same observed with the three least abundant species. This contradicts Hutchinson (1959) who noted that sympatric congeners usually differ in their food resources among the most common bat species they studied.
Cluster analysis: two groups of frugivorous bat food habits in LSBS were revealed (Fig. 1). The first is composed of four species, three from the genus Carollia and G. commissarisi. The second consists of five species from the subfamily Stenoderminae. These groups coincide with functional understory and canopy bat frugivore guilds described by Bonaccorso (1979) for Barro Colorado Island, Panama. However, more trophic similarity exists between species with similar weights. Perhaps differences in the size of food items consumed by bats could explain differences in bat diets when similar weights and plants are consumed (Bonaccorso 1979). Among the Stenoderminae, Dermanura sp. had a more diverse diet and one less dominated by members of the genus Ficus. Dermanura sp., together with G. commissarissi, constituted a group of understory generalists.
Seasonality of fruiting patterns: a possible factor explaining seasonality of fruiting patterns in tropical forests is seed disperser competition (Snow 1965). This study partially confirms the hypothesis that seed dispersers have seasonal food habits. Bat diet in LSBS was more diverse in the dry season (January-April) then wet season (May-August). Also, there were fewer items/feces found during the dry season because it is the season of least fruit abundance in LSBS (Opler et al. 1980, Newstrom et al. 1994. This coincides with optimal foraging strategy predictions (Pyke et al 1977, Anderson 1983).
Bat reproductive cycles: an important consequence of phenological seasonality is found in bat reproductive cycles (Racey 1988). Kunz (1980) found pregnancy and lactation were more energetic expensive for female bats. During lactation in rodents, females increase energetic demands between 66-133% (Randolph et al. 1977, Millar 1978. Although Dinerstein (1981) came to different conclusions working in cloud forest environments, insects as a food item were associated with frugivorous bats' reproductive events in LSBS. Correlations observed between female bat reproductive events and insect (high energy and protein sources) and V. panamensis consumption suggests that both can be critical resources for frugivorous bats during reproduction in LSBS, particularly during lactation (Bonaccorso 1979, Autino andBarquez 1993). We feel that this important discovery with frugivorous bats has been underemphasized in the literature.
Potential sources of error: lack of collecting data for at least a year could contribute to underestimating the value of some food sources for certain bat species and not finding "keystone" food resources such as fig (Korine et al. 2000). Also subestimating or not accounting for undigested fruits because seeds were undetected was a possible source of error. Four species of seeds found under feeding stations and tents were not detected in fecal samples. However, large-seeded species (> 5 mm) were more frequently represented under tents and feeding stations than from feces because they were ejected and not swallowed. Perhaps bats use feeding perches to manipulate large fruits or those requiring more time to be ingested (Thomas 1988). Another source of error is differential defecation rate of bat species. For example, 80% of captured C. castanea individuals yielded feces, compared to only 42% of A. jamaicensis individuals. A similar tendency is seen with the other carolliine species (C. sowelli, C. perspicillata) and Stenodermatini (A. lituratus, Dermanura sp., Chiroderma villosum, V. nymphaea, V. helleri). Perhaps these individual bat species manipulate fruit differently. Fleming (1986b) found that Carollia species consume fruits in two minutes or less, ingesting most seeds. However, A. jamaicensis take over ten minutes to process a fruit and ingests few seeds. This partially explains differences in fecal sample size when similar numbers of a species were captured and has implications for bat seed dispersal efficacy. A fourth source of error may be related to differences in nutritional values of food sources (Wendeln et al. 2000), which could greatly influence food preferences, reproduction and other variables. A final source of error was not accounting for vertical stratification of bat communities (Bernard 2001, Kalko andHandley 2001) as we only sampled the lower strata.
Conservation of bats, rain forests and regeneration: conserving and managing plants used as food by frugivorous bats is important to maintain populations of frugivorous bats and ensure plant species dispersal. This study found that bats consume many pioneer plant species and thus, some bat species could be favored by increases in secondary forest areas. However, bats also depend on primary forest areas that provide needed resources, such as refuge, feeding sites and food. As Kalko and Handley (2001) and Cosson et al. (1999) have discussed, habitat alteration affects understory fruit bat species more than canopy species. Brosset et al. (1996) found that over 60% of bat species found in forested habitats were absent from deforested areas in French Guiana. Both habitats are necessary for bat conservation. And regeneration of tropical forests is likewise dependent on dispersers such as bats. of Agriculture (Cooperative Agreement 1275-22000-189-04S with the Milwaukee Public Museum). This publication is part of the "Theobroma cacao: Biodiversity in full and partial canopies" research project, coordinated by the Milwaukee Public Museum. | 2017-03-31T04:00:41.164Z | 2003-05-17T00:00:00.000 | {
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233870154 | pes2o/s2orc | v3-fos-license | Myelodysplastic syndrome patients display alterations in their immune status reflected by increased PD‐L1‐expressing stem cells and highly dynamic exhausted T‐cell frequencies
Little data are available for the expression of immune checkpoint (IC) molecules within myelodysplastic syndrome (MDS). Here, we report increased PD‐L1+CD34+CD38− and PD‐L1+CD34+CD38+ stem cell frequencies within MDS patients compared to stem cell recipients in remission. Additionally, we observed exceedingly similar PD1+ and Tim‐3+ T‐cell frequencies between acute myeloid leukaemia (AML) and MDS samples that were elevated compared to patients in remission. Furthermore, we found highly dynamic Tim‐3+ and PD1+ T‐cell frequencies within serial samples of relapsing MDS with excess blasts (MDS‐EB II) patients, correlating with further disease markers. These findings support the idea of a potential successful implementation of IC inhibitor treatment in suitable MDS patients.
Introduction
Myelodysplastic syndromes (MDS) are a heterogeneous group of haematological malignancies characterized by ineffective haematopoiesis and an increased risk for progression to acute myeloid leukaemia (AML). Considering the mostly advanced age of MDS patients, alternative, less invasive treatment options, besides haematopoietic stem cell transplantations (hSCT), are urgently needed. Cancer pathogenesis is associated with exhaustion of cytotoxic lymphocytes and administration of immune checkpoint inhibitors (ICIs), that restore the function of cytotoxic cells, proved to be successful in various tumour entities (reviewed in Refs 1,2). Importantly, ICIs have less severe side effects than conventional therapies, even in elderly patients (reviewed in Refs 3,4). Several studies report an exhausted T-cell phenotype in AML, especially in relapsing patients (i.e. Refs 5,6). Interestingly, only very few studies have analysed immune checkpoint (IC) molecules in MDS. Furthermore, these studies are generally based on peripheral blood (PB) cells and/or cryopreserved samples. We therefore aimed to analyse the expression of IC molecules on haematopoietic stem cells and bone marrow (BM)-infiltrating T cells that were freshly isolated via red blood cell lysis and directly subjected to flow cytometry. The study was approved by the local ethics committee (study number 3973, 3768 and 2020-1222) and informed written consent was obtained from all participants.
Seven MDS and nine AML samples were obtained at the Department of Haematology, Oncology and Clinical Immunology from the University Hospital D€ usseldorf. Six hSCT recipients who remained in remission for more than six months served as controls ('hSCT remission >6 months'). Patient characteristics are shown in Table SI.
We observed significantly increased proportions of PD-L1 + CD34 + stem cells in MDS compared to hSCT recipients in remission for both the CD38 À and CD38 + subset (CD38 À subset: P = 0Á0127 and CD38 + subset: P = 0Á0336; Fig 1A, B). This is in accordance with the study from Yang and colleagues, who reported increased levels of PD-L1 mRNA transcripts in sorted CD34 + BM cells of MDS patients. 7 The trend of increased PD-L1 expression could also be observed for AML patients, although not significantly. Interestingly, in MDS/AML patients the CD38 + subset showed significantly higher PD-L1 + cell proportions compared to the CD38 À subset (P = 0Á0135, Fig 1C), from which leukaemic stem cells (LSC) originate in both AML and MDS. 8,9 This is in line with previous reports stating that in AML and MDS PD-L1 is more commonly expressed by non-tumour cells. 10 Nevertheless, these findings imply that a PD1/PD-L1 blockage could be successful not only in the treatment of AML, but also of MDS, as the LSCs comprising the CD34 + CD38 À subset still showed higher PD-L1 levels than in hSCT recipients in remission. Myelodysplastic syndrome (MDS) samples showed increased levels of PD-L1-expressing CD34 + cells. White blood cells (WBCs) from MDS, acute myeloid leukaemia (AML) and haematopoietic stem cell transplantation (hSCT) recipients who remained in remission for more than six months were isolated via red blood cell lysis and directly subjected to flow cytometry analysis. Calculated cell frequencies were based upon to the 'gate of interest' (GOI), which contained all WBCs except for granulocytes and cell debris that were excluded by size and granularity via side scatter area (SSC-A) and forward scatter area (FSC-A). The gating strategy can be found in Figure S1. (A, B) A significant increase of PD-L1-expressing CD34 + cells in MDS compared to hSCT recipients in remission could be observed for both the CD34 + CD38 À (A, P = 0Á0127) and the CD34 + CD38 + cell subset (B, P = 0Á0336). (C) CD34 + progenitor cells of MDS and AML patients were analysed. PD-L1 + CD34 + CD38 À cells showed significantly decreased proportions compared to PD-L1 + CD34 + CD38 + cells (P = 0Á0135). (D) T cell frequencies were comparable between all three groups. (F-H) There were no significant differences in the Tim-3 + CD4 T cell (E), Tim-3 + CD8 T cell (F), PD1 + CD4 T cell (G) and PD1 + CD8 T cell frequencies (H). (I, J) Frequencies of PD1-expressing CD8 T cells of all participating hSCT recipients were correlated with the corresponding frequencies of PD-L1-expressing CD34 + stem and progenitor cells. PD-L1 + CD34 + CD38 À (I) or PD-L1 + CD34 + CD38 + (J) stem and progenitor cells showed a moderate positive correlation with PD1 + CD8 T cells. Statistical analyses were performed using GraphPad Prism 9.0.0 software (GraphPad Software, San Diego, CA, USA). For the comparison of two groups a parametric paired t test was performed. Three groups were compared by Welch's ANOVA followed by Dunnett's T3 multiple comparison. Correlation between two variables was determined by simple linear regression. P < 0Á05 was considered statistically significant.
Overall T-cell as well as PD1 + and Tim-3 + CD4 and CD8 T-cell frequencies were comparable between all three groups ( Fig 1D-H). In turn, several studies reported increased PD1 + and Tim-3 + CD8 T-cell frequencies in AML. 5,6 In line with these findings, we could also observe a trend towards elevated PD1 + and Tim-3 + CD8 T-cell frequencies within AML. Additionally, the corresponding T-cell frequencies within MDS were at the same levels as in AML (MDS vs AML: P = 0Á9992 for both PD1 and Tim-3, Fig 1F, H). This high similarity of PD1 + CD8 T cells between MDS and AML let us assume that exhausted CD8 T cells might also be a frequent characteristic within both AML and MDS patients. This could be supported by the finding, that PD1 + CD8 T cells and PD-L1 + stem and progenitor cells in hSCT recipients showed a moderate positive correlation (CD34 + CD38 À subset: R 2 = 0Á2682, P = 0Á0162 and CD34 + CD38 + subset: Fig 1I, J).
Considering the afore-mentioned data, we questioned whether PD1 + and Tim-3 + T-cell and PD-L1 + CD34 + stem cell frequencies are associated with disease progression. We therefore correlated flow cytometry data with disease markers like BM blast count, occurrence of dysplastic cells and minimal residual disease (MRD) markers in longitudinal samples of three hSCT recipients with MDS-EB II. The first patient (Fig 2A) received conditioning regimens but did not reach remission pre hSCT. Simultaneously, 11% of CD34 + progenitors were PD-L1 positive. As early as 30 days post hSCT, the patient showed a molecular relapse when the initially diagnosed trisomy 8 was detectable again. At this time point, the blast count remained below 5% but PD-L1 + CD34 + progenitors were similar to the pre-hSCT value (13%). Here, the PD1 + T-cell frequencies were at their highest level in the monitoring period and decreased with administration of eight cycles of azacytidine from 55Á7% to 17Á3% within the CD4, and 41Á0% to 19Á8% within the CD8 T-cell subset. Wilms tumour protein (WT1) PB expression decreased in the same manner. Lastly, PD1 + and Tim-3 + T cells as well as WT1 expression slightly increased again. Here, PD-L1 + CD34 + progenitors were hardly detectable. We therefore would recommend closer monitoring of this patient as increased Tim-3 + PD1 + T cells were described in the PB of relapsed AML patients. 6 The second patient (Fig 2B) also suffered from an ongoing disease at the time point of hSCT. Already 30 days post hSCT, he showed trilineage dysplasia and a donor chimaerism drop to 72Á5% (not shown). Consequently, immunosuppressive treatment was reduced to initiate a graft-versus-leukaemia effect. Thereupon, the patient reached remission with no signs of dysplasia or increased blast count. Moreover, donor chimaerism was at 93Á5% at day 60 post hSCT. However, 96 days post hSCT the blast count was at 5% and dysplastic cells could be observed once again. Finally, the patient relapsed into AML 136 days post hSCT with a blast excess of 47 % and detection of the initially diagnosed RUNX1 mutation. PD1 + and Tim-3 + T cells, PD-L1 + CD34 + blasts, blast count and WT1 expression tightly correlated inasmuch as they all increased simultaneously and donor chimaerism dropped to 12Á8% (not shown). Administration of the first cycle of decitabine and hydroxycarbamide led to a drop in all mentioned parameters except donor chimaerism, which increased again (71Á4%). Nevertheless, the proportions of Tim-3 + and PD1 + CD4 and CD8 T cells remained unexceptionally higher than the proportions at 96 days post hSCT when the first signs of dysplasia were detected before the patient relapsed into an AML.
The third patient stayed in remission over the entire monitoring period (Fig 2C). PD1 + and Tim-3 + T cells, blast count and WT1 expression were comparable between the three analysed time points. Especially PD1 + T-cell frequencies remained quite constant with frequencies fluctuating between 36Á7% and 39Á6% and 20Á1% and 24Á7% within CD4 and CD8 T cells respectively. The data presented here impressively showed that MDS-EB II disease progression included changes in the immune status. Independent of baseline levels and in contrast to the patient in remission, the relapsing patients showed highly dynamic Tim-3 + and PD1 + T-cell frequencies. Importantly, we observed these fluctuations not only within haematological but also in molecular relapse. Despite their successful administration in certain tumours, preliminary results of ICI monotherapy as single-agent treatment with nivolumab (anti-PD1) did not show an effective response in MDS patients. Yet, ICI treatment in combination with hypomethylating agents (HMAs) showed promising clinical activity in untreated higher-risk MDS and AML. 11,12 HMAs are known to increase PD-L1 expression in both AML and MDS. 7,13 Furthermore, it has been shown that tumour mutational burden (TMB) correlated with a clinical benefit from ICI therapy in several tumours, i.a. in non-small-cell lung carcinoma. 14 Interestingly, PD-L1 expression seems to be thereby independent of TMB, e.g. in squamous cell lung carcinoma. 15 Consequently, more criteria should be discussed for how to preselect AML/MDS patients who could benefit from ICIs in order to improve efficacy and to reduce therapy-related side effects. A therapy accompanying close monitoring of exhausted T cells could be a tool for preselection of suitable AML/MDS patients who could benefit from an ICI therapy. Furthermore, it could be used for resistance control in patients treated with HMAs. In addition, algorithms such as vi-SNE or FlowSOM should be considered in the analysis of high-dimensional flow cytometry data of serial samples, as they are able to identify residual leukaemic stem cells. 16 Finally, as BM aspirations are more invasive, cost-intensive and time-consuming than blood sampling, we were interested in determining if T-cell subsets in PB and BM are comparable and could therefore be used for a therapy accompanying monitoring of exhausted T cells. As there were no matched MDS samples available, we analysed matched samples from four therapy-na€ ıve AML patients. There were no significant differences in the proportions of exhausted CD4 and CD8 T cells as well as CD8 memory T-cell subsets Figure S2). Consequently, we suggest that PB samples are in most cases sufficient for disease monitoring of exhausted T cells.
In summary, our data indicate that the inhibition of T cells by haematopoietic stem cells could be an underlying mechanism not only in AML, but also in MDS development. Furthermore, we could show that an intra-individual analysis of T-cell populations could be an easily applicable approach to preselect MDS and AML patients who might benefit from the use of ICIs, including resistance monitoring. Despite these encouraging findings, the present study has some limitations such as the small group size and the heterogeneous group characteristics in terms of MDS and AML subtype, treatment history, genetic aberrations and blast proportions (see Table SI). To ensure that the observations are reproducible in other subtypes and therapies, serial samples from AML/MDS patients beyond MDS-EB II who received hSCT or other types of treatment need to be analysed prospectively. As stated by Daver et al., pre-therapy PD-L1 on blasts does not correspond with a response to combination therapy of HMAs with PD-1 inhibitors within de novo and secondary AML. 12 Therefore, the prospective analysis Exhausted T-cell frequencies are highly dynamic in MDS with excess blasts (MDS-EB II) relapse and correlate with disease markers. Bone marrow (BM) WBCs from three MDS-EB II patients were isolated via red blood cell lysis at serial time points around hSCT and were directly subjected to flow cytometry analysis. Flow cytometry data of PD1 + (blue lines) and Tim-3 + (purple lines) CD4 (light lines) and CD8 (dark lines) T-cell frequencies, as well as PD-L1 + CD34 + blasts (red lines) were retrospectively correlated to BM blast count (green lines) and PB Wilms tumour protein (WT1) expression which were routinely elicited during disease monitoring. (A) Disease course of a MDS-EB II patient who suffered from molecular relapse is shown. Trisomy 8 (detected at diagnosis) was observed as early as the first bone marrow aspiration 30 days after hSCT. At this time point, PD1 + T cells showed the highest frequencies, which decreased with administration of eight cycles of azacytidine. PB WT1 expression decreased in the same manner. Lastly, PD1-and Tim-3-expressing T cells as well as WT1 expression slightly increased again. (B) Disease course of a MDS-EB II patient who suffered from haematological relapse and further progression to acute myeloid leukaemia (AML) is shown. Proportions of PD1-and Tim-3-expressing T cells, PD-L1-expressing blasts, blast count and WT1 expression are tightly correlated in this patient, increasing simultaneously with signs of dysplasia and peaking when the patient progressed to AML. Administration of the first cycle of decitabine and hydroxycarbamide led to a drop in all mentioned parameters. (C) Disease course of a MDS-EB II patient who stayed in remission over the entire monitoring period is shown. PD1-and Tim-3-expressing T cells and blast count were relatively comparable between the three analysed time points.
should also include patients receiving ICI treatment in order to confirm the practicality of real-time monitoring for preselecting patients for ICI treatment. Additionally, a functional analysis should be performed to determine whether the increased exhausted immunophenotype reported here directly correlates with a dysfunctional or impaired immune system. Cooperation with specialists in outpatient health care settings, where lower risk MDS is treated more frequently than in the clinics, should be therefore highly encouraged.
Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Table SI. Patient characteristics. Fig S1. Gating strategy for the identification of immune checkpoint molecules on T and CD34 + cells. The gate of interest (GOI) contained all cells with the exception of granulocytes and cell debris, which were excluded by size and granularity via forward scatter area (FSC-A) and side scatter area (SSC-A). T cells (CD3 + SSC-A low ), CD34 + CD38 À and CD34 + CD38 + haematopoietic stem and progenitor cells were identified prior to gating of (A) PD-L1 on CD34 + cells and (B) PD1 and Tim-3 on CD4 and CD8 T cells. Fig S2. T cell subsets in peripheral blood and bone marrow sample of therapy-na€ ıve AML patients are comparable. T-cell subsets of matched PB and BM samples from four therapy-na€ ıve AML patients were analysed. There were no significant differences in A CD4 T cell, B CD8 T cell, C PD1 + CD4 T cell, D PD1 + CD8 T cell, E Tim-3 + CD4 T cell and F Tim-3 + CD8 T-cell frequencies. G A representative staining of CD8 Central Memory (CM, CCR7 + CD45RA À ), Effector Memory (EM, CCR7 À CD45RA À ), na€ ıve-like (CCR7 + CD45RA + ) and Terminally differentiated Effector Memory (TEMRA, CCR7 À CD45RA + ) T cells is shown. H CD8 CM, EM, na€ ıve-like and TEMRA T cells were comparable between matched PB and BM samples. Each symbol represents one patient. | 2021-05-07T06:22:55.901Z | 2021-05-06T00:00:00.000 | {
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119104205 | pes2o/s2orc | v3-fos-license | Odor lateralization and spatial localization: Null effects of blindness
People can navigate through an environment using different sensory information, including olfactory cues. Correct intranasal localization and external location of odors can be learned, and some people are able to lateralize olfactory stimuli above chance, which raises the question: What determines the spectrum of olfactory localization abilities. Here, we explored whether odor lateralization and localization abilities are increased in the course of sensory compensation. In a series of studies, we combined two different aspects of odor localization. Study 1 compared abilities of 69 blind people (Mage = 41 ± 1.6 years; 32 females) and 45 sighted controls (Mage = 38.3 ± 2.1 years; 25 females) to correctly lateralize eucalyptol, an odorant with a strong trigeminal component, presented to either nostril. Studies 2 and 3 involved a more ecologically valid task, namely spatial localization of olfactory stimuli. In Study 2, 13 blind individuals (Mage = 28.5 ± 3.5 years; seven females) and 16 sighted controls (Mage = 34.9 ± 3.2 years; ten females) tried to localize a single odorant, while in Study 3, 97 blind individuals (Mage = 43.1 ± .5 years; 48 females) and 47 sighted controls (Mage = 38.7 ± .7 years; 27 females) attempted to localize a single target odor in an experimental olfactory space comprising four different odorants. Blind and sighted subjects did not differ in their abilities to lateralize and to localize odors, and their performance across all tasks suggests that odor lateralization and localization are important for navigation in an environment regardless of visual status.
Introduction
People can navigate through an environment using different sensory information. Although for senses like vision and audition it is obvious that bilateral perception contributes to accurate spatial orientation, the functions of bilateral olfactory perception are not fully clear. Von Békésy (1964) suggested that time delay and internostril intensity differences can help people determine the location of an odor source. A higher concentration of an odorant would then indicate shorter distance from the odor source. In this case, bilateral olfactory cues would contribute to accurate spatial orientation and localization.
Studies investigating human ability to localize odors intranasally suggest that lateralization (i.e., correct identification of the nostril receiving olfactory stimulation) of odorants activating exclusively the olfactory receptors is difficult. While a few studies showed that people are able to lateralize phenyl ethyl alcohol (PEA) (Kobal & Hummel, 1992;Porter, Anand, Johnson, Khan, & Sobel, 2005), most studies yield opposite results (Frasnelli, Charbonneau, Collignon, & Lepore, 2009;Frasnelli, La Buissonnière Ariza, Collignon, & Lepore, 2010;Moessnang, Finkelmeyer, Vossen, Schneider, & Habel, 2011;Radil & Wysocki, 1998;Wysocki, Cowart, & Radil, 2003). Thus, it seems that selective activation of the olfactory system is not enough for people to lateralize odors correctlyboth olfactory and trigeminal receptors of the nasal mucosa need to be involved in order to increase perception accuracy (Kleemann et al., 2009). Consequently, stimuli with a Btrigeminal component^were quite consistently found to be lateralized correctly (Frasnelli et al., 2010;Hummel, Futschik, Frasnelli, & Hüttenbrink, 2003;Kleemann et al., 2009;Kobal, Van Toller, & Hummel, 1989;Porter et al., 2005;Wysocki et al., 2003). Schneider and Schmidt (1967) observed that performance in different olfactory localization tasks was the lowest for an odor possessing the lowest trigeminal qualities of the three stimuli applied in their research (coffee). Relatedly, Croy et al. (2014) found that a few people who were able to correctly lateralize PEA in their study exhibited a significantly enhanced activation of cerebral trigeminal processing areas in response to this odorant. This finding suggests that the trigeminal system of some individuals is so sensitive that it responds to odorants that most other people find to be purely olfactory, consequently increasing the lateralization abilities of this group (Croy et al., 2014). This could also explain the inconsistencies in literature, as PEA in high concentrations can also induce trigeminal perceptions (Yang et al., 2003), a notion supported by a study showing detection of PEA by two out of 15 anosmic individuals (Doty et al., 1978).
Another group of studies explored a different spatial localization ability, namely directional smelling. For example, newborns turn away from a source of an unpleasant odor (ammonium hydroxide) (Rieser, Yonas, & Wikner, 1976), which suggests that they know where it is located. Further, in an early study by Szymanski (1920), humans were able to determine the location of an odor sourcean ability potentially relevant for creating spatial representations based on olfactory cues. This result was extended by Porter and collaborators (Porter et al., 2007), who showed that people are able to follow scent trails. Jacobs and collaborators (Jacobs, Arter, Cook, & Sulloway, 2015) demonstrated that it is possible to navigate to a certain location using only olfaction, and a study of location memory across modalities (Schifferstein, Smeets, & Postma, 2009) showed that people can learn to remember locations by smell to the same degree as by audition and touch. Participants in a study by Welge-Lussen, Looser, Westermann, and Hummel (2014) could also localize an odor source while sitting in a central position with quite high accuracy (85°at a 2-m distance), and this accuracy increased to approximately 40°at closer distances. Interestingly, the results of Welge-Lussen and collaborators (2014) were the same for a predominantly olfactory odorant (PEA) and predominantly trigeminal substance (cineol). Thus, even in the absence of intranasal location mediated exclusively by the trigeminal system, humans may be able to exploit differences in intensity or timing (von Békésy, 1964) to achieve relatively accurate direction smelling (spatial localization), even with purely olfactory stimuli. This is further suggested by decreased scenttracking accuracy in a monorhinal compared to a birhinal tracking condition (Porter et al., 2007).
Correct localization of olfactory stimuli can, however, be learned (Negoias, Aszmann, Croy, & Hummel, 2013), as following olfactory training, women were better able to correctly localize a purely olfactory stimulus (PEA). Training can also improve scent tracking (Porter et al., 2007). In addition, some people are able to lateralize olfactory stimuli above chance (Croy et al., 2014;Frasnelli et al., 2010), which raises the question of what determines the spectrum of olfactory localization abilities. Possibly, some people can develop this skill based on an exposure to odors in daily situations. An example of such a group could be blind people. Some authors hypothesize that the highly developed olfactory functions observed for some blind individuals might result from daily Bsmell training^ (Gagnon, Ismaili, Ptito, & Kupers, 2015). Here, in a series of studies we explored whether odor lateralization and localization abilities are increased in this group. Following the idea of Welge-Lüssen et al. (2014), we combined two different aspects of odor localization. First, we used the classic method used in intranasal localization tasksin Study 1 we compared the abilities of blind and sighted groups to correctly lateralize eucalyptol, an odorant with a strong trigeminal component, presented passively to either nostril. Studies 2 and 3 involved a more ecologically valid task, namely spatial localization of an external olfactory stimulus. In Study 2 subjects tried to localize a single odorant, while in Study 3 they attempted to localize a single target odor in an experimental olfactory space comprising four different odorants.
Materials and methods
Each study began with a short interview regarding the medical history of the subjects and any potential olfactory problems. Further, to ensure normal olfactory function, the participants completed a three-item olfactory screening test (Lötsch, Ultsch, & Hummel, 2016). In order to maintain identical testing conditions for all subjects, in all studies both blind and sighted participants were fitted with a Mindfold mask eliminating all incoming light and visual input without blocking the nose or forcing the eyes closed (Mindfold Inc., Boulder, CO, USA). All three studies took place in a quiet, large room under constant conditions. All doors and windows were always closed during testing, and no one entered or exited the room during the experiments. The room was thoroughly ventilated after each testing.
Ethics statement
The studies were approved by the ethics board of the Institute of Psychology, University of Wroclaw. The work was performed in accordance with the Declaration of Helsinki for Medical Research involving human subjects. All participants provided written, informed consent prior to their inclusion in the study.
Data analysis
In all studies we used IBM SPSS v. 24 software to analyze data with the alpha level set to .05.
In Study 1, for each participant we computed the proportion (percent) of correct answers by dividing it by the total number of trials taken and multiplying by 100. We compared sighted and blind individuals' mean proportions of correct answers in a eucalyptol lateralization task utilizing an independent sample t-test and referred it to the expected level of chance (50%). In Studies 2 and 3, the localization performance was defined as the absolute value of a difference between the actual position of the target odorant and supposed position of the target odorant, indicated by the participants' index finger (in degrees).
In Study 2, localization accuracy of sighted and blind individuals was compared using an independent samples t-tests. In Study 3, we investigated the spatial localization across multiple trials by means of mixed linear models (LMM) with Bsightedness^(blind vs. sighted), Btask^(head moves vs. no head moves), and Blocation of target odor^(lateral vs. central) as fixed factors, and age and ordinal number of trials as covariates. The data and applied scripts are available upon request.
Study 1
In Study 1, we investigated subjects' ability to lateralize eucalyptol, a mixed olfactory/trigeminal stimulus.
Procedure
We tested the subjects using a hand-held squeezing device (for details, see Hummel et al., 2003) consisting of two 250-ml bottles placed in a special stand. One bottle contained 15 ml of 50% eucalyptol solution (with propylene glycol used as a solvent) and one contained 15 ml of odorless propylene glycol. Each bottle had a soft, plastic spout on topthese parts were placed in the subject's nostrils. During the testing, bottles were pressed simultaneously with the squeezing device in order to deliver an identical air puff into both of the participant's nostrils at the same time. The participant's task was to answer in a two-alternative (left/right) forced choice paradigm the side of which had been stimulated with an odorous substance. The subjects received no feedback from the experimenter on the correctness of their evaluation. Stimulation of the left or right nostril followed a pseudorandomized sequence; the participants received between ten and 20 air puffs, and each nostril was stimulated between five and ten times. The subjects were told that they could report fatigue and withdraw from further participation in the experiment after the minimum number of ten air puffs had been reached.
Results
The results of the blind and sighted participants are illustrated in Fig. 1 (panel A). The tested model revealed no significant differences between sighted and blind participants in terms of their ability to lateralize eucalyptol, t(1,112)=-.13, p=.89. Both groups significantly exceeded the 50% level of chance when lateralizing eucalyptol. Results obtained by blind and sighted individuals were 77.6 ± 2.3 (t(44)=8.5, p<.001) and 77.1 ± 23.2 (t(68)=11.9, p<.001), respectively.
Study 2
In Study 2, we examined subjects' ability to localize olfactory sources in an experimental setup more similar to everyday life. The subjects were required to localize one odor that was placed on a table in front of them.
The participants were asked to localize the source of an odor in a 180°space in front of them. The study was conducted by two assistantsone was responsible for handling the odorants (from now on referred to as BAssistant 1^) and the second assisted the participants (BAssistant 2^). Assistant 1 placed one 50-ml bottle containing 15 ml of the odorant (orange) on a large table. Assistant 1 marked a space where the odor was placed and noted the exact angle where the odor was presented (it was supposed to be placed at approximately 0, 60, 120, or 180°). To facilitate the exact calculation of the presentation angle, a 2 x 1 m print-out scale was placed on the table.
The blindfolded participant was invited to the room and was seated by Assistant 2 on one side of the table, in the center of the print-out scale. The subject sat comfortably upright in a rotating chair with his/her head resting on an adjustable head rest and was instructed to keep the head fixed in this position. Initially, the participant was seated with his/her back turned towards the table. Assistant 2 explained the task. The participant was asked to insert earplugs after confirming that he/she understood the task. The earplugs were to be removed after the participant was turned to face the table and received a tap on the shoulder. During a few seconds when the participant was already facing the table, but his/her ears were still blocked, Assistant 1 opened the bottle containing the odorant. Ear blocking was employed in order to avoid auditory localization of the odor source during bottle opening.
Approximately 30 s after the odorant bottle was opened, Assistant 2 asked the subject to localize the odor source. The participants were strictly instructed not to move their heads during this task, and the research assistants controlled this aspect during the task performance. The subject was instructed to sniff and indicate the location of an odor source with an extended arm and index finger; Assistant 2 noted the exact angle indicated by the participant's index finger. Task performance was calculated by subtracting the angle that a subject indicated from an angle that a smell was presented. We further analyzed the absolute value of this difference, as we did not have a hypothesis regarding the enhanced abilities on the location of the odor source to the left versus the right side of the target odor. Participants were seated approximately 50 cm from the odorant source (a distance similar to the Bclose distal condition^in Welge-Lussen et al., 2014).
Results
The results of the blind and sighted participants are illustrated in Fig. 1 (panel B), and Fig. 2 shows the distribution of scores of both participating groups. We found no significant difference between the localization accuracy of the blind (M = 37.9 ± 5.9°) and sighted participants (M = 37.5 ± 8.1°), t(27)=-.04, p=.97.
We further attempted to estimate the chance performance based on random pointing locations. We compared actual errors made by the participants with errors of a random distribution. We generated a variable consisting of random numbers ranging from 0 to 180 that described theoretical random indications of subjects and we subtracted these values from the actual locations of the odorants (it needs to be noted that the Fig. 1 Comparison between sighted and blind individuals in terms of their accuracy in lateralization (Study 1) and localization tasks (Studies 2 and 3). In the graph presenting the results of Study 1, axis Y depicts a proportion of correct lateralizations (0-100%), whereas in the graphs illustrating the findings of the Studies 2 and 3, the scale reflects 180°, which is a maximum theoretical difference between the actual and the indicated locations of the odorous substance. Empirically, the values of this difference ranged between 0 and 160°. *** denotes a significant difference (p<.001) between scores obtained by the sighted and the blind individuals as compared to the expected level of chance (50%) mean by-chance error estimates obtained in this way will vary slightly when the randomization is rerun). We used absolute values of errors of the applied random distribution and compared them with the absolute observed errors made by the subjects by means of a paired-sample t-tests. Although the difference between the mean absolute observed error (M = 37.7, SD = 27.4) and the mean absolute random error (M = 52.4, SD = 30.1) equaled almost 15°, this difference did not reach statistical significance, t(28)=-.18, p=.08. However, the number of trials for which the participants made an error below or equal to the average random error was considerable (20 out of 29), and it did not differ between the participating groups (nine trials out of 13 made by the blind subjects and 11 out of 16 made by the sighted subjects, χ 2 (1)=.01, p=.98).
Study 3
In Study 3, the same experimental setup was applied as in Study 2. This time, however, the subjects were simultaneously Fig. 2 The distribution of scores obtained by the blind and the sighted participants in Study 2. The y-axis depicts the absolute value of a difference between the actual and the indicated location of the odorous substance in angular degrees presented with four smells placed at four distinct places of the experimental board (at approximately 0, 60, 120, and 180°). We selected odors that were intense, perceptually distinct and trigeminally stimulatingcinnamon, jasmine, orange, and cloves in one condition and chocolate, eucalyptol, freesia, and strawberry in the second. The participants were assigned to odor conditions randomly, and the order of substances was pseudorandomized. Every odor was dissolved in propylene glycol in order to obtain concentrations that would seem equally intense in a pretest involving smelling the solutions from a distance of 50 cm. For every odorant bottle, we prepared a corresponding Sniffin' Stick. Assistant 2 presented the sticks to the participant one by one in a pseudorandomized order, asking him/her to indicate where the corresponding odor source was located. The participants were told that the location of the odor samples might be changed each time they pointed to a certain direction, but in reality, the positions remained the same in order to avoid odor mixing.
The participants were asked to complete two tasks. First, they were asked to localize the target odorant without moving their heads (the face was directed towards the middle of the scale and kept on a headrest; a research assistant ensured that the participants did not move). Then, they completed the same task again, but this time they were allowed to move their heads sideways.
Results
The results of the blind and sighted participants are illustrated in Fig. 1 (panels C and D) and Fig. 3 shows the distribution of scores of both participating groups across two tasks. General linear model with Bsightedness^(blind vs. sighted), Blocation of target odor^(central [60 or 120°] vs. sided [0 or 180°]), Btask^(no head movements vs. head movements) yielded a significant main effect of Blocation of target odor^in terms of accuracy in spatial localization, wherein odors located in the central positions were localized significantly more accurately (M = 37.8 ± 2.1°) than odors located in the sided positions (M = 62.1 ± 2.1°; F(1,675)=65.1, p<.001). There were no other main or interaction effects (Fs<.75,p>.39).
Similar to Study 2, we further compared our subjects' performance to the estimate of the chance performance based on random pointing locations. We found that across two tasks, the participants' actual average error (M=50, SD=38.9) was significantly lower than the randomly generated average error (M=54.7, SD=40.3), t(682)=-2.4, p=.016. The number of trials for which the participants made an error below or equal to the average random error did not differ between the blind and the sighted groups (271 out of 452 trials made by the blind subjects and 136 out of 231 trials made by the sighted subjects, χ 2 (1)=.074, p=.79).
To further explore the significant effect of the target odor location, we estimated the chance performance based on random pointing locations separately for the central and sided locations. For sided locations, the participants' actual average error (M=62.3, SD=43.2) did not differ significantly from the randomly generated average error (M=65.5, SD=45.7), t(344)=-.97, p=.34. The number of trials for which the participants made an error below or equal to the average random error of 65.5°when the target was on the side did not differ between the blind and the sighted groups (133 out of 234 trials made by the blind subjects and 61 out of 111 trials made by the sighted subjects, χ 2 (1)=.11, p=.74).
For central locations, the participants' actual average error (M=37.3, SD=28.8) was significantly lower than the randomly generated average error (M=43.8, SD=30.3), t(337)=-2.77, p=.006. The number of trials for which the participants made an error below or equal to the average random error of 43.8°w hen the target was in the central position did not differ between the blind and the sighted groups (127 out of 218 trials made by the blind subjects and 64 out of 120 trials made by the sighted subjects, χ2(1)=.76, p=.38).
Discussion
Our study showed that a large sample of blind subjects did not differ from sighted people in lateralization and localization of odorants. This was true for a trigeminal stimulus (eucalyptol) presented monorhinally and various odors presented birhinally in three different conditions. The level of performance of both groups across all tasks suggests that odor lateralization and localization are very important regardless of visual status, most probably because these abilities can protect the respiratory tract and aid navigation in an environment.
We observed no differences between the blind and the sighted participants in odor lateralization. Lateralization abilities correlate with overall olfactory sensitivity . It seems then that this aspect of olfactory function is yet another element of sensory perception where no compensation is observed in blindness. However, it should be noted that both blind and sighted participants scored significantly above the level of chance when lateralizing eucalyptol. This finding is consistent with previous works showing that trigeminally stimulating substances can be accurately lateralized intranasally (Frasnelli et al., 2010;Hummel et al., 2003;Kleemann et al., 2009;Kobal et al., 1989;Oleszkiewicz et al., 2017;Porter et al., 2005;Wysocki et al., 2003). As trigeminal stimuli are usually associated with burning, tickling, stinging, and even painful sensations (Doty et al., 1978), an evolutionary point of view suggests there is an advantage in precisely localizing the source of these threats (e.g., environmental hazards). This notion is further confirmed by studies showing that somatosensory sensitivity to trigeminal stimuli is higher in the anterior than in the posterior part of the nasal cavity (Scheibe, Schmidt, & Hummel, 2012), which suggests Fig. 3 The distribution of scores obtained by the blind and the sighted individuals across two tasks in Study 3. The y-axis depicts the absolute value of a difference between the actual and the indicated location of the odorous substance in angular degrees that the trigeminal system could protect the olfactory system (and the entire respiratory tract) from toxic stimuli. Further, habituation effects are much lower for trigeminal than for olfactory stimuli (Sinding et al., 2017), so even repeated (like in our experiment) or prolonged exposure to such stimuli should not affect perception accuracy. In the case of our paradigm it is also important that intensity of trigeminal stimulation is associated with the accuracy of odor localization (Frasnelli, Hummel, Berg, Huang, & Doty, 2011;Frasnelli & Hummel, 2005;Hummel et al., 2003;Schneider & Schmidt, 1967). Dangers associated with trigeminal stimuli can be detected independent from visual input, and because of a direct pathway between the olfactory receptors and the human brain (Stockhorst & Pietrowsky, 2004), accurate reactions can be possible without the mediating effect of sight. No compensatory effect of blindness in this case might thus result from equally high importance and processing abilities of trigeminal stimuli for blind and sighted people. It would, however, be of interest to compare results when using a pure odor with the findings on trigeminal stimuli. Given that olfactory stimuli do not need to be associated with avoidance of hazards (beneficial both to blind and sighted people), the potential compensation-driven differences depending on the visual status might be more pronounced in the case of pure olfactory stimuli.
In a series of experiments, we observed no differences in odor localization between blind and sighted participants. Both groups were able to localize applied odors with quite high accuracy. In a task involving only one odor, the accuracy reached a mean level of 37 (Study 2), and for four odors this was 50°(Study 3), a result very similar to that of Welge-Lussen and collaborators (Welge-Lussen et al., 2014), where localization accuracy in a near-field condition (a distance of 40 cm) ranged between 36 and 47°. This means that although our subjects were not able to precisely point to the source of odor, they were capable of indicating a rough direction where it was coming from. It should also be mentioned that in Study 3, we compared localization abilities for Bcentral^(60°, 120°) and Blateral^(0°, 180°) odor sources. We assumed it would be easier to roughly distinguish the side of stimulation when the olfactory stimulus was placed laterally than to decide if it is placed more on the left or right when it was in front of a subject. This would also be expected following the line of thinking presented by Von Békésy (1964), and previous findings in the area of auditory processing in the blind people (Röder et al., 1999). However, quite contrary to our hypothesis, we found significant differences in localization accuracy for these two conditions, wherein odors located in the central positions were localized significantly more accurately than odors located in the sided positions (the performance of the blind and the sighted people was still similar in these two cases). This finding is also very intriguing from an evolutionary perspective. We could assume that it is more useful to precisely distinguish the lateral locations of olfactory stimulation, as olfactory perception could in such a case supplement information about, for example, potential dangers present in the far peripheral visual field.
It is very interesting that there seemed to be no transfer of sensory function for odor localization in most of the blind people. Our results are consistent with numerous studies regarding null effects of visual impairment on performance in various olfactory tasks (e.g., Cornell Kärnekull, Arshamian, Nilsson, & Larsson, 2016;Guducu, Oniz, Ikiz, & Ozgoren, 2016;Luers et al., 2014;Majchrzak, Eberhard, Kalaus, & Wagner, 2017;Smith, Doty, Burlingame, & McKeown, 1993;Sorokowska, 2016; for a recent review and metaanalysis on this issue, see Sorokowska, Sorokowski, Karwowski, Larsson, & Hummel, 2019). This pattern of findings suggests that the effect of blindness on olfactory functions is not a simple reaction of the organism to a sensory loss (for a review see: Kupers & Ptito, 2014). Nevertheless, some blind individuals do have a better sense of smell than sighted people (Rombaux et al., 2010). Possibly there exist some other factors, other than visual impairment itself, that affect potential enhancement of olfactory processing. We have a few hypotheses as to why we did not observe any superiority of blind people specifically in odor-localization tasks. First, blind people could rely on audition and touch to a greater extent than on olfaction in the case of motor skills and spatial orientation, especially because sensory compensation for audition and touch is observed more consistently than for olfaction (Kupers & Ptito, 2014). Additionally, materials and aids for blind and visually impaired pupils and students are mostly based on tactile and auditory stimulation. Even if the brains of blind people are reorganized and olfaction of blind people is theoretically better than that of the sighted individuals (Kupers et al., 2011;Renier et al., 2013;Rombaux et al., 2010), reliance on auditory and tactile sources of information might diminish the relative importance of olfaction in navigation. Indeed, tactile cues are often used for blind people's spatial orientation (Leonard & Newman, 1970), and although odors might be used for localization, the olfactory system is generally a poorer localizer than vision and hearing (Jacobs et al., 2015). Some authors suggest that, contrary to vision and audition, olfaction is a sense used primarily for proximate stimuli, and it might play a less important role in spatial orientation based on distal stimuli (Köster, 2002).
We could also hypothesize that our null findings were due to the experimental designs that were not sensitive enough to capture the effect of sensory compensation. However, it should be noted that the localization abilities have never been tested before in the context of olfactory compensation, and that similar studies in the area of auditory processing showed enhanced localization skills in blind individuals (Lessard, Paré, Lepore, & Lassonde, 1998;Röder et al., 1999). The tasks completed by our participants were not easy.
Performance of our subjects in the studies that were designed especially for the purpose of this research (Studies 2 and 3) was similar in the blind and sighted groups, but it was still quite low. There was no ceiling effect in our samples, and should the compensatory-driven enhancement of odor localization abilities be observed, the blind subjects could easily perform much better.
Finally, it is also possible that although we intended our spatial localization studies to be as ecologically valid as possible, focusing selectively on odors might not have been very natural for our subjects. A general property of navigation is that locations are encoded redundantly, often using more than one sensory system (Jacobs et al., 2015). Further, we focused the attention of the participants on olfactory stimulation, regardless of their visual status. In a wayfinding experiment of Passini and Proulx (1988), the authors showed that, compared with sighted individuals, totally blind people noticed and processed more information (including olfactory cues) as landmarks during wayfinding. Future studies could focus on two different aspectsfirst, whether sensory compensation in blindness is found in tasks involving cross-modal processing, and second, whether sighted and blind individuals also perform equally well in tasks where their attention is not directed towards olfactory information. | 2019-04-18T13:03:33.234Z | 2019-04-16T00:00:00.000 | {
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94555180 | pes2o/s2orc | v3-fos-license | Evaluation of solar fraction (Fn) for the north wall of a controlled environment greenhouse: an experimental validation
In this communication, an attempt has been made to define and evaluate the solar fraction (Fn) for a north wall on the basis of annual experimental studies. An even‐span greenhouse has been considered for experimental studies for the climatic conditions of Delhi. Experiments were conducted from September 1999 to August 2000. A model based on Auto‐CAD 2000 has also been developed for validation. It has been observed that the solar fraction mainly depends on angle of incidence, solar altitude angle and shape and size of the greenhouse. It is inferred that solar fraction (Fn) plays a significant role in thermal modelling of an even‐span type greenhouse. Further, it was noted that there is a fair agreement between experimental and theoretical results. Copyright © 2002 John Wiley & Sons, Ltd.
INTRODUCTION
The controlled environment greenhouse may be a better option for vegetable production to meet the national need. Higher crop productivity can be achieved, in addition to other bene"ts, by maintaining a favourable environment in a greenhouse. There are various types of greenhouses, which can be classi"ed on the basis of shape and size. Some of them are of even and uneven span, saw tooth and hillside (Tiwari and Goyal, 1998). In even and uneven span-type greenhouses, the solar radiation falling on the north wall inside the greenhouse after transmission through a canopy cover is further transmitted to atmosphere ( Figure 1). This becomes a loss particularly during the months of winter, for the area falling on the north of north tropic sun line. This thermal loss can be prevented by providing a brick north wall instead of canopy cover on the north of a greenhouse. It can also act as a thermal storage wall. The fraction of solar radiation falling on brick north wall inside the greenhouse is referred to as solar fraction (F ) for the present study. On the basis of literature survey (Yang et al., 1990;Jamal, 1994;Jolliet, 1994;Sutar and Tiwari, 1995;Sharma et al., 1998;Ismail and Goncalves, 1999), it is observed that this factor has not been considered for thermal modelling of even and uneven span type greenhouse. Recently, Wang and Boulard (2000) measured solar radiation distribution in full-scale greenhouse tunnels and observed high radiative heterogeneity in tunnels. Analysis indicated that solar radiation inside the greenhouse at ground level was higher in N}S oriented tunnels than E}W oriented tunnels in March and June, respectively. Later on, Singh and Tiwari (2000) have introduced the concept of solar fraction (F ) for thermal modelling. They found that the solar fraction (F ) plays a signi"cant role in thermal modelling of even span-type greenhouse. Then it was decided to determine the solar fraction (F ) experimentally as well as theoretically. A theoretical model based on Auto-CAD 2000 was developed by incorporating the solar altitude angle ( ) and the angle of incidence ( ) on the walls and roofs for even-span greenhouse. Solar fraction (F ) was evaluated for the typical months of September, December, March and June. On the basis of the experimental and theoretical results it is inferred that: (i) There is a fair agreement between experimental and theoretical results and (ii) The solar fraction (F ) on the north wall is maximum during winter months and minimum during summer months, respectively due to minimum and maximum latitudes.
EXPERIMENTAL SET-UP
An even-span greenhouse with an e!ective #oor area of 6.00 m;4.00 m and a central height of 3.00 m was erected by the galvanized iron (GI) pipes in the campus of IIT, Delhi (latitude (): 303 N). A door of 1.80 m;0.92 m has been provided in the east wall. A brick wall of 0.275 m thickness was constructed on the north side to avoid the thermal losses as shown in Figure 2(a). The brick wall was painted black to absorb maximum solar radiation. An ultraviolet stabilized plastic sheet was also used as a canopy cover. The orientation of the greenhouse is E}W and the inclination of north and south roofs are 153 and 273, respectively, from the horizontal plane Figure 2(b).
EXPERIMENTAL OBSERVATIONS
In order to get distribution of solar radiation inside the greenhouse, it was decided to observe the solar radiation at six points on the north wall and "ve points on the #oor as shown in Figure 2 Each point on the north wall was located in the central part of each zone having equal area. This arrangement covered the availability of solar radiation falling on the north wall. The solar radiation falling on the #oor of the greenhouse was measured along the central length and breadth of the greenhouse. Solar radiation distribution was measured with a calibrated portable &Solarimeter' or Suryamapi (local trade name) at these points of the north wall and #oor of the greenhouse. Experiments have been generally conducted every week starting from 8 a.m. to 4 p.m. on the hourly basis. The observations of hourly variation of solar radiation for di!erent points for typical days of di!erent months have been given in Table I.
The total radiation on the north wall has been calculated by multiplying each observation in W m\ by the area of the corresponding zone (2 m). Similarly, the total intensity of the #oor was calculated by multiplying each observation by the area of the zone (8 m). For zone 8, average of A, B and C has been taken. It is important to note that the value of solar intensity measured at B point of zone 8 is nearly equal to the average of value measured at A and C, respectively. The hourly variation of solar radiation (beam and di!use) were also measured on horizontal surface outside of the greenhouse.
SOLAR FRACTION (F )
The solar fraction (F ) for the north wall is de"ned as follows: F " Solar radiation available on north wall inside the greenhouse for a given time Solar radiation measured on the north wall and floor of the greenhouse for same time (1) The data of Table I for total solar radiation on the north wall and the #oor have been used to calculate F (Equation (1)). The calculated values of solar fraction for a given time of typical days have been given in the same table.
THEORETICAL MODELLING
The calculation for solar fraction (F ) for a three-dimensional geometry is a very di$cult task due to di!erent values and directions of solar radiation on walls and roofs at the same time. To simplify the problem, a three-dimensional model of even-span greenhouse for a given dimension (6.00 m;4.00 m;3.00 m) has been developed by using Auto-CAD 2000. In order to get the variation in solar fraction (F ) for a complete year, solar fraction (F ) has been calculated for days, i.e. 15 September 1999, 15 December 199915 March 2000 and 22 June 2000 for di!erent months of the year at 8 a.m., 10 a.m., 12 noon, 2 p.m. and 4 p.m. The following main steps have been taken to calculate the value of solar fraction (F ): (1) Methodology for computation of solar fraction (F ) for each wall and roof by using Auto-CAD: For a given south wall, a ray making an angle equal to incidence (normal to the south wall) at top view has been made by using Auto-CAD for a time of the day. Further, a line from the ground making an angle equal to solar altitude at the same time was made from the right view. Parallel lines were drawn at top and bottom of south wall. Parallel lines have been extended inside the greenhouse as shown in Figure 3. The north wall will intercept the extended line. The solar fraction due to south wall (F ) and south roof (F ) are de"ned as (1!F ) and (1!F ) will be a solar fraction for the #oor of greenhouse due to south wall and south roof, respectively, for that time. Similarly, the solar fraction (F ) at same time can be evaluated from each wall and roof of model. Thus, the rays were drawn for 8.00 a.m., 10.00 a.m., 12.00 noon, 2.00 p.m. and 4.00 p.m. of 15 September 1999, 15 December 199915 March 2000 and22 June 2000. The theoretical values of solar fraction (F ) were calculated for each wall and roof by using Equations (2a) and (2b).
The solar fraction (F ) will vary with time of day. Solar fraction (F ) should be calculated for each wall and roof facing solar radiation for a given time of day. The solar fraction (F ) for the north wall will be the average of a solar fraction due to each wall and roof, which is expressed as follows: Similarly, solar fraction due to di!erent walls/roofs was worked out for these typical days and various times.
(2) Determination of incidence angle ( ), solar altitude angle ( ) and solar radiation for each wall and roof for known latitude ((): 303 N): In order to calculate solar fraction (F ) by Equations (2a) and (2b), one should calculate incidence angle ( ) and solar altitude angle ( ) for a given latitude (), hour angle ( ), declination ( ), surface azimuth angles ( ) and angle of inclination ( ). This incidence angle ( ) for each wall and roof can be calculated by using the following expression: (Du$e and Beckman, 1991): cos "(cos cos #sin sin cos ) cos cos #cos sin sin sin #sin (sin cos !cos sin cos ) Here, hour angle ( ) and solar declination ( ) are calculated by using the following formula: "15(t !12) (6) "23;45 sin 360(284#n) 365 (7) sin "cos X "cos cos cos #sin sin (8) The surface azimuth angles ( ) and inclination angle ( ) for di!erent walls and roofs have been given in Table II. The values of angle of hour, declination, incidence and solar altitude for di!erent walls and roofs for di!erent times of the day are presented in Table III.
The solar radiation on each wall/roof was calculated by using the Liu and Jordan formula (1960) as follows: where, R G "cos /cos X , R "(1#cos )/2 and R " (1!cos )/2. The total solar radiation available on canopy cover of a greenhouse can be computed by the following expression: Total solar radiation available inside the greenhouse can be obtained by multiplying Equation (10) by transmissivity ( ) of the canopy cover. From Table IV, it is clear that the calculated total solar radiation is close to the sum of measured solar radiation on the wall and #oor of the greenhouse in some of the cases. However, there is also signi"cant deviation in other cases. It was due to the presence of some cloudy patches during the observations at typical hours.
(3) Computation of solar fraction (F ): By using the step mentioned in the methodology, isometric, plan and north elevation at 8 a.m., 10 a.m., 12 noon, 2 p.m. and 4 p.m. on 15 September 1999 have been shown in Figures 4(a)}(e). With the help of Auto-CAD software, the distance for AC and AB were measured 1995 and 3082 mm, respectively. Thus, the solar fraction (F ) due to the south roof at 8.00 a.m. was worked out to be 0.69 by Equation (2b). Similarly, the solar fraction due to the east wall, south wall, north wall, west wall and north roof was worked out to be 0.5, 0.0, 0.0, 0.0 and 1.0, respectively. Then, an average solar fraction (F ) was computed from Equation (4) as follows: 0.69#0.5#0.0#0.0#0.0#1.0 6 "0.36 The same exercise was repeated to get solar fraction (F ) at 10:00 a.m., 12:00 Noon, 2:00 p.m. and 4.00 p.m. on 15 September 1999, 15 December 1999, 15 March 2000 June 2000. The obtained solar fraction (F ) for beam and di!use radiation will be the same for total radiation too.
RESULTS AND DISCUSSION
The hourly variation of solar fraction (F ) for di!erent days (15 September 1999, 15 December 1999, 15 March 2000and 22 June 2000 of the year has been shown in Figures 5 and 6. These "gures indicate that the values of solar fraction are the same (i) for 8 a.m. and 4 p.m. and (ii) 10 a.m. and 2 p.m. In these cases, incidence angle ( ) at 8 a.m. on the east wall and 4 p.m. on the west wall are equal. Similarly, incidence angle ( ) for the east wall at 10 a.m. and 2 p.m. for the west wall are also equal. Table I 15 September 1999 8.00 a.m. The value of solar fraction is maximum in early morning and late evening due to low solar altitude angle. From these "gures one can also conclude that the value of solar fraction is a minimum at 12:00 noon irrespective of summer and winter due to high solar altitude. It is also further noted that solar fraction is a minimum at noon in comparison with the value at the morning and evening for a given dimension of greenhouse. The results are summarized as follows: At early morning and late evening, the north wall is exposed completely due to the lower position of the sun and the #oor is exposed in a better way during noon due to the higher position of the sun.
Further, it is important to note that the value of solar fraction during the winter month at any time is more than the value of solar fraction during the summer period due to the low solar altitude (Figure 7).
The present studies show the importance of solar fraction (F ) for the north wall particularly during the winter period. The solar fraction (F ) is about 0.24 for the north wall. If this factor has not been taken into account properly in the thermal modelling, this means that 24 per cent of transmitted solar radiation inside the greenhouse is further transmitted to the outside atmosphere through the north wall canopy cover. This factor mainly depends on the following factors: (i) angle of incidence ( ), (ii) solar altitude angle ( ), (iii) shape and size of the greenhouse.
CONCLUSIONS
On the basis of the present study, it is concluded that the solar fraction (F ) for the north wall should be evaluated in the designing of a greenhouse for a given location. This will also helps in maintaining the di!erent temperature zone in the large greenhouse by providing concrete north wall for higher temperature zone.
A
"area (m) F "solar fraction for the north wall I "solar radiation (W m\) n "day of the year | 2019-04-04T13:15:10.854Z | 2002-03-10T00:00:00.000 | {
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263665683 | pes2o/s2orc | v3-fos-license | Futures contracts as a means of hedging market risks.
— This academic article examines derivative instruments, their role in financial markets
I. INTRODUCTION
Trade and commercial interactions have always been part of human history.But while this need is remained the same, markets have evolved along with the complexity and development of the society.Modern society is characterised by industrialized nation that heavily relies on welldeveloped financial markets to sustain its economic growth.It can be said that they are the mechanisms that govern capital allocation and investments of the society.Companies that succeed over time are those that have established and maintained adequate systems of monitoring and supervising their internal operations as well as keeping commitments to investors.Obviously, risks are still a big part of trading, but the new financial instruments can be a good tool through which risk can be reduced and unbundled into manageable forms.
Derivatives markets have become more and more used by companies.They appear to be making a big impact on global financial markets, given their incredible and perhaps unexpected growth across the globe.Their use is mainly connected to find a way to manage risks or because investors and regulators are finding a way to stay competitive in a constantly changing world characterized by widely fluctuating prices and new opportunities.
Commodity was the first and the main underlying asset that developed derivatives or custom-designable Over-the-Counter contracts were linked to.Despite their persistent use by companies, there are still doubts among the public and regulators for two main reasons.The first one is connected to the fact that hedging is a tool that can also reduce the gain if in the spot market the price goes in the opposite direction than expected.For example, from the perspective of the seller who has signed a futures contract, the investor can face losses if the final price is higher than the one agreed.This happens because without contract the asset could have been sold at a higher value and the vendor could have received more money for the same quantity of products.The second one is that this debate has been carried on and enhanced when serious scandals brought severe losses because of the "hedging activity".It is true that several companies have suffered from heavy damages or even bankruptcy.One of the key roles of the futures market is indeed hedging, which is a perfect risk management tool for businesses to prevent wildly fluctuating commodity prices.Hedging can change the corporations' passivity structure, enabling them to keep production costs under control, secure profits, and provide high-quality development.
This research wants to prove how futures contracts and the hedging activity in the futures markets can be a really useful tool to smooth market risks.In this paper we try to gather enough evidence to convey and support our point of view.The main discrepancy that we found is between theory and practice.The former is supportive of how these instruments can bring benefits, instead the latter is poor of optimistic evidence as the example made above.But even so, it is reported that more than 90% of the Fortune Global 500 companies [1] use financial derivatives to control risks, so we can say that even though hedge activity with derivatives is a risky game, these cases remain an isolated example.
a. Derivatives instruments 1. Derivative markets
Financial markets are crucial to the efficient running of company's economies, and they are also a big part of many companies' life, whether they are big or small, new, or well known, in primary or third sector.Financial markets is a word used to identify any marketplace where securities' trading take place, including for example the stock market, bond market, forex market, and derivatives market, and some others.
The volume of international companies and international trading has increased exponentially thanks to the wave of globalization and liberalization that has characterized the world these last two decades.Moreover, scientists assert that the financial innovations created these recent years are more impressive and futuristic than ever.[1].
A study made by Abolhassan Julivand in his paper "Why Firms Use Derivatives: Evidence from Canada" using USA, New Zealand, and Canadas's firms as sample, shows that the use of derivatives for hedging purpose can produce the economies of scale, and it can be a strong motive for companies to use them.[2].
To understand the concept of 'derivatives' its definition is presented: "The term refers to a broad class of financial instruments.These instruments derive their value from the price and other related variables of the underlying asset.They do not have worth of their own and derive their value from the claim they give to their owners to own some other financial assets or security."[3].
At the beginning Over-the-Counter derivatives markets were a free-rule place of trading, but after the crisis of 2008 the regulation has changed, and new rules and limits have been introduced.Following the failure of Lehman Brothers and its consequential credit crisis, a new wave of regulations changed the operation of derivatives markets.The goal was and still is to increase OTC market transparency, boost market performance, and lower systemic risk.As a result, it might be claimed that this kind of market has been "has been forced to become more like the exchange-traded market".[4].
Derivative risks
Risk is one of the main reasons that can clarify this unexpected growth and use of financial instruments.For this reason, risk management is more crucial than ever due to the extremely uncertain environment.Most of the commodity and capital markets are characterised by different kind of risks.These can conduct to a rapid and unexpected changing on the financial assets prices, interest rates, and exchange rates, and consequently, to expose businesses and investors to a shattering economic crisis.Individuals or companies that do this kind of transactions bear high probability of losses, because there are high chance to lose significantly more than one's initial capital.
In this way, derivatives assets are traded to reduce rather than raises the risks, even though it does not eliminate the risks associated with the portfolio of financial instruments.As Table 1 shows, two main different risk affect the most: systematic and unsystematic risk.If this risk is less in the derivatives than in the underlying asset, the first tool has lower loss exposure than the one in the spot market.[5].
Nevertheless, "default risk, operational risk, liquidity risk, legal risk, and market risk" [6] are just a few of the different kind of obstacles that a company can face in the derivatives markets.
b. futures contract
A futures contract is a binding agreement between two parties, supplier or short position and consumer/buyer or long position.The agreement is a regulated contract where the seller and the buyer agree to sell and to buy a specific underlying commodity, in a specific day in the future and with pre-agreed payment condition.
Futures trading has a long history because the official futures exchange was created in USA with the Chicago Board of Trade (CBOT) in 1848 [7].This kind of trade has been formed because the agricultural dealers needed a way to reduce price risks.The fluctuation of the price of corn, wheat and butter made it difficult for business owners to run their operations: sellers wanted to reduce the priced linked to the cost of storing the raw materials, and purchasers wanted to set fixed pricing for these goods before delivery.
The composition
Thanks to the widely use of future exchange in the current markets, trading in commodities futures is now a tightly controlled industry, contrary to the past.The regulation's goal is to preserve its "fairness" and "competitiveness".[6].
Almost all the details existing in the futures contract are standardized, such as: 1) Delivery month or expiring date: it settles the day in which the contract will expire and the way in which the delivery will be made, through cash or physically.
2) Contract size: it represents the number of underlying assets that the contract is linked to, and it can vary depending on the underlying asset.
3) Quality of the commodity.4) Margins: it represents the sum of money that the company has to pay in advance and keep in order to open a position in a contract.Usually the range is between "3-12% of the contract's value".[8].5) Delivery location.
Talking about the parties of the contract's trading, an investor can take two types of positions: long, when there is the will to buy, or short, when the asset is sold.Moreover, an investor can use both position at the same time to combine hedging strategies.[9] Long Positions Here the investor who wants to buy the asset is hoping for the price to rise because she or he can have profit from it.The typical stock purchased is a long stock asset, since the possible disadvantage is just the paid price, and the benefits are infinitive.
Short Positions
This situation is the exact opposite of the one above.Here the investor is expecting for a downward on the price direction.An example can clarify this position.If an investor borrows n number of shares and then he/she decides to sell them in the spot market, this action can produce a decrease of the stock price.Now the investor has an open position for n number of shares with the broker, from whom the shares have been bought, which has to be closed in the future.If there is a reduction on prices, she/he can buy back the same number of stock shares, but this time for a cheaper value than before.The excess cash is the profit.
These positions can be used from the tree main categories that can be identified as future traders [10]: Hedgers These are organizations or people who use futures contracts to insulate themselves against exposure to unfavourable changes in asset prices.
In this situation, the investor is a buyer, better said is covered a long position, because he/she gains from price growth and loses from price decrease.The investor can go in the futures market as a short position by selling the assets, in order to be protected from a possible decline in the value.A short hedger is a person who fits this description.A long hedger would be an investor who does exact the opposite actions.
Speculators
Speculators are the ones who takes up price risk in order to make profits.They are independent person who can trade with their own funds or on behalf of customers or brokerage businesses.In this situation, both the potential benefits and losses they could face are very significant.
Arbitrageurs
Arbitrageurs are the third most important group, and their activity involves lock-in risk-free profit by concurrently engaging in transactions in two or more marketplaces.This happened when a commodity is bought in a market and simultaneously sold in another one with a difference in prices with the intention of making money.The difference with the other two main participants described above, it is that they trade only in order to make returns from discrepancies in the market.
The arbitrageur holds the duty of make sure that the value of the futures contracts does not go in conflict with the level determined by supply and demand and if pricing anomalies happen, he or she has to quickly correct them.Another figure is very important in the trading as Figure 2 shows:
Clearing house
This figure is a financial institution that has been created intentionally to ease the transaction of derivatives.Its main task is to keep track of all the transactions that take place in the futures market because in this way it can calculate the net position of each of its members.The net position is known as 'the initial margin', better said a deposit that each traders requires to put before any kind of trade to ensure contract performance.Its role is very important, because it makes sure that there is enough money to cover each trader's obligations and at the same time it embodies a guarantee because it bears the credit risk connected to the transaction.This clarifies an essential point: is the Clearing House who is the counterpart of each contract, and the two parties never enter in contact with each other.[11].The following formula expresses the relationship between several factors influencing a future price: F=S(c-s+r)^t [12] F = the future price of the commodity S = the spot price of the commodity r = the risk-free interest rate or Expected Dividend yield s = the storage cost, expressed as a percentage of the spot price c = the convenience yield, usually 1 t = time to delivery of the contract [12] Suppose company Agua buy an index in cash market at 8000.Here the value s is 12% and the free-risk interest rate is 4% per annum.With t of three months (90 days) the result of the equation should be:
If the price of the index future is higher than 81,5326€, an index stock can be bought in the spot market and consequently sold in the future market in order to take advantages of the discrepancies in prices.
In the case of a currency future, so a future written on currencies, the theoretical formula is: [12] r = represents the risk-free foreign rate S = the spot exchange rate.
III. METHODOLOGY
In order to support the initial theory, this research is divided in 3 sections: two mainly theoretical and the last one practical.
The theoretical framework gives a theoretical but deep understanding of how derivative instruments work.In the first part, great attention is given to the risks that a company can face in the financial markets and which derivative tools can alleviate.The second part focuses on futures contract: its composition, the main actors, and the calculation of the price.
Mainly theoretical research is a research approach that emphasizes the development of new theories, concepts, or models rather than collecting and analyzing data.This type of research is often used in economics and finance to develop new models that can be used to predict future market trends.The methodology for mainly theoretical research involves a comprehensive review of the literature, identification of gaps in the current knowledge, and development of new theories or models to fill those gaps.The primary goal of mainly theoretical research is to contribute to the advancement of knowledge in a particular field of study.
The analysis and interpretation talks about the advantages of futures contracts, and they are compared with others financial instruments.Finally, some knowledges are given about how hedging works and a study is used to support the initial theory.Hedging is a risk management strategy used to offset potential losses from adverse price movements in the future markets.Hedging can be done using various financial instruments such as futures contracts, options contracts, and forward contracts.Mainly theoretical research in this field may involve developing new models or theories to predict the movements of future markets and identify the best hedging strategies to minimize risks.
The methodology in this research involves a comprehensive review of the existing literature on futures and options trading.This review is used to identify gaps in the current knowledge and develop new hypotheses or theories to fill those gaps.Theoretical research in this field may also involve developing new mathematical or statistical models to predict market movements and identify the best hedging strategies.real data from a finance site, we made a model that describes the different scenarios that a company can face if it hedges.In this part we analysed the side of the buyer, as the seller's perspective is exactly the opposite.This provides empirical support to the theoretical parts and, at the same time, confirm how and why hedging futures contract can be profitable, always bearing in mind that it is a risky activity.
This methodology enables us to give a general knowledge of the futures contract as the main topic, to ease the comprehension of their use in the examples.Then the paper of Wang, N., & Zhou, Q. [13] show graphically and numerically what the theoretical part try to transmit and support empirically my initial hypothesis of the advantages of derivative instruments.
The level of investigation can be categorized as a theoretical research study.The paper focuses on providing a theoretical understanding of derivative instruments, specifically futures contracts, and their role in managing risks in financial markets.It discusses the evolution of markets, the use of derivatives by companies, the risks associated with derivatives, and the composition of futures contracts.This paper also mentions the discrepancy between theory and practice and aims to gather evidence to support the authors' viewpoint.
The paradigm employed in this article is a positivist paradigm.The researchers aim to gather evidence and support their point of view through a comprehensive review of the literature, identification of gaps in current knowledge, and the development of new theories or models to fill those gaps.This article relies on existing research and knowledge in the field of finance and economics to build a theoretical framework and provide insights into the advantages of futures contracts and hedging strategies.
Finally, the following variables can be identified to give structure to the results obtained: Derivative Instruments: • Use of complex derivative instruments (options, forward contracts, futures) • Reasons for the growth in derivative instrument usage (economies of scale, interdependence between markets, investment decisions, risk reduction, speculation) Derivative Risks: • Systematic risk
a. The convenience of futures 1. Contract and the hedge activity
As we have said before, derivative instruments represent a wide range of tools.Even though there are different types in the market, the most common ones are futures, forwards, and options.This chapter will give some hints on why futures contracts and the activity of hedging them can be profitable.The first section will briefly explain the main differences between the three main derivatives tools and also between futures contracts and stocks.The second part gives some trustworthy and professional studies that support the main idea of the benefit of hedging futures contracts.
Futures and Forwards
Futures and forwards are both contracts which include the physical exchange of an underlying asset on a fixed date in the future and at a fixed price stipulated in a previous contract.But they have dissimilarities and the main are [4]: Forwards are popularly known as OTC contracts; better said these contracts are typically executed over the phone and not traded.
Meanwhile futures are traded on the exchange.
Futures are relatively safer and more secure compared to forwards.This thanks to the regulation that lately is getting applied to this kind of financial transaction and mainly thanks to the guarantee of the Clearing House.So, if one of the party defaults then it would be the Clearing House itself that will honour the trade and then initiate recovery from the other party.
Futures are standardized and hence liquidity creation become a lot easier.First of all, if we are talking about futures contracts on index, the delivery is done only with cash.Secondly, every contract details are previously settled like the place where to trade the underlying asset, the price or the expiration date.Thirdly, in the stock exchange there are considerable number of buyers and sellers, and the price discovery also happens through a very transparent market mechanism.Lastly, they are also standardized in terms of the underlying.The underlying has to either be an index or a specific stock.
The liquidity of futures is one of the main advantages compared to forwards where the liquidity is a major challenge.That's because if you get into a forward contract and want to exit midway then the only hope is to find someone with similar requirement otherwise you are stuck in the contract until the maturity.
Futures and Options
An investor can purchase an asset at a certain price and date by using both option and future.But despite the fact that both instruments can be used to provide an investor the chance to make profit and hedge, the way in which they work and the level of risky change.
Below are explained the main advantages of futures over options [14]: Futures have an initial fixed trading cost, called a margin [15].This is a requirement for major commodity and currency futures that have not been changed for years so investors know in advance the amount of the initial margin.
On the contrary, the option premium can differ greatly because it is connected to the vast market and the underlying asset's volatility.The initial premium increases if the price of the underlying assets or the markets itself are unstable Options are "wasting assets" [16], which means that as time passes, their value decreases, a condition known as "time decay".The expiring time is one aspect that affect the "time decay" [17].This factor is essential to take into consideration otherwise it has the potential to drastically reduce an option position's profitability and make the investor lose.This characteristic is not part of a future contract.
Futures pricing is quite straightforward, because using the cost-of-carry model the price of the futures contract is the same as the underlying asset in the spot market adding the cost of storing.If the two prices are out of alignment, arbitrage activity would occur and rectify the imbalance.Instead, the value of an option includes more elements, like the current stock price, the intrinsic value, time to expiration or the time value, volatility, interest rates, and cash dividends paid.
Futures and Stocks
Until now future contracts have been compared with other derivative instruments, but they have several advantages over trading stocks.[18]. Futures are highly leveraged investments: in order to trade in futures market an investor has to pay the "initial margin".This is a fraction of the value of the underlying asset that it is used as guarantee.The amount of money is a guarantee that the investor has to maintain through the whole position, or it can be exchanged if the price fluctuation is negative for the investor.The loss that investors can face could be much more than the margin amount.But at the same time the profits can multiply if the prices move as expected.Thus, an investor can trade a larger quantity of underlying assets even though he/she does not have the whole sum.
Future markets are very liquid, because this kind of derivatives instruments are traded in huge amount every day.Moreover, orders may be done rapidly on these markets since there are always buyers and sellers there.
Speculators can receive fast money with futures because they are traded with more exposure than with stocks.However, as the profits can come quicker and at a great amount, with futures there are high chances of loss because of the great volatility within them.In general, the fluctuation of prices is higher in the futures market than in the stock market.
The two side of the coin are: futures bring elevated risk but at the same time futures are seen as chances to gain with short-term price fluctuations [19].
Futures are crucial tools for hedging or diversification in order to manage risks.They are used by businesses engaged in international trade to manage three types of risk: foreign exchange risk, interest rate risk, and price risk.Moreover, they can improve the efficiency of the spot market thanks to their ability to reduce unexpected costs expenses associated with outright asset purchases.
Futures contracts are an easy way for investors to raise finance against stocks of commodities.This because of the standardisation of contracts: traders have a guarantee of quality of the asset, quick liquidity and there is a protection against risk of depreciation.
Hedging with futures contracts
As it is explained above, one of the main reasons futures contract and hedging are getting so popular is because they are a good way to avoid or at least smooth price fluctuation.
The use of commodity derivatives as a risk management technique is commonly accepted.But one of the most controversial debated between theories and also in practice is whether derivative instruments can lessen a company's exposure to price risk.Indeed, the idea of company hedging began when theories about risk management were developed.The known professors Smith and Stulz [20] proposed that firms hedge because it can avoid possible distress and bankruptcy cost; therefore, hedging can increase firm value.
According to them, the firm value can rise with hedging because of a combination of linear relationship between shareholder value and low risk.Therefore, risk mitigation is unquestionably a key goal of commodities hedging.
Financial futures can be used by firms as a hedging tool to safeguard their assets against unexpected mutation in the underlying.Both the possibility of loss and gain are decreased by hedging, because it fixes the price even though in the spot market it increases or decreases.This is an example.A firm has a stock portfolio and decides to hedge its value by selling stock index futures contracts.In the situation where prices drop, both the portfolio's value and futures contract's price decrease.Here the company can buy back the contract at a cheaper price because it is in the short position.In the end, the profit from the hedging has partially offset the loss on the portfolio.[21].
Another good reason to hedge is that these financial instruments can boost the portfolio achievement.This because the two main risks explained above, can be reduced using futures contracts.Playing with long and short positions in futures contracts in the right way can reduce the transaction costs and it can be more profitable then trading big amount of securities.
A study made by Walid S. Abanomey and Ike Mathur [22] shows the potential gains of commodities futures contracts in the risk/return trade-offs of a portfolio of international stocks and bonds.This kind of practice is used because the former's constant convenience yields have the potential to generate positive returns with no relation to the latter's returns, which promotes diversification.Moreover, reliable hedge against the risk associated with investing in stocks and bonds is provided by the fact that commodity spot returns are inflation and interest rate related, as well as inversely correlated with stock and bond returns.
Unfortunately, real empirical cases offer controversial answers.Some well-known finance and economics professors recognized all over the world provide positive findings, while some others offer negative evidence.[23].
The difficulty is to analyse real and truthful information.For example, if the study focuses on commodity derivatives, different obstacles can be found.Firms usually trade these kinds of derivatives frequently within a short period so there are none or few positions left on the balance sheet.Or companies are keen to use various types of commodity derivatives at the same time including futures, forwards, swaps, options, so it is challenging to develop an acceptable proxy to assess the price risk management impact of a combination of several derivatives with different complexity and structure.
All of these problems made it really difficult to find empirical evidence that could back up my theory with truthful information and data to build on.
For the purpose of strengthen the initial hypothesis of the dissertation empirical research has been analysed.Ningli Wang and Qichong Zhou [13] have released a paper where they prove that under some conditions hedging can positively impact on companies' performances.They have collected data on NYSE and NASDAQ markets from 2010 to 2019 from the SEC website.As table 3 shows, the total sample is composed of 32876 firms.In the Table above the companies' sample are grouped in 10 main industries.The result of the study is shown in Annex 1 where it is proven that the hedge of commodity in these industries can diminish the volatility of stock price in 7 of them.
Following there is a list of how much each industry experiences a decrease in stock price volatility thanks to commodity hedging: Hedging has reduced volatility by 45.7%, 32.1%, and 23.5% for independent power and renewable electricity providers, electric utilities, and gas utilities Utilities show a 20.3% drop in the volatility of stock price, and it is notable statistically (99% confidence level) and economically speaking. Consumer sectors experience an average of 7,45% decrease Healthcare also decrease an 11.6% Communication service has a decreased of 8.9% Information technology experiences a 3.5% decrease Materials sector a 3.1% These results verify how in 8 different industries the volatility of the stock price is reduced thanks to the hedge and these outcomes are statistically significant in 5 of them.
Thanks to this research, the key initial theory of this dissertation is supported by the previous results.It can be said that the hedge activity of commodity futures is a successful tool of risk management (even though the results are opposite for speculation).Hedging has a different impact on industries, where for example the utilities have a lot of benefit from it, and it also has been proven that small companies and company with better profitability can take more advantages from the hedge of commodity.
V. CONCLUSIONS
In conclusion, the article highlights the potential of futures contract markets and their effectiveness in managing risks.However, despite the theoretical acceptance of these concepts, their practical implementation has proven to be challenging.This raises the question of why financial markets do not fully reflect the reality of futures trading.To shed light on this question, Professor Emeritus Roger W. Grey, expresses uncertainty about the success or failure of futures trading and remarks on the limited understanding of futures markets among economists [24].This statement suggest that futures contract markets have not been fully comprehended yet.Some markets, particularly those involving certain commodities, remain relatively unknown, lacking comprehensive literature or economic studies to explain their dynamics fully.Risks associated with futures trading are not well categorized, as they vary in quantity and intensity across different markets and time periods.It is not uncommon for firms to experience losses despite their hedging efforts.
The recent past serves as a reminder that attempts to reduce risk through hedging are inherently complex, given the potential hurdles and uncertainties involved.Even well-established and financially stable organizations like Morgan Stanley have faced costly losses due to hedging activities.The example of Morgan Stanley's subprime hedge, which resulted in a $9 billion loss [25], illustrates the challenges of predicting the future and the high potential for losses.
Derivatives contracts, including hedging strategies, are associated with a significant probability of losses, often close to 50%.This indicates that the damages caused by derivatives do not depend on the quality of the hedge itself or the corporation's choice to hedge, as the unpredictable nature of the future and the inherent risk contribute to potential high losses.While there have been historical failures with derivative instruments, they represent a small proportion of overall hedging activities.
Ultimately, the paper underscores the theoretical potential for high profits through hedging.However, given the high risks involved, companies must implement robust monitoring and control methods to minimize losses.By doing so, they can mitigate damage and capitalize on the significant opportunities offered by futures markets and hedging strategies.
Figure 1 :
Figure 1: Unsystematic and systematic risk in a Portfolio of derivatives.Source: Own elaboration based on contributions from [4].
Figure 2
Figure2describe who demand-supply dynamics can be the cause of changes in the prices of agricultural and non-agricultural goods throughout time.
Figure 3 :
Figure 3: Demand-Supply dynamics in the market.Source: Own elaboration based on contributions from [11].
Table 3 :
The hedge of commodity and the speculation activity analysed by industries. | 2023-10-05T15:21:45.749Z | 2023-09-01T00:00:00.000 | {
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227239932 | pes2o/s2orc | v3-fos-license | Multilocular Thymic Cyst in a Young, Otherwise Healthy Woman: A Case Report
Thymic cysts are rare lesions, accounting approximately for 1% of all mediastinal masses. We report a case of a 36-year old woman who presented preoperatively with a calcified mass shadow found on a routine chest radiograph X-ray. After further investigation with chest computed tomography (CT), magnetic resonance imaging (MRI), and tests for Myasthenia gravis, a benign mediastinal cyst was diagnosed and the patient underwent median sternotomy and complete surgical excision of the lesion. The histological examination described a multilocular thymic cyst. Thymic cysts are usually associated with thymic epithelial tumors, such as thymomas, or multisystemic morbid conditions such as human immunodeficiency virus (HIV) infection, rheumatologic disease, and Myasthenia gravis. At all follow-up examinations to date, the patient remains healthy.
Introduction
Thymic cysts are rare lesions, accounting for approximately 1% of all mediastinal masses. They are classified into two types, namely, unilocular and multilocular, with the latter presenting with a lower incidence [1][2][3]. The pathogenesis of these two subsets differs significantly. Indeed, unilocular mediastinal cysts are mostly present at birth, whereas multilocular cysts are thought to be developed under the effect of an underlying inflammatory process [4]. Furthermore, the presence of rim calcification in a mediastinal cyst is most likely to be associated with chronic inflammation or a cystic tumor such as cystic teratoma [5].
Histologically, thymic cysts consist of a thick and fibrous wall of epithelium and they include serous fluid or gelatinous content. Inflammation is also evident [2][3].
In general, thymic cysts are asymptomatic and consist of a random finding. Symptoms such as dysphagia, dyspnea, cough, thoracic pain, or Horner syndrome may exist, depending on the size and location of the cyst [1,3,6].
Despite the fact that thymic cysts may be present in any anatomic place between the neck and diaphragm, the majority of them are localized in the anterior mediastinum. Differential diagnosis includes mesothelial and bronchogenic cysts, cystic teratomas or lymphangiomas, and malignant tumors [1,3].
Broadly, thymic cysts are radiologically diagnosed. While they may be presented in chest radiograph X-ray, the chest computed tomography (CT) is the most diagnostic method, as it describes the size, shape, characters, and the relations with adjacent tissues [7].
As there is no consensus on the appropriate therapeutic approach for thymic cysts, surgery seems to be the gold standard, as it decreases the possibility of infections or recurrence [7][8]. Surgical procedures, such as thoracotomy, sternotomy, video-assisted thoracoscopic surgery (VATS), and robot-assisted thoracoscopic surgery (RATS), have been applied [3,9]. When operation cannot be performed, less invasive methods, such as sclerosing with ethanol, have been reported [10].
Herein, we present a case of a young, otherwise healthy woman with an incidental finding on routine chest X-ray that was eventually diagnosed as a multilocular thymic cyst. Notably, clinical and laboratory examinations failed to reveal the presence of either a chronic inflammatory process or any underlying malignancy.
Case Presentation
A 36-year-old woman was referred to our department for further investigation of a calcified mass shadow found on a routine chest radiograph X-ray ( Figures 1A-1B). Her physical examination and medical history were unremarkable. Chest computed tomography (CT) revealed an ellipsoid, well-defined, encapsulated cyst with low attenuation and rim calcification located in the anterior mediastinum ( Figure 1C). Magnetic resonance imaging (MRI) showed a well-circumscribed cystic lesion with fluid content of low intensity on T1-weighted images and with no adhesion to the surrounding tissues ( Figure 1D). Edrophonium test (Tensilon test) and serological test for acetylcholine receptor antibodies and muscle-specific tyrosine kinase antibodies were negative for Myasthenia gravis. Serological testing for HIV infection was negative as well. A benign mediastinal cyst was established as the working diagnosis but due to the observation of a nonaccessible lymph node proximal to the left brachiocephalic vein, the patient underwent median sternotomy ( Figure 2A) and complete surgical excision of the lesion ( Figure 2B). The histological examination described a multilocular cystic lesion with a dense fibrous wall containing calcification and fragments of thymic tissue. The wall of the cyst was heterogeneously lined with single or multiple layers of squamous epithelial cells with foci of epithelial hyperplasia. Cholesterol granuloma formation and inflammatory cells, such as lymphocytes and epithelioid cells, were also observed locally ( Figures 2C-2D). The overall histological examination was compatible with the diagnosis of a benign multilocular thymic cyst. Applying the Stanford diagnostic criteria of surgical pathology for the lesion indicates that it is most probably acquired rather than congenital due to the focal areas of hyperplasia as well as the presence of cholesterol granulomata and the observed inflammatory elements. The lymph node had non-specific reactive changes.
Discussion
Thymic cysts are rare lesions, most often incidentally diagnosed [1]. In general, multilocular thymic cysts are usually associated with thymic epithelial tumors, such as thymomas or underlying multi-systemic morbid conditions, and autoimmune diseases such as HIV infection, rheumatologic diseases, or Myasthenia gravis [11][12][13]. Although a few cases of pure calcified thymic cysts have been reported in the literature, most cysts with calcification concern cystic teratomas [4]. Besides the presence of a tumor, calcification may be attributed to an underlying chronic inflammatory process. In our case, however, no evidence of pathology was found neither at the time of surgery nor after three years of follow-up.
According to retrospective clinical data, thymic cysts consist of 1%-5% of all mediastinal masses and 5% of anterior mediastinal masses [14][15][16]. According to a recent study, the median age of diagnosis is 49.5 years [17]. Although generally asymptomatic, thymic cysts have been associated with symptoms such as chest pain, superior vena cava syndrome, wheezing, or dyspnea due to either mass effect or infection. Pathologically, thymic cysts are classified as congenital (in association with a branchial cleft defect) or acquired [18]. The former, lined by single-layered epithelium contain Hassall corpuscles and may be located in both neck and mediastinum [19]. On the other hand, acquired cysts -as in the presented case -contain reactive immune cell populations and are characterized by the presence of cholesterol granulomas in pathology examination [2,[18][19].
With regard to the treatment, no consensus exists on the appropriate approach for benign thymic cysts. Surgical removal seems to be the preferred therapeutic strategy, as it decreases the possibility of complications or recurrence [7][8]. Surgical treatment includes thoracotomy, sternotomy, VATS, and RATS [3,9]. In our case, the size of the cyst and a non-accessible lymph node proximal to the left brachiocephalic vein led to the complete surgical excision of the lesion through a median sternotomy.
Conclusions
In conclusion, an acquired thymic cyst represents a rare entity, usually harboring diseases that need to be excluded, namely, HIV infection, lymphoma, and autoimmune diseases. In our case, however, no such diagnosis was established, raising questions regarding the initiating factors contributing to the disease progress. The lack of broad surgical experience and the association with other mediastinal diseases has also prevented the development of a standardized surgical approach with a level of evidence beyond the expert's opinion.
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. | 2020-10-29T09:03:23.018Z | 2020-10-01T00:00:00.000 | {
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201086348 | pes2o/s2orc | v3-fos-license | Vertical Versus Horizontal Foreign Direct Investment and Technology Spillovers
Foreign direct investment (FDI) and international trade are two major channels of international diffusion of technological knowledge (Keller 2004,2010). While a number of empirical studies confirm significant spillover effects of knowledge through imports, the empirical findings on technology spillover effects through FDI are conflicting. In particular, there is relatively little evidence of spillovers of knowledge from inward FDI to the host country’s firms in the same industry. For example, Haskel et al. (2007) examine the situation in the United Kingdom and find significantly positive productivity spillovers from FDI.
Introduction
Foreign direct investment (FDI) and international trade are two major channels of international diffusion of technological knowledge (Keller 2004(Keller ,2010. While a number of empirical studies confirm significant spillover effects of knowledge through imports, the empirical findings on technology spillover effects through FDI are conflicting. In particular, there is relatively little evidence of spillovers of knowledge from inward FDI to the host country's firms in the same industry. For example, Haskel et al. (2007) examine the situation in the United Kingdom and find significantly positive productivity spillovers from FDI. In addition, Keller and Yeaple (2009) show similar results for the United States. By contrast, Aitken and Harrison (1999) and Haddad and Harrison (1993) find no significant or weak productivity spillovers from FDI for developing countries hosting FDI (the former and the latter obtain results on Venezuela and Morocco, respectively). 1 Addressing the endogenous nature of the FDI decision, Lu et al. (2017) examine the spillover effects of FDI in China and find that the presence of FDI in the same industry has a significantly negative effect on the productivity levels of domestic firms due to the competition effect, while it has no significant effect on the exporting performance and research and development (R&D) investment of domestic firms. Todo (2006) and Todo and Miyamoto (2006) derive that R&D activities play an important role in productivity spillovers from FDI to local firms in the same industry: for example, a positive, statistically significant spillover effect is observed only for R&D-performing foreign firms (in Indonesia) or foreign firms' R&D stock (in Japan). In contrast, a number of studies find significant productivity spillovers from inward FDI to the host country's upstream firms through backward linkages. These studies include Javorcik (2004) for the case of Lithuania, Javorcik and Spatareanu (2008) for that of Romania, Blalock and Gertler (2008) for that of Indonesia, and Newman et al. (2015) for that of Viet-nam. 2 Moreover, Branstetter (2006) and Singh (2007) find evidence of technology spillovers from outward FDI. That is, firms investing in foreign countries acquire technological knowledge from other firms in the host countries.
When a firm establishes business enterprises in two or more countries through FDI, it becomes a multinational enterprise (MNE). Canonical FDI theory defines two types, namely horizontal and vertical, of FDI and an MNE's activities. 3 Horizontal FDI (HFDI) replicates a subset of the production process in foreign countries to serve local markets (Brainard, 1993(Brainard, , 1997Helpman et al., 2004;Markusen, 1984Markusen, , 1995Markusen andVenables, 1998, 2000). It is often motivated by an intention to reduce transportation costs. In contrast, vertical FDI (VFDI) involves geographical fragmentation of the production process and is often motivated by an intention to take advantage of factor cost differentials (Helpman, 1984(Helpman, , 1985Helpman and Krugman, 1985;Venables, 1999). However, the actual patterns of FDI and an MNE's activities are much more complex than the simple dichotomy of "horizontal" and "vertical". Yeaple (2003a) constructs a model in which horizontal, vertical, and complex (i.e., both horizontal and vertical) FDI arises endogenously. Grossman et al. (2006) also analyze MNEs' integration strategies that may simultaneously involve horizontal and vertical FDI. Ekholm et al. (2007) propose the type of "export-platform FDI" as another type of FDI. Moreover, utilizing the information on sales and sourcing patterns of foreign affiliates of MNEs, Baldwin and Okubo (2014) support the importance of "networked FDI." A number of empirical studies provide support to the predictions for HFDI. For example, Brainard (1993Brainard ( , 1997 obtains evidence of HFDI but little evidence of VFDI. Markusen and Maskus (2002) argue that a large proportion of FDI takes place among developed countries and is characterized by the horizontal type of FDI. Looking at the location decisions of MNEs, however, Yeaple (2003b) and Hanson et al. (2005) find evidence consistent with comparative advantage. Moreover, Alfaro and Charlton (2009) point out that the share of VFDI is much higher than previously thought even within developed countries. They argue that a significant amount of VFDI has been misclassified as horizontal in the previous studies and find that a substantial amount of VFDI between developed countries emerges in high-skill sectors, because parent firms own the stages of production proximate to their final production and source raw materials and inputs in low-skill production stages from outside of the firms.
A literature that is separated from studies on FDI has shown the importance of international fragmentation of production and the involvement of firms in international production networks (or global value chains (GVC)). 4 Depending on the type of FDI or the degree of involvement in GVC, affiliates of MNEs conduct different activities in their host economies, affecting the extent of technology spillovers between MNEs and domestic firms in the host economies. For example, when MNEs conduct VFDI, foreign affiliates engage in intra-firm trade with their parent firms in the source country. On the other hand, in the case of networked FDI, foreign affiliates are involved in GVC and are likely to trade with other affiliates in the same region. Ramondo et al. (2016) document that intra-firm trade is concentrated on a small group of large foreign affiliates of MNEs headquartered in the United States. They find that the median manufacturing foreign affiliate of the US MNEs has no transaction of goods with its parent in the United States. This finding suggests that VFDI is concentrated on large firms and large affiliates. 5 To the best of our knowledge, no existing studies have investigated how the type of FDI or the degree of involvement in GVC affects technology spillovers between MNEs and their host economies.
An empirical work by Branstetter (2006) is closely related to the analysis in this chapter. He defines the term "technology spillovers" as "the process by which one inventor learns from the research outcomes of others' research projects and is able to enhance her own research productivity with this knowledge without fully compensating the other inventors for the value of this learning" (pp. 327-328). In this sense, technology spillovers must be distinguished from the term "productivity spillovers" which measures how productivity of a firm is affected by other firms' one or R&D activities. Then, in the literature with respect to spillovers, patent citation data have been used as a proxy of technology spillovers in the above definition (Jaffe et al. 1993). 6 Branstetter (2006 analyzes firm-level data on Japanese MNEs in the United States and patent citations at the United States Patent and Trademark Office (USPTO) and obtains evidence that FDI facilitates technology spillovers both from investing firms to local firms in the host country and from the local firms to the investing firms. Although he examines whether different types of FDI, such as acquisition, greenfield investment, and R&D facilities, have different effects on spillovers, he does not distinguish various production activities of foreign affiliates. Atalay et al. (2014) for production chains within the United States. In particular, they show that the ownership of vertically linked affiliates is not related to the transfer of goods within the boundaries of the firm. Ramondo et al. (2016)) argue that the vertical ownership promotes efficient intra-firm transfers of intangible inputs. 6 There are a number of existing empirical studies on technology spillovers based on patent citations (Cappelli and Montobbio, 2020;Haruna et al., 2010;Hu and Jaffe, 2003;Jaffe and Trajtenberg, 1999;Jinji et al., 2013Jinji et al., , 2015Jinji et al., , 2019aLi, 2014;MacGarvie, 2006;Mancusi, 2008;Murata et al., 2014;Peri, 2005).
In this chapter, we attempt to identify how the structure of MNEs' activities in terms of horizontal and vertical FDI affects technology spillovers between MNEs and host countries. Then, we combine a comprehensive firm-level dataset of the business activities of Japanese MNEs' foreign affiliates and information on the patent citations between MNEs and their host countries. Following Branstetter (2006), we define "technology spillovers" as the effects on the research productivity from the outcomes of others' research activities without full compensation for the value of research productivity enhancement. 7 Alternatively, we use firm-level data on Japanese firms' FDI and patent citations at the USPTO. 8 Our firm-level dataset includes information on the sales and purchases of the foreign affiliates, classified according to the destination and source countries. We exploit this information to construct new measures of horizontal and vertical FDI based on the shares of the host and home countries in their transactions. Now we define a measure of "pure horizontal FDI" as the extent to which affiliates' purchases of intermediate inputs and sales of final goods are concentrated in the local market. We also define a measure of "pure vertical FDI" as the extent to which their affiliates' purchases of intermediate inputs and sales of final goods are linked to the home country. We, furthermore, define measures of "partially horizontal" and "partially vertical" FDI. We then estimate how different types of FDI affect technology spillovers from Japanese MNEs to the host country and from the host country to Japanese MNEs. As for the empirical methodology, we follow Branstetter (2006). Since the dependent variable (i.e., patent citations) is the count data, we utilize a negative binomial model developed by Hausman et al. (1984). Moreover, to deal with a potential endogeneity problem we employ an endogenous switching model discussed by Miranda and Rabe-Hesketh (2006).
Our main findings are as follows. We find that an increase in the degree of pure VFDI has significantly positive effects on technology spillovers captured by patent citations when technologically advanced economies host Japanese MNEs. Technology spillovers occur in both directions between the MNEs and their host countries. These positive effects of pure VFDI on technology spillovers are robust for different specifications as well. Partially VFDI (i.e., FDI with a higher share of purchase of intermediate inputs in the local market and a higher share of sales of outputs to the home country) also has significantly positive effects on technology spillovers from the (high-income) host countries to the MNEs. 9 By contrast, an increase in the degree of pure HFDI has no significant effect or significantly negative effects on technology spillovers between the MNEs and their host countries. Partially HFDI (i.e., FDI with a higher share of purchases from the home country and a higher share of sales to the local market) has significantly positive effects on technology spillovers from the MNEs to the (high-income) host countries, but the result is not robust for different estimations. From these results, we conclude that pure VFDI plays a dominant role in technology spillovers in both directions between Japanese MNEs and the high-income host countries.
To explain the mechanism for the observed relationship between the structure of FDI and technology spillovers, we develop a simple partial-equilibrium model of FDI and technology spillovers among developed countries. A differentiated good is produced in three stages. 10 The product market is characterized by monopolistic competition. Depending on parameter values, firms may have an incentive to engage in horizontal or vertical FDI. Assuming that factor costs are the same in the two countries, there is no possibility of VFDI in the usual sense. Nevertheless, VFDI does occur if there are technology gaps in some production stages between the two countries and/or if firms can take advantage of the superior technology by fragmenting their production process abroad. Technological differences in some production stages are considered to be the source of technology spillovers through FDI. We show that technology spillovers occur in one way or two ways if firms engage in VFDI, depending on how the three production stages are located in the two countries. Besides, we show that HFDI does not necessarily induce technology spillovers, because it is mainly motivated by saving transportation costs and hence appears even in the absence of technological differences.
The rest of this chapter is organized as follows. Section 6.2 describes the data employed in our empirical analysis. Section 6.3 introduces estimation methods. Section 6.4 provides empirical results. Section 6.5 develops a simple theoretical model of FDI and technology spillovers to explain the observed relationship in our empirical study. Section 6.6 concludes this chapter.
Data
In this section we describe the data employed in our empirical analysis. Following Jaffe et al. (1993), Jaffe and Trajtenberg (1999), and other studies, we use patent citation data as a proxy for technology spillovers. The patent citations are collected from the dataset compiled by the National Bureau of Economic Research (NBER) patent database for patents at the USPTO. 11 The dataset includes information on the application date, the country name of the assignee, the main US patent class, and citations made and received for each patent. From the dataset we extract information on the patent applications and citations by Japanese MNEs and their host countries. Because of the truncated problems of citations in the NBER dataset, we concentrate our analysis on the period before 2003, though the NBER patent dataset includes data until 2006.
Data on Patent Citations and Japanese Firms' FDI
Our data on Japanese MNEs' activities abroad are obtained from the Basic Survey on Overseas Business Activities (BSOBA) or Kaigai Jigyo Katsudo Kihon Chosa conducted by the Japanese Ministry of Economic, Trade and Industry (METI). This data source provides detailed data on affiliate-level FDI activities such as the sales and purchases of affiliates of Japanese MNEs, classified by their destinations and sources, i.e., sales to (or purchases from) the local market or exports to (or imports from) the home country and a third country.
The foreign affiliates listed in the BSOBA are either foreign affiliates with at least 10% of their capital held by a Japanese parent company or those with at least 50% of their capital held by a foreign subsidiary, which in turn has at least 50% of its capital held by a Japanese parent company. These affiliates exclude those that run businesses in the financial and insurance industry or the real estate industry in host countries. According to the METI, there were approximately 15,000 foreign affiliates that responded to the survey in 2000. 12 Table 6.1 shows the top 30 host economies for Japanese MNEs in 2000, based on the number of affiliates that had completed the BSOBA. 13 As shown in the table, the United States attracts the largest number of Japanese firms' affiliates, followed by China. Asian economies as well as developed countries are popular host economies for Japanese MNEs.
Our sample covers the period between 1995 and 2003. All countries in which Japanese MNEs have at least one affiliate are included in our sample. Since the number of countries varies greatly depending on that of patent applications made, we divide our sample of the countries into two groups, Groups I and II, according to the number of USPTO patent applications made by the sample countries during the period of 1995-2003. We call Group I countries/economies "Technologically Advanced Economies," which mainly include high income countries/economies. In contrast, we call Group II countries/economies "Technologically Less Advanced Economies," which mainly include middle and low income countries. The number of patent applications in Group I is larger than 1,000, while it is less than that in Group II. 14 All countries that are categorized into each group are listed in 13 About 10,100 affiliates reported full or partial information on their sales and purchases classified by the destinations and the sources in 2000. 14 Although the number of USPTO patent applications made by China, India, Russia, and Singapore is more than 1,000 during the period, a large jump in the applications is observed after 2000, We use the Nikkei company code system to link the two data sources and collected the data on 1,445 parent companies that run at least one affiliate during the sample period. 279 parent companies out of 1,445 ones made at least one citation to USPTO patent applications from 93 countries and, on the other hand, 301 ones received at least one USPTO patent citation.
Types of FDI
In the literature, FDI and MNEs' activities are usually categorized into horizontal and vertical cases. In the empirical studies, there are a number of ways to measure horizontal and vertical FDI. Hummels et al. (2001) and Alfaro and Charlton (2009) use the industrial classifications to define the types of FDI. Hanson et al. (2001Hanson et al. ( , 2005 utilize the firm-level database of the United States Bureau of Economic Analysis (BEA) to characterize VFDI as intra-firm flows of inputs that they observed flowing from parent companies in the United States to affiliates abroad. The method of Hanson et al. (2001Hanson et al. ( , 2005 enables them to measure one-way US bilateral intra-firm trade. Using the same BSOBA dataset as in this chapter, Fukao and Wei (2008) employ the local sales ratio of the affiliates to classify vertical and horizontal FDI. In particular, if a local sales ratio of a foreign affiliate is less than the average ratio in the sample, then FDI to the affiliate is classified into VFDI. By contrast, if a local sales ratio is larger than the average ratio, FDI to the affiliate is classified into HFDI.
An advantage of the BSOBA dataset is that it allows us to measure vertical and horizontal FDI by using information on the sale of outputs and the purchase of inputs by foreign affiliates. 15 The local sales and local purchases ratios of foreign affiliates of Japanese MNEs are denoted by ShSa H F D I and Sh Pu H F D I , respectively. Similarly, the sales and purchases ratios to and from Japan for foreign affiliates of Japanese MNEs are denoted by ShSaV F D I and Sh PuV F D I , respectively. Table 6.3 shows the average values of both ratios during the sample period for the subsamples of technologically advanced and less advanced economies. Looking at the ratios over the years, no evident trend is observed during the sample period. Interestingly, the table also shows that the values of ShSaV F D I , which indicates the vertical structure of sales from foreign affiliates, are around 10-12% in technologically advanced economies and around 20-22% in technologically less advanced economies. If we focus on Sh PuV F D I (i.e., the vertical structure of purchases by foreign affiliates), then the values climb up to around 40-42% in technologically advanced economies and 37-40% in technologically less advanced economies. With respect to the purcompared with a very limited number in the early years for these countries. We therefore categorize these four countries into the second group. 15 One limitation of the BSOBA dataset is, however, that it does not track transactions between foreign affiliates or between foreign affiliates and the parent companies. As a result, there may exist some biases for measuring the types of FDI by using information on sale and purchase because we cannot examine flows within the boundary of a firm from our dataset. 1995 1996 1997 1998 1999 2000 2001 2002 2003 Tech chase of inputs, Japanese MNEs engage in VFDI more actively in technologically advanced host countries than in technologically less advanced host countries. This evidence is consistent with Alfaro and Charlton (2009), who showed that VFDI emerges as far more prevalent between developed countries. By exploiting information on the horizontal and vertical structures in sales and purchases of foreign affiliates, we construct new indexes of horizontal and vertical FDI, i.e., H F DI , V F DI , P H F DI , and PV F D I , in the following way: As is evident from the definition of the index, H F DI measures the extent to which affiliates' purchases of intermediate inputs and sales of final goods are concentrated in the local market. Now, H F DI captures the degree of "pure" HFDI. If H F DI = 1, a foreign affiliate makes all purchases and sales in the local market, whereas, if H F DI = 0, either or both of purchases and sales of the foreign affiliate become zero in the local market. Note that H F DI = 0 does not necessarily mean that the foreign affiliate engages in vertical activities because there is a possibility of transactions with third countries. Next V F DI measures the extent to which affiliates' purchases of intermediate inputs and sales of final goods are linked to the home country (i.e., Japan), so that V F DI captures the degree of "pure" VFDI. On the other hand, P H F DI and PV F D I capture "partially" horizontal and "partially" vertical FDI, respectively. The value of P H F DI rises if an affiliate buys more intermediate goods from the home country and sells more final goods to the local market. Since the structure of sales is more important to distinguish the type of FDI than the structure of purchases, we consider that P H F DI measures the degree of "partially" horizontal FDI in terms of its horizontal sales structure. Similarly, the value of PV F D I becomes large if an affiliate buys more intermediate goods from the local market and sells more final goods to the home country. Since the structure of sales is vertical in PV F D I , we consider that it measures the degree of "partially" vertical FDI.
We then test whether there are any differences in the effects on technology spillovers among these types of FDI.
The Empirical Model
In this section, let us explain our empirical model. Although the BSOBA is conducted every year, there are many blanks in the data on a particular firm because in some years certain respondents did not report to the METI. For this reason, we use only a pooled data in our estimation. Consequently, we run the following specification as in Branstetter (2006), where i refers to the affiliate i, and C i is the number of citations made (or received) by USPTO patents of the Japanese parent company that owns affiliate i. Note that C i = C i holds for affiliate i and affiliate i if the same parent company owns affiliates i and i . We expect that citations made by Japanese parent companies capture the technology spillovers flowing from the host countries to Japanese companies, while the citations received by them reflect the flows from Japanese companies to host countries. F DI i in Eq. (6.1) is one of the alternative measures of the FDI types, i.e., H F DI , V F DI , P H F DI , and PV F D I for affiliate i. In Eq. (6.1), β 1 is a constant term and u i is an error term. As indicated in Branstetter (2006), patent citations may rise as the "citable" host invention increases. At the same time, the higher absorptive capacity in the home country may be associated with a higher ability to understand and exploit external knowledge, and cite more external patents (Mancusi, 2008). Thus, for elucidation of the assertions we use L P Parent i and L P Host i , which refer to the logarithm of the count of the USPTO patent applications made by affiliate i's Japanese parent company and the host country where the affiliate i runs its business, respectively, to proxy the home absorptive capacity and "citable" host invention. Note that L P Parent i is the same across affiliate i for the same parent company, and L P Host i is the same across affiliate i for the same host country.
The focus of interest in Eq. (6.1) will be the coefficient β 2 . Hence, we examine if the FDI types of Japanese firms in host countries have an influence on patent citations made and received by the firm. We also investigate if there is a difference in the magnitude and sign of the coefficients between the citations made and received by the home and host countries and across the types of FDI that Japanese firms implemented.
Since the observations of a dependent variable (i.e., patent citations) are the count data, we utilize a standard estimation technique, namely, a negative binomial model discussed in Cameron and Trivedi (1998), where the data are Poisson process, but more flexible modeling of the variance to account for overdispersion than the Poisson is allowed. We use this estimation technique to acquire our basic findings and alternative estimation results.
The other challenge of estimating the effects of each of the FDI types on technology spillovers arises from the fact that patent citations may be endogenous, because unobservables in determining the types of FDI may be correlated with those in determining the citations. Besides, certain geographic factors such as distance and language may influence the citations as well as the types of FDI. Neglecting these unobserved or endogenous factors may cause biased and inconsistent estimators. 16 To address this issue, we employ an endogenous switching model. 17 In that model, the citation C i follows a Poisson distribution, and the probability distribution for count data is given by so that a log-linear model for the mean of C i , μ i , can be specified as where L Dist i is the logarithm of the distance (measured as kilometers) between Japan and the host economy of affiliate i, LG D P i is the logarithm of GDP of affiliate i's host economy, LCost i is the logarithm of salary per employee of affiliate i, and i is an unobserved heterogeneity term.
LG D P i measures the market size of the host economy, and LCost i measures the labor cost of the affiliate. Instead of F DI i in Eq. (6.1), here we use a dummy D i (D H F DI i , D V F DI i , D P H F DI i , or D PV F DI i ) for the types of FDI, which equals one for a particular type, and zero otherwise. Following Fukao and Wei (2008), we construct the dummy for a particular type of FDI such that it equals one when the value of an FDI type's index (H F DI , V F DI , P H F DI , or PV F D I ) for affiliate i is greater than the average value of the particular FDI type's index in the full sample, and zero otherwise. We then use a probit model to examine how a parent firm determines its FDI type. The logic we use is that the type decision on FDI depends on factors that favor a particular type of FDI or not. The probit model can be formulated as where D * i is an auxiliary random variable, z i is a vector of factors which may influence the particular type of FDI. As usual, we have u i ∼ N (0, 1), and u i is independent of i . In the so-called endogenous switching model, V ar( i ) = σ 2 , and total variance is λ 2 σ 2 + 1. If λ = 0, then D i is considered to be exogenous. Although a Poisson distribution is used, the variance of C i is not necessarily equal to the conditional mean, and overdispersion is allowed in this model. Using the normality assumption for i , we have where x i is a vector of explanatory variables in Eq. (6.3) (i.e., L P Host i , L P Parent i , L Dist i , LG D P i , LCost i , and the constant term), which implies that if σ = 0, then the model exhibits overdispersion, as we would expect from the negative binomial model in Eq. (6.1). 18 In the estimation, following Fukao and Wei (2008), we include L Dist i (distance), LG D P i (market size), and LCost i (labor cost) in z i in Eq. (6.4). Among those variables, the data on salaries and the number of employees of foreign affiliates are obtained from the BSOBA. The data on distance are collected from the database of the CEPII Research Center and the data on GDP in host countries are obtained from the Penn World Table. 19
Empirical Results
In this section, we report our estimation results. We first show the basic findings obtained by the negative binomial model. We then report the results by the endogenous switching model and discuss whether the endogeneity issue matters in our analysis. Finally, we discuss the robustness of our findings by showing the results of alternative estimations with additional explanatory variables.
Basic Findings
We first estimate Eq. (6.1) by the negative binomial model, and the results are reported in Table 6.4. The upper panel of Table 6.4 shows the estimated results for the sub- Notes: (a) ***, **, and * indicate significance at the 1%, 5%, and 10% levels, respectively (b) The values in the parentheses are standard errors (c) A constant term is included in the estimations sample of technologically advanced economies. We observe from the results that the estimates of H F DI are significant and negative, whereas they are significantly positive for V F DI both for the citing and cited. As for P H F DI , the estimated coefficient is insignificant for the citing, but significantly positive for the cited, whereas the coefficient of PV F D I are significant and positive for the citing and insignificant for the cited. The lower panel of Table 6.4 presents the estimated results for the subsample of technologically less advanced economies. Unlike the case of the technologically advanced economies, only the coefficients on PV F D I show significantly positive, whereas the coefficients of the other types of FDI reveal negative or insignificant effects on the citing as well as the cited.
These results show that an increase in the degree of the "pure" VFDI has a significantly positive effect on technology spillovers in both directions between Japanese parent companies and their host countries if Japanese MNEs invest in high-income countries. This implies that VFDI plays a dominant role in technology spillovers with mutual effects in technologically advanced economies. When middle-and lowincome countries host Japanese MNEs, an increase in the "partially" vertical FDI has a significantly positive effect on the number of patent citations in both directions between the Japanese parent companies and firms in their host countries.
Estimating with an Endogenous Switching Model
To deal with endogeneity issues, we simultaneously estimate both an endogenous switching model described by Eqs. (6.2) and (6.3) for technology spillovers and a probit model based on Eq. (6.4) for the decision on FDI types. We focus on the subsample of technologically advanced economies. The estimated results are summarized in Table 6.5. We first observe that the coefficient of LCost is significantly positive for both "pure" VFDI (D V F DI ) and "partially" vertical FDI (D PV F DI ). This implies that VFDI to technologically advanced economies is not motivated by wage cost saving.
In terms of the endogeneity between technology spillovers and the decision on FDI types, the estimates of ρ in Table 6.5 show strong significance against the null hypothesis in two cases out of eight estimations for technologically advanced economies and in all cases for technologically less advanced economies. 20 Consequently, neglecting the endogenous issues may cause biased and inconsistent estimators (Miranda and Rabe-Hesketh, 2006). The estimations in Table 6.5 reveal that D V F DI based on the endogenous switching model for both the citing and cited cases provides results similar to those based on the negative binomial model. For D PV F DI , the two models also provide similar results, suggesting that more local purchases and more sales in Japan may favor Japanese parent companies with more technology spillovers from Our findings imply that, for technologically advanced economies, "pure" VFDI is associated with significant technology spillovers even if we control for endogenous issues.
Alternative Estimations
To check the robustness of the basic findings in Sect. 6.4.1, we conduct alternative estimations for Group I countries by adding explanatory variables. We include P RO X (technological proximity), Cap Ratio (capital ratio), and Close (a dummy for industrial classification, which is one for the same sector and zero otherwise). We also include L Dist (the logarithm of the distance between Japan and host countries) and Y ear which captures the changes in citations.
Japanese parent companies and firms in their host countries may increase their citations of each other just because Japanese parent companies and firms in their host countries change the focus of their research activities in ways that bring them "closer" to each other in the technology space (Branstetter, 2006). To control for this issue, we include a measure of technological proximity (P RO X) in the regression. As suggested by Jaffe (1986) andBranstetter (2006), P RO X is constructed by where F i = ( f 1i , · · · , f ki ) is a vector of the cumulative count of patents obtained by affiliate i's parent firm in kth technical area 21 and F host,i is a vector of the aggregate count of patens obtained by all firms in the host country in which affiliate i is located. The literature on the role of affiliate ownership in technology spillovers is limited. There are a few studies focused on the correlation between productivity and the ownership of affiliates. Javorcik (2004) and Javorcik and Spatareanu (2008) found that the correlation of productivity with FDI is stronger if the affiliate is only partially, and not fully foreign owned, because joint ownership generates more technology transfer, and wholly owned affiliates employ more sophisticated technology that is out of reach of the average domestic supplier. As indicated by Keller (2010), however, the technology gap may be a key reason for differential effects for wholly versus partially owned affiliates. Cap Ratio, which is the share of affiliate capital owned by Japanese parent companies, is included to test the effects of ownership of affiliates on technology spillovers.
As in Sect. 6.4.1, we use patent citations at the USPTO as the dependent variable and employ the negative binomial model for our estimation. The estimated results are presented in Table 6.6. 22 The coefficients of P RO X are significantly positive in all cases of citing and cited. These results confirm the findings in Branstetter (2006). The coefficients of Close are significantly positive in technology spillovers from host economies to Japanese MNEs, which implies that Japanese parent companies cite more patents of host economies when their affiliates run a business that is the same as or close to that of the parents. However, this is not the case for technology spillovers from Japanese MNEs to host economies, since the coefficients of Close are significantly negative. The ownership variable, Cap Ratio, has positive coefficients that are mostly significant, which implies that a higher share of ownership of foreign affiliates by Japanese parent companies tends to facilitate technology spillovers in both directions between Japanese MNEs and their host economies.
The estimates of H F DI , V F DI , P H F DI , and PV F D I for the USPTO give similar results to those we observed in Table 6.4, except for P H F DI and PV F D I in the cited. Specifically, the significantly positive effect of PV F D I is not robust for some combinations of explanatory variables.
A Simple Model of FDI and Technology Spillovers
To explain the observed relationship between the structure of FDI and technology spillovers in the previous section, we develop a partial-equilibrium model of FDI and technology spillovers among developed economies. We consider a world of two countries, home and foreign. Foreign variables are denoted by an asterisk.
We focus on the market for a differentiated good x. Consumers in the two countries share the same preference. The preference of the representative consumer takes the standard Dixit-Stiglitz form (Dixit and Stiglitz, 1977): , where x j is the consumption of a variety j of good x, α = 1 − 1/σ , σ > 1 is the elasticity of substitution across varieties, and N is the total number of varieties supplied in the home market. We also assume that the market size is the same in the two countries.
The demand for a variety j of good x in the home country is given by where E is the total expenditure on good x in the home country (which is the same for the foreign country), p c j is the CIF price of variety j produced by a home firm and P is the price index for the x sector goods, which is defined as where n and n * are the numbers of varieties produced by the home firms and the foreign firms, respectively, with n + n * = N , and p * c k is the CIF price of variety k produced by a foreign firm.
In each country, there is one primary factor of production, labor, denoted by l. The wage rate, w, is the same in the two countries, i.e., w = w * ≡ ω. Labor is immobile across countries.
Production and Supply
Good x is differentiated by variety and is supplied by monopolistically competitive firms. Each firm produces one variety. The nationality of a firm is identified by the location of its headquarters.
Good x is produced in three sequential stages. 23 Intermediate inputs for good x are specific to varieties, and hence there is no market for its intermediate inputs. For simplicity we assume away the possibility of outsourcing production of intermediate inputs. All of the three production stages must be conducted in house, although firms can offshore some or all of the production stages by establishing affiliates in the other country.
The first stage of production is simply conducted by using only labor, so that one unit of an intermediate input m 1 is produced by one unit of labor: m 1 = l 1 . The quality of the intermediate input may be differentiated. It is θ 1 if the first stage is performed in the home country and θ * 1 if it is in the foreign country, where 0 < θ 1 ≤ 1 and 0 < θ * 1 ≤ 1. The quality of m 1 matters when it is used in the second stage of production. The production function of an intermediate product is given by m 2 = (θ 1 m 1 ) γ l 1−γ 2 , where m 2 stands for an intermediate product in the second stage and γ ∈ (0, 1). In the production function of m 2 , θ 1 is replaced by θ * 1 if m 1 produced in the foreign country is used. Moreover, in the final stage of production, m 2 and labor are used to produce a variety j of good x: x j = (θ 2 m 2 ) γ l 1−γ 3 , where the quality of m 2 is θ 2 (θ * 2 ) if the second stage of production is performed in the home (foreign) country, where 0 < θ 2 ≤ 1 and 0 < θ * 2 ≤ 1. A difference in the quality of intermediate inputs m 1 and m 2 reflects the technology gap for the particular production stage between the two countries. 24 Note that the location of the production stage rather than the nationality of firms determines the quality of intermediate inputs. This may be because the information on the technology of producing intermediate inputs is locally spilled over, while it is not spilled over across countries. 25 Since the final good x is not differentiated in quality, the location of the final production stage does not affect characteristics of varieties.
Iceberg transportation costs apply to cross-country shipment of both intermediate inputs and final goods. For one unit of an intermediate input and a final good to arrive at a foreign destination, τ ≥ 1 units of an intermediate input and t ≥ 1 units of the final good must be sent, respectively.
When a firm sets up a production stage in the country different from the country in which its headquarters is located, it engages in FDI and incurs an extra fixed cost of Φ/3 units of labor per stage, where Φ > 0 is exogenously given and constant.
Each monopolistically competitive firm chooses both the location of the three production stages and the price for its own variety in each market, taking the price index as given. As is well known, a monopolistically competitive firm charges a constant mark-up over the unit cost of the final good, which is given by p j = C j /α, where p j is the FOB price of variety j and C j is the unit cost of producing variety j, which will be shown in detail below.
Technology Spillovers
We introduce the possibility of technology spillovers. Suppose that the quality of intermediate inputs can be upgraded by R&D. We suppose that the outcome of R&D is stochastic. Consequently, the difference in the quality of intermediate inputs arises, depending on whether R&D was successful or not. 26 We take up the effects of spillovers from one firm's R&D outcome to other firms' product stage. As is well known, technology spillovers occur from a firm with higher technology to a firm with lower technology. We assume that when a firm uses a better quality of an ith-stage intermediate input for the production of the (i + 1)th stage, the information on the better quality of an ith-stage intermediate input is spilled over to the (i + 1)th stage and improves the productivity of R&D for the (i + 1)th-stage intermediate input.
Under the above assumptions, the following lemma on spillovers holds. 24 For our purpose of this chapter, we do not need to specify the cause of the technology gap between the two countries. Then, we assume that there is a technology gap between the two countries. 25 This is different from technology spillovers that we will discuss below. 26 The purpose of our analysis in this section is to investigate the relationship between types of FDI and technology spillovers. For this, we want to show how the technology gap between the two countries is related to FDI types and how it is also related to technology spillovers. But, we do not need to specify the details of R&D and the production in the next period after spillovers occur.
FDI and Technology Spillovers: HFDI
We next analyze how firms locate production stages and how international technology spillovers are associated with FDI.
We define horizontal and vertical FDI in the following way. If a firm conducts the final stage in both the home and the foreign countries, it engages in HFDI. On the other hand, if a firm conducts either or both of the first two stages in the country that is different from the country in which its headquarters is located without conducting the final stage in that country, it engages in VFDI.
We focus on the representative firm whose headquarters is located in the home country. We call it "the home firm." All firms with the same nationality behave in the same way.
We first look at HFDI. We denote the combination of locations for three production stages by three capital letters. For example, if the first stage is located in the home country, the second stage is in the foreign country, and the final stage is in the home country, then we denote this combination by H F H. Then, the possible patterns of HFDI for the home firm are {F F F, H F F, H H F, F H F}. The unit cost function to serve the foreign market by the pattern of F F F is given by Similarly, the unit cost functions to serve the foreign market by the patterns of H F F and H H F are given by, respectively, Moreover, the unit cost function to serve the foreign market by exporting from the home country is (6.8) where the transport costs for the shipment of the final good are not included.
In the analysis of the optimal configuration of production for the foreign market, it turns out that the configuration for the home market does not matter. Thus, we just focus on the profits from supplying to the foreign market. Given the demand for a variety and pricing policy under monopolistic competition, profits of the home firm from supplying a variety to the foreign market by the patterns F F F, H F F, H H F, and H H H are given by, respectively, Φω, (6.10) Note that since F H F is dominated by H H F and H F F for θ i = θ * i = 1 for i = 1, 2, we can exclude F H F from our analysis. Then, the following lemma is obtained.
Lemma 6.2 Suppose that θ i = θ * i = 1, i = 1, 2. The home firm still has an incentive to engage in HFDI if t > τ γ and Φ is sufficiently low.
This lemma implies that even without technology differences, firms have an incentive to engage in HFDI if transportation costs for the cross-border shipment of the final goods are sufficiently high and the additional fixed costs for FDI are sufficiently low. This is just the standard motive for the HFDI. Lemma 6.2 yields the following proposition.
Proposition 6.1 HFDI does not necessarily induce technology spillovers.
Proof From Lemma 6.2, HFDI occurs even in the case of no technology difference. If the technology level is the same across countries, then there is no possibility of technology spillovers. Q.E.D.
When the production configuration is H F F, the first and the second stages are located in different countries. Thus, Lemma 6.1 suggests that technology spillovers may occur. However, the proof of Lemma 6.2 shows that H F F can be the optimal configuration even in the absence of a technology gap. If this is the case, technology spillovers do not occur in the case of H F F.
FDI and Technology Spillovers: VFDI
We turn to the case of VFDI. In this case, the home firm conducts the final stage only in the home country, so that the possible patterns of VFDI are {F H H, F F H, H F H}. We obtain the following lemma. Lemma 6.3 Suppose that θ i = θ * i = 1 for i = 1, 2. Then, the home firm has no incentive to engage in VFDI as long as τ and Φ are positive.
Proof By definition, the home firm conducts the final stage in the home country for any type of VFDI. We need to examine the supply to the home market. Then, compared to the national integration (i.e., the pattern of H H H), FDI in either or both of the first two stages incurs additional transportation costs and fixed costs of FDI. Thus, in the absence of technology advantage in the foreign production, VFDI always increases production costs as long as τ and Φ are positive. Q.E.D.
This lemma implies that in the absence of a factor cost differential, firms have no incentive to engage in VFDI without a technology difference as long as there are positive transportation costs for intermediate inputs and FDI requires additional fixed costs. Now, the unit cost functions for the home firm in VFDI are given by In order to simplify the analysis, we assume that t = 1, so that firms have no incentive to locate the final stage of production for the foreign market separately from that for the home market. It is then shown that as long as the final stage is performed in a single location, firms have no incentive to conduct either or both of the first two stages at more than one location. Given this, profits of the home firm in production configurations k = H F H, F F H, F H H, and H H H are given by, respectively.
This proposition shows that if home firms engage in VFDI in the configuration of H F H, technology spillovers occur from the foreign country to the home country. Technology spillovers in the opposite direction may also occur but do not necessarily. For other types of VFDI, we expect that technology spillovers occur only in one direction from the foreign country to the home country.
Note that if the wage rates between the two countries are sufficiently different, then firms may have an incentive to engage in VFDI even in the absence of a technology difference, but VFDI does not induce international technology spillovers. This situation corresponds to the typical North-South VFDI rather than the North-North VFDI. Then, this never undermines our argument on the relationship between VFDI and technology spillovers. That is, North-South VFDI does not necessarily involve international technology spillovers.
Conclusion
In this chapter, we have investigated how the structure of MNEs' activities affects technology spillovers between MNEs and their host countries by using detailed firmlevel data on Japanese MNEs and patent citation data. We propose new specifications of FDI by information on sales and purchases of foreign affiliates of MNEs. We define pure HFDI as FDI with a high share of both purchases of intermediate inputs and sales of outputs in the local market and pure VFDI as FDI with a high share of transactions (i.e., both purchases of intermediate inputs and sales of outputs) with the home country. In addition, partially horizontal and partially vertical FDI are defined.
Our estimation results reveal that when a technologically advanced country hosts Japanese MNEs, an increase in the degree of pure VFDI has a significantly positive effect on technology spillovers as measured by patent citations in both directions between the host country and Japanese MNEs. In contrast, pure HFDI has no significant effect or significantly negative effects on technology spillovers in either direction. We also find that VFDI by Japanese firms to technologically advanced countries is not based on factor price differentials.
To explain the mechanism for the observed relationship between the structure of FDI and technology spillovers, we have developed a simple model of FDI and technology spillovers, in which a good is produced in multiple stages. Our model reveals that VFDI among technologically advanced economies would be associated with international technology spillovers, while HFDI does not necessarily induce technology spillovers.
The results indicate that technology spillovers from FDI occur among technologically advanced economies. In particular, VFDI plays an important role in technology spillovers. We come to the conclusion that technologically advanced countries can gain knowledge flow from MNEs' activities both as the home country and as the host country when FDI involves the geographical fragmentation of the production process.
Another finding of this chapter is that when the host country is a technologically less advanced country, any types of FDI do not have positive effects on technology spillovers. One possible explanation for this finding is that we have focused on technology spillovers measured by patent citations. Since patent applications are made to be counted as "spillovers," indigenous firms in technologically less advanced countries do not largely benefit from technology spillovers in our definition. Another possible explanation for the finding is that it reflects the stringency of intellectual property right (IPR) protection in host countries. Branstetter et al. (2006) and Wakasugi and Ito (2009) find that the stronger protection of IPR in host countries has a positive effect on technology transfer from parent firms to their foreign affiliates. Nagaoka (2009) also finds a positive effect of stronger patent protection on expanding the scope of the recipients of technology transfer. Taking these empirical findings into account, we notice that the weaker protection of IPR in developing countries generally hinders technology spillovers from FDI in our measurement.
Since our findings are based on Japanese MNEs' data, we suggest testing whether our findings could be applicable to other countries' MNEs through examining detailed data on MNEs in other countries.
Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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.
The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. | 2019-08-15T06:34:12.296Z | 2021-11-11T00:00:00.000 | {
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13732150 | pes2o/s2orc | v3-fos-license | A plasmonic colorimetric strategy for visual miRNA detection based on hybridization chain reaction
In this work, a novel colorimetric strategy for miRNA analysis is proposed based on hybridization chain reaction (HCR)-mediated localized surface plasmon resonance (LSPR) variation of silver nanoparticles (AgNPs). miRNA in the sample to be tested is able to release HCR initiator from a solid interface to AgNPs colloid system by toehold exchange-mediated strand displacement, which then triggers the consumption of fuel strands with single-stranded tails for HCR. The final produced long nicked double-stranded DNA loses the ability to protect AgNPs from salt-induced aggregation. The stability variation of the colloid system can then be monitored by recording corresponding UV-vis spectrum and initial miRNA level is thus determined. This sensing system involves only four DNA strands which is quite simple. The practical utility is confirmed to be excellent by employing different biological samples.
act as the stabilizer of the colloid system. Nevertheless, double-stranded DNA exposes no nitrogen-containing bases and cannot interact with AgNPs. After HCR initiated by target miRNA, the transformation of fuel strands with single-stranded DNA tails to double-stranded DNA changes the stabilization ability of DNA molecules in AgNPs colloid system, which can be reflected by UV-vis spectrum. This simply designed method for the detection of miRNA achieves the limit of detection as low as 1 pM, which is lower than or at least comparable to other enzyme-mediated colorimetric assays.
Results and Discussion
The detailed strategy for colorimetric assay of miRNA is illustrated in Fig. 1. Briefly, Blocker DNA is immobilized on a gold solid interface via thiol-gold interaction 32 . By partial hybridization with another DNA probe named HP0, double-stranded DNA is formed. The gold solid interface is then immersed in the mixture of AgNPs and two DNA probes named as HP1 and HP2, which are not only two fuels strands for hybridization chain reaction 33 , but also act as stabilizers of AgNPs colloid system. The presence of HP1 and HP2 makes AgNPs much more stable. The nanoparticles disperse well in 0.1 M MgCl 2 and the UV-vis spectrum peak barely changes. Since a toehold exists at the duplex on the gold solid interface, the introduced target miRNA could initiate strand displacement reaction, releasing HP0 back to the solution, which further triggers the hybridization chain reaction, generating long double-stranded DNA product. Thereby, due to the lack of single-stranded tails of HP1 and HP2, the colloid system of AgNPs is less stable against the treatment of salt. By analyzing UV-vis spectrum of the system, the phenomenon of miRNA-induced aggregation of AgNPs could be quantified.
The prepared AgNPs suspension is transparent and yellow colored. From the TEM image in Fig. 2a, it is observed that AgNPs disperse well in water and the diameter is about 5 nm. Moreover, a well characteristic absorbance peak at the wavelength around 400 nm can be confirmed in the UV-vis spectrum of AgNPs (Fig. 2b). After miRNA-induced HP0 release and the following HCR, the aggregation of AgNPs can be observed, which confirms the effectiveness of the proposed strategy (inset in Fig. 2a).
To achieve the optimized aggregation condition, we have performed preliminary experiments. Divalent cations are much more effective to neutralize negative charges on the surface of AgNPs than monovalent cations like Na + . A series of Mg 2+ concentrations have been used to check the salt-tolerance of AgNPs. More Mg 2+ may lead to the larger decrease of the absorbance peak. The ratio of net decreased peak (Δ A/A 0 ) is used to represent the distribution state of AgNPs. Figure 3a shows the relationship between Δ A/A 0 of bare AgNPs and the concentration of Mg 2+ . The fitting curve is a Boltzmann sigmoid and the equation is as follows: The slope of this fitting curve reaches a maximum value at the point of 0.0062, indicating that AgNPs is most sensitive to the changes of salt when the concentration of Mg 2+ is 6.2 mM, which is then used for the following colorimetric investigation. However, in the case of AgNPs protected by HP1 and HP2, Δ A/A 0 barely increases after the treatment of Mg 2+ , which demonstrates the high stability of the AgNPs stabilized by DNA probes with single-stranded DNA tails (Fig. 3b). UV-vis spectra are recorded for the experiments using miRNA of different concentrations to trigger strand displacement reaction and further HCR. HCR could be directly confirmed by a gel electrophoresis experiment. The bands representing much larger molecular weight are observed after the reaction, indicating the formation of assembled long double-stranded DNA (inset in Fig. 4a). Furthermore, the presence of miRNA leads to the decrease of absorbance peak, which is shown in Fig. 4a. More miRNA releases more HP0, and leads to the consumption of more stabilizers. Finally, the absorbance peak decreases accordingly. Optimized reaction time of hybridization chain reaction is identified to be 100 min by comparing the recorded absorbance peaks (Fig. 5). + . y x 1 1135 0 0858 (2) in which y is Δ A/A 0 , x is the concentration of target miRNA, n = 3, R 2 = 0.9869. The limit of detection is calculated to be 1 pM (S/N = 3), which is sufficient for the application of clinical diagnosis. For example, influenza A virus H1N1 diagnostics can be performed by the detection of the biomarker of miR-29a-3p using the proposed colorimetric strategy.
A representative member of miR-29 family, miR-29c, is chosen to be detected by the proposed method, which contains only single base mismatch compared with miR-29a. The resulted Δ A/A 0 is negligible, which confirms the high specificity of this method for the detection or target miRNA.
In addition, we have carried out experiments involving real samples to check the practical utility of this colorimetric biosensor. Throat swabs from healthy and infected individuals are collected and treated before the colorimetric assay. Moreover, a commercial qRT-PCR Kit is employed to obtain the control values. The results listed in Table 1 reveal that the detected values of the two methods are consistent with each other. The proposed colorimetric assay could distinguish infected individuals by accurately determining the level of the biomarker. Sampling and colorimetric assay operations are both quite simple, which could promise great practical utility.
In summary, we have herein developed a novel colorimetric strategy for visual miRNA detection based on hybridization chain reaction. Target miRNA could release HP0 to the solution to trigger HCR, which hinders the protection of AgNPs by single-stranded DNA tails of HP1 and HP2 from divalent cation-induced aggregation. The information of UV-vis spectra can then be used to reveal the initial miRNA level. This non-enzymatic method involves only four DNA strands, which is quite simple. Moreover, it also has high sensitivity towards the clinical diagnosis applications.
Materials and Chemicals.
The methods were carried out in accordance with institutional and national guidelines and with the approval of the Medical Ethics Committee of The First Affiliated Hospital of Soochow University (no.2012076). Informed consent was obtained from all subjects. The study was also conducted in accordance with the Declaration of Helsinki. Silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), trisodium citrate, diethypyrocarbonate (DEPC), mercaptohexanol (MCH), ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma (USA). All other reagents were of analytical grade. Human samples were from local hospital. Water used was previously purified by a Millipore system to 18 MΩ cm resistivity and treated with DEPC. All DNAs and miRNA were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). The sequence were listed in Table 2. TEM images were taken by using a FEI Tecnai G20 transmission electron microscopy (FEI, USA). UV-vis spectra were measured by an Agilent Cary 300 Scan UV-vis absorption spectrophotometer (Agilent Technologies, USA).
Preparation of AgNPs.
Bare AgNPs were synthesized according to a previously reported protocol 34 . Briefly, AgNO 3 solution and trisodium citrate solution were prepared and mixed. The concentrations of AgNO 3 and trisodium citrate were both 0.25 mM. NaBH 4 solution was then prepared with the concentration of 10 mM. 3 mL of NaBH 4 solution was added to 100 mL of the mixture of AgNO 3 and trisodium citrate. Then, the reaction solution was stirred vigorously for 0.5 h. Afterwards, the solution was kept in dark and left to sit for 24 h. The formed AgNPs were later purified by three cycles of centrifugation at 12,000 g for 0.5 h.
Optimization of Mg 2+ Concentration. Bare AgNPs could keep stable due to presence of negatively charged citrate. However, with the disturbance of salt in the colloid system, aggregation occurred and the color of the solution changed correspondingly. Mg 2+ with a series of concentrations were prepared to induce the aggregation of AgNPs. Absorbance peaks were recorded and ratios of net decreased peak values (Δ A/A 0 ) were calculated. After analysing the data, the critical value of Mg 2+ to induce aggregation was found to represent the Mg 2+ -tolerance of AgNPs 35 .
Gel electrophoresis Experiments. Different DNA samples were monitored for comparison by 4% agarose gel electrophoresis for 30 min (100 V). The photograph of gel stained with ethidium bromide was taken under UV light by a Gel DocTM XR+ System (Bio-Rad, USA).
UV-vis Spectroscopy Analysis of miRNA.
Plasmonic colorimetric detection of miRNA was carried out as follows. 2 μ M HP1 and HP2 were added to AgNPs solution to protect the colloid system. Standard miRNA solutions with the concentration from 1 pM to 100 nM was prepared to treat Blocker DNA/HP0 duplex modified gold solid interface for 1 h. The released HP0 in the solution was then transferred to HP1 and HP2 protected AgNPs. Since HP0 could initiate HCR and make colloid system less stable, after the addition of Mg 2+ with the critical value, AgNPs aggregated, which was recorded by UV-vis absorption spectrophotometer.
Determination of miRNA in Real Samples.
To verify the practical utility of this method in human samples, throat swabs from healthy and infected (H1N1 influenza A virus) individuals were collected and diluted by sterile saline for miRNA analysis. Total RNA were extracted by a RNA extraction kit from Qiagen according to manufacture's instructions. The levels of miR-29a-3p were then calculated referring to the linear curve of the proposed colorimetric assay. In addition, the results obtained from commercial Quant One Step qRT-PCR Kit were used as the controls. | 2018-04-03T05:44:19.148Z | 2016-08-18T00:00:00.000 | {
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24271407 | pes2o/s2orc | v3-fos-license | Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.
The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.
was combined with the inorganic phosphate assay of Itaya and Ui (12) and the ashing procedure of Ames (13) for measurement of protein-bound phosphate. All materials were previously washed in 6 N HCl to remove contaminating phosphate.
Protein samples containing 0.2 to 1.5 nmol of phosphate were added to borosilicate glass tubes (6 x 50 mm) and were precipitated with 10% trichloroacetic acid at 0". The supernatant solution was carefully removed after centrifugation and the protein was washed twice with 5% trichloroacetic acid. The prote-in was suspended in 16% trichloroacetic acid, heated for 20 min at 90", and centrifuged after cooling to 0". The protein pellet was solubilized quickly in 0.1 N NaOH, 0", and immediately precipitated with 20% trichloroacetic acid. After two more washes in 5% trichloroacetic acid, the protein was dissolved in 25 ~1 of 10% MgNO, .6H,O in 95% ethanol and gently heated to dryness. The samples were then ashed over an intense flame. After dissolving the residue in 150 ~1 of 1.2 N HCl, 50 ~1 of filtered phosphate reagent was added, and after 5 minutes, the developed color was measured at 660 nm. The phosphate reagent contained 1 volume of 10% (NH&Moo,, .4H,O in 4 N HCl to 3 volumes of 0.2% malachite green. Phosphate standards (0.2 to 1.5 nmol of KH,PO,) were also ashed. Recovery of protein-bound phosphate in "'P-phosphorylase a and J'P-troponin previously phosphorylated by phosphorylase kinase and cyclic AMP-dependent protein kinase, respectively, was generally 97%. The variation on multiple assays of the same protein sample was 5% or less. Polyacrylamide Gel Electrophoresis -The purity and apparent molecular weight of protein samples were estimated bv nolvacrvlamide gel electrochore& in the presence of SDS as descr"ided "by *eber et al. (14) Chromatography on CM-agarose in the presence of 6 M urea and EDTA was used to separate the individual protein components of the troponin . tropomyosin complex (Fig. 2). The protein in the first peak was not adsorbed to the CM-agarose and was identified as a mixture of tropomyosin and TN-C by gel electrophoresis (Fig. 1, Gels ZJ and c). Occasionally this fraction was contaminated with a small amount of TN-T. The tropomyosin was separated from TN-C by adjusting the pH to 4.6 in the presence of 1 M KC1 and collecting the precipitated tropomyosin by centrifugation. The subsequent two peaks obtained by elution with KC1 were identified as TN-T and TN-I, respectively, by their electrophoretic mobilities on polyacrylamide gels (Fig. 1, Gels d and e). Quantitative densitometry of the purified protein fractions showed a purity of 99% for tropomyosin, 93% for TN-C, 97% for TN-T, and 97% for TN-I. Sulfopropyl Sephadex was also used to separate the protein components, but the distinct separation of TN-T and TN-I was not as consistently successful as when CM-agarose was used. These results show that the troponin subunits may be purified without the prior separation of tropomyosin from the troponin complex. 7). Actin at a molar ratio to troponin . tropomyosin of 7:l did not appear to affect the rate or extent of phosphorylation of troponin by cyclic AMP-dependent protein kinase. Identical results were obtained with molar ratios of 3.5:l and 14:l (data not shown).
The presence or absence of Ca'+ did not significantly alter the phosphorylation reaction (Fig. 7) in the presence of actin.
DISCUSSION
Beef cardiac troponin is a phosphoprotein of the myotibrils whose function may be regulated by phosphorylation and dephosphorylation reactions. Although skeletal muscle troponin also contains protein-bound phosphate (18, differences between the cardiac and skeletal muscle proteins suggest different roles for the phosphorylation. The subunit of skeletal muscle troponin which contains most of the phosphate was identified as 20). This is in contrast to results reported here and observations by others (3) where the TN-I subunit from cardiac muscle contained most of the phosphate. A significant portion of the phosphate in cardiac troponin was found in TN-T as well. Skeletal muscle troponin is not phosphorylated in response to tetanic contractions (5, 21) or after be an important criteria for indicating a possible role for a phosphorylation reaction in a particular cellular process.
We have found that TN-I is specifically phosphorylated by cyclic AMP-dependent protein kinase in the heterogeneous protein complex of troponin which is in agreement with others (3). In addition we found tropomyosin added to cardiac troponin was not phosphorylated and did not alter the maximal amount of :VzP incorporated into TN-I. The maximal rate of phosphorylation of cardiac troponin by cyclic AMP-dependent protein kinase was about 3.5 times greater than the rate of phosphorylation of unfractionated 3 In the experiments described in this report, cyclic AMP-dependent protein kinase purified from porcine skeletal muscle was used. Cyclic AMP-dependent protein kinase has been shown to exist in two general isozymic forms (28-30). The relative amounts of the two forms may vary within a specific tissue, but the properties of any one form isolated from many tissues appear to be similar.
Both isozymic forms of cyclic AMP-dependent protein kinase contain a dimeric regulatory subunit and two catalytic subunits which dissociate when cyclic AMP binds to the regulatory subunits. Differences in physical properties have been identified between regulatory subunits, but no differences between the catalytic subunits of either enzyme have been noted (28, 31, 321. The catalytic subunits demonstrate identical specificities and relative rates of phosphorylation with a variety of protein substrates. Likewise no differences were noted in regard to the phosphorylation of beef cardiac troponin by either isozymic form. Therefore "cyclic AMP-dependent protein kinase" has been used in this report without qualification and refers to either isozymic form. | 2018-04-03T02:39:25.834Z | 1977-01-01T00:00:00.000 | {
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247055414 | pes2o/s2orc | v3-fos-license | Analysis and Experimental Verification of Model Solutions of the Pekeris Boundary Problem in the Infrasonic Frequency Range
A comparative assessment of the known solutions of the Pekeris boundary problem is carried out. A detailed analysis of the generalized solution, which is constructed in a non-self-adjoint model formulation, is carried out. One of the features of the generalized solution is the presence of the vortex component of the intensity vector, the level of which increases with decreasing frequency. The vortex component is recorded in the channels of the vector receiver during an experiment with a noisy object.
Introduction
The model description of sound fields is based on the solution of boundary problems for the Helmholtz operator. A classical example of such a boundary problem is the Pekeris boundary problem [1]. However, this solution was not the only one, three more solutions were obtained in the class of analytic functions [2][3][4]. Comparative analysis of solutions in the class of analytic functions, the integral solution in the class of functions that can be represented by the Fourier-Bessel integral, and the generalized solution constructed in the non-self-adjoint model formulation (NMF) was performed in work [5]. A rather detailed analysis of the generalized solution was carried out in works [5][6][7]. The fundamental differences between these solutions are as follows.
The solution represented by the Fourier-Bessel integral describes the sound field of the standing wave type in the waveguide, and the sound field of the diverging spherical wave type, modulated by the angular spectrum of the source, in the half-space. Only the decision of L.M. Brekhovskikh [2] is correct in the all domain of definition of a waveguide -half-space of the four solutions [1][2][3][4] constructed in the class of analytic functions. It defines the sound field, represented by diverging waves, as eigenfunctions of a self-adjoint operator. In this model solution, the waveguide and the halfspace are considered as two energetically independent subsystems, and the power flow through the waveguide -half-space interface is identically equal to zero. This circumstance contradicts the fact that the Pekeris waveguide is an open system with energy losses for radiation into the half-space at angles of incidence less than the critical value. The rest of the solutions of this class are correct only when describing the sound field in the waveguide itself, since they all contain leakage waves of the complex spectrum, the amplitude of which grows exponentially in the half-space.
The generalized solution constructed within the framework of the NMF is correct and limited in the all domain of definition. It is discontinuous in pressure and the normal component of the particle 2 velocity at the transformation horizons, but continuous in impedance, determined through the ratio of integral quantities such as force and volume particle velocity. The alternating normal component of the intensity vector with a half-wavelength period is generated at the transformation horizons due to the discontinuity of the solution. The latter circumstance means that the integral power flow is equal to zero and the generalized solution is correct. The most popular is the classic solution of L.M. Brekhovskikh, built in a self-coupled model formulation. However, the generalized solution constructed within the framework of the NMF is most consistent with the experiment, and this correspondence grows with decreasing frequency.
Analysis of the model solutions
Differences in the model description become significant at frequencies lower than the first critical frequency of the model Pekeris waveguide, when leaky waves and the zero mode of the generalized solution become dominant in the total sound field. The zero mode is an inhomogeneous (slow) wave localized at the source horizon. Its speed с0 is less than the speed of sound in water с1. This velocity corresponds to zero reflection coefficient of the water -liquid seabed interface, which is realized in the region of complex angles of incidence [5].
All poles corresponding to the eigenvalues of the non-self-adjoint operator describing the Pekeris boundary problem appear on the top sheet of the Riemann surface if the cut corresponding to the condition is used ( is the vertical wavenumber in the half-space). Such a cut was first used in [4], and the transition to the NMF is a regularization of the solution [4] in the domain of definition of the waveguide -half-space. The real and complex poles, which correspond to the fours of the eigenvalues of the horizontal wave number ( , , , ), appear on the top sheet of the Riemann surface when using this cut. The location of the poles on the plane of the complex variable with a cut corresponding to the condition , and the migration of the poles with a change in the frequency parameter are shown by an arrow in Figure 1. for diverging waves and symbols for backward waves, also included in the total solution. The subset includes normal waves that continue into the half-space as an inhomogeneous wave with a decreasing amplitude. For such normal waves, the half-space input impedance is reactive, inertial. The subset includes normal waves that continue into the halfspace as an inhomogeneous wave with an increasing amplitude. For such normal waves, the half-space input impedance is reactive, elastic. The subset includes normal waves of the complex spectrum (leakage waves), which continue into the half-space as an inhomogeneous wave, the amplitude of which decreases along the horizontal coordinate and increases along the vertical coordinate. For such normal waves, the input impedance of the half-space is active-reactive, elastic with a positive definite real part.
The main differences in the structure of the sound field for the generalized solution and the classical solution are shown in Figure 2 for a frequency greater than the first critical frequency, and in Figure 3 for a frequency less than the first critical frequency. The Figures 2, 3 show the sound pressure fields (with the excluded cylindrical divergence) in coordinates normalized to the waveguide depth . , . , .
The appearance of backward waves with a positive definite group velocity in the total solution is a condition for the realization of longitudinal resonance. Such a mechanism is well known in the theory of solid waveguides with a free surface [8], in which quasi-transverse waves are generated at transverse resonance frequencies, quasi-longitudinal waves are generated at longitudinal resonance frequencies, and the boundary value problems for solid waveguides are always described within the framework of the NMF. The unity of the formation mechanism of wave motion in liquid and solid waveguides indicates that the phenomenon of longitudinal resonance is a fundamental phenomenon. A model description of longitudinal resonances in a wedge-shaped waveguide with a small opening angle is given in work [6], where experimental confirmation of this phenomenon is described.
Experimental verification of model solutions
Receiving systems based on combined receivers (CR) are widely used in the study of sound fields generated by natural or simulated sources. An example is the work [10][11][12][13][14][15], in which extensive experimental studies of the main characteristics of sound fields with the use of combined receivers were carried out. In this work, the emphasis of experimental studies is placed on the comparison of experimental data and various model descriptions of the sound field in the Pekeris waveguide, which are listed above.
An experimental study of the sound field generated by a noisy object in a shallow sea was carried out using a receiving system equipped with combined receivers. The sea depth at the site of the experiment was 105 m, the combined receiver was located at a horizon of 50 m. The noisy object moved along a straight line relative to the receiving system at a constant speed. The traverse point corresponds to the moment of time 08 hours 05 minutes. The pass-through characteristics in the channels of the combined receiver are shown in It is clearly seen that at frequencies of the infrasonic range of 4-8 Hz in all vector channels, the vortex (alternating) component of the intensity vector prevails, which is generated in structures such as standing waves. This effect can only be described within the framework of the NMF, and is well confirmed experimentally. With increasing frequency, the level of the vortex component of the intensity vector decreases and the level of the horizontal (potential) component increases. The presence of horizontal power flows in the waveguide at frequencies lower than the first critical frequency is confirmed by experiment, and also requires a transition to the description of the sound field in the framework of the NMF.
Conclusion
The comparison of the model description of sound fields in the framework of the generalized solution constructed in the non-self-adjoint model formulation and the classical solution has been completed. The fundamental differences between these solutions, which become significant in the infrasonic frequency range, are noted. The experimental data obtained using the CR, which confirm the preference of the model solution constructed in the framework of the NMF, are presented. | 2022-02-23T20:07:29.723Z | 2022-02-01T00:00:00.000 | {
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237363815 | pes2o/s2orc | v3-fos-license | Stochastic Discontinuous Galerkin Methods for Robust Deterministic Control of Convection Diffusion Equations with Uncertain Coefficients
We investigate a numerical behaviour of robust deterministic optimal control problem subject to a convection diffusion equation containing uncertain inputs. Stochastic Galerkin approach, turning the original optimization problem containing uncertainties into a large system of deterministic problems, is applied to discretize the stochastic domain, while a discontinuous Galerkin method is preferred for the spatial discretization due to its better convergence behaviour for optimization problems governed by convection dominated PDEs. Error analysis is done for the state and adjoint variables in the energy norm, while the estimates of deterministic control is obtained in the $L^2$--norm. Large matrix system emerging from the stochastic Galerkin method is addressed by the low--rank version of GMRES method, which reduces both the computational complexity and the memory requirements by employing Kronecker--product structure of the obtained linear system. Benchmark examples with and without control constraints are presented to illustrate the efficiency of the proposed methodology.
Introduction
In many phenomena in physics or engineering applications, certain parameters of a model are optimized in order to reach the desired target, for instance, the location where the oil is inserted into the medium, the temperature of a melting/heating process, or the shape of the aircraft wings. Such real-world phenomena can be modelled as optimal control problems or optimization problems with PDE constraints. However, in reality, the input parameters of these simulations, such as the wind speed or material properties, are not often known due to the missing information or inherent variability in the problem; see, e.g., [1]. Therefore, in the last decade, the idea of uncertainty quantification, i.e., quantifying the effects of uncertainty on the result of a computation, has become a powerful tool for modeling physical phenomena in the scientific community.
PDE-constraint optimization problems with uncertainty have been studied in various formulations in the literature, such as mean-based control [2,3], pathwise control [4,5], average control [6,7], robust deterministic control [8][9][10][11][12][13], and robust stochastic control [14][15][16][17]. Robust deterministic control is more practical and realistic since randomness cannot be observed during the design of the control. Therefore, we are here interested with the following robust deterministic control problem y(x, ω) = y DB (x) on ∂D × Ω, (1.2b) where S : Y → Y ′ is a linear operator that contains uncertain parameters, D ⊂ R 2 is a convex bounded polygonal set with a Lipschitz boundary ∂D, and Ω is a sample space of events. The cost functional including a risk penalization via the standard deviation std(y) is denoted by J (y, u). The first term in (1.1) is a measure of the distance between the state variable y and the desired state y d in terms of expectation of y − y d . Without loss of generality, we assume that the state y ∈ Y is a random field, whereas the desired state y d ∈ Y is modelled deterministically. The second term measures the standard deviation of y, which is added since it is desirable to have a control for which the state is more accurately known, leading to a risk averse optimum. The last term corresponds to distributive deterministic control. The constant µ > 0 is a positive regularization parameter of the control u, whereas γ ≥ 0 is a risk-aversion parameter. Deterministic source function and Dirichlet boundary conditions are denoted by f and y DB , respectively. We note that the cost functional J is a deterministic quantity although it contains uncertain inputs.
Further, the closed convex admissible set in the control space U is defined by where constants u a , u b ∈ R with u a ≤ u b .
Finding an approximate solution for the optimization problems containing uncertainty (1.1)-(1.2) is extremely challenging and requires much more computational resources than the ones in the deterministic setting. In the literature, there exist various competing methods to solve such kinds of problem, for instance, Monte Carlo (MC) [5,18,19], stochastic collocation method (SCM) [13,17,20,21], and stochastic Galerkin method (SGM) [9,12,13,22,23]. Although the MC method is popular for its simplicity, natural parallelization, and broad applications, it features slow convergence, which does not depend on the number of uncertain parameters [24,25]. For the SCMs, the crucial issue is how to construct the set of collocation points appropriately because the choice of the collocation points determines the efficiency of the method. In contrast to the MC approach and the SCM, the SGM is a nonsampling technique, which transforms the problem into a large system of deterministic problems. As in the classic (deterministic) Galerkin method, the idea behind the SGM is to seek a solution for the model equation such that the residue is orthogonal to the space of polynomials. Since the random process is expressed as an expansion with the help of orthogonal polynomials, the SGM is considered as a variant of the generalized polynomial chaos approximation [26][27][28] as the stochastic collocation method. An important feature of the SGM is the separation of the spatial and stochastic variables, which allows a reuse of established numerical techniques. The results obtained in [13] also show that the SGM generally displays superior performance compared to the SCM for the robust deterministic control problems. Within the framework of the aforementioned features, the stochastic Galerkin method is preferred as a stochastic method in this study. On the other hand, for the discretization of the spatial domain, we use a discontinuous Galerkin method due to its better convergence behaviour for the optimization problems governed by convection dominated PDEs; see, e.g., [29][30][31]. We also refer to [32,33] and references therein for more details on the discontinuous Galerkin methods.
In spite of these nice properties exhibited by the stochastic discontinuous Galerkin method, the dimension of the resulting linear system increases rapidly, called as the curse of dimensionality. As a remedy, we apply a lowrank variant of generalized minimal residual (GMRES) method [34] with a suitable preconditioner. With the help of a Kronecker-product structure of the obtained large matrices, we reduce both the computational complexity and memory requirements; see, e.g., [35][36][37]. Low-rank approximation of the optimal control problems with uncertain terms have been also studied in [14,38,39] for unconstrained control problems and in [40] for control constraint problems. In the aforementioned studies, randomness is generally defined on the diffusion parameter; however, we here consider the randomness on diffusion or convection parameters by applying the discontinuous Galerkin method in the spatial domain. In addition, according to the best of our knowledge, a lowrank approximation of the optimal control problems governed by convection dominated equations containing randomness has not been discussed before in the setting of discontinuous Galerkin discretization in the spatial domain.
We organize our paper by first discussing the existence of the solution in the next section. In Section 3, we reduce the problem into finite dimensional setting via Karhunen-Loève (KL) expansion, stochastic Galerkin method, and symmetric interior penalty Galerkin method. Error analyses are done in Section 4. In Section 5, we construct the matrix formulation of the underlying optimization problem by proceeding the optimize-then-discretize approach, and then discuss implementation of the low-rank GMRES solver. Results of the numerical experiments are provided in Section 6 to illustrate the efficiency of the proposed methodology. Finally, we end the paper with some conclusions and discussions in Section 7.
Existence and uniqueness of the solution
Let Ω be a sample space of events, F ⊂ 2 Ω denotes a σ-algebra, and P is the associated probability measure that maps the events in F to probabilities in [0, 1]. A generic random field η on the probability space (Ω, F , P) is denoted by η(x, ω) : D × Ω → R. For a fixed x ∈ D, η(x, ·) is a real-valued square integrable random variable η(x, ·) ∈ L 2 (Ω, F , P), i.e., Then, the mean E[η], the standard deviation std(η), and the corresponding variance V(η) for any random field η, are given, respectively, by Recalling the tensor-product space H k (D) ⊗ L 2 (Ω) equipped with the norm the state and control spaces are defined as follow, respectively, We also set X := L 2 (D) ⊗ L 2 (Ω) and W = L 2 (D). In order to show existence of the solution, it is assumed that the operator S satisfies the following conditions: a) S is coercive such that P-a.s., (Sv, v By following the standard arguments in the theory of optimal control, see, e.g., [41,Theorem 1.3] and [42,Theorem 2.14], the existence and uniqueness of an optimal solution for the optimization problem (1.1)-(1.2) can be proved. With the definitions above, Y and U are Hilbert spaces, the functional J is strictly convex, and the admissible set U ad is a closed and convex set. Then, according to Lion's Lemma [41,Theorem 1.3], a unique optimal controlū ∈ U exists and the variational inequality holds Now, we can state the first order optimality system of the optimization problem containing uncertain coefficients (1.1)-(1.2).
Proof Rewrite the objective functional J as .
By the definition of directional derivative, we obtain that By well-posedness of the state equation (1.2) followed from the Lax-Milgram lemma, one can easily show that the operator S is invertible so that, by taking directional derivative, one gets Thus, (2.4) gives us To guarantee the existence and uniqueness of the solution from Lion's Lemma [41,Theorem 1.3], we need the following requirement Next, we introduce the adjoint state p(u) ∈ Y by Multiplying both sides of (2.7) by y(v) − y(u) , integrating over D, and taking the expectation of the resulting system, we obtain Inserting (2.8) into (2.5) and combining with (2.6) give us which is the desired result.
In this study, we consider S as the convection-diffusion operator where a : (D × Ω) → R and b : (D × Ω) → R 2 are random diffusivity and velocity coefficients, respectively, which is assumed to have continuous and bounded covariance functions. In addition, we make the following assumptions on the uncertain coefficients: i) ∃ a min , a max such that for almost every (x, ω) ∈ D × Ω, 0 < a min ≤ a(x, ω) ≤ a max < ∞. In addition, a(x, ω) has a uniformly bounded and continuous first derivatives. ii) The velocity coefficient b satisfies b(·, ω) ∈ L ∞ (D) 2 for a.e. ω ∈ Ω and Then, the well-posedness of the state equation (2.11) can be shown by following the classical Lax-Milgram lemma; see, e.g., [43,44]. Now, we give the corresponding weak formulation of the optimization problem containing uncertainty (1.1)-(1.2) as follows min u∈U ad Moreover, the optimality system in (2.3) can be stated in the weak formulation as follows: where the adjoint p ∈ Y solves the following convection diffusion equation containing uncertain inputs In the following, we introduce the techniques, that is, Karhunen-Lòeve (KL) expansion, stochastic Galerkin, and discontinuous Galerkin method, to recast the infinite-dimensional model problem (2.12)-(2.13) into the finite dimensional.
Finite representation of stochastic fields
To solve (2.12)-(2.13) numerically, it is needed to reduce the stochastic process into finite mutually uncorrelated random variables. Therefore, the coefficients a(x, ω) and b(x, ω) are approximated by finite uncorrelated components , called as finite dimensional noise [43,45]. Introducing the probability density functions of where Γ represents the support of such probability density, B(Γ) is a Borel σ-algebra, and ρ(ξ)dξ corresponds to the distribution measure of ξ. Moreover, ρ(ξ) denotes the joint probability density function. Hence, we can state the tensor-product space H k (D) ⊗ L 2 (Γ) endowed with the following norm Following the well-known KL expansion [46,47], a random field η having a continuous covariance function as follows admits a proper orthogonal decomposition where η(x) and κ are mean and standard deviation of η, respectively, and ξ := {ξ 1 , ξ 2 , . . .} are uncorrelated random variables. The pair {λ k , φ k } is a set of the eigenvalues and eigenfunctions of the corresponding covariance operator C η . Then, we approximate η(x, ω) by truncating its KL expansion of the form The truncated KL expansion (3.4) is a finite representation of the random field η(x, ω) in the sense that the mean-square error of approximation is minimized; see, e.g., [48]. To guarantee the positivity of the truncated KL expansion (3.4) for the diffusivity coefficient a(x, ω), it is also assumed that the mean of random coefficient exhibits a stronger dominance; see, e.g., [49]. By the assumption on the finite dimensional and Doob-Dynkin lemma [50], the solution of (2.11) can be expressed in the finite dimensional stochastic space, that means, y(x, ξ(ω)) ∈ Y ρ = L 2 (H 1 0 (D); Γ) with ξ = ξ 1 (ω), . . . , ξ N (ω) . Then, setting E[y] = Γ y ρ(ξ)dξ, the optimization problem (2.12)-(2.13) becomes min u∈U ad Then, the optimization problem (3.5)-(3.6) has a unique solution pair (y, u) ∈ Y ρ × U ad if and only if there is an adjoint p ∈ Y ρ such that the following optimality system holds for the triplet (y, u, p): Next, we present the representation of stochastic solutions, i.e., y(x, ξ), p(x, ξ), by using a polynomial chaos (PC) approximation [26].
Stochastic Galerkin Method
The state solution y(x, ξ) ∈ L 2 (Γ, F , P), as well as the adjoint solution p(x, ξ) ∈ L 2 (Γ, F , P), can be represented by a finite generalized polynomial chaos (PC) approximation as stated in Cameron-Martin theorem [51], where y i (x) and p i (x) are the deterministic modes of the expansion and the total number of PC basis is determined by the dimension N of the random vector ξ and the highest order Q in the basis set of ψ i By following [49,52], we then define the stochastic space as For simplicity, we only deal with the state equation since the procedure for the adjoint equation is similar to the state ones. By inserting KL expansions (3.4) of the diffusion a(x, ω) and the convection b(x, ω) coefficients, and the solution expression (3.9) into the variational form of the state equation (3.6) and projecting onto the space of the PC basis functions, we get a linear system, consisting of J deterministic convection diffusion equations for j = 0, . . . , J − 1 Here, we apply the same distribution for both diffusion and convection random coefficients in order to reduce the computational effort. However, it can be possible to use different distributions; see, e.g, [53] for more discussion. We also note that the quantity of interest is the statistical moments of the solution y(x, ω) rather than the solution y(x, ω).
Symmetric interior penalty Galerkin (SIPG) method
We briefly recall the SIPG discretization following the studies in [30,54]. A shape-regular simplicial triangulations of D is denoted by For a fixed realization ω and the unit outward normal n K to ∂K, we decompose the boundary edges of an element K into the inflow ∂K − and outflow ∂K + parts such that ∂K + = ∂K\∂K − . Jump and average operators of y and ∇y for a common edge E = K ∩ K e are given, respectively, by where y| E (or ∇y| E ) and y e | E (or ∇y e | E ) are traces from inside K and K e , respectively. For a boundary edge E ∈ K ∩ ∂D, the operators are defined by { {∇y} } = ∇y and [[y]] = yn, where n denotes the unit outward normal to ∂D.
Defining the discrete space as follows where P(K) is the set of linear polynomials and following the standard discontinuous Galerkin structure discussed in [32,33], the (bi)-linear forms for a finite dimensional vector ξ can be stated as follow: where the parameter σ ∈ R + 0 , called as the penalty parameter, should be sufficiently large to ensure the stability of the SIPG scheme; independent of the mesh size h. However, as discussed in [33, Sec. 2.7.1], it depends on the degree of polynomials used in the DG discretization and the position of the edge E. In our numerical experiments, we choose σ as σ = 6 on the interior edges E 0 h and 12 on the boundary edges E ∂ h . Then, the (bi)-linear forms of the stochastic discontinuous Galerkin (SDG) for the state equation correspond to governed by where the discrete admissible set (1.3) is defined by is a unique solution of the control problem (3.14)-(3.15) if and only if an adjoint p h ∈ Y h exists such that the optimality system holds for ( one can easily obtain the following expression for the discrete directional derivative of functional J h (u h ):
Error analysis
We provide an a priori error analysis of the optimization problem (2.12)-(2.13), discretized by the stochastic discontinuous Galerkin method. Before deriving the corresponding estimates, we define the associated energy norm on D × Γ as where y(., ξ) e is the energy norm on D, given as By the standard arguments as done in deterministic case, one can easily show the coercivity and continuity of a ξ (·, where the coercivity constant c cv depends on a min , whereas the continuity constant c ct depends on a max . Next, we state the estimates on the finite dimensional probability domain Γ and the physical domain K ∈ T h . Let a partition of the support of probability (r γ n , s γ n ), with (r γ n , s γ n ) ⊂ Γ n for n = 1, . . . , N so that the mesh size k n becomes k n = max γ |s γ n − r γ n |, n = 1 . . . N . For the multi-index q = (q 1 , . . . , q N ), the (discontinuous) finite element approximation space having at most q n degree on each direction ξ n is denoted by S q k ⊂ L 2 (Γ). Then, for v ∈ H q+1 (Γ), ϕ ∈ S q k , we have, see [43], For v ∈ H 2 (K) and v ∈ P(K), where K ∈ T h , the following discontinuous Galerkin approximation [33, Theorem 2.6] also holds where the constant C is not depending on v and h. Further, we define the following projection operators, which are needed in the rest of the paper: with the following estimate In addition, taking ζ = Π n (ξ) and χ = Π h (ν) in (4.5a) and (4.5b), respectively, it holds that With the help of the H 1 -projection operator in (4.8a), the Cauchy-Schwarz inequality, the L 2 -projection operator in (4.5a), and the approximation in where the constant C does not depend on v, h, and k n . To recognize error contributions emerging from the spatial domain D and the probability domain Γ, separately, a projection operator P hn mapping onto the tensor product space Y h is given by (4.10) and the decomposition Then, it follows from (4.7a), (4.7b), and (4.10) that Before the derivation of a priori error estimate, we state the following auxiliary problem where p h (u) ∈ Y h solves the following auxiliary system: It is also noted that we prefer to use u L 2 (L 2 (D); Γ) in the derivation of error estimates instead of u L 2 (D) for better readability in terms of notation.
Next, we derive an upper bound for the error between the discrete solutions (y h , p h ) and the auxiliary solutions (y h (u), p h (u)).
Lemma 4.2.
Assume that (y h , p h ) and (y h (u), p h (u)), respectively, are the solutions of (3.17) and (4.14). Then, the following estimates exist for positive constants C 1 and C 2 independent of h Proof By subtracting (4.14a) from (3.17a) and taking v h = y h − y h (u), we have that With the help of the coercivity of a ξ (4.2) and the Cauchy-Schwarz inequality, we obtain which yields the desired result (4.20a). Analogously, by subtracting (4.14b) from (3.17b) and It follows from the coercivity of a ξ , Cauchy-Schwarz inequality, and Jensen's inequality that We note that the procedure applied in (4.18) is also used in the derivation of (4.21). Hence, by (4.21) and (4.20a), we deduce the desired result (4.20b).
To obtain an upper bound for the control, we divide the domain D into pieces by considering the active and inactive parts of the control u as done in [55,56]: It is assumed that these sets are disjoint, D = D + ∪ D ∂ ∪ D − , and D − satisfies the following inequality related to the regularity of u and T h which is valid if the boundary of the D ∂ is represented by finite rectifiable curves [57]. Further, we define a set such that D + ⊂ D * = {x ∈ D : u a < u(x) < u b } [58].
Now, we finalize the error analysis by combining the findings in Lemmas 4.3 and 4.4.
Theorem 4.5. Assume that (y, u, p) and (y h , u h , p h ), respectively, are the solutions of (2.15) and (3.17). Then, it holds that Thus, by combining (4.37) and (4.38), we deduce the desired result (4.36).
Matrix Formulation
In this section, we first construct the matrix formulation of the underlying problem (2.12)-(2.13) by employing the "optimize-then-discretize" approach; see, e.g., [42]. In this methodology, one first obtains the optimality system (2.15) of the infinite-dimensional optimization problem, and then discretizes the optimality system by a stochastic discontinuous Galerkin method discussed in Section 3. Later, we propose a low-rank variant of generalized minimal residual (GMRES) method with a suitable preconditioner to solve the corresponding linear system.
State system
After an application of the discretization techniques discussed in Section 3, one gets the following linear system for the state part of the optimality system (2.15): where y = (y 0 , . . . , y J−1 ) T and u = (u 0 , . . . , u J−1 ) T with y i , u i ∈ R N d , i = 0, 1, . . . , J − 1 and N d corresponds to the degree of freedom for the spatial discretization. The mass matrix M ∈ R N d ×N d , the stiffness matrices K i ∈ R N d ×N d , and the right-hand side vectors f i ∈ R N d are given, respectively, by . Then, G 0 becomes an identity matrix, whereas G k , k > 0, contains at most two nonzero entries per row; see, e.g., [49,52]. On the other hand, g i is the first column of G i , i = 0, 1, . . . , N .
Matrix formulation of the optimality system
The discrete optimality system in (3.17) can be represented as a block matrix system including the state, adjoint, and variational equations in the finite dimensional setting. To solve the underlying block linear system, "the primaldual active set (PDAS) methodology as a semi-smooth Newton step" is applied; see, e.g., [60] for more details. After a definition of the active sets and the inactive set the block formulation becomes where and ½ A − , ½ A + , and ½ I correspond to the characteristic functions of A − , A + , and I, respectively. Equivalently, M γ can be rewritten as which is a saddle point system. We note that since Legendre polynomials are used, G 0 = I, and hence, M I = M.
In practical implementations, the saddle point system (5.5) typically becomes very large, depending on the length of the random vector ξ and the number of refinement in the spatial discretization. We break this curse of dimensionality by using a low-rank approximation, which reduces both the computational complexity and memory requirements by using a Kroneckerproduct structure of the matrices defined in (5.5).
Low-rank approach
We first introduce the notation and some basic properties of the low-rank approach. Let Θ = [θ 1 , . . . , θ J ] ∈ R N d ×J and let the operators vec(·) and mat(·) be isomorphic mappings between R N d ×J and R N d J as following where N d and J are the degrees of freedom for the spatial discretization and the total degree of the multivariate stochastic basis polynomials, respectively. The matrix inner product is defined by U, V F = trace(U T V ) with U F = U, V F . Further, the following relation holds, see, e.g., [36]: Now, we can interpret the system (5.5) as follows where Y = (y 0 , . . . , y J−1 ) , U = (u 0 , . . . , u J−1 ) , P = (p 0 , . . . , p J−1 ) , B 1 = mat F d , By the identity (5.6), we have Assuming that the matrices Θ and B have the following low-rank representations, see, e.g., [37,61,62], with r Y , r U , r P , r B1 , r B2 , r B3 ≪ N d , J, (5.8) can be stated as follows where vec operator is ignored. Moreover, the three block rows in (5.10) can be written as in low-rank formats W i V T i for i = 1, 2, 3. By the usage of (5.11), the low-rank approximate solutions to (5.7) can be obtained; see Algorithm 1 modified from [62] for details of the low-rank implementation of GMRES. Moreover, with the help of the following fact can be computed as following During the iteration process, the rank of low-rank factors can increase either via matrix vector products or vector (matrix) additions. Thus, the cost of rank-reduction techniques is kept under control by using truncation based on singular values [36] or truncation based on coarse-grid rank reduction [63]. Our approach is based on the discussion in [37,64], where a truncated SVD of U = W T V ≈ B diag(σ 1 , . . . , σ r )C T is constructed for the largest r singular values, which are greater than the given truncation tolerance ǫ trunc . In Algorithm 1, this process is done by the truncation operator T . Further, in the numerical simulations, a rather small truncation tolerance ǫ trunc is used to represent the full-rank solution as accurate as possible.
We know that iterative methods such as GMRES exhibit a better convergence in terms of the number of iterations when they are used with a suitable preconditioner. The low-rank variants also display the same behaviour so that we use a block diagonal mean-based preconditioner of the form T corresponds to the approximated Schur complement with K 0 = K 0 + 1+γ µ M diag(½ I ); see, e.g, [14,49].
Numerical Results
This section contains a set of numerical experiments to illustrate the performance of proposed discretization techniques and a low-rank variant of GMRES approach. All numerical simulations are done in MATLAB R2021a on an Ubuntu Linux machine with 32 GB RAM. Iterative approaches are ended when the residual becomes smaller than the given tolerance value ǫ tol = 5 × 10 −3 or the maximum iteration number (#iter max = 250) is reached. The truncation tolerance ǫ trunc = 10 −8 is chosen, such that ǫ trunc ≤ ǫ tol ; otherwise, one would iterate the noise during the low-rank process.
Algorithm 1 Low-rank preconditioned GMRES (LRPGMRES)
Input: Coefficient matrix L : R 3N d ×J → R 3N d ×J , inverse of the preconditioner matrix P −1 0 : R 3N d ×J → R 3N d ×J , and right-hand side matrix B in the low-rank formats. Truncation operator T with given tolerance ǫtrunc.
Output: Matrix Θ ∈ R 3N d ×J satisfying L(Θ) − B F / B F ≤ ǫ tol . 1: Choose initial guess Θ if |γi+1| ≤ ǫ tol then 32: Compute y from My = ξ, where (M)j,i = mj,i. 33: with the correlation length ℓ n . Linear elements are used to generate discontinuous Galerkin basis, whereas Legendre polynomials are taken as the stochastic basis functions since the underlying random variables have uniform distribution over [− √ 3, . . , N . Explicit eigenpairs (λ j , φ j ) of the covariance function (6.1) can be found in [44]. Further, all parameters used in the simulations are described in Table 1.
Unconstrained problem with random diffusion parameter
As a first benchmark problem, we consider an unconstrained optimal control problem, that is, U ad = U, having a random diffusion coefficient defined on D = [−1, 1] 2 with the source function f (x) = 0, the convection parameter b(x) = (0, 1) T , and the Dirichlet boundary condition The random diffusion parameter is chosen as a(x, ω) = ν η(x, ω), where the random field η(x, ω) has the unity mean with the corresponding covariance function (6.1) and ν is the viscosity parameter. The desired state (or target) y d corresponds to the stochastic solution of the forward model by taking u(x) = 0. The desired state exhibits exponential boundary layer near x 2 = 1, where the solution changes in a dramatic manner. Therefore, the boundary layer becomes more visible as ν decreases; see Figure 1 for the mean of the state E[y h ], the desired state y d , and the corresponding control u h for various values of the viscosity parameter ν. Table 2 shows the values of the cost functional J (u h ) and tracking term y h − y d 2 X obtained by L\B for various values of the viscosity parameter ν and the regularization parameter µ. We observe that the tracking term and the objective functional become smaller as µ decreases. Moreover, Table 3 exhibits that the peak values of states' variance can be reduced by increasing the value of the parameter γ. Next, we display the performance of L\B in terms of total CPU times (in seconds) and storage requirements (in KB) in Table 4. However, we could not report some numerical results since the simulation is ended with "out of memory", which we have denoted as "OoM". To handle the curse of dimensionality and so increase the value of truncation number N , we need effective numerical approaches or solvers such as a low-rank variant of GMRES iteration with a mean based preconditioner discussed in Section 5.3. Table 5 reports the results of the simulations by considering various data sets in the low-rank format. By keeping other parameters fixed, we show results for varying truncation number N in KL expansion and regularization parameter µ for κ = 0.5 in Table 5. When N increases, the complexity of the problem increases in terms of the number of rank, memory, and CPU time. Another key observation is that the relative residual decreases independently of the value of µ while increasing N . Next, we investigate the effect of the standard deviation parameter κ on the numerical simulations. Figure 2 displays the behaviours of the cost functional J (u h ), the tracking term y h − y d 2 X , and the relative residual for various values of κ. We observe that the values of J (u h ) and y h − y d 2 X decrease monotonically as the value of κ increases. Moreover, the low-rank variant of preconditioned GMRES method yields convergence behaviour for all values of κ. Lastly, Figure 3 shows that the speed of convergence of relative residual decreases by increasing the value of risk-aversion parameter γ in the beginning of the iteration.
Unconstrained problem with random convection parameter
Our second example is an unconstrained optimal control problem containing random velocity input parameter. To be precise, we set the deterministic diffusion parameter a(x, ω) = ν > 0, the deterministic source function f (x) = 0, and homogeneous Dirichlet boundary conditions on the spatial domain D = [−1, 1] 2 . On the other hand, the random velocity field b(x, ω) is defined as b(x, ω) = (η(x, ω), η(x, ω)) T , where the random input η(x, ω) has the unity mean, i.e., η(x) = 1. Further, the desired state y d is given by . Figure 4 and 5 display, respectively, the mean of state E[y h ] and the control u h for varied values of the regularization parameter µ obtained by solving the full-rank system L\B. As the previous example, we observe that the state y h becomes closer to the target solution y d while µ decreases. Next, we compare the full-rank solutions obtained by solving the system L\B with the low-rank ones. Figure 6 exhibits behaviours of the cost functionals J (u h ) (left), the tracking term y h − y d 2 X (middle), and the relative residual (right) for varying values of the regularization parameter µ. The key Fig. 6: Example 6.2: Behaviours of the cost functional J (u h ) (left), the tracking term y h −y d 2 X (middle), and the relative residual (right) with N d = 6144, N = 3, Q = 3, κ = 0.05, ℓ = 1, ν = 1, γ = 0, and the mean-based preconditioner P 0 for varying µ. Table 6: Example 6.2: Simulation results showing total number of iterations, ranks of the truncated solutions, total CPU times (in seconds), relative residual, and memory demand of the solution (in KB) with N d = 6144, N = 3, Q = 3, ℓ = 1, ν = 1, µ = 10 −6 , and the mean-based preconditioner P 0 for varying γ. observation is that the low-rank solutions display the same pattern with the full-rank solutions as µ increases. Moreover, Table 6 reports the results of the simulations by considering various values of the risk-aversion parameter γ. As the previous example, the relative residual becomes smaller as decreasing the value of γ. (middle), and y f − y l 2 X (right), where the full-rank and low-rank solutions are denoted by y f and y l , respectively, computed by solving the full-rank and low-rank systems with N d = 6144, N = 3, Q = 3, ℓ = 1, µ = 10 −6 , γ = 0, ν = 1, and κ = 0.05 for varying values of the mean of random input η(x).
Last, we investigate the effect of the mean of random input η(x) on both full-rank and low-rank solutions. Denoting the full-rank solution and the lowrank solution by y f and y l , respectively, the behavior of the differences y f − y d 2 X , y l − y d 2 X , and y f − y l 2 X computed by solving the full-rank and lowrank systems is displayed in Figure 7. As increasing the mean of random input η(x), the difference between the full-rank and low-rank solutions becomes smaller.
Constrained problem with random convection parameter
Last, we consider a constrained optimal control problem containing a random velocity parameter. Except from the set up of Example 6.2, we have an upper bound for the control variable such as u b = 100. Taking the results in the ), the objective function J (u h ), the tracking term y h − y d 2 X , the difference of the full-rank and low-rank y f − y l 2 X , ranks of the truncated solutions, and the relative residual with N d = 6144, Q = 3, ℓ = 1, ν = 1, and the mean-based preconditioner P 0 . previous example into account, the regularization and risk-averse parameters are chosen as µ = 10 −6 and γ = 0, respectively. Figure 8 displays the desired state y d , the mean of state E[y h ], and the control u h obtained by L\B. We observe that the upper bound of the control constrained is satisfied. In Table 7, we compare the low-rank solutions with the full-rank ones. As increasing the truncation number N , we obtain better results as expected.
Conclusions
In this paper, we have numerically studied the statistical moments of a robust deterministic optimal control problem subject to a convection diffusion equation having random coefficients. With the help of the stochastic discontinuous Galerkin method, we transform the original problem into a large system consisting of deterministic optimal control problems for each realization of the random coefficients. However, we could not obtain some numerical results when increasing the value of truncation number N . Therefore, to reduce computational time and memory requirements, we have used a low-rank variant of GMRES iteration with a mean based preconditioner (LRPGMRES). It has been shown in the numerical simulations that LRPGM-RES can be an alternative to solve such large systems. As a future study, randomness can be considered in different forms, for instance, in boundary conditions, desired state, or geometry. Moreover, to handle curse of dimensionality, reduced order models, see, e.g., [65,66], can be an alternative to the low-rank approximations.
Funding. This work was supported by TUBITAK 1001 Scientific and Technological Research Projects Funding Program with project number 119F022. The authors would also like to express their sincere thanks to the anonymous referees for their most valuable suggestions.
Declarations
Conflict of interest. The authors declare no competing interests. | 2021-09-01T01:16:05.494Z | 2021-08-31T00:00:00.000 | {
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258064463 | pes2o/s2orc | v3-fos-license | The combination of multiple plant growth promotion and hydrolytic enzyme producing rhizobacteria and their effect on Jerusalem artichoke growth improvement
Rhizobacteria are well recognized for their beneficial multifunctions as key promoters of plant development, suppressing pathogens, and improving soil health. In this study, experiments focused on characterizing the plant growth promotion (PGP) and extracellular hydrolase production traits of rhizobacteria, and their impact on Jerusalem artichoke growth. A total of 50 isolates proved capable of either direct PGP or hydrolase-producing traits. Two promising strains (Enterobacter cloacae S81 and Pseudomonas azotoformans C2-114) showed potential on phosphate and potassium solubilization, IAA production, and 1-aminocyclopropane-1-carboxylic acid deaminase activity and hydrolase production. A hydrolase-producing strain (Bacillus subtilis S42) was able to generate cellulase, protease, amylase, β-glucosidase, and phosphatase. These three selected strains also gave positive results for indirect PGP traits such as siderophore, ammonia, oxalate oxidase, polyamine, exopolysaccharide, biofilm, motility, and tolerance to salinity and drought stress. Colonization was observed using a scanning electron microscope and rhizobacteria appeared at the root surface. Interestingly, inoculation with consortia strains (S42, S81, and C2-114) significantly increased all plant parameters, including height, biomass, root (length, surface, diameter, and volume), and tuber fresh weight. Therefore, we recommend that potential consortia of PGP and hydrolase-producing rhizobacteria be employed as a biofertilizer to improve soil and boost crop productivity.
. Characteristics of PGP and hydrolase traits exhibited by rhizobacterial isolates. N nitrogen fixation, P phosphate, K potassium, IAA indole-3-acetic acid, ACC 1-aminocyclopropane-1-carboxylic acid, − negative result, + positive result; Production of β-glucosidase: + halo below 1 cm, ++ halo greater 1 cm; Production of cellulase, protease, and amylase presented as relative index value with standard deviation. Different letters represent significant differences (p < 0.05) according to the LSD test. www.nature.com/scientificreports/ actions including siderophore and ammonia production, oxalate oxidase, and polyamine synthesis. Three strains proved capable of producing EPS and biofilm. C2-114 presented greatest tolerance to abiotic stresses such as salts, drought, and high temperature. S42, S81, and C2-114 were capable of motility, with S42 exhibiting the largest diameter among those examined. There was evidence that all selected strains possess indirect PGP characteristics and abiotic tolerance. The scanning electron microscope (SEM) revealed the colonization effectiveness of Jerusalem artichoke roots (Fig. 3). Our results showed the root surface was colonized by bacterial cells. Cells of three strains were distributed in the vicinity of the epidermal rough surface. (Fig. 4). There was a reduction in the photosynthetic rate, stomatal conductance, and transpiration rate both treated with bacteria and untreated. The selected isolates had different effects on plant height and tuber fresh weight (Fig. 5). The consortium treat- www.nature.com/scientificreports/ affected root length, root diameter, root surface, and root volume (Fig. 7). Moreover, the consortium treatment significantly increased tuber fresh weight in 24.2%.
Discussion
Rhizobacteria that possess multiple PGP traits can be utilized for agricultural application. The aim of this work was to screen rhizobacteria with a variety of direct and indirect PGP properties for stimulating the growth of Jerusalem artichoke. The data suggested that PGP consortium isolates (S42, S81, and C2-114) had significantly increased the development of Jerusalem artichoke by enhancing plant biomass and tuber fresh weight.
Interestingly, according to our observation of PGP and enzyme properties, some PGP isolates possessed direct PGP multifunctional traits, yet all hydrolase productions of indirect PGP traits were not consistently found. It is possible that some bacteria might not necessarily require the production of the enzyme in order to synthesize the nutrients. However, the best of our best knowledge, the common PGP and hydrolytic enzyme characteristics of bacteria isolated from the Jerusalem artichoke rhizosphere were recognized and examined for the first time. Rhizobacterial isolates produced hydrolase production, including cellulase, β-glucosidase, protease, phosphatase, and urease enzymes. These enzymes played the important role in the C-cycle, N-cycle, and P-cycle in the soils. Cellulose is the most abundant component of organic amendments. It is a main precursor for the synthesis of organic matter and organic carbon 15,18 . Moreover, these enzymes can also hydrolyze the cell walls of pathogens in order to protect the host plants 2 . Our results agreed with El-Deeb et al. 19 , Hazarika et al. 14 and Magotra et al. 20 , who reported that Bacillus exhibited the highest levels of hydrolytic enzyme activities which could use for characterizing PGP candidates 12,14,[19][20][21][22][23][24] .
The root plant absorbs phosphorus and potassium in soluble form to be utilized in growth 14,25 . Our findings in this study suggest that many isolates could solubilize both inorganic phosphate and potassium. The capacity of bacteria to solubilize phosphate and potassium was demonstrated by their utilization and conversion of sugar to organic acid, which lowered the pH of the media [26][27][28] . Moreover, several studied found that EPS-producing bacteria was able to support the high efficiency of solubilization 27,28 . Our isolate C2-114 was showed the ability to produce EPS, indicating that EPS enhances the efficiency of inorganic solubilization. Furthermore, it is possible for the P and K solubilizer to release zinc and silicate sources through the same mechanisms 22,23,28 .
IAA has the direct effect on the root elongation and indirect effect on improving of water and nutrient uptake efficiency 28 , respectively. The excreting of IAA in soils differs from those in vitro, depending on various factors. In a previous study by Sritongon et al. 29 , the same strain of P. azotoformans produced 78.75 µg mL −1 of IAA in www.nature.com/scientificreports/ the presence of l-tryptophan. However, varying amounts of IAA in vitro depend on several factors including the culture conditions, strains of species, and tryptophan concentrations 30 . E. cloacae produced the greatest amount of IAA. Our results are in agreement with the study by Kavamura et al. 12 , which revealed high concentrations of IAA produced by members of the Enterobacteriaceae family. Plant growth is regulated by the interaction between IAA and ACC levels 16,31 . Our findings suggest that S81 produces the highest amount of IAA and ACC deaminase. The high concentration of IAA in the plant roots influences the ACC precursor, resulting in the formation of increased ethylene 31 . Excessive ethylene is the triggering signal with which plant growth is inhibited. ACC deaminases that are produced by PGP rhizobacteria hydrolyze the precursor of ethylene into α-ketobutyrate and ammonia to decrease ethylene [31][32][33] Under normal and water-limited conditions, both IAA and ACC deaminase as the signal particularly enhances length, diameter, and volume of sunchoke root 16 . According to the research of Khan et al. 34 , both IAA and ACC deaminase showed the effect on increasing of plant biomass can increase plant biomass. ACC deaminase showed outstanding activity over others in stress condition. However, under normal conditions, ACC deaminase activity was greater than under stress 35 . ACC deaminase was required for PGP rhizobacteria to efficiently promote plant development under both conditions. Three strains (S42, S81 and C2-114) were examined for various actions that could indirectly promote the plant. They presented positive results for secretion of oxalate oxidase, which leads to the destruction of phytopathogens such as Sclerotinia sclerotiorum and Sclerotium rolfsii 36 . S81 and C2-114 produced the siderophore, a chelator that scavenges ferric in environment soil. Iron is a necessary co-factor for metabolic enzymes, thus causing competition that suppresses plant pathogen growth 37 . However, we did not find an interaction between the siderophore and the inhibition of S. rolfsii growth. S42 revealed positive inhibition of S. rolfsii, which may have been caused by either protease cellulase, amylase, or the other hydrolase, in addition to bioactive compounds. The enzymes produced were part of a triggering role to protect the host plant. Moreover, hydrolases break the cell walls of plant pathogenic fungus which infect Jerusalem artichoke; these pathogens include Fusarium oxysporum, Colletotrichum capsici, and S. rolfsii 2,17 .
Two PGP strains gave a positive result of polyamine production. Polyamine (putrescine, spermidine, and spermine) improves plant development. Besides, it alleviates plants in adverse environments such as metal toxicity, oxidation, drought, salinity, and cold 38 . Three strains were capable of producing EPS and biofilm. They retain moisture on the surface of their cells to occupy and protect the root surface from dehydration, hence reducing the impacts of variable dryness, salinity and temperature 12 . Bacillus ap. and Pantoea sp. were the most prolific EPS producers 12,16,23 . Microbial biofilm increases the efficacy of microbe-root connection 14,23,24,39 , reduces infection from plant pathogen, and defends plants from stress 23,39 . It was proved that our selective strains could survive environmental stresses such as high temperature, salt, and drought. Microbes employed cellulase and pectinase to hydrolyze root cell wall and colonize the root tips and roots branch at the interior and exterior of the roots 19,30 . Our findings also showed that three strains displayed motility, such as swimming, swarming, and twitching. Selected strains are therefore candidates for strain possessing multiple functions in PGP, colonization, and survival in environmental soils.
PGP rhizobacteria activity in soil is not driven by a single species but by numerous species operating simultaneously 12,40 . The absence of an inhibitory zone at the intersection of perpendicular streaks indicated that the hydrolase-producing strain and PGP strains were compatible. It was possible to combine strains and use them to promote the growth of Jerusalem artichoke. Thus, they were conducted in a non-sterile soil meaning their performance may differ from that exhibited in vitro.
According to the physiological of plant results, the photosynthetic rate, stomatal conductance, and transpiration rate were showed the decreasing trends from 30 to 90 DAT. Stomatal conductance and transpiration responded to preventing water loss in water deficit situations 6 . Significant differences were observed in photosynthetic rate during drought stress 1,16 . These parameters indicated variable responses to stress.
In this case, the height of plant, shoot dry mass, root dry mass and total biomass were increased when it treated with a single inoculant of bacterial isolate C2-114. Similarly, to Sritongon et al. 29 , C2-114 increased in sunchoke biomass at initial growth, which showed a strong correlation with IAA. In addition, enhanced root elongation, diameter, and surface were regarded as positive effects of S81 and C2-114 inoculation. This may be the result of the presence of IAA and ACC deaminase. ACC and IAA were cooperate to modulate the ratio of ACC to IAA in modified root systems 16,27,34 .
The consortium of selected isolates revealed a significant difference in their capacity to increase height, shoot dry weight, root dry weight, biomass at 60 DAT to harvest and tuber fresh weight. Similar to Hazarika et al. 14 PGP rhizobacteria consortia had more effect on plants than individual inoculum. Consortia of bacterial isolates alleviated the negative effect of salinity stress and increased biomass of French bean 27 . Our data indicates that mixed culture seems to contribute to synergetic activity by enhancing the Jerusalem artichoke. Therefore, a consortium of both PGP (two strains) and hydrolase producing rhizobacteria could improve plant biomass and tuber productivity better than a single inoculant via multiple direct and indirect PGP activity as well as extracellular enzyme synthesis. Moreover, applying the mixed inoculant showed higher number of survival cells in their natural environments than using a single inoculant 12,16,[40][41][42] . It can be utilized as bio-fertilizer in sunchoke. However, this work has limitations which potential consortium isolates need to be confirmed in the field conditions. For further research, we will focus on the efficacy of field-applied organic amendments in promoting plant growth for the development of biofertilizers.
Conclusion
Our study revealed rhizobacteria possess multifaceted PGP direct and indirect activities that supported their capacity to enhance plant productivity. Two PGP strains (Enterobacter cloacae and Pseudomonas azotoformans) and hydrolase producing strain (Bacillus subtilis) were confirmed to enhance the growth of plants. These interesting results suggested that the mixed three isolates stimulated the growth of plants such as the biomass and the tuber of Jerusalem artichoke. Thus, this consortium demonstrated high potential for plant growth promotion. In order to verify soil improvement; the consortium of inoculants can be used as a supplementation of soil amendment or compost and developed to the bio-fertilizer product and used it infertile soils for sustainable improvement.
Materials and methods
Plant materials and rhizobacterial isolation. Samples of Jerusalem artichoke roots with adhering soil were collected from 0 to 10 cm depths at farm of the field crop department, faculty of Agriculture, Khon Kaen University, Khon Kaen Thailand (16°47′45′′N, 102°81′07′′E). The roots were stored in an ice box before being transferred to the laboratory. The root of each sample was cut into approximately 0.5 cm segments.
Screening of plant growth-promoting (PGP) traits.
All isolates were screened for PGP traits such as nitrogen fixation, phosphate solubilization, potassium solubilization, synthesis of indole3-acetic acid and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Regarding nitrogen fixation, each isolate was inoculated into Ashby's nitrogen free broth 44 . NBRIP agar 45 and Aleksandrov agar 46 were used to evaluate the solubilization of phosphate and potassium, respectively. Each isolate was cultured in tryptic soy broth (TSB) (Himedia) supplemented with 1 g L −1 l-tryptophan (Acros Organics) for 24 h, and Salkowski's reagent added 47 . ACC deaminase activity was screened on DF minimal salt agar 48 with 3 mM of 1-aminocyclopropane-1-carboxylic acid (ACC) instead of ammonium sulfate 32 . Pseudomonas azotoformans C2-114 (Accession number LC130639), which possessed nitrogen fixation, phosphate and potassium solubilization, IAA production, and siderophore traits 29 was used to investigate ACC deaminase activity and other PGP and hydrolase activities.
Quantification of PGP activities. Phosphate solubilization. Phosphate solubilization of each PGP isolate was determined using NBRIP medium 45 . PGP isolate was grown in TSB medium at 30 °C, 150 rpm for 24 h. The bacterial cell (consisting of approximately 10 8 CFU mL −1 as inoculum (100 µL) of each isolate was transferred to a 5 mL sterile NBRIP broth tube containing 0.5% (w/v) tricalcium phosphate powder as the insoluble phosphate. Tubes were done in triplicate, and incubated at 30 °C, 150 rpm for 7 days. Aliquots were of 1 mL supernatant of each sample and control, 1 mL of molybdate-vanadate reagent 49 . All tubes were incubated in darkness for 20 min. The development of a yellow color was used to quantify soluble phosphorus. The sample was measured at 420 nm (U-5100, Hitachi). A standard curve was constructed using KH 2 PO 4.
Potassium solubilization. Potassium solubilization of each PGP isolate was determined using Aleksandrov medium 46 . Inoculum (100 µL) was transferred to 5 mL of sterile Aleksandrov broth containing 0.2% (w/v) potassium aluminium silicate powder (Himedia) as an insoluble potassium source. The tubes were done triplicate and incubated at 30 °C, 150 rpm for 7 days. Aleksandrov broth medium without inoculation served as the control. Soluble potassium in the supernatant was measured at 776.5 nm using an atomic absorption spectrometer (AAnalyst 100, Perkin Elmer) with flame air C 2 H 2 and a slit of 0.7 nm. A standard curve was constructed using KCl.
Indole-3-acetic acid (IAA) production. IAA production of each PGP isolate was determined using Salkowski's reagent 47 . Inoculum (100 µL) of each isolate was added into 5 mL sterile TSB medium supplemented with 1 g L −1 l-tryptophan (Acros Organics). The tubes were done in triplicate and then incubated at 30 °C, 150 rpm for 48 h. Aliquots of 1 mL of supernatant were mixed with 2 mL of Salkowski's reagent 47 , before being incubated in the darkness for 30 min. The appearance of red coloration was used to quantify IAA production. The sample was read at 530 nm. A standard curve was constructed using IAA (Acros Organics).
ACC deaminase activity. ACC deaminase activity was determined according to Penrose and Glick 32 . The isolates were cultured in 5 mL of DF minimal salt broth medium 48 www.nature.com/scientificreports/ vortexed. Aliquot of the supernatant (1 mL) was mixed with 800 µL of 0.56 N HCl and 300 µL of the 0.2% (w/v) 2,4 dinitrophenylhydrazine reagent (dissolved in 2 N HCl), respectively, before incubation at 30 °C for 30 min. 1 mL of 2 N NaOH was then added and thoroughly mixed prior to being measured at 540 nm. Standard curve was constructed using α-ketobutyrate (Sigma-Aldrich). Soluble protein was performed according to Bradford assay 50 . In order to create a standard protein curve, bovine serum albumin (Acros Organics) was used.
Screening of hydrolytic enzymes activities. All isolates were examined for cellulase, β-glucosidase, α-amylase, protease, urease, and phosphatase traits. For evaluation, secreted cellulase and β-glucosidase were tested on solid minimal medium 51 : 6.8 g Na 2 HPO 4 , 3.0 g KH 2 HPO 4 , 0.5 g NaCl, 1.3 g (NH 4 ) 2 SO 4 , 0.5 g MgSO 4 ·7H 2 O, 15.0 g agar, 1000 mL distilled water, to which was added 0.5% (w/v) cellulose for cellulose hydrolysis and 0.1 mL of 0.04% (w/v) 4-methylumbelliferyl-β-d-glucopyranoside (MUG) (Acros Organics) to spread on the agar before inoculation. After incubation at 30 °C for 48 h, Gram's iodine solution was used to evaluate cellulose hydrolysis. Fluorescent halo of MUG hydrolysis appeared under UV light. For the assessment of proteolytic production, each isolate was inoculated on skim milk agar (Peptone 5 g, beef extract 3 g, skim milk 10 g, agar 15 g-1000 mL). A clear zone was visible around the colony after incubation at 30 °C for 48 h. To evaluate α-amylase, production was performed on starch agar (Peptone 5 g, beef extract 3 g, starch 10 g, agar 15 g-1000 mL). Starch hydrolysis was examined using Gram's iodine solution. For phosphatase production, the culture was spotted on the TSA medium containing 0.01% (w/v) phenolphthalein bisphosphate tetrasodium salt (Sigma-Aldrich), examined by 8.4% (v/v) ammonium hydroxide 26 . Urease production was carried out on Christensen's medium urea agar (Himedia) containing sterile 2% w/v urea. The colony was observed displaying a pinkish-red color on the agar 52 .
Other PGP, motility, and stress tolerance. Selected isolates were examined for indirect PGP traits including siderophore production, antagonistic test, polyamine production, ammonia production, oxalate oxidase enzyme production, biofilm, exopolysaccharide production and the microbial characteristic under environmental stress conditions. Chrome Azurol S (CAS) agar was used to evaluate siderophore formation 53 . The suppression of Sclerotium rolfsii was investigated following the dual method described in Sritongon et al. 29 . The percentage of inhibition was calculated from center to the edge of fungal mycelia length: (A − B)/A × 100 (A: Control, B: bacterial inoculation). Moeller's decarboxylase broth base (Himedia) pH 7.0 supplemented with 1% (w/v) l-arginine hydrochloride (Acros Organics) was used to test polyamine production. Ammonia production was evaluated using 4% (w/v) peptone water and examined with Nessler's reagent 54 . Oxalate oxidase was tested on the agar plate containing potassium oxalate as the sole carbon source 55 . Motility assay that included swimming, swarming, and twitching was carried out using nutrient medium (0.5% (w/v) peptone and 0.3% (w/v) beef extract) having 0.3%, 0.5%, and 1.0% (w/v) agar, respectively. Biofilm production was performed using crystal violet staining with a modified version of Latorre et al. 56 . Exopolysaccharide (EPS) production was performed in accordance with Kavamura et al. 12 by replacing saccharose with 10% (w/v) glucose. The capability of isolates to tolerate a stressful environment was tested by culturing in nutrient broth with varied NaCl concentration, 20% (w/v), polyethylene glycol (PEG6000), and growth at 40 °C.
16S rRNA identification and phylogenetic analysis. Selective (16°47′59′′N, 102°81′61′E). The soil was air-dried, passed through a 2-mm sieve, and then added to a pot (15 inches in diameter and 11 inches in depth) to fill approximately 21 kg. The soil was classified as sandy loam with pH 6.57 and electrical conductivity of 0.63 dS m −1 . Soil chemical properties were organic carbon 0.20%, organic matter 0.35%, total nitrogen 0.02%, available phosphorus 12.00 mg kg −1 , available potassium 24.50 mg kg −1 , and cation exchange capacity 3.70 c mol kg −1 .
A pot experiment was conducted in a randomized complete block design (RCBD) with four replicates. The pot experiment was performed during the early rainy season (April to September 2019). The experiment consisted of the following seven treatments: control without bacterial inoculation (W), inoculated with hydrolase-producing isolates (S4), inoculated with PGP isolate (S8), inoculated with PGP isolate (C2), inoculated with PGP isolate and hydrolase producing isolate (S4S8), inoculated with PGP isolate and hydrolase producing isolate (S4C2), inoculated with two PGP isolates and hydrolase producing isolate (S4S8C2). Jerusalem artichoke variety HEL65 was prepared as described above. All methods were performed in accordance with relevant guidelines and regulations. In each pot, one plant with two true leaves was transplanted and established at the same depth. Each isolate was individually cultured in TSB medium. The isolates were grown with agitation of 150 rpm at room temperature for 18 h. The bacterial cells were collected by centrifugation at 6000 rpm for 10 min, then washed twice with sterile distilled water. The cell pellets were resuspended in 0.85% (w/v) NaCl and adjusted to a concentration 10 9 CFU mL −1 . For co-inoculation and consortium inoculation, equivalent volumes of each isolate were mixed as the inocula. Bacterial inocula (20 mL) were inoculated using a syringe to the vicinity of the roots of the seedling. Plant analysis. Plant height, shoot and root dry weights were recorded at 30, 60, 90 days after transplanting (DAT) and harvest time (140 days). The shoot and root samples were separated. The roots were placed on a sieve and carefully rinsed with tap water to eliminate soil particles. Using a scanner (Perfection V800™ Photo, Epson), root morphology such as total length, surface area, volume, and average diameter was scanned and analyzed using Win-Rhizo Pro2004a software (REGENT Instruments Inc., Canada). The shoots and roots were dried in a hot air oven at 70 °C for 72 h for constant mass. The biomass of the plant was presented using the sum of the dried shoot and root mass. The photosynthetic parameters were measured from its true leaves using a portable photosynthesis system (LI-6400XT, LI-COR Bioscience, USA). Fresh weight of the tubers for each treatment was recorded at harvesting. Statistical analysis. The in vitro data and pot experiment were subjected to a one-way analysis of variance (ANOVA). All statistical analysis was performed using the Statistix 10 software. The mean of treatments of all tests was identified by least significant difference (LSD) test at p < 0.05. | 2023-04-12T14:08:23.382Z | 2023-04-11T00:00:00.000 | {
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15294627 | pes2o/s2orc | v3-fos-license | Hidden E type structures in dilute A models
The hidden E_{7} (E_{6}) structure has been conjectured for the minimal model ${\cal M}_{4,5} ({\cal M}_{6,7}$) perturbed by $\Phi_{1,2}$ in the context of conformal field theory(CFT). Motivated by this, we examine the dilute A_{4, 6} models, which are expected to be corresponding lattice models. Thermodynamics of the equivalent one dimensional quantum systems is analyzed via the quantum transfer matrix approach. Appropriate auxiliary functions, related to kinks in the theory, play a role in constructing functional relations among transfer matrices. We successfully recover the universal Y- systems and thereby Thermodynamic Bethe Ansatz equations for E_{6,7} from the dilute A_{6,4} model, respectively.
Introduction
The impact of perturbed conformal field theory (CFT) has many aspects [1,2]. In this communication we explore one of its predictions; the "Trinity" among minimal unitary CFT theory M p,p+1 , p = 3, 4, 6 perturbed by Φ 1,2 , lattice models off criticality, (the Ising model in a field, the tricritical Ising model off the critical temperature and the tricritical 3 state Potts model) and the dilute A 3,4,6 models. Many results have already been accumulated on the equivalence in universality [3]- [18]. The scaling exponents of the dilute A 3 model in periodic or open boundary conditions have been evaluated analytically [12]- [17]. They agree with numerical results for the Ising model in a magnetic field. Masses for eight elementary excitations of the dilute A 3 model are found to be proportional to components of the (largest) eigenvector of Cartan matrix for E 8 [16,18]. Vertex operators of A (2) 2 , which is the symmetry of the dilute A 3 model at criticality, satisfy a set of relations indicating the hidden E 8 structure [19].
Especially, we like to call attention to thermodynamics of a 1D system related to the dilute A 3 model in [20]. A set of solutions to the eigenvalue problem of the 1D Hamiltonian has been identified in exquisite "string" forms [20,21,22]. Nine of them are expected to contribute nontrivially in the thermodynamic limit. This observation leads to a set of integral equations (Thermodynamic Bethe ansatz , TBA ) which determines the free energy. Remarkably, TBA exhibits the underlying E 8 structure [23].
In [24], we have attacked the same problem in a different setting. By following general frameworks, one represents the free energy by the largest eigenvalue of the "quantum transfer matrix" (QTM) acting on a virtual space [25] - [31]. We have managed to solve the (single) eigenvalue problem of commuting QTM by introducing auxiliary functions related to fusion of QTM [32,34,35]. We will simply call them fusion QTMs. Eight fusion QTMs are found to satisfy a closed set of functional relations related to E 8 . A quantum analogue of the Jacobi-Trudi formula [36,39], as well as combinatorial aspects in terms of "Yangian analogue" of Young tableaux [36,37,38,39,40] play a fundamental part in the proof of the relations. Nice analytic properties of fusion QTMs allow for the transformation of functional relations into coupled integral equations. The resultant thermodynamic Bethe ansatz (TBA) equation yields a direct evaluation of free energy. Again it coincides with a hypothetical TBA for the E 8 theory.
As promised in [24], we carry out this program for the dilute A 4 and A 6 models. A novel feature lies in the fact that "a box" of the "Young tableaux" is no longer the fundamental constituent. This may be natural in view of the representation theory; the vector representation is no longer minimal. In the language of the S-matrix theory, boxes present breathers, rather than kinks. We conjecture explicit forms of QTMs related to these kinks. The investigation on these kink QTMs reveals connections between U q (A (2) 2 ) modules of symmetric tensors of the vector representation and U q ( E 6 ), U q ( E 7 ) modules when q equals to proper root of unity. With help of this observation, closed functional relations among fusion QTMs are also found for the dilute A 4,6 models. Quite parallel to the dilute A 3 model, one recovers TBAs expected for E 6,7 [23].
The paper is organized as follows. As the subject may not be so familiar to readers, we present a brief survey on the QTM approach, together with the sketch of the idea of analytic Bethe ansatz in section 2. The dilute A L model is briefly described in section 3. The sl 3 fusion structure of the model is presented in view of analytic Bethe ansatz, the "Yangian analogue" of Young tableaux and the quantum Jacobi-Trudi formula in section 4. We concentrate on the dilute A 4 model in sections 5 and 6. The explicit form of QTM related to the "kink" is proposed in section 5. Yangian homomorphisms among Y (E 7 ) modules serve as a useful guide in search of the form. The rest of QTMs are defined and their functional relations are examined in section 6. The result coincides with prediction in [58]. Similar results for the dilute A 6 model are given in section 7. With piece of information on analyticity of these QTMs, supported by numerics, the desired TBAs are recovered in section 8. We conclude the paper with a short summary in section 9.
2 Survey on the QTM approach Exact evaluation of physical quantities at finite temperatures poses serious difficulties even for integrable models. One has to go much beyond mere diagonalization of a Hamiltonian; summation over eigenspectra must be performed.
The string hypothesis brought the first breakthrough and success. It postulates dominant solutions to the Bethe ansatz equation (BAE) in the thermodynamic limit. In a sense, the method tackles the combinatorial aspect of the problem directly.
QTM
The quantum transfer matrix (QTM) method takes a different route. It utilizes the famous mapping between the Hamiltonian H M of a 1D quantum system and the row to row transfer matrix T RT R (u) of the corresponding 2D classical model [25] - [31]. In the present context, the latter is given by Here the box represents the RSOS weight and M is the number of sites. See appendix for explicit weights for the dilute A L models. The parameter u represents the anisotropy of interactions between horizontal and vertical directions, and is called the spectral parameter. The explicit relation between H M and T RT R (u) reads, where ǫ is the normalization parameter of the Hamiltonian. The essential idea in the QTM approach is encoded in the following identity, Namely, the partition function of the original problem is transformed into that of the 2D classical models on M ×N sites. The fictitious dimension N is sometimes referred to as the Trotter number. We can interpret T ′ RT R (u) as the row to row transfer matrix in the "vertical" direction. Similarly, one can construct a transfer matrix propagating in the "horizontal" direction It may be better to rewrite this in the form, The exchangeability of two limits are proven in [25]. The gap opens up between the largest and the second largest eigenvalues of T ′ QT M (u). In the thermodynamic limit M → ∞, we only have to deal with the largest eigenvalue of QTM. This strongly contrasts to the spectra of T ′ RT R (u). One observes almost degenerate low lying excitations in the latter case as M → ∞. The evaluation of free energy per site of the 1D quantum system is thus reduced to the largest eigenvalue problem of T ′ QT M (u). We are free from the summation problem. This is, unfortunately, not the happy end of the story. The Trotter number should be sent infinity at the end. The diagonalization of QTM is accomplished by the application of the Bethe ansatz method. The BAE depends nontrivially on N, which originates from the local interaction parameter u. Thus we can not retort to the simple-mind application of the usual scheme of converting the transcendental equation into the integral equation. This makes the extrapolation N → ∞ quite nontrivial.
commuting QTM
Instead of dealing with BAE roots directly, we employ a different idea. The integrable structure of the underlying model allows for the introduction of one parameter family of commuting QTMs which is labeled by a novel complex parameter x. For the explicit demonstration of this, we adopt a more sophisticate approach [32,33] than the one presented above. One introduces a "staggered manner" QTM to avoid (T RT R (0)) −1 factor in the definition of T ′ RT R (u).
The following relation is still valid, As is emphasized above, we find the intriguing fact, commutativity of QTMs, Generally, one can construct a "higher spin" QTM T fusion (u, x) by the fusion procedure. The Yang Baxter integrability also assures commutativity among these generalized QTMs, Note that the factor u is in common. Hereafter we will sometimes drop this common factor in commuting QTMs. We also sometimes use same notations for transfer matrices and their eigenvalues as we are considering them on the identical eigenspace.
We utilize the existence of the complex x plane in which QTMs are simultaneously diagonalizable. There exist functional relations among these fusion QTMs in the x plane. Our idea is to utilize these functional relations in place of BAE. See [52,53] for the discussion in the case of usual row to row transfer matrices. Our motivation is simple. The number of roots is of order N, the Trotter number. All these locations are changing with N, while functional relations depend on N weakly. The dependence can be summarized in the known scalar factors in functional relations. One may expect the tractable limit N → ∞ for functional relations. The problem of combinatorics (summation over eigenspectra ) is then reduced to the study of functional relations 1 and analytic structures of fusion QTMs, as will be discussed soon below.
Analytic Bethe ansatz and functional relations
The QTMs should not possess singularities in the x plane as Boltzmann weights are regular functions of x. BAE can be interpreted as the polefree condition of QTMs in the complex x plane. Conversely, the analyticity requirement imposes restrictions on explicit eigenvalues of QTMs.
The analytic Bethe ansatz was proposed in [55], as a tool in deriving expressions of eigenvalues of transfer matrices. It starts from a simple observation; the eigenvalue at the "vacuum sector" is determined by diagonal elements of R matrix, which are referred to as vacuum expectation values.
In general sectors, eigenvalues should be modified such that each vacuum expectation value is "dressed" by appropriate combination of Baxter's Q operators. See eq.(4) for a typical example. The combination is determined by requiring analyticity of the transfer matrix. We call the resultant expression, Dressed Vacuum Form (DVF).
A "universal" BAE has been proposed in [55,56]; one can write down BAE for the model based on U q (ĝ) using only algebraic data of U q (g). Starting from a properly chosen "highest weight term", we can construct a pole-free set of functions under BAE. In [38] a similarity has been pointed out between the above procedure and the construction of the highest weight module of Lie algebra. It leads to an assumption that there exists a set of functions, pole-free under BAE, corresponding to an irreducible module of quantum affine Lie algebra. The set is naturally identified with the eigenvalue of the transfer matrix of which trace is taken over the irreducible module. This has been promoted as an axiom in [38] and subsequent papers [39,40] producing fruitful results. We take sl 2 as the simplest example. By V m (x) (T m (x)), we mean the m+1-dimensional sl 2 module, and the associated transfer matrix. The DVF of T 1 (x) consists of two terms. We do not specify their forms and represent them by boxes with letters 1 and 2, Each box carries spurious poles, which are actually canceled due to BAE. We represent this situation graphically as, The eigenvalue of a fusion QTM can be apparently represented by sum over products of "boxes" with various letters and spectral parameters. 1 One can adopt different auxiliary functions from the fusion hierarchy. Results on other choices of auxiliary functions for several models, see [33], [45]- [51] For example, one can construct a transfer matrix of which auxiliary space acts on the symmetric subspace of V 1 × V 1 . We associate to this the set of glued boxes, i 1 x−i i 2 x+i , (i 1 ≤ i 2 ). The difference in spectral parameters is fixed so as to match the singularity of R matrix. The cancellation of spurious singularities is again depicted as where we omit spectral parameters. The eigenvalue of T 2 (x) is given by the sum of three diagrams in the above. Extension to general T m is now obvious. Starting from the "highest weight" term, 1 1 · · · 1 , we have a cancellation diagram, The sum over them, regarded as expressions, yields the eigenvalue of T m (x). One can easily identified the diagram with the crystal graph of the m-fold tensor of U q (sl 2 ) representing the irreducible module.
Suppose that we have a short exact sequence among tensor products of irreducible modules of quantum affine Lie algebra, then a functional relation follows, Remark that spectral parameter dependencies are implicit in W i s 2 . The desired functional relations are derived as a consequence of relations among affine modules. Even if exact sequences are not available, one can still check the validity of hypothetical functional relations using explicit forms of transfer matrices, which can be derived by applying the analytic Bethe ansatz. Indeed, functional relations for sl 2 are easily derived without knowledge on exact sequences. By using the above box-representation, one can derive graphically, where g m (x) is a known scalar function which depends on N.
Such functional relations are sometimes referred to as the T − system.
Functional relations and Thermodynamic Bethe Ansatz
Unfortunately, functional relations alone do not provide enough information on the explicit eigenvalues. This can be easily seen from the fact that excited states' eigenvalues satisfy same algebraic relations. One needs additional information on analyticities of fusion QTMs. Let us again demonstrate this for the sl 2 case. One conveniently rewrites the where a known property of the g− function, Consider functions in the largest eigenvalue sector of T 1 (x). We have convincing numerical evidences for the conjecture that zeros of T m (x) approximately lie on the curve ℑx ∼ ±(m + 1). Then both sides of (2) are analytic, nonzero and asymptotically constant (ANZC) within the strip ℑx ∈ [−1, 1]. Strictly speaking, we must modify the lhs for m = 1. We will not go into such detail in this introductory part.
This piece of information is now sufficient to transform the algebraic relations to integral equations which enable the explicit evaluation of Y m and then T m .
Take the logarithmic derivatives of both sides of (2) and perform Fourier transformations. LetdlY m [k] be the Fourier transformation of the logarithmic derivative of Y m (x). Thanks to the ANZC property, the Cauchy theorem applies. The resultant equation is simply given by Remarkably, both sides only contain functions with the same Fourier mode. Dividing both sides by 2 cosh k, performing inverse Fourier transformation and integrating once over x, we reach the integral equation, which is identical to TBA, K(x) denotes the Fourier transformation of 1/2 cosh k. Though we have omitted above, the rhs of the equation (3) for m = 1 has a nontrivial scalar factor originated from g function. It thus brings a N− dependency, however, by the combination uN in the N → ∞ limit. Remembering that u is inversely proportional to N, we can send N → ∞ analytically! The resultant drive term depends only on β.
In this way, we take a completely different route from the string hypothesis but reach the same conclusion. In the absence of appropriate conjectures on dominant patterns of roots, our method has an explicit advantage in attacking the problem. This is the case with the dilute A 4,6 models. 3 In the rest of this paper, we shall extensively apply the above ideas to these cases. Before that, we repeat lessons from the above. To find functional relations is not enough. One must find them having ANZC property in appropriate domains of the complex x plane. This is the most crucial step in the present approach.
The dilute A L model
The dilute A L model is proposed in [12,13] as an elliptic extension of the Izergin-Korepin model [41]. (See [42] for an elliptic extension of the different type.) The model is of the restricted SOS type with local variables ∈ {1, 2, · · · , L}. The variables {a, b} on neighboring sites should satisfy adjacency condition, |a − b| ≤ 1. The solvable weights contain parameters u, q and λ. We supplement their explicit forms in appendix. The model exhibits four different physical regimes depending on parameters, , L ≥ 2 We are interested in regimes 2 and 3. As in section2, one defines the Hamiltonian of the associated 1D quantum chain by as in [20]. ǫ = −1, (1) corresponds to regimes 2 (3), respectively. The one particle excitations for the dilute A L case have been examined in [43,44] for L =3,4 and 6. (See also [18] for another derivation for L = 3) Eight, seven and six particles are identified respectively, and their masses are summarized by a single formula in the trigonometric limit, where g ∨ is 30, 18, 12 for L =3,4, 6 and is nothing but the dual Coxeter number for E 8 , E 7 and E 6 , respectively. We present sets of allowed a's in table 1,2 for L =4, 6, which are of our current interest.
A number k in the bracket means that it corresponds to the k− th light particle. Note that vectors of the form (m 1 , m 2 , · · · ), coincide with the eigenvectors of Cartan matrices for E 7 and E 6 , respectively. The exponents {a} will re-appear in a novel context later. The leftmost numbers, which are just indices up to the present, will be connected to indices for nodes in the Dynkin diagrams of E 7 and E 6 . The sl 3 type fusion structure in the dilute A L model has been discussed in [54]. This comes from the singularity of RSOS weights at u = ±3; the face operator becomes a projector related to sl 3 at these points. One picks up a desired subspace from tensor products of spaces using these projectors. The adjacency conditions of local states are described by combinatorics of tableaux.
We are interested in eigenvalues of fusion QTMs. Then the most relevant is the fact that these eigenvalues are again expressible in terms of "Young tableaux" depending on spectral parameters, as exemplified in section 2.3 for the sl 2 case.
Explicitly, the eigenvalue T 1 (u, x) of T QTM (u, x) is given by and w = exp(iπ ℓ L+1 ) where ℓ = 1 for the largest eigenvalue sector. θ 1 (ix) is defined in appendix. The parameters, {x j } are solutions to BAE, As in section 2, we represent these three terms by three boxes with letters 1,2 and 3, One infers from the sl 2 example that a combinatorial aspect may also appear. This turns out to be true. The eigenvalues of fusion QTMs are given by the sum of combinations of boxes, which can be identified with semi-standard Young tableaux for sl 3 . On each diagram, the spectral parameter changes +2i from the left to the right and −2i from the top to the bottom. (Fig 2). We restrict ourselves to diagrams of the rectangular shape for a while. Firstly we note that QTM associated to 2 × m ( 3 × m) Young diagram can be reduced to the one associated to 1 × m (or just scalars). This is x x+2i x-2i x due to identities, Second, eigenvalues of 1 × m fusion QTMs have the "duality" in the following sense. Let us denote a renormalized 1 × m fusion QTM by T m (x); The spectral parameters are assigned x − i(m − 1) · · · x + i(m − 1) from the left to the right. The renormalization factor, common factor to all expressions of length m tableaux, is given by Hereafter we sometimes denote f (x ± iy) for the dilute A 4 (A 6 ) model. From the sl 3 structure, together with the above property, one can prove the following functional relations, The periodicity for the dilute A 4 model , φ(x + 10/3i) = φ(x), leads to g m+10 (x) = g m (x) m ≥ 0 and g 7−m (x) = g m (x), (0 ≤ m ≤ 7). From the adjacency matrix, one concludes T 8,9 (x) = 0.
Thus functional relations are invariant under the transformation, . The symmetry of functional relations is not necessary inherited to its solution in general. We verify, however, the "duality", and T m+10 (x) = T m (x), m ≥ −1 numerically for the largest eigenvalue sector, only which we are interested in. These duality relations are also observed and numerically verified for A 6 models with change in the period. See section 7. Functional relations among them, however, do not possess the desired analytical property, as discussed in [24]. We thus introduce other class of QTMs related to skew Young diagrams.
Let µ and λ be a pair of Young diagrams satisfying µ i ≥ λ i , ∀i. We subtract a diagram λ from µ. We call the result a skew Young diagram µ − λ, consisted of (µ 1 − λ 1 , µ 2 − λ 2 , · · · ) boxes. In the theory of symmetric 2) polynomials, the Jacobi-Trudi formula tells that a complex Schur function associated to a skew Young diagram can be expressed by a determinant of a matrix, of which elements are given by elementary Schur functions associate to "one-row" diagrams or " one-column" ones. The quite parallel formula holds for the present situation, which we call the "quantum" Jacobi-Trudi formula [36,38,39].
Consider a set of semi-standard skew Young tableaux of the shape µ − λ. We assign an expression to each table. The spectral parameter of the "top-left" box is fixed to x + i(µ ′ 1 − µ 1 ) 4 where µ ′ 1 denotes the depth of the tableaux.
One identifies each box in a table with an expression under the rule (6) with the shift of the spectral parameter. Then the product over all constituting boxes yields the desired expression for the table. Theorem 1 Let T µ/λ (x) be the sum over the resultant expressions divided by a common factor, . Then the following equality holds, where T m<0 := 0.
The proof is quite similar to the one for Young tableaux [38,39]. One must only keep in mind that the allowed position of a box is restricted by its spectral parameter.
The crucial fact is that the so defined T µ/λ (x) is an analytic function of x due to BAE, and contains T 1 (x) as a special case. The former is not so obvious from the original definition by the tableaux, but it follows trivially from the quantum Jacobi-Trudi formula.
Kink Transfer Matrix
In following few sections, we restrict our discussion to the dilute A 4 model. We will summarize results for the dilute A 6 model in section 7.
For the E 8 case, the vector representation is minimal and other representations are constructed by fusion of it. Eigenvalues of associated transfer matrices of the dilute A 3 model are thus derived from products of T 1 (x + some shift). This is no longer true for the dilute A 4 model. Let W a (x) be the Yangian highest weight module associated to the node a in Figure 1. Through several evidences, we identify T 1 (x) of the model with the transfer matrix connected to W 1 (x), which is not minimal. The most fundamental object in E 7 is W 6 (x) rather than W 1 (x). Any other object may be constructed from T (6) (x), the transfer matrix of W 6 (x), of which explicit form is not known from the dilute A 4 model. The determination of explicit form of T (6) (x) is thus vital in the present approach. For this purpose, homomorphisms of U q (ĝ) modules deserve attentions. They lead to non-trivial algebraic relations among T (6) (x) and other QTMs. Although such information is not available for U q (Ê 7 ), there exists a list of homomorphisms of Y (E 7 ) modules in [57], Note that the normalization of spectral parameter is different from [57]. For example, the second relation implies, After trials and errors, we find the following ansatz compatible with the above homomorphisms , As explicit RSOS weights are not yet derived, it may be inappropriate to call T (6) (x) as the eigenvalue of transfer matrix. In the following discussion, however, we do not use the assumption that it coincides with the actual eigenvalue of transfer matrix of W 6 . Rather, we simply use facts (1) it is pole free under BAE (2) it satisfies the desired relations expected from eqs. (14) and (15). Readers should understand this terminology just as a "nickname".
The following functional relations between T (6) (x) and T m (x) will facilitate discussions in later sections.
Fusion Quantum transfer matrices and the T − system for the dilute A 4 model
Having defined the "kink" QTM T (6) , we are in position to introduce other QTMs and explore functional relations among them. First we present QTMs defined by skew Young tableaux.
The explicit forms of DVFs in eq.(4) and eq.(18) then conclude T (1) (x) = T (1) (x), T (6) (x) = T (6) (x). By remembering 5/3i equals to the half period of our elliptic function θ 1 (x), one also verifies T (5) (x) = T (5) (x). The conjugate property of T (2) (x) and T (3) (x) is less obvious. One can nevertheless show this by the use of the quantum Jacobi-Trudi formula. T (6) comes into expressions of remaining QTMs. The Yangian homomorphisms turn out to be useful in deriving their explicit forms. We take T (4) (x), related to W 4 (x), for instance. As argued in section 2.3, we identify an analytic set with an affine irreducible module. Thus eq. (13) for a = 2 implies, That is, the product of DVFs T (5) (x − I/6)T (6) (x + i/3) decomposes into two (or more) subsets and each subset is analytic within itself. We look at the explicit DVF of the lhs and find that it contains analytic subset given by φ( The sum of remaining terms must be analytic under BAE. We identify them as T (4) (x). In a similar way, we deduce T (7) (x).
Definition 2
The common factor . We note that the conjugate property, discussed for other T (a) s, is also verified for T (7) (x) when it is written in terms of its explicit DVF. On the other hand, the property is not so apparent for T (4) (x), although one can prove it by a different route. See the discussion after the proof of eq.(36).
We are now ready to describe the statement as to functional relations among QTMs .
Proposition 1
The above QTMs enjoy the following T −system, These coincide with the T − system for E 7 proposed in [58] in a different context.
Proof of eq.(36). The proof utilizes (19) as follows. Consider the product T (4) (x)T (6) (x). Substituting eq. (30), we have One applies the rule (19) to the product of two T (6) s by shifting x ± i/6. The result leads to which coincides with eq.(36). As mentioned previously, the complex conjugate property of T (4) (x) is not obvious from its DVF. Instead, we can now shown it from the established relation (36) with the help of the conjugate property of T (5) (x) and T (6) (x) commented below (27).
To prove remaining relations, we need to prepare further lemmas.
Lemma 2
The following decompositions are valid , Proof of Lemma 2. The relation (39) is checked by comparing DVFs of both sides directly. To prove eq.(40), we rewrite T (5) in the lhs in terms of T (6) by using eq. (19), By applying eqs. (19) and (20), one reaches the rhs of (40 ). For later use, we rearrange the sum of eqs. (39) and (40), Lemma 3 This is the analogue of the relation, and it can be shown in a similar manner. In proving eq.(34), one needs decomposition of a huge QTM.
Proof of Lemma 5. We use a trick; the complex conjugate property, established soon below the proof of eq.(36), allows us to replace T (4) (x) by T (4) (x) in the lhs. After this, we substitute definitions (30) and (31) into the lhs, where we have used definitions (24) and (27) to rewrite T (1) and T (5) by T 1 and T 2 . Thanks to the trick, the differences in arguments of products of T (6) are such that one can apply eqs. (19), (20) and (21). The result of the application then agrees with the rhs of the equality in Lemma 5. By comparing the rhs of Lemma 4 and Lemma 5 with use of the duality T 3 = T 4 , one notices the following equality.
Let us rewrite the lhs by substituting the definition (30). In doing so, we employ a similar trick to the above; we replace T (4) (x + 1 6 i) in the lhs by T (4) (x − 1 6 i). Then it follows, In this form, eq. (19) applies to the first three terms of the rhs, and eq. (20) can be used in the fourth. Also we use eq. (39) the first term of the rhs. The result reads, where we represent T 2 (x) by T (5) (x + 5/3i) and use the duality T 3 = T 4 . One notices that the content of bracket in the rhs reduces to T 0 (x ± 1 3 i) due to eq.(41), which completes the proof. Proof of (38). In the same manner, we start from the equivalent expression T (7) (x − 1 6 i)T (7) (x − 1 6 i) and substitute (31). After rearrangement using eqs. (21), (22) and (23), we find this expression equal to Let us subtract T (3) (x) from the above. Note that T (3) (x) should be understood as the result of the application of (12) to (26) and of the duality The content of the bracket is identical to the lhs of Lemma 3 with the shift x → x + 10/6i by noticing the periodicity (8). Immediately, one verifies that the rhs reduces to T 0 (x ± 1/3i) , and ( 38) is proven. Proof of ( 34). The decomposition of Λ (11,6,6)/(5,5) (x)Λ (6,6,1)/(5) (x + 7i) can be done by the formula (12). Equivalently, we can argue it in a graphic manner as shown in Fig. 7, just as in Fig. 5 .
In next section, we summarize similar results for the dilute A 6 model without proof.
7 The T − system for the dilute A 6 model We firstly comment on the "duality property" in the dilute A 6 model, and T 12 (x) = T 13 (x) = 0. This is again compatible with the symmetry of functional relations but is still a conjecture supported by numerics. The Yangian representation theory asserts that irreducible modules of Y (E 6 ) are made by tensoring minimal objects, W 1 (x) and W 5 (x ′ ). On the other hand, we assume as QTM for W 6 (x). Thus the situation is similar to the E 7 case; one must figure out eigenvalues of QTMs associated to W 1 (x) and W 5 (x ′ ) independently from the knowledge of the dilute A 6 model. We conjecture that eigenvalues of QTMs for these are same and its explicit form reads, The following relations hold in parallel to eqs.(19)- (23), which are key ingredients in the proof of the T − system.
(T (1) (x)) 2 = T 2 (x + i 7 4 ) (45) Similarly for the dilute A 6 model, Therefore, we have a lemma parallel to the dilute A 3 case. where τ ′ = 5τ ( 7 2 τ ) for the dilute A 4 (A 6 ) model. The significance of the above property is clear when one considers these relations (to be precise, logarithmic derivatives of them) in Fourier space, or "k" space. Cauchy's theorem assures that all quantities satisfy algebraic equations at same k , i.e., without mixing of modes. Thus they can be solved in an elementary way. We omit the explicit procedure, for it has been given for other models [34,35,24]. The resultant coupled integral equations read lnY (a) (x) = −ǫδ a,t βs(x) + C a,b * ln(1 + Y (b) )(x), s(x) = δ 2π n e iknx 1 2 cosh k n , where t = 1(6), β = 12πβ(8πβ), and C g denotes the Cartan matrix for E 7 (E 6 ). δ also depends on whether we are dealing with the dilute A 4 model or the dilute A 6 model through δ = π 2 /(2τ ′ ) and k n = nδ. We also adopt the abbreviation, A * B(x) := 2τ ′ /π −2τ ′ /π A(x − x ′ )B(x ′ )dx ′ . This is nothing but the conjectured TBA for the E 6,7 RSOS model at level 2 [23].
The free energy is expressed via Y − functions with the aid of eq.(32). We shall only give the result for ǫ = 1.
Conclusion
We have seen that the E 6,7,8 structure appears in the dilute A 6,4,3 model; exponents of mass scale , zeros of QTMs and TBAs. These results strongly support the underlying E type symmetry in the dilute A L model. A Yangian analogue of Young tableaux arises in proof of the T − system. The combinatorial aspects provide interesting problems on their own. We thus believe that the subject is worth of further research. − θ 4 (±2a − 5) θ 4 (±2a + 1) θ 1 (u)θ 1 (±4a − 1 + u) θ 1 (3)θ 1 (±4a − 4) , otherwise . | 2014-10-01T00:00:00.000Z | 1999-09-15T00:00:00.000 | {
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22596729 | pes2o/s2orc | v3-fos-license | Reflectance confocal microscopy of red blood cells: simulation and experiment
: Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient’s health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology.
Introduction
The properties of red blood cells (RBCs) are remarkable indicators of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology [1] is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease [1,2]. In vitro imaging and analysis of human RBCs have been demonstrated using a conventional blood smear [1], light scattering from an unstained blood smear [3], holographic microscopy [4,5], photo-acoustic microscopy [6,7], and confocal microscopy [8]. In vivo imaging of RBCs, which is ideal for measuring the physiological properties of the cells within their natural environment, was first demonstrated using reflectance confocal microscopy (RCM) [9,10] at high-resolution and without the need for fluorescence labeling [9]. RCM has also been demonstrated promising for clinical diagnosis of the oral mucosa [11], cornea [12], uterine cervix [13], and the skin [9,10,14], including the characterization of the various skin layers [14], small capillaries [9,10] and blood cells [10,15]. Spectral encoding techniques [16] have improved several aspects of RCM by eliminating the need for rapid mechanical beam scanning; spectrally encoded confocal microscopy (SECM) has been shown useful for high frame rate imaging [17] and for imaging large tissue areas [18,19] using simple, compact imaging probes. By taking advantage of the unidirectional flow of blood within small capillary vessels, spectrally encoded flow cytometry (SEFC) [20], a form of SECM that does not require any beam scanning, has been demonstrated promising for measuring hematocrit and counting white blood cells in vivo [21,22].
A main challenge of all reflectance confocal imaging techniques, including laser-scanning RCM, SECM and SEFC, are interference effects that stem from the high spatial coherence of the focused beam. In most tissue types, these effects are manifested as speckle noise that significantly deteriorates imaging quality. When imaging RBCs, however, the rounded, smooth plasma membrane and the relatively homogenous cytoplasm result in a characteristic appearance of smooth rings and curved features [20]. These features stem directly from the three-dimensional shapes of these cells, and could thus provide invaluable information on their morphology and physiological state. In this work, we develop a numerical model that simulates the confocal reflectance image of a single RBC for various relative axial positions and tilt angles, and compare our results to in-vitro SEFC images of flowing RBCs. This method allows us to evaluate the shape of the cells in three dimensions, with potential diagnostic capabilities that are based on accurate measurement of RBC morphology. Fig. 1. Schematic description of the simulated imaging system. L1, L2 -lenses. An image of the cell is acquired by scanning in the lateral x-y plane. The small black and red triangles represent the origin of the optical system and the cell coordinates, respectively.
Theoretical model and numerical simulation
A schematic of a simplified reflectance confocal imaging system used for our simulation is shown in Fig. 1. According to the Fresnel approximation, the electromagnetic field U 2 after the lens L1 that is illuminated by a monochromatic plane wave U 1 , is given by: where λ denotes the wavelength, k = 2π/λ, and f 1 and P L1 denote the focal length and the pupil function of the lens L1, respectively. The simulated field amplitude distributions around the focal plane for a water immersion NA = 0.6 objective lens and 840 nm wavelength ( Fig. 2(a), blue circles) agrees well with the analytical expression (dashed lines) given by [23]: where J 0 is zero-order Bessel function, ρ denotes the normalized radial coordinate at the pupil plane, and u and v denote the normalized radial and axial coordinated [23]. A cross sectional (x-z) distribution of the optical field at the focal region is shown in Fig. 2(b) (red colormap). Assuming that the reflections from an RBC originate primarily at the cell's plasma membrane, the reflected wavefront would have phase structures that follow the detailed curvature of the cell membrane. In their work from 1972 [24], Evans and Fung have derived an approximate analytical expression for the front Z c + and back Z c surfaces of an RBC: where R 0 , C 0 , C 2 and C 4 are specific shape parameters [24] and the subscript 'c' denotes coordinates in the cell's frame of reference. A cross-sectional view of the simulated cell with a 20° tilt angle and axial displacement of 1 μm below the focal plane is shown in Fig. 2(b) (grey meshgrid). For a given lateral position (x c ,y c ) of the illumination optical axis, the waves U + and Ureflected from the front and back cell-medium interfaces, respectively, are given by: where z + (z -) denotes the axial coordinate of the intersection between the front (back) cell interface and the optical axis, P cell denotes the cell pupil function (see Fig. 1), and the digit 2 at the exponential term results from the roundtrip optical path of the reflected wave. For simplicity, and to avoid numerical artifacts, P cell was slightly smaller than the actual cell size, allowing to neglect reflections from the edges of the cell where angles between the membrane's normal and the optical axis are much larger than the maximum illumination and collection cone angles (26.8°). The wave U 3 reflected back from the cell is thus the sum of U + and Uafter propagating a distance of f 1 -z + and f 1 -z -, respectively, toward the objective lens L1: The wavefront U 4 immediately before the lens L2, was calculated by multiplying U 3 by the lens L1 transfer function and propagating a distance f 2 toward the lens L2: The complex amplitude of the wave U 5 , just before the pinhole, was calculated by multiplying the wave U 4 by the transfer function of the lens L2 and propagation by a distance f 2 : Finally, assuming an infinitesimally small pinhole, the signal measured by the detector was calculated as the wave intensity only at the optical axis, i.e. I(x c ,y c ) = |U 5 (0,0)| 2 . The complete confocal image of the entire cell I(x c ,y c ) was calculated by following Eqs. (4)-(7) for all lateral positions of the imaging beam.
Using our model, confocal images of a single cell were simulated for different axial locations Δz and tilt angles α (Fig. 3). The field symmetry around the focal plane resulted in similar images for positive and negative axial displacements, while the cell's radial symmetry resulted in similar images for positive and negative tilt angles. The simulated images were comprised of various bright rings and curves generated by interference between the two waves reflected from the top and bottom cell-water interfaces. In general, high-brightness images with partial radial symmetry were obtained for Δz<2 μm and tilt angles below 30°. The relative brightness of the rings varied for different axial shifts: in perfect focus (Δz = 0) the inner ring was the brightest, while for Δz = 1 μm the outer ring was more visible. High (>20°) tilt angles resulted in a characteristic bowtie pattern with low-contrast superimposed interference rings. At high tilt angles and large defocusing, the cell appeared as a very dim arc. Additional simulations with different cell morphological parameters revealed (data not shown) that the number of rings or arcs is determined primarily by the overall cell thickness variations; thicker cells with a thin central region have more transitions between constructive and destructive interference, hence a higher number of concentric rings. Reflectance confocal imaging of RBCs was conducted using a benchtop SEFC system that was described in detail in previous publications [20,21]. Briefly, SEFC uses a diffraction grating to separate broadband illumination light into its components across a single transverse line within a flow channel. The spectrally encoded reflections from the flowing cells are collected through the same optical path into the core of a single-mode fiber and directed to a fast spectrometer that decodes the spectral reflectance. As a result, SEFC enables instantaneous confocal imaging of an entire line, resulting in a fast, compact imaging system that does not require any beam scanning. A two-dimensional confocal image of the blood is obtained as the cells flow in the direction (y-axis) perpendicular to the imaged line (x-axis).
Experiment -cell imaging using SEFC
Our bench-top SEFC system, schematically illustrated in Fig. 4, comprised of a fibercoupled broadband super-luminescence diode array (Superlum, 840 nm center wavelength, 53 nm bandwidth), two lenses (−20 mm and 75 mm focal lengths for lenses L3 and L4, respectively) for beam magnification, a transmission diffraction grating (1200 lines/mm, Wasatch photonics) and a × 60 water-immersion NA = 1.2 objective lens. Light reflected from the blood cells was propagated back through the same optical path and deflected by a cubic beam-splitter to a fiber-coupled custom-build spectrometer (5000 spectra/s). An additional transmission-mode imaging channel for widefield imaging of the cell flow was comprised of a white-light illumination, a cold mirror (680 nm cut-on wavelength) and a CCD camera (UI-2230SE, IDS). A sample of blood was drawn from a healthy volunteer into a collection tube containing an anticoagulant (K2-EDTA), diluted by adding phosphate buffered saline with 2% bovine serum albumin, inserted into a syringe within a syringe pump (Syringe pump 11 Elite, Harvard Apparatus), and pushed at a velocity of 1 mm/s through a plastic flow channel with a 5 mm x 0.2 mm rectangular cross section (Fig. 4). A typical SEFC image (Fig. 5) of the diluted (1:20) blood within the flow chamber contained scattered spots that correspond primarily to individual RBCs (see magnified rectangles in Fig. 5). Table: The parameters used to simulate the patterns that correspond to each cell image. Cells' profiles (red curves) are derived using Eq. (3). Dashed curves represent the averaged cell shape in Ref [24]. CV -corpuscular volume. Scale bars denote 1 µm.
By varying the different size parameters, it was possible to match the simulated images to SEFC images of the cells. First, the number of concentric rings in the SEFC image was matched in the simulated image by choosing appropriate C 0 and C 2 parameters. Second, adjustment of all C 0-4 parameters was performed to fine-tune ring width and spacing. Third, a look-up table similar to that in Fig. 3 was constructed, from which the tilt angle α and the axial displacement Δz where chosen that best match the SEFC image. Finally, the cell radius R 0 was set to match the transverse dimensions of the imaged cell. Examples of six cells from our raw SEFC data and their corresponding simulated images are shown in Fig. 6 (top panels). The resulting fit parameters for these cells are presented in the table below, next to the cell profile function Z c + (red curves). The profile of a typical average cell derived in Ref [24]. is plotted by dashed black curves for reference. Assuming rotational symmetry, the corpuscular volume (CV) of each cell was estimated by calculating the volume between the cell's top and bottom interfaces using: Fig. 7) revealed that such patterns had occurred whenever additional nearby cells were present within the optical path. While some of the distorted cell images could be attributed to abnormal cell morphologies due to sample mishandling, most of the observed irregular patterns were likely caused by wavefront distortions induced by the neighboring RBCs.
Discussion and conclusion
The unique interference patterns between the front medium-cell and back cell-medium interfaces of a single RBC allow direct sensing of the cell's three-dimensional shape. In this work, we have established a simple approximate model that simulates the reflectance of a tightly focused beam in a confocal microscope configuration. Due to interference between the reflections from the two cell-water interfaces, the resulting images comprise of several concentric rings and curved features. Similar features were also seen in experimental SEFC images of RBCs as they flow in a diluted medium through a flow channel; SEFC is ideal for such comparison as it allows confocal imaging of individual cells without a nearby glass [3] interface that would modify the interference patterns or disturb the natural cell shape. By using this approach, it may be possible to compute the volume of each individual cell and consequently the mean corpuscular volume, which is an important and clinically useful RBC index. This method could also be used for distinguishing between healthy and abnormal RBCs in autosomal recessive blood diseases such as thalassemia and sickle cell anemia.
At present, the presented method has some limitations due to the simplified simulation model and the wide diversity of cell shapes. The Fresnel approximation is valid for wavefronts propagating with relatively small angles; the approximation is good for NA<0.6, remains practical (up to 10% error in PSF lateral dimensions) for 0.6<NA<0.9, and generally fails for higher numerical apertures. Our experimental SEFC system utilized a high-NA (1.2 water immersions) objective lens; however, its measured lateral and axial resolutions (0.7 μm and 1.5 μm, respectively) were much lower than expected, most likely as the beam diameter was smaller than the back aperture of the objective lens. Based on these parameters, we assumed that the effective NA of the imaging experiment was approximately 0.6, within the validity range of the Fresnel approximation. High-NA systems would most likely require a more sophisticated optical model for error-free morphological measurements; however, such system would probably exhibit less interference effects between the two cell interfaces due to the shallow depth of field. The wide diversity of cell shapes makes effective matching between experimental and theoretical images challenging; while the errors in the approximated cell profiles and volumes (Fig. 6) were difficult to estimate in this work due to the manual matching procedures, future development of automatic algorithms may improve processing speed and accuracy and reduce measurement errors. Another limitation of the system is its sensitivity to the presence of other cells within the optical path, which distort the beam wavefront and modify the resulting interference patterns. This issue could be addressed by using a highly diluted blood and/or by neglecting measurements of cells that do not show the expected characteristic patterns. Finally, the presented method may not be suitable for in vivo experiments [21] due to the high density of cells within blood vessels and the degrading effects of the scattering tissue and vessel walls on the beam wavefront.
In summary, a model for simulating reflectance confocal images of RBCs has been demonstrated, revealing the relation between cell morphology and the resulting interference patterns. By comparing the simulated cell images to experimental confocal images, the model could be useful for measuring individual cell morphology and for estimating its volume. Potential applications for this technology include in vitro measurement of the mean corpuscular volume of RBCs and diagnosis of hematological disorders such as thalassemia and sickle cell anemia. | 2018-04-03T06:12:58.534Z | 2015-11-01T00:00:00.000 | {
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41912771 | pes2o/s2orc | v3-fos-license | Spontaneous subclavian vein thrombosis — a source of pulmonary embolism in young man manifested by pleural effusion: A case report
ABSTRACT Introduction: Spontaneous upperextremity deep venous thrombosis is associated with exercise, occurs in young healthy individuals and can be source of pulmonary embolism. Case Report: This case illustrates a 26yearold Caucasian, lefthanded, healthy man with overlooked subclavian vein thrombosis, which was source of pulmonary embolism. Pulmonary embolism manifested with pleural effusion and at the beginning was treated as tuberculous pleuritis. Conclusion: In this case, pulmonary embolism manifested with repeating chest pain due to pleural effusion. Source of embolus was an overlooked left subclavian vein thrombosis. Underlying anatomical abnormalities in this case was adherent first and second ribs, causing compression of the subclavian vein. Incorrect diagnosis at the beginning was the cause of delayed treatment.
INTRODUCTION
Sir James Paget first described thrombosis of the subclavian veins in 1875 [1] and in 1884, von Schrötter postulated that this syndrome resulted from occlusive thrombosis of the subclavian and axillary veins [2]. In recognition of the work of these pioneers, in 1949, Hughes coined the term Pagetvon Schrötter syndrome [3]. It usually results from the excessive use of the involved arm by predisposed individuals. Upper extremity deep vein thrombosis (UEDVT) is an increasingly important clinical entity with potential for considerable morbidity. Complications include post phlebitic limb syndrome from resultant valvular incompetence, venous gangrene and pulmonary embolism (PE). PE is present in up to one third of patients with UEDVT [4].
CASE REPORT
A 26yearold lefthanded healthy man presented with a three month history of repeated bilateral chest pain. Six months previously, he started heavy muscular activity of upper limbs (work out at a gym). Two months before he had been examined by his pulmonologist because chest Xray revealed bilateral small effusion and tuberculin test had been positive. The patient started treatment as suspected tuberculous pleuritis and Chest radiography revealed small pleural effusion. Electrocardiography and echocardiography were normal. Suspected pulmonary embolism was confirmed by computed tomography. DVT in lower limb was excluded by compression ultrasonography. Because the patient had prominent superficial veins on arm and left part of chest, we suspected left subclavian vein thrombosis, which was established by venography ( Figure 1). Aortography revealed aneurysm of left subclavian artery ( Figure 2) and also confirmed left thoracic outlet syndrome due to adherent first and second ribs ( Figure 4). Patient was administered low molecular weight heparin as initial step, followed by the acenocoumarol therapy. A thrombophilia screen revealed prothrombin 20210A mutation, which was detected by polymerase chain reaction. Adequate medical anticoagulant treatment with low molecular weight heparin followed by coumarin led to significant clinical upturn and coumarin was stopped after six months. After six months the patient presented to the vascular surgeon, who performed resection of adherent first and second ribs ( Figure 5). For two weeks after surgery, the patient was administerd low molecular weight heparin followed by aspirin. Nowadays, nine years after surgery, the patient has experienced complete resolution of his symptoms, his prominent superficial veins on arm and left part of chest have disappeared and he has used no medication. The CTangiography confirmed complete resolution DVT of left subclavian vein ( Figure 6).
DISCUSSION
Spontaneous subclavian or axillary vein thrombosis comprises 2% of all depp vein thrombosis (DVT). The mean age is 30 years, both genders are affected equally and there is a predilection for the dominant arm. Patients with subclavian vein thrombosis may describe a history of trauma or more frequently, strenuous use of the arm (>50% cases). Common precipitating activities involve repeated hyperabduction and external rotation of the arm or backward and downward rotation of the shoulder [5]. The mechanism for subclavian vein thrombosis is thought to be extrinsic compression of the subclavian vein within a tunnel comprising the first rib inferiorly, the subclavius muscle superiorly, the IJCRI -International Journal of Case Reports and Images, Vol. costoclavicular ligament medially and the anterior scalene muscle laterally. Abnormalities such as a laterally inserting costoclavicular ligament or cervical rib (10%) can compress the subclavian vein, predisposing to thrombosis. This is termed "effort thrombosis" or "thoracic outlet obstruction syndrome" [6]. In this case, there was a clear precipitant, gymnastics maneuvers for last six month and there was the adherent first and second ribs as cause of exstrinsic compression of subclavian vein. Annual incidence of upper thoracic outlet syndrome ranges between 3-80 new cases for 1000 individuals, and it typically affects younger individuals aged between 20 and 50 years [7]. Not all patients with subclavian vein thrombosis are symptomatic. Those with symptoms may present with mildtomoderate nonpitting edema and mild cyanosis of the hands and fingers on the affected side. Dilatation of subcutaneous collateral veins may be present over the upper arm and chest. This later sign may be the only clue to UEDVT in otherwise asymptomatic patiens. In a few cases, in which the diagnosis was missed or delayed or the patient presented late, the thrombus may have extended to the superior vena cava or be the cause of PE. In addition, patients with UEDVT less often have clinically overt PE than those with lowerextremity DVT [8]. PE is a common and a potentially life threatening condition associated with considerable morbidity and mortality. Pleural effusions are a common occurrence in patients with PE, which is also a disorder commonly overlooked in the workup of a patient with pleural effusion [9]. In this case, the patient presented with no edema on afected side, had only dilatation of subcutaneous collateral veins over the left upper arm and chest and diagnosis of UEDVT was overlooked. PE manifested with pleural effusion and diagnosis was overlooked too. Patient was administered antituberculous drugs and corticoids because of incorrect diagnosis of tuberculous pleuritis at the beginning. Incorrect diagnosis was made, because patient had positive tuberculin skin test, which can be false positive [10]. Prompt diagnosis of UEDVT allows right treatment and impoves the clinical outcome. The presence of thrombus may be established by duplex ultrasonography examination, venography, magnetic resonance angiography or computerized tomography (CT). Colour duplex ultrasonography is the best initial evaluation because it is noninvasive and has a high sensitivity and specificity. In turn, venography is still the 'gold standard' in evaluating subclavian and axillary vein thrombosis. Upper extremity venography is a cost effective imaging examination, and a normal image for upper extremity venography should reveal bilateral, symmetric, rapid, and smooth radiolabeled radiotracer flow from the upper extremities to the central venous systém [11]. In this case, venography revealed left subclavian vein thrombosis, thoracic radiography showed left adherent first and second ribs. The initial measures commonly used are based on rest and elevations of the affected limb followed by anticoagulation. Both conservative and surgical therapy (anticoagulation, systemic or catheter directed thrombolytic therapy, thrombectomy, balloon angioplasty followed by stenting) are common alternative treatments for UEDVT. From the physician's standpoint, the optimal management of subclavian vein effort thrombosis is still a problem because of missing approved treatment quidelines. Anticoagulation is the basis of the management of UEDVT with the main objective of preventing further propagation of thrombus. Consensus statements advocate catheterdirected thrombolysis within five days of symptoms. Hence, if there is evidence of thoracic outlet compression on imaging or symptomatic venous stenosis, consensus opinion advocates thoracic outlet decompression. The physicians are advised to use unfractionated heparin or low molecular weight heparin as initial step in DVT therapy followed by the acenocoumarol therapy. The current standard is six months of oral anticoagulation [4,12]. In this case, patient experienced PE with low risk and was administered the same treatment as treatment for UEDVT i.e. low molecular weight heparin as initial step, followed by acenocoumarol therapy for six months [13]. After six months the patient was referred for thoracic outlet decompression. A review and treatment algorithm for PagetSchröetter syndrome has recently been published [14].
CONCLUSION
Pulmonary embolism presenting with pleural effusion is difficult to diagnose. Asymptomatic subclavian vein thrombosis as source of embolus can be overlooked. Physicians and other health care professionals must be vigilant with young healthy individuals who have pleural effusion, because this symptom may be a 'forgotten sign' of lifethreatening pulmonary embolism. *********
Author Contributions
Margita Belicová -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published Hubert Poláček -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Guarantor
The corresponding author is the guarantor of submission. | 2019-03-11T13:05:05.355Z | 2012-07-07T00:00:00.000 | {
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268789963 | pes2o/s2orc | v3-fos-license | Mobile Health App and Web Platform (eDOL) for Medical Follow-Up of Patients With Chronic Pain: Cohort Study Involving the French eDOL National Cohort After 1 Year
Background Chronic pain affects approximately 30% of the general population, severely degrades quality of life and professional life, and leads to additional health care costs. Moreover, the medical follow-up of patients with chronic pain remains complex and provides only fragmentary data on painful daily experiences. This situation makes the management of patients with chronic pain less than optimal and may partly explain the lack of effectiveness of current therapies. Real-life monitoring of subjective and objective markers of chronic pain using mobile health (mHealth) programs could better characterize patients, chronic pain, pain medications, and daily impact to help medical management. Objective This cohort study aimed to assess the ability of our mHealth tool (eDOL) to collect extensive real-life medical data from chronic pain patients after 1 year of use. The data collected in this way would provide new epidemiological and pathophysiological data on chronic pain. Methods A French national cohort of patients with chronic pain treated at 18 pain clinics has been established and followed up using mHealth tools. This cohort makes it possible to collect the determinants and repercussions of chronic pain and their evolutions in a real-life context, taking into account all environmental events likely to influence chronic pain. The patients were asked to complete several questionnaires, body schemes, and weekly meters, and were able to interact with a chatbot and use educational modules on chronic pain. Physicians could monitor their patients’ progress in real time via an online platform. Results The cohort study included 1427 patients and analyzed 1178 patients. The eDOL tool was able to collect various sociodemographic data; specific data for characterizing pain disorders, including body scheme; data on comorbidities related to chronic pain and its psychological and overall impact on patients’ quality of life; data on drug and nondrug therapeutics and their benefit-to-risk ratio; and medical or treatment history. Among the patients completing weekly meters, 49.4% (497/1007) continued to complete them after 3 months of follow-up, and the proportion stabilized at 39.3% (108/275) after 12 months of follow-up. Overall, despite a fairly high attrition rate over the follow-up period, the eDOL tool collected extensive data. This amount of data will increase over time and provide a significant volume of health data of interest for future research involving the epidemiology, care pathways, trajectories, medical management, sociodemographic characteristics, and other aspects of patients with chronic pain. Conclusions This work demonstrates that the mHealth tool eDOL is able to generate a considerable volume of data concerning the determinants and repercussions of chronic pain and their evolutions in a real-life context. The eDOL tool can incorporate numerous parameters to ensure the detailed characterization of patients with chronic pain for future research and pain management. Trial Registration ClinicalTrials.gov NCT04880096; https://clinicaltrials.gov/ct2/show/NCT04880096
Introduction
Chronic pain affects approximately 30% of the general adult population [1,2] and was one of the top 5 leading causes of years lived with disability in 2016 [3], especially among older people [4].Moreover, 60% of people with chronic pain are less able or unable to work, and 20% report having lost their job as a result [5].The overall cost of chronic pain is estimated to be approximately €441 billion (US $468 billion) in Europe and US $560 to $635 billion in the United States [6][7][8].At the same time, the market for analgesic drugs represented approximately US $68 billion in 2016, and an increase from 2% to 5% was forecast for 2021, with a further 5% increase by 2025 [9].Unfortunately, available analgesics often have limited efficacy, with undesirable effects and without any real pharmacological innovation [10], despite prolific basic research [11].
One of the solutions could be based on better patient characterization to offer physicians decision aids in the management and initial choice of treatment for chronic pain.A preliminary step that would make it possible to identify patient trajectories and factors capable of predicting treatment success.This characterization should cover the entire biopsychosocial field.It should also include a temporal dimension, with real-life monitoring of different parameters, and subjective and objective markers of chronic pain.Indeed, the current assessment of patients with chronic pain provides only fragmentary data on their daily pain experiences due to memory bias [12].This monitoring strategy is currently being developed by several research teams [13][14][15][16][17][18].It is also necessary to make the patient an active participant in their own care, with the goal of improving compliance and adherence.Indeed, evidence suggests that the self-management of chronic diseases reduces hospitalizations, the use of emergency services, and overall care management [19,20].
The use of innovative digital technologies (eHealth) appears to be a solution.eHealth can improve the characterization and monitoring of pain symptoms, and the management of pain and associated comorbidities [21][22][23].eHealth can also offer remote interventions without the difficulties inherent in long-distance travel or regular visits to the general practitioner or specialist [24].Thus, recent publications have highlighted the urgent need to develop, assess, and use validated eHealth programs for chronic pain [13,14,18,[25][26][27][28][29][30][31].eHealth technologies encompass a wide range of diverse tools, offering potential applicability for chronic conditions [32,33].These technologies can be categorized into 6 types: virtual visits, electronic health records, digital therapeutics, artificial intelligence and machine learning, wearable monitors, and mobile health (mHealth) apps [32].This last category can vary in its content, which can generally be grouped into 3 objectives: education, monitoring, and treatment (often including self-management strategies), sometimes with a combination of these objectives [34].However, there are many obstacles in putting this management into practice and monitoring it via digital tools, including the lack of training of patients in the management of their pathology and the use of digital tools, the lack of interest or training of some physicians for this new management integrating new digital technologies, the lack of integration of these digital strategies in medical management, the over-medicalization of pain that can result from the use of these digital tools, and the lack of time and information in health care structures [35].
Change is therefore necessary to integrate digital technology in the management of chronic diseases, including pain [36].Among digital tools, mHealth offers significant opportunities for patients to foster communication with their practitioners or other patients (ie, through forums).mHealth offers distraction strategies, provides information and therapeutic education, promotes self-expression, improves access to health care, and facilitates social support.Moreover, mHealth can provide patients with greater convenience and more regular access to information about their conditions compared to traditional care.Although many studies have examined the use of innovative mHealth to assist in the management of chronic diseases, less attention has been paid to chronic pain [37].However, the context is favorable, with growing interest from health agencies and funding organizations for these solutions.Moreover, in France, patient demand was evidenced by the results of a survey on compliance conducted in early 2017 by the French Institute of Public Opinion.The results showed a strong demand by patients for the development of digital tools for the self-management of their chronic diseases.
In response to this situation, the Analgesia Institute (Clermont-Ferrand, France) has recently created an mHealth tool (called eDOL) for data collection and monitoring of chronic pain and its impacts on patient well-being.Following a feasibility study on the use and acceptability of eDOL Version 1 [38] and the implementation of Version 2, we provide in this article an overview of eDOL's ability to retrieve a significant amount of medical data of interest for physicians and researchers on the subject of chronic pain.This study was carried out after more than a year of existence of the eDOL national cohort, and it included more than 1000 patients.This study did not assess the impact of the eDOL tool on patients' medical management or on patients' pain symptoms and their consequences.
Ethical Considerations
The study was approved by the research ethics committee (Comité de Protection des Personnes Ouest II Angers; reference number: 2020-A02027-32) and the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés [CNIL]; n° 921059).The study has been registered at ClinicalTrials.gov(NCT04880096).The study was conducted in accordance with French laws and regulations on research on human beings and data protection, and in accordance with the Declaration of Helsinki [39].
Data Collection
Data were collected and managed using the eDOL tool (mobile app + web platform) developed by Bepatient (Paris, France) and hosted by Avenir Télématique (Villeneuve-d'Ascq, France).In accordance with the provisions relating to the confidentiality of information concerning, in particular, the people who took part in the research and the results obtained [40], individuals with direct access took all the necessary precautions to ensure the confidentiality of the information relating to the participants.These persons and the investigators themselves are subject to professional secrecy [41].All data collected and transmitted to the sponsor (University Hospital of Clermont-Ferrand, France) were anonymized, and each patient had an individual numerical code.The head of research ensured that each patient was informed of which data were collected and that they did not object to their use or disclosure.Answers to questionnaires and medical data were transmitted in spreadsheet format (Excel 2013; Microsoft Corporation).All anonymized data were accessible to the biostatisticians (BD and BP), coordinator (ND), and project manager (NK).Only the investigators could access their patients' personal data to identify them.A dashboard linking patients' identities and individual numerical codes was available only on the investigators' professional interface on the eDOL web platform.The final database, which was used for statistical analyses, included only individual numerical codes to preserve anonymity.
Data Entry and Processing
To manage data processing, a data warehouse has been set up in the Laboratory of Informatics, Modelling and Optimization of the Systems (LIMOS) Mixed Unit of Research (UMR 6158) (Clermont Auvergne University, France).This warehouse has made it possible to retrieve the anonymized data collected by BePatient regularly and automatically.The data were retrieved via a secure application programming interface (API).Files were saved and read to extract the data.These data were then cleaned according to a set of rules to remove erroneous data (patient input errors), integrate them into a database, and provide an interface to view and export the cohort data.
Study Design and Population
This cohort study provides a snapshot after 1 year of a national cohort of patients with chronic pain using the eDOL app.The characterization and real-life monitoring of patients from 18 pain clinics and follow-up by 80 investigators in France took place between September 14, 2021, and January 31, 2023.The study was offered to all physicians and allied health professionals in the investigating centers.Participation in the study was offered to patients with chronic pain, who were owners and regular users of a smartphone and who were followed up in a pain clinic.All adult (≥18 years old) patients able to read and understand French and provide consent to integrate the cohort study were included.Participants were free to withdraw their consent at any time by informing the sponsor.Each patient had access to the information document (paper or electronic) detailing the purpose, content, and conduct of the cohort.If they agreed to participate, they were asked to download the eDOL app and complete questionnaires using the eDOL app.The URL to access this app was sent by email from physicians to their patients.After downloading the app and creating their profiles, patients could accept the general terms and conditions of use and confirm that they agree to the use of their medical data in this study.Each patient made only 1 initial study visit during which the physician introduced the study to them, checked their eligibility, explained the eDOL tool, and gave them a brief training document on how to use the eDOL app.Participants completed several questionnaires and assessments over a period of 1 month (initial patient characterization) and then repeatedly (or not) quarterly, half-yearly, and yearly depending on the questionnaire.Throughout the duration of the study, physicians saw their patients at several follow-up visits (optional), according to the patient's health pathway, after the inclusion visit.
eDOL Digital Tool eDOL is a digital health tool used for data collection and monitoring related to pain and its impacts on patient well-being.It comprises 3 main components: a smartphone app for patients, a web interface for health care professionals (Figure 1), and a data repository for researchers.The tool provides various features and modules to support patients in managing their chronic pain while allowing physicians to access and monitor real-time data for clinical and therapeutic purposes.
Patient Mobile App
The app allows patients to regularly input data regarding their pain and its effects and to access educational content.Patients were invited to complete various contents, but none of them were compulsory.
Questionnaires
Patients complete questionnaires and assessments on a regular basis.These questionnaires cover general aspects, specific chronic pain symptoms, and associated comorbidities.They are filled systematically at different intervals, such as once only, quarterly, half-yearly, or yearly.Some questionnaires address specific types of chronic pain.For a detailed list of all the questionnaires used, refer to the previous publication by Kerckhove et al [38].
Weekly Meters
The app facilitates the real-life monitoring of various parameters related to pathology.Patients can assess the evolution of their pain and its impacts using an 11-point numeric rating scale (from 0 to 10) for intensity assessment in different areas, such as pain, stress and anxiety, fatigue, sleep, morale, and bodily comfort.
Body Map
Each month, patients can use a body map to select and indicate the painful areas on their body, assigning corresponding intensity levels.The map provides front and back views (Figure 1).
Chatbot
Patients can download a chatbot (conversational agent called Eva) to integrate it into the app.The Eva chatbot has been developed in collaboration with the company Aliae (Metz, France) and with a medical group of the Analgesia Institute.The chatbot prompts participants to complete their meters on a weekly basis.
Educational Modules
The app offers educational modules that cover therapeutic education on pain and its broader aspects.These modules consist of informative sheets with images for better comprehension.They have been developed with the assistance of a group of physicians working in various pain clinics.Each module starts with a "What you need to know" section, defining the chapter's scope, followed by educational sheets comprising an image corresponding to the subsection, a short testimonial or quote, an explanatory text, and a personalized image with a concluding statement.Modules include understanding pain, understanding treatments, moving better, improving fatigue and sleep, functioning in daily life, and managing emotions.
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In the event of technical problems or questions regarding the app or the completion of questionnaires or meters, participants could refer to contact details provided via an email, and for each investigator center, a clinical research assistant was available by phone.
Web Interface for Health Care Professionals
The eDOL web platform enables health care professionals to access real-life patient data (questionnaire scores and graphical presentation of weekly meters) and input consultation data (clinical examinations, treatments, etc).The platform features an ergonomic dashboard with tabs for management (patient medical records), health measures (graphical display of weekly assessments), and questionnaires (all completed with scores and answers).
Big Data Repository for Researchers
The development of eDOL V2 involved the creation of a data repository in collaboration with the LIMOS.The data repository interfaces directly with eDOL V2 and automatically collects patient cohort data (via the app) and data from health care professionals (via the web platform) on a weekly basis.The transferred data are anonymized to comply with the French data protection authority for analysis purposes.Information on the data contained in the database is available to investigators on request.In the event of a clinical study requiring eDOL data, a written request describing the study must first be sent to the data controller (Analgesia Institute).The Analgesia Institute's Scientific Committee will assess the relevance of the project before giving its approval.
Study Outcomes
The primary objective of this study was to describe the tool's ability to retrieve a large amount of longitudinal medical data and its relevance to cohort patient follow-up.
The secondary objectives were to assess the characterization of participating patients, pain disorders, pain medications, and related comorbidities, and evaluate the acceptability of the eDOL app with a satisfaction survey (based on the Patient Satisfaction Questionnaire Short Form [42] and the Client Satisfaction Questionnaire [43,44]) for patients (10 questions).The satisfaction survey (in French language) was sent to each included patient via the eDOL tool.Response options for each question ranged from 0 (strongly disagree with the statement) to 10 (strongly agree with the statement).The questionnaire/weekly meter completion rate and center participation (inclusion rate) were also calculated.
As an exploratory study, the profile of patients adhering to the eDOL program was analyzed.
Statistics
Patients have been described according to the following variables: compliance with eligibility criteria, epidemiological characteristics, and clinical and treatment characteristics.Categorical data are expressed as numbers and associated percentages, and continuous data are expressed as means with standard deviations or medians (25th-75th percentiles), according to the statistical distribution.The assumption of normality was studied using the Shapiro-Wilk test.Comparisons according to observance were conducted using the chi-square test or Fisher's exact test for binary variables, whereas comparisons concerning quantitative variables were performed using ANOVA or the Kruskal-Wallis test.When appropriate (omnibus P value <.05), a post-hoc test for 2×2 multiple comparisons was applied: Tukey-Kramer test after ANOVA or Dunn test after the Kruskal-Wallis test.Different analyses were carried out according to the level of use (compliance) of the eDOL app, which was defined by quartiles of percentage weekly meters completed during follow-up (Q1: <5%, Q2: <50%, and Q3: >50%).Statistical analyses were performed using Stata software (Version 15; StataCorp).All tests were 2-sided, with an α level set at 5% for statistical significance.
Generalities
All the 18 pain clinics participating in the study included at least one patient.The median rate of inclusion per center was 5.5 patients/month, and an average of 84.5 (SD 31.9)patients were consistently included every month (Figure 2).During the study period, from September 14, 2021, to January 31, 2023, 1427 patients were included.
Collected Data
Of the 1427 patients included in the study, 249 (17.4%) were not analyzable (no installation of the eDOL app or no completion of the baseline sociodemographic questionnaire).Thus, data from 1178 patients were analyzed (Figure 3).
The eDOL tool also enabled us to collect data on comorbidities related to chronic pain and its psychological and overall impact on patients' quality of life.High numbers of patients were considered to have kinesiophobia (
The completion rate of the weekly assessments for the real-life monitoring of the different meters (pain, moral, anxiety, fatigue, sleep, bodily comfort, and physical activity) was 75.1% (885/1178) at patient inclusion.Among the 1007 patients with at least 3 months of follow-up, the completion rate decreased over the course of follow-up to 49.4% (497/1007) after 3 months of follow-up and then stabilized at 39.3% (108/275) for patients with at least 12 months of follow-up (Multimedia Appendix 10).An acceptable meter completion rate was defined as at least 50% completion of all theoretical meters over the follow-up period (1 meter/week).Univariate analysis was performed to characterize patients according to their percentage of weekly meter completion (<5%, ≥5% to <50%, and ≥50%).Univariate analysis showed very little difference between compliant and noncompliant patients.In fact, women and patients with an intermediate profession or no activity appeared to be slightly more compliant.Age, level of education, family situation, and alcohol or tobacco consumption had no impact on compliance.Surprisingly, the same observation was noted for pain characteristics.It was found that the length of time the pain had been present, the frequency or duration of painful paroxysms, the pain intensity and interference, and the presence of nocturnal awakenings or difficulty in falling asleep did not interact with patient compliance.There was no difference in the inclusion scores for the various questionnaires, depending on the level of compliance of patients.Finally, analysis of the type of pain and treatments received showed no difference between patients irrespective of their level of compliance.
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The eDOL tool also includes educational modules on chronic pain and its repercussions.These modules were freely available to the patients but were not compulsory.Overall, 16.3% (192/1178) of patients viewed at least one educational module.The educational modules were viewed in their entirety by around 65.2% (minimum: 44.6%, maximum: 85.5%, according to the educational module) of patients who opened them.The most frequently viewed educational module was the one on "improving fatigue and sleep."Conversely, the educational module on "living with pain" was the least viewed (Table 1).
Satisfaction With the Use of the eDOL Tool
The satisfaction questionnaire was filled in by 27.1% (319/1178) of patients at the end of the study.These patients had similar characteristics to all the patients in the cohort (average age 49.4 years; 258/319, 80.1% female; 213/319, 66.9% with pain for more than 5 years; and 137/319, 42.8% with nociplastic pain).The median acceptability score was 7.2/10 (95% CI 6.9-7.7), with only 7.2% (23/319) of patients providing a rating less than 5/10.Moreover, 83.4% (267/319) of patients who responded wanted to participate in the further development of the eDOL tool.The items with the lowest scores were "I enjoy my exchanges with the Eva chatbot and I think it's a tool that can assist me on a daily basis" (mean 6.0, SD 3.1), "I think the Eva chatbot is a relevant tool for collecting my meters every week" (mean 6.7, SD 2.8), and "I believe that the information I have entered in eDOL enables my doctor to better understand my pain and improve its management" (mean 6.9, SD 2.6) (Multimedia Appendix 11).
The interest of adding a chatbot in the eDOL tool was also assessed.Among the 1178 participating patients, 775 (65.8%) downloaded the chatbot, and among these, all 775 (100%) used the chatbot at inclusion.This percentage halved (403/775, 52.0%) at the 6-month follow-up and appeared to stabilize at 40.0% (310/775) at the 12-month follow-up (Figure 4).The use of the chatbot allowed us to recover 9456 weekly meters and 12,300 conversations.With regard to satisfaction, of the 775 patients, 418 (53.9%) responded to the questionnaire, and among these patients, 230 (55.0%) had a satisfaction score of at least 7/10, which corresponds to high satisfaction, and only 35 (8.4%) had a score below 5/10, which corresponds to low satisfaction.The main positive points of the chatbot indicated by the participants were "easy to use," "responsive," "moral support," "converse with a positive message," and "availability."Conversely, the main difficulties related to the use of the chatbot indicated by the participants were "forgetfulness of use," "answers sometimes not adapted," and "repetitive."
Principal Findings
This study presents the most extensive cohort of patients using an mHealth follow-up app and living with chronic pain, irrespective of the type of pain, worldwide.Indeed, studies in the literature on the use of an mHealth app in a cohort of patients have mainly focused on specific types of pain conditions, predominantly osteoarthritis and chronic low back pain [34].Most studies have concentrated on digital interventions directed toward the patient-provider interface [46], which makes comparisons with the available literature challenging.This is also due to the typically shorter duration of such studies.
In this cohort study, 82.6% (1178/1427) of eligible patients consented to use the eDOL tool, and among them, about 86.7% (1021/1178) of participants used the eDOL app to complete different questionnaires during the initial phase.Thus, the rate is in line with the rate identified in our previous feasibility study (89.3%) [38] and the rate reported in the study by Stoppok et al [47] wherein 85.5% of participants expressed a willingness to use the app for more than a month when surveyed beforehand.Nevertheless, attrition appears to be high in internet interventions for chronic pain [48], and this has been reflected in our study, with about 60% (167/275, 60.7%) loss of users (according to weekly meter filling) among patients with at least 12 months of follow-up.A comprehensive American study [49] involving a large sample revealed that over half of the participants discontinued participation within the first week.Attrition is not solely confined to mHealth, and a systematic review of longitudinal studies on chronic spinal pain identified attrition as the most significant risk of bias [50].Notably, attrition rates varied widely across these longitudinal studies, with a rate of 63% after 12 months in a study assessing pain after intensive care unit discharge [51] and 83% after 12 months in a study assessing pain self-management (chronic musculoskeletal pain and comorbid depression) [52].Moreover, in the longitudinal study conducted by Bicego et al [53], attrition rates varied within the range of 11% to 22% 1 year after treatment.
With the loss of patient adherence over time, it is necessary to include a huge number of patients (inclusion of a minimum of 5000 patients is planned in the eDOL cohort) to have enough longitudinal data available.Despite this, the eDOL tool is already making it possible to collect a significant volume of data to characterize patients experiencing chronic pain, and it opens the door to future clinical studies to better understand the biopsychosocial aspect of chronic pain, the therapeutic management (drug or nondrug) implemented, the patient's care pathway (type of consultation, frequency of consultation, medical wandering, etc), and the cost of management (treatment prescriptions, consultations, work stoppage, etc).All these possibilities make the eDOL cohort an enormous potential source of data on chronic pain that is available to clinicians and researchers.
Limitations and Difficulties Encountered
The main limitation of our study was the attrition rate described previously.This attrition could influence the results of patient satisfaction with the eDOL tool, as only 30% responded to the XSL • FO RenderX satisfaction questionnaire.However, it is difficult to know whether the patients who did not respond were dissatisfied, or simply forgot or did not feel like answering the questionnaire despite their effective satisfaction, especially since compliant patients and those who responded to the satisfaction questionnaire had similar characteristics to all the patients in the cohort.In our study, the chatbot was an attempt to add a social role to this tool in order to establish a good therapeutic alliance, as recommended for promoting engagement and reducing attrition in digital interventions [54].However, its algorithms were trained in the United States, which might make it impersonal and irrelevant for the French population, which are characteristics identified as barriers to the use of such tools [55].The lack of use of the chatbot could also be explained by patients' lack of interest in "chatting" with a robot, as such a robot has shown several limitations in its exchanges.One solution would be to replace it with a chat or message involving health care staff, who have already shown an interest in adhering to the digital tool [56].However, this strategy inevitably requires additional time for caregivers to respond to patients' expectations, and such time is rarely available in view of caregivers' current workloads in pain clinics.An additional possibility is to add a forum for patient users to exchange information with each other.This strategy has been proven to be beneficial [31].
Educational modules have been integrated into the eDOL app to make it more informative for patients and thus increase their adherence.Nonetheless, patients made very little use of them.Their low use can be partially explained by the fact that the modules were not available on the homepage but were present on a dedicated page of the menu.Another explanation is that patients only received 2 smartphone notifications informing them of the existence of these modules in the app.We can hypothesize that patients might have had difficulties in finding them.
Additionally, in the absence of compulsory follow-up visits in the eDOL protocol, patient visits to the pain clinics were those scheduled as part of their usual care pathway (ie, not focused on the app itself); thus, some patients had visits 3, 6, or even 12 months apart.This may have acted as a hindrance to user engagement, as noted in telemedicine interventions [57].Furthermore, although the use of an app and a mobile phone appears to be widely preferred over other interface options to access pain-related resources among individuals living with chronic pain, less than 60% of them expressed a desire to use online pain management tools permanently, according to a German survey [47].This finding could also explain the significant attrition commonly observed in mHealth interventions.
Various strategies are available to mitigate this adherence issue observed within this study, including reducing the number of questionnaires and the number of questions per questionnaire, simplifying navigation (user experience/user interface design), adding activities to help patients manage their symptoms (therapeutic education for sleep, mindfulness for stress, positive psychology for morale, etc), ensuring greater involvement of the care team, etc.Our research team has already taken steps to mitigate attrition rates in the ongoing novel version of the eDOL tool (Version 3; since November 2023) by reducing the number of questionnaires, replacing some questionnaires with shorter ones (PROMIS [Patient-Reported Outcomes Measurement Information System] questionnaires), enhancing user experience by designing a new interface, and deactivating the chatbot.Additionally, the reminders to complete meters, body schemes, or questionnaires were changed into notifications instead of emails as patients did not solely rely on their phones to read their emails.The limited interaction with educational modules suggests that there is an opportunity to enhance user engagement and accessibility features within the app.The new design should facilitate increased engagement with therapeutic education activities by making them more prominently visible.Additional activities are currently being prepared and will be incorporated into the new version soon.Finally, to limit missing medical data, the new version of eDOL enables us to link our data with the national databases of the French health insurance system.This linkage will make it possible to obtain data on all the care received by patients in our cohort, including treatment reimbursements, doses and quantities of treatments prescribed, hospitalizations or consultations, sick leave benefits, etc.This linkage will also enable us to carry out medicoeconomic studies on the cost of chronic pain management.
The second limitation pertains to the representativeness of the sample in this cohort regarding the general population living with chronic pain.The participants in this study were recruited exclusively from pain clinics; however, only 3% of people living with chronic pain benefit from this type of health care setting in France.The vast majority seek assistance in primary care settings or in other specialized services such as rheumatology, neurology, and oncology.Nonetheless, the characteristics of the patients included in the eDOL cohort demonstrate resemblances to people living with chronic pain in Europe in terms of various factors such as age, pain intensity, duration of pain, and comorbidities [2,[58][59][60].These data reveal a higher proportion of women, which is not surprising as this difference in prevalence is well documented in the literature [61][62][63].This female-to-male ratio is also observed in remote research studies utilizing mobile apps (median 56.9%) [49].However, as in our feasibility study [38], this ratio is exacerbated in our cohort, reaching nearly 8/2.There are other examples of digital health studies showing a similar gender distribution [64][65][66].
A selection bias was evident, primarily due to the prerequisite of smartphone utilization, which inherently excludes individuals who lack access to or proficiency with this technology.This criterion could potentially result in the exclusion of older patients or patients with a more precarious status.Lastly, another limitation could be measurement bias, a phenomenon commonly encountered in observational studies [67].It can emerge from factors such as recall period, selective recall, social desirability, or approach to sampling.Within our study, the recall period emerged as the primary potential source of risk.Given that all the inquiries focused on the present moment or, at the most, events occurring within the preceding 1 to 2 weeks, the likelihood of recall bias can be considered marginal.
Conclusion
Overall, the eDOL tool collected a large volume of medical and biopsychosocial data from 1178 patients over a short period of just over 1 year.The quantity of data collected demonstrated the effectiveness and interest of the eDOL tool with regard to the collection of medical data for research and for caregivers.This interest will continue to grow as more patients are included in the cohort, enabling clinical studies to be carried out on eDOL data to assess care pathways, management costs, patient trajectories, patient profiles, benefits or risks of treatments, and other aspects.©Noémie Delage, Nathalie Cantagrel, Sandrine Soriot-Thomas, Marie Frost, Rodrigue Deleens, Patrick Ginies, Alain Eschalier, Alice Corteval, Alicia Laveyssière, Jules Phalip, Célian Bertin, Bruno Pereira, Chouki Chenaf, Bastien Doreau, Nicolas Authier, ePAIN, Nicolas Kerckhove.Originally published in JMIR mHealth and uHealth (https://mhealth.jmir.org),12.06.2024.This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR mHealth and uHealth, is properly cited.The complete bibliographic information, a link to the original publication on https://mhealth.jmir.org/,as well as this copyright and license information must be included.
Figure 1 .
Figure 1.Interface of eDOL Version 2. Screenshots of the various modules of the eDOL smartphone app for patients (top) and the eDOL web interface for clinicians (bottom).
Figure 2 .
Figure 2. Recruitment rate by month and cumulative frequency.Rate of patient recruitment in the eDOL study is represented in terms of patient frequency/month (histogram) and cumulative frequency (curve).The numbers at the bottom of the histogram bars represent the number of active investigator centers.
Figure 3 .
Figure 3. Study flowchart.Graphical representation of the various stages in patient recruitment and analysis.
Figure 4 .
Figure 4. Chatbot use over time.Visual representation of chatbot use among the 775 patients who downloaded the chatbot.Patients were classified according to their month of inclusion, and chatbot use (at least one conversation in the month) was assessed for each month of follow-up.A darker green color represents a higher utilization rate, and a darker red color represents a lower utilization rate.
Table 1 .
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216633067 | pes2o/s2orc | v3-fos-license | Teacher Dimensions in Technical Higher Education – A Student Perspective
3 Lecturer, The Faculty of Engineering in Foreign Languages, Politehnica University of Bucharest, Bucharest, Romania, anca.greculescu@upb.ro Abstract: The current paper is based on a survey conducted on 321 engineering students studying at University Politehnica of Bucharest and University Politehnica of Timişoara. The research aims at outlining the profile of the teacher in technical higher education as depicted by engineering students. The study surveyed students‟ opinions regarding the teacher-student relationship, teacher‟s role as an evaluator, professional expertise, education in general, personality traits, teachers‟ roles and managerial skills. The results of the study highlight the importance of the student-teacher relationship, the teacher‟s roles as evaluator and examiner and his/her professional expertise. Teachers‟ personality traits, managerial skills and roles seem to be less important to students. Moreover, the t test data show significant statistical differences between the real and ideal teacher in technical higher education from a student perspective. Nevertheless, students and society as a whole have different perceptions of teachers. Regarding the dimensions of the teacher in technical higher education, based on the statistics, on a 1 to 5 scale, the results show similarities of the two models. For instance, students rank first intelligence and last elitism and competitiveness as teacher attributes. Furthermore, the present study also demonstrates that universities comply with the system proposed by Marlene and Lee Canter that allows teachers to apply positive support for students.
Context
Didactic planning and management within secondary and pre-tertiary education are teacher-oriented, the teacher being considered its "living tool". (Surdu, 1999: 110). Over the course of time, the teacher has been considered the living tool of educational infrastructure and has faced permanent alterations and restructuring based on different assumptions about education. Subsequently, the teacher has been depicted as apostle, missionary, propedeutic (supplying the knowledge or discipline introductory), maieutic (eliciting new ideas from another, fostering the truth), hermeneutic (concerning interpretation of the truth), manager, consultant, advisor, assessor/evaluator, model, partner, agent, learning colleague (for the schoolchild), illuminated guide who descends from his/her pedestal to seek solutions and truths together with the educables. (Jinga & Istrate, 1998: 88;Surdu, 1999: 110) Over the past quarter of the century, the teacher"s mission, roles and functions have constantly and significantly modified. Hence, in order to meet the economic, social, political and sustainable development (Sterling, 2010) requirements of the knowledge-based society and the needs and interests of both schoolchildren and students, there has been a change in their perception of the teacher"s characteristics within the pre-tertiary and higher education.
What is more, over the course of the past decades, within tertiary education, there has been a high focus on conducting research likely to outline a profile of the real university teacher as opposed to the ideal one from a student perspective. The aim of these studies is to underline the evolution of the real and ideal teacher"s profile in higher education. Thus, there is a constant interest in identifying the characteristics of the ideal teacher, considered important by students and in outlining the dimensions of the real university teacher (performing nowadays in tertiary system). As a consequence, the current studies deem fit to highlight the gap between real and ideal in profiling the higher education teacher. (Mannan & Traicoff, 1976;Harley, Barasa, Bertram, Mattson & Pillay, 2000;Arnon & Reichel, 2007;Rusu, Şoitu, & Panaite, 2011;Haamer, Lepp & Reva, 2012;Douna, Kyridis, Zagkos, Ziontaki, & Pandis, 2015;Nartgüna & Sezghin, 2015) The literature aforementioned as well as studies conducted by Karpouza & Emvalotis, 2018;Kyridis, Avramidou, Zagkos, Christodoulou, & Pavli-Korre, 2014;Kyridis, Dinas, Vlachaiti, Ioannitou, & Lambropoulou, 2002;Kusto, Afful, & Mattingly, 2010;Leondari & Kyridis, 1999;Nurul, Sri, Nur, Chung, Ivanova, Mohd, Ummi, Masitah, & Lawrence, 2014;) focus on the personality traits that depict the ideal university teacher, his/her professional competences (specialized knowledge and a wide range of competences such as: methodological, communication, student-teacher relationship, technical and technological, psychological and social, evaluation and career management, research competences), poise and posture (outfit and social behaviour) as well as ethical behaviour. The results show that to students" mind, the ideal university teacher"s profile is based on a series of variables with regard to gender, age and political beliefs.
The purpose of the studies conducted in the field of education is not to turn the real university teacher into an ideal one (since a teacher will never become an ideal, for an ideal is nothing else than the assumption of perfection never found in practice yet, hence, we are always on constant pursuit of it). The aim is to continuously bridge the gap between the real and the ideal teacher, both under ongoing change so as to comply with sustainable pedagogy and education as well as to constantly enhance the relationship between these two educational actors: professor-student. (Haamer, Lepp, & Reeva, 2012).
In pre-higher education, the teacher-student relationship is extremely important (Manyu & Stone, 2018), and higher education is just an extension since interpersonal relationships are the basis of coherent and sustainable education.
According to the grid of symbolic interaction (Blumer, 1969;Gavreliuc, 2002: 46) applied to the educational system, any social agent engaged in a relationship continuously negotiates the meaning of the situation in which he/she is part of. Hence, students and teachers act in compliance with: what they understand of each other; the rapport built within educational and social interactions; the meanings altered, renewed and passed over within educational interactions.
The above mentioned assumptions are also supported by related studies carried out in Romania. Rusu, Șoitu andPanaite (2011: 1019) assume that students from Alexandru Ioan Cuza University in Iași, studying at the Humanities and the Faculty of Philosophy and Political Sciences respectively, attach significance to the teacher"s ability to create rapport, considered one of the prerequisites for the ideal teacher (71. 42%). Among other characteristics of the teacher-student relationship mentioned in the research referred to: "…a sympathetic man, a second parent, who communicates with his students, to be a relationship based on trust and genuine interest…", "…a calm man, patient, temperate, gentle, who knows how to approach students, turning them into his own children…".
Moreover, Voicu and Ciolan (2008: 7), in their study ˝The School as it is", argue that ˝from schoolchildren"s point of view, the way a teacher relates to them is crucial". ˝Indulgence, friendliness, fairness or patience are highly sought in a teacher by schoolchildren".
A positive teacher-student relationship implies commitment, passion, specialized knowledge, trust, respect, openess, authenticity, transparency, availability, responsibility, interest, engagement and equitable and fair treatment for each and every student. A teacher-student relationship characterized by the teacher"s inappropriate behavior, disinterested and unmindful of his/her relationship with the students, unavailable, unapproachable, unfair and biased and who teaches an unfamiliar difficult subject, whose outfit and posture are inappropriate, may a negative impact on the didactic process and on the relationship between the educator and the ones to be educated. In higher education, teachers and students alike interact constantly and this interaction counts irrespective of any experiences. Unfortunately, the teacher-student relationship is still pending further research, since only few studies address this topic (Karpouza & Emvalotis, 2018).
Nowadays, another important feature of an ideal teacher, from a modern perspective, is his/her evaluation ability (Jinga & Istrate, 1998). Likewise, the study beforementioned conducted by Rusu, Șoitu andPanaite (2011: 1019), also emphasizes the teacher"s role as evaluator. Hence, June, 2019 Educaţie Multidimensională Volume 11, Issue 2 teachers" fair assessment ranks second in students" opinions by 66.23%, and third comes ˝knowledge of the subject" by 58.44%. The fourth place as importance in students"views is the teacher as a ˝facilitator of students' intellectual development" by 55.84%.
Research Methodology
The current survey addressed the teacher profile in Romanian technical higher education, from two different perspectives: teachers wieved and perceived by students in general; teachers viewed and perceived by the society as a whole.
The current survey was conducted by means of an isondaje platform on-line questionnaire, and targeted 321 students: 176 male students (54.8%) and 145 female students (45.2%); 207 (64.5%) students studying at University Politehnica of Bucharest and 114 students from University Politehnica of Timişoara (35.5%); respondents" age between 18 and 40.
Q1. What three words would you use to describe teachers (in general)?
The teacher-student relationship ranks first in Romanian engineering students" opinions (28.7%), since it is regarded as highly important and teaching is by far a relational activity, one with psychological, social and didactic effects between two educational agents: the teacher and the educable (schoolchild or student). What is more, teaching plays a vital role in the quality assurance of the educational process.
By and large, didactic performance is substantially determined by the existing pedagogical relationships. Bad relationships between teachers and students can trigger learning difficulties, students" poor education and training, challenges of classroom management. As a consequence, students are aware of this and fairly appreciate the importance of their relationship with the teacher for the learnig process.
It is worth mentioning that the current research reveals differences in respondents" opinions with regard to the teacher-student relationship. Therefore, on one hand, this is perceived as a relaxed, efficient relationship, since it is depicted in terms of "respect, commitment, dedication, engagement, motivation, patience, discipline, seriousness, empathy, calmness, friendliness, understanding, inventivity, courtesy etc." On the other hand, it is perceived as a less positive and efficient relationship and it is characterized by :˝communism, boredom, superiority, disinterest, authority etc.".
In line with the latter general opinion, the school, the teacher and the teacher-student relationship are likely to be outdated and old-fashioned, unlikely to overcome the old obsolete methods and attitudes, reluctant to new challenges and changes. It deems necessary to understand students" different opinions, since, over the course of their study years, they meet dozens of teachers with different personalities, different teaching styles and attitudes and different levels of training and education.
Engineering students" second choice -teacher as an evaluator/assessor (21.2%) -reveals the students" threat of evaluation and, especially, of the evaluator as well as the students" misunderstanding of the teacher"s role as an evaluator. Traditionally, the teacher is considered the one who criticizes, evaluates and judges unfairly and this role is described in terms of "marks/grades, tests, examination, unfair, to sit pass or fail, nervousness, stress etc".
Ranking third in students" views, by 17. 1%, the teacher's professional expertise means intellectual authority, possession of specialized knowledge, swapping of know-how with students and development of specialized competences necessary for students" future career. What is more, this opinion is expressed in terms of ˝information, knowledge, expertise, culture, capability, professionalism, literate, scholar, capable, smart, new things well explained, hardworking, experience, future, prosperity, engineer etc".
The last ranks in the current research belong to the roles of the teacher (2.4%) and managerial skills (0.6 %), labelled as ˝teacher, model, advisor, friend, parent, inspirational, guide, planner, mentor". This explains the existence of an environment in which the teacher still plays traditional roles and acts less as a planner, guide or facilitator. At the same time, it becomes worth mentioning the new modern roles performed by the teacher in line with nowadays educational dynamics: advisor, guide, inspirational, facilitator of learning, planner and organizer. Hence, the focus is gradually shifted from knowledge-based product to knowledge-based process, the teacher attaching greater importance to the students" learning needs, motivation, guidance and disregarding the delivery of prefabricated knowledge.
Q2. How do you think teachers are perceived in Romania? Taking into consideration students" answers to the above question, it deems fit to notice their high appreciation of teachers provided the direct contact with them (also shown from the statistical means in Table 2), whereas the rest of society reveals unfair low appreciation of teachers. The difference is statistically significant as proved by the test t = 75,360 (p<0,01).
Therefore, direct contact and relationship that students have with the educational system and teachers bring about students"empathy to their trainers, thus, appreciating their effort and commitment likely to assist them in becoming specialists in various domanins. On the contrary, those who have lost contact with education and teachers will no longer consider this profession important and, unfortunately, they become unmindful of the importance of teaching and teachers considered the basis of all professions and jobs. Moreover, it is worth mentioning that further development of society relies heavily on teachers and their dedication.
Q3. To what extent do you think the following attributes fit to the real/ideal teacher"s profile? There are statistically significant differences for all attributes listed in Table 3, regarding the profile of the real and the ideal teacher in technical higher education from a student perspective. On one hand, the results show the real is not tentamount to ideal, since the ideal does not exist in practice, it is more a target to be achieved. On the other hand, there is always room for teaching development inasmuch as we are well aware of the students" preferences and opinions about the characteristics of their desired teacher or trainer. Therefore, there is a constant need to bridge the gap between real and ideal, provided a clear profile of the teacher as outlined by students. As a consequence, the teacher"s future bahaviour is likely to change so as to fit properly into the educational and instructional processes. Moreover, this is likely to bring about alterations of the curricula for future teachers" psychopedagogical training.
In order to highlight a few essential teacher dimensions regarding the real and ideal model in higher education, we have observed the results of the research as illustrated in Table 4 below. In this sense, one observation is that on a 1 to 5 marking scale, intelligence ranks first for both teacher models, real (4.35) and ideal (4.61). More than anything, for students, intelligence is crucial when it comes to their teachers. And this is because teaching, as a profession, makes the teacher face challenges, deal with diverse and unexpected situations in the classroom, never encountered before. Hence, intelligence, as a superior form of optimal and efficient adaptation to new difficult puzzling situations, by means of restructuring of previous experiential data, proves a prerequisite for any teacher likely to overcome this kind of predicament.
Second in the ranking comes responsible/ responsibility (4.07), proving students" perceptions of the real teacher as a responsible person who shows respect for the profession and assumes consequent responsibilities and duties. Punctual ranks third (3.94), which explains students"s perceptions of the higher education teacher regarded as a person mindful of efficient time keeping in the classroom. With regard to didactics, punctuality is considered a form of respect for the educables and their efficient training alike. Ranking fourth, the attribute of trustworthy (3.91) underlines the importance for keeping one"s promise and taking the teacher"s words for granted.
As far as the ideal teacher is concerned, the following attributes rank second and third: good communicator (4.57) and trustworthy/a person of his/her word (4.45). Thus, students highly appreciate a teacher who is a good communicator, who is able to efficiently transmit specialized information so as to make himself/herself clear to the students, who is able to create rapport with them and create a congenial positive interpersonal atmosphere likely to stimulate and support each and every student in their learning process. To students" mind, the ideal academic teacher is capable of understanding their needs, behaviour, a trustworthy person, of his/her word, never changing mind or ideas, demands or expectations, nor mood. Responsible, as an attribute of the ideal academic teacher, ranks fourth (4.44).
Passionate is next in ranking, on the fifth position, the real teacher model (3.87) and the ideal (4.44) respectively. In fact, in this case, the real is tentamount to ideal in the sense that students would prefer a passionate academic teacher both in practice and theory. Passion implies a calling for this profession, vocation, dedication, hard work and commitment. It is worth mentioning the positive aspect of this students" observation.
In the aftermath of the aforementioned, the first five positions of the ranking concerning the attributes assigned to the academic teachers, both profiles of the real and ideal teacher share 4 common characteristics (intelligent, responsible, trustworthy, pasionate), two of them being placed on the same position in both models (intelligent -first rank and passionatefifth rank). As a consequence, there is a huge similarity between the students" expectations from a higher education teacher and the way they perceive their real teachers, surprisingly and encouraging at the same time.
Among the last ranking adjectives are elitist (3. 21), competitive (3. 19) and approachable (3.16). This fact illustrates that students do not consider the higher education teacher an elitist, a distinguished superior and competitive person, in the pursuit of professional affirmation. Nor is the academic teacher perceived as approachable. Hence, even if the teacher is intelligent, responsible and punctual (the first three ranked attributes), they June, 2019 Educaţie Multidimensională Volume 11, Issue 2 82 are not available off-duty and ready to provide students with additional information, nor likely to offer learning support for their students. With regard to the adjectives assigned to the ideal academic teacher, the last three ranking positions are occupied by transparency in actions (3.93), elitist (3.59) and competitive (3.53). We can assume that a transparent teacher in actions is not important for students. Furthermore, for them, it does not matter whether their teacher is an elitist, cultivated, superior or prone to self-sufficiency or competitive.
We notice that 2 adjectives (elitist and competitive) are common for both the real and ideal models, hence such a teacher is neither wanted nor does he/she exist in reality. Q4. To what extent do you agree with the following assumptions about the real higher education teacher? The items listed in Q4 were analyzed according to the correlations of all the 16 selected assumptions. Three factors with values higher or equal to 1.00 were selected. The rotation of factors led to the structure provided in the previous table. The first factor counts for 21. 66% of the variant, the second for 21.61% and the third for 7.36% of the variant. Summing up, the four factors count for 50.65% of the variant.
Factor 1 (Communicative-pedagogical dimension) correlates with the initial variables as follows: teachers do not make sexist or misogynistic remarks to female students (0.757); Teachers possess updated specialized knowledge (0.678); Teachers are open to communication with students (0.654); Teachers are not arrogant and treat students fairly (0.649); Teachers are methodical and structured in teaching (0.604); Teachers are available offduty (0.603); Teachers evaluate students objectively (0.551).
Factor 2 (Motivational dimension) correlates with the initial variables as follows: Teachers are concerned about quality assurance in teaching (0.763); Teachers put theory into practice in teaching (0.740); Teachers show enthusiasm (0.735); Teachers use interactive teaching methods (0.710); Teachers encourage students to speak their mind freely (0.709); Teachers provide students with feedback on evaluation (0.621).
Factor 3 (Moral dimension) correlates with the initial variables as follows: Teachers do not make vulgar remarks to students (0.757); Teachers attend appointments with students (0.574).
Q5. To what extent do you agree with the following assumptions about the real higher education teacher? Table 7. No. crt.
Assumptions
Statistical mean 1 Teachers successfully overcome didactic and professional challenges in educational and instructional activity 3.6 2 Teachers encourage students to speak their mind freely 3.57 3 Teachers are concerned about quality assurance in teaching 3.55 4 Teachers do not make vulgar remarks to students 3.43 5 Teachers show enthusiasm for the taught subject 3.4 6 Teachers are methodical and structured in teaching 3.37 7 Teachers provide students with feedback on evaluation 3.3 8 Teachers use interactive teaching methods 3.24 9 Teachers put theory into practice in teaching 3.22 10 Teachers do not make sexist or misogynistic remarks to female students 3.07 The profile of the real academic teacher has been outlined by means of a range of assumptions with regard to the didactic activity, the teacherstudent relationship extra school hours and students" evaluation methodology.
This has led to a hierarchy of answers ranked on a 1 to 5 scale, in a decreasing order from 3.6 to 2.39. The average is set to range between 2.74 and 3.24, namely around 3.
The top ranking address the following assumptions: Teachers successfully overcome didactic and professional challenges in educational and instructional activity (3.6) ; Teachers encourage students to speak their mind freely (3.57); Teachers are concerned about quality assurance in teaching (3.55) ; Teachers do not make vulgar remarks to students (3.43) ; Teachers show enthusiasm for the taught subject (3.4) ; Teachers are methodical and structured in teaching (3.37) ; Teachers provide students with feedback on evaluation (3.3).
These research findings reveal students" appreciation for their teachers" openness, the quality of the subjects taught and their feedback on evaluation. Moreover, there is positive appreciation for the other aspects mentioned, to be investigated in further research: communication with students during breaks or off-duty and extra school hours, arrogant attitude towards students and lack of objective evaluation.
Q6. To what extent do you agree with the following rights and obligations of teachers and students alike? Another objective of the current study is to sustain Marlene and Lee Canter"s suggestion that teachers should display positive attitude. Therefore, the questionnaire also addresses some rights and obligations of teachers and students in line with Canters" proposal (Stan, 2006: 58-78): Teachers" fundamental rights: to ensure an optimum learning environment; to expect appropriate behaviour from their students; to expect appropriate support from parents and school administration if need be.
Students" fundamental rights: to have a teacher likely to assist them in developing and prevent them from self-destruction due to inappropriate behaviour; to receive support from the teacher so as to acquire appropriate behavior; to adopt a certain type of behaviour provided previous awareness of their choice and subsequent consequences.
The rights of both academic parties (teachers and students) as formulated by the Canters are validated as follows:
Conclusions
The current study addresses the profile of the teacher in technical higher education in Romania and it was conducted at University Politehnica of Bucharest and University Politehnica of Timişoara. The target group consisted of 321 interviewees, 176 male students and 145 female students. The survey was based on an on-line questionnaire. The results of the research illustrate the following: Students rank first the teacher-student relationship (28.7%), thus explaining their rapport based on continuous interaction between the two educational agents. To students" mind, the relationship between the trainer and the trainee, between the master and the apprentice is more important than the knowledge provided; The teacher as an evaluator ranks second (21.2%) and argues for the stress students experience during examination periods. Moreover, it underlines the evaluator"s authority bound to certify the competences of the one who studies and who wishes to obtain a "trip ticket", that is the diploma likely to ensure confidence in your own capacity and career opportunities; The teacher as knowledge provider ranks third. The teacher is depicted in terms of know-how, expertise, culture and considered a reliable and worthy icon for students; The students who are directly engaged in the educational and instructional process and in their relationship with the teachers are likely to appreciate them more than the ones who have lost contact with the teachers; The ranking of attributes assigned to academic teachers emphasizes intelligence, in case of the real teacher the statistical mean is (4.35) and for the ideal one (4.61). Furthermore, the last three positions tackle adjectives such as elitist and competitive, hence students will look for intelligent higher education teachers, capable to face professional challenges. Nevertheless, they should not be bound to elitism, perfection, selfsufficiency nor competitivness or on a constant pursue of self-affirmation.
The current research is in line with the model proposed by Marlene and Lee Canter who advise teachers to take on a positive attitude in education, thus observing the fundamental rights of teachers and students alike.
The challenges of the current study are the limited sample of the target group and the application of the questionnaire to both schoolchildren and engineering students, at the same time.
With the advent of modern technology, the importance of teacherstudent relationship prevails (as shown by the findings of the current research). Moreover, such a face-to-face teacher-student, trainer-trainee relationship is endangered, nowadays, by the excessive threat of technology in all educational aspects. Thus, our assumptions are not against information technology and communication within the scope of education, on the contrary, we deeply understand its importance as well as its benefits for the development and consolidation of educational systems worlwide and its contribution to the development of certain digital competences -June, 2019 Educaţie Multidimensională Volume 11, Issue 2 prerequisites of a knowledge-based society for graduates. Nevertheless, we must raise awareness about the constant tendency to undermine interpersonal relationships within educational process nowadays. In the aftermath of ongoing advancement of technology and communication, students" high appreciation of interpersonal relationships comes by surprise. This proves they may have become aware of the problematics of technology to human alienation. As a result, information technology and communication prove a sine qua non condition for educational systems as long as their impact on students and learning process is positive.
Ranking second in the current study, teacher"s role as evaluator unveils the fear and stress such an assessment process may cause as well as the inappropriate manner evaluation is conducted at present. Evaluation must be perceived as a crucial aspect of the educational process, an extension of the teaching-learning activity and, thus, it must be planned by the teacher well in advance and in collaboration with the educables. Students and school children alike should not be afraid of evaluations, the language of assessment should not remain in the realm of judgement. The teacher must not criticize or give negative appraisals of their professional performance. Consequently, they must get accustomed to evaluations and understand that evaluation is an essential element in the learning cycle, likely to provide feedback useful for their professional development. Teachers are thus recommended to include formative evaluation in their activity. It is worth mentioning that formative evaluation proves efficient when it leads to selfassessment and corrects and guides further learning. Corrective advice is ensured through students and not through the teacher"s didactic strategies. Furthermore, the students themselves are capable of self-correction, selfevaluation and further self-planning of learning. They are capable of selfautonomy in learning, they set their learning objectives and improve their performance based on the results of the evaluation. By conducting formative evaluation, teachers are likely to ensure its therapeutic benefits and enhance the positive impact of evaluation on the educables. Viewed as a therapy, assessment is no longer a diagnosis. It becomes the springboard for solid projects and plans likely to overcome any educational predicament in education.
The teacher"s professional expertise ranks on the third place in the current study, stressing the importance of specialized knowledge. In modern times of technology and computers, the teacher still plays the vital role of information provider as compared to artificial intelligence. Although teachers and students alike may easily resort to artificial intelligence for educational purposes, it is human intelligence that makes the difference between man and machine, man and computer, man and technology.
We must argue that teachers must undergo continuous training in order to acquire and develop multidimensional competences and successfully tackle educational challenges and queries.
Students" willingness to show a pro-active attitude towards their teachers in class is commonsensical. The teacher as a human being is the one to create the educational atmosphere the student needs. By way of conclusion, the ideal teacher is intelligent, multidimensional, fair and a humane evaluator, an expert in his/her field of expertise and capable of ensuring a congenial learning environment. | 2018-11-20T18:43:21.446Z | 2018-11-14T00:00:00.000 | {
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11191118 | pes2o/s2orc | v3-fos-license | Self-prioritization and perceptual matching: The effects of temporal construal
Recent research has revealed that self-referential processing enhances perceptual judgments — the so-called self-prioritization effect. The extent and origin of this effect remains unknown, however. Noting the multifaceted nature of the self, here we hypothesized that temporal influences on self-construal (i.e., past/future-self continuity) may serve as an important determinant of stimulus prioritization. Specifically, as representations of the self increase in abstraction as a function of temporal distance (i.e., distance from now), self-prioritization may only emerge when stimuli are associated with the current self. The results of three experiments supported this prediction. Self-relevance only enhanced performance in a standard perceptual-matching task when stimuli (i.e., geometric shapes) were connected with the current self; representations of the self in the future (Expts. 1 & 2) and past (Expt. 3) failed to facilitate decision making. To identify the processes underlying task performance, data were interrogated using a hierarchical drift diffusion model (HDDM) approach. Results of these analyses revealed that self-prioritization was underpinned by a stimulus bias (i.e., rate of information uptake). Collectively, these findings elucidate when and how self-relevance influences decisional processing.
two outstanding issues merit consideration and further empirical scrutiny. These pertain to the generality and basis of the reported self-prioritization effect. First, how sensitive is this effect to differences in the operationalization of the self? At least among healthy individuals, the overarching message in the available literature is that stimulus prioritization is an obligatory consequence of self-referential processing. To optimize functioning in complex and challenging environments, attention is automatically tuned to favor self-relevant stimuli . But is this inevitably the case or, as is perhaps more likely, is stimulus prioritization sensitive to differences in self-construal (Trope & Liberman, 2003? Second, what is the basis of the self-prioritization effect? Despite widespread evidence that self-relevant stimuli facilitate perceptual judgments , the precise mechanism through which this effect arises remains unknown. Accordingly, the goals of the current research were twofold-to explore the extent and source of the selfprioritization effect during perceptual matching (Sui et al., 2012).
Self-construal and perceptual matching
In exploring the self-prioritization effect, research to date has adopted a rigid operationalization of the self that potentially underestimates the nuanced ways in which it may (or indeed may not) impact task performance . Rather than comprising a unitary, monolithic entity, the self is a multifaceted, flexible construct shaped by the collective influence of long-term knowledge, situational forces, and temporary processing goals (Conway & Pleydell-Pearce, 2000;McConnell, 2011;Roberts & Donahue, 1994). For example, it is widely acknowledged that the self comprises multiple social identities (with associated beliefs, expectations, and values)-derived from membership in various social groups (Tajfel, 1978)-that influence behavior in a context-specific manner. When a particular subcomponent of the self is active, individuals make sense of the world through the lens of the associated knowledge structure (e.g., McConnell, 2011;Oyserman, 2009). Similarly, and of relevance to the current inquiry, it is well established that thinking and doing are highly susceptible to temporal influences on self-construal (see Trope & Liberman, 2003. A central tenet of construal-level theory (CLT) is that forthcoming events (e.g., going on vacation) can be represented in either a super-or subordinate manner, what matters is when an event is scheduled to occur (e.g., next month vs. next year). Whereas impending events (e.g., a trip to Hong Kong) are characterized in a concrete, detail-rich manner (e.g., booking a hotel, acquiring local currency, finding one's passport), distant events comprise abstract, decontextualized representations that convey only the gist or general meaning of an episode (e.g., enjoying a well-earned break). In other words, as events become temporally distant, representations decrease in detail and increase in schematic content (see also Vallacher & Wegner, 1985). Importantly, characterizations of the self display comparable temporal shifts in complexity (i.e., concrete to abstract), a structural phenomenon that has implications for assessments of self-continuity (i.e., the perceived similarity between one's current and future self). Specifically, overlap between one's current and future self fluctuates as a function of temporal distance, such that a person feels greater affinity with her potential self in the near than distant future (e.g., Parfit, 1971Parfit, , 1987Pronin & Ross, 2006;Schelling, 1984;Thaler & Shefrin, 1981;Wakslak, Nussbaum, Liberman, & Trope, 2008). Indeed, travel deeply into the future and psychological connectivity between these constructs can be severed altogether, resulting in one's future self acquiring the status of a stranger (Hershfield, 2011;Mitchell, Schirmer, Ames, & Gilbert, 2011;Parfit, 1971Parfit, , 1987Pronin, Olivola, & Kennedy, 2008;Pronin & Ross, 2006;Wakslak et al., 2008). Intriguingly, this self-becomes-other effect has also been reported for events in the not-so-distant future (e.g., next month, next semester; Pronin et al., 2008;Pronin & Ross, 2006). These temporal influences on self-construal may act as an important determinant of the self-prioritization effect . Such a boundary condition would have important implications for contemporary theoretical accounts of self-prioritization, as it would challenge the viewpoint that selfbiases are a mandatory characteristic of perceptual processing . Of course, what has also yet to be established is the specific mechanism that underpins the self-prioritization effect during perceptual matching (Sui et al., 2012).
In exploring the origins of decisional bias, prior research has identified two distinct pathways through which top-down knowledge (e.g., self-relevance) can influence task performance (Ashby, 1983;Leite & Ratcliff, 2011;Link & Heath, 1975;Summerfield & de Lange, 2014;van Ravenzwaaij, Mulder, Tuerlinckx, & Wagenmakers, 2012;White & Poldrack, 2014). During decision making, adjustments can be made to how the stimulus under consideration is evaluated (i.e., stimulus bias) or the manner in which a response is prepared (i.e., response bias). That is, biases can be traced to how sensory information is converted into decisional evidence or how that evidence is used to generate a decision (White & Poldrack, 2014). In terms of the self-prioritization effect, the prevailing viewpoint is that self-relevance influences the perceptual operations that underpin decision making and thus represents a bias in stimulus processing. To demonstrate such a bias, however, necessitates the decomposition of task performance during perceptual matching to isolate the specific cognitive process that underpins the self-prioritization effect (Sui et al., 2012). Importantly, in the context of binary decision tasks, drift diffusion models provide just such an opportunity (e.g., Ratcliff, 1978;Ratcliff & Rouder, 1998;Ratcliff, Smith, Brown, & McKoon, 2016;Voss, Nagler, & Lerche, 2013;Voss, Rothermund, & Brandtstädter, 2008;Voss, Rothermund, & Voss, 2004;Voss & Voss, 2007;Voss, Voss, & Lerche, 2015;Wagenmakers, 2009).
Decomposing the self-prioritization effect
Drift diffusion models decompose behavioral data (i.e., response times & accuracy) into a set of latent parameters that represent the cognitive operations underlying decisional processing (Voss, Nagler, et al., 2013;Voss et al., 2015). A variant of continuous sampling approaches, these models assume that information is continuously gathered during a decision phase until sufficient evidence is acquired to initiate a response. A schematic depiction of the model is provided in Fig. 1. The model describes evidence accumulation unfolding over time and fits accuracy and response time distributions. The duration of the diffusion process is known as the decision time and the diffusion process itself can be characterized by several important parameters (Voss et al., 2004;Voss, Rothermund, et al., 2013;Voss et al., 2015). 1 The drift rate (v) maps the speed and quality of information acquisition (i.e., larger drift rate = faster information uptake), thus provides a measure of stimulus processing (i.e., stimulus bias) during decision-making (White & Poldrack, 2014). Threshold separation (a) estimates the distance between the decisional boundaries, hence indexes how much evidence is sampled before a decision is made (i.e., higher estimates represent thresholds that are most distinct from each other, suggesting greater certainty is required before a response is initiated). The starting point (z) defines the position at which evidence accumulation begins. If z is not centered between the thresholds, this indicates an a priori bias in favor of the outcome that is closer to the starting point (i.e., response bias). Finally, the duration of all nondecisional processes (i.e., operations occurring pre/post decision making) is given by the additional parameter t 0, and is taken to indicate biases in stimulus encoding and response execution (Voss, Nagler, et al., 2013;Voss et al., 2015).
Drift diffusion modeling is useful in the current context as it has the capacity to isolate the cognitive processes underlying decision making (White & Poldrack, 2014), thereby elucidate the origin of the self-prioritization effect. If, as has been suggested, self-relevance influences the perceptual component of the decisional process , then self-prioritization should be underpinned by differences in the rate of information uptake (i.e., stimulus bias, v) during the standard shape-label matching task (Sui et al., 2012).
Overview
In three experiments, the goals of the current research were to explore when and how self-relevance impacts the emergence of the self-prioritization effect during perceptual matching . Noting fundamental temporal influences on self-construal (Hershfield, 2011;Mitchell et al., 2011;Pronin et al., 2008;Pronin & Ross, 2006;Wakslak et al., 2008)-specifically, that representations of the self increase in abstraction as a function of temporal distance-we expected the self-prioritization effect during perceptual matching to be restricted to geometric shapes associated with temporally proximate (vs. distant) representations of the self. We explored this hypothesis in a standard perceptual matching task in which participants associated labels pertaining to the self (e.g., self now, self tomorrow) and a stranger with various geometric shapes (e.g., circle, diamond), then judged (as quickly and accurately as possible) whether subsequent shape-label pairings matched or mismatched the previously learned associations (see Sui et al., 2012). To identify the decisional processes underlying task performance, data were submitted to a HDDM analysis (Wiecki et al., 2013). Of theoretical interest was establishing the extent to which self-relevance influences the rate at which evidence is acquired (i.e., drift rate, v) during perceptual matching. 2 Experiments 1 and 2 explored prospective representations of the self, Experiment Experiment 3 representations of the self in the past.
Experiment 1 Participants and design
Sixteen undergraduates (one male, M age = 19.69 years, SD = 1.89) took part in the research, for which they received £5 (~$6.20). 3 All participants had normal or corrected-tonormal visual acuity. Informed consent was obtained from participants prior to the commencement of the experiment, and the protocol was reviewed and approved by the Ethics Committee at the School of Psychology, University of Aberdeen. The experiment had a 4 (shape category: selfnow vs. self-year vs. self-forty vs. stranger) × 2 (trial type: matching vs. nonmatching) repeated-measures design.
Stimulus materials and procedure
Participants arrived at the laboratory individually, were greeted by an experimenter, and told they would be performing a perception task. Following Sui et al. (2012), the experiment had two phases. The first phase comprised a learning task in which participants were required to associate geometric shapes (i.e., circle, horizontal rectangle, cross, diamond) with four targets: self-now, self-in-one-year, self-inforty-years, and a stranger. To bolster the temporal manipulation and trigger construal-based processes, participants engaged in periods of guided imagery (i.e., a simulation for each target) during which they formed target-shape associations. Based on prior research, participants were instructed to close their eyes and imagine the self (now, in-one-year, in-fortyyears) or a stranger walking along a quiet beach (Macrae et al., 2015). 4 After 20 seconds had elapsed, participants were instructed to represent the target of the respective simulation (i.e., self-now, self-one, self-forty, stranger) with a specific geometric shape (i.e., circle, horizontal rectangle, cross, diamond). The shapes were not presented at this stage. The order of the mental simulations and shape-target associations were counterbalanced across the sample.
Next, participants were seated in front of a desktop computer and informed they would be performing a perceptualmatching task. Using two buttons on the keyboard (i.e., N & Fig. 1 Schematic version of the drift diffusion model, adapted from Voss, Rothermund, et al., (2013, p. 4). An information gathering process begins at starting point z and continues with a mean slope v until it reaches an upper (a) or lower (0) threshold. The process durations and outcomes vary from trial to trial because of random noise. Outside the threshold boundaries the decision-time distributions are shown M), participants had to report whether a series of shape-label pairings (e.g., circle & self-now, cross & stranger, rectangle & self-year, diamond & self-forty) were correct (or incorrect) on the basis of the associations learned previously. Each trial began with the presentation of a central fixation cross for 500 ms, followed by the pairing of a shape (i.e., circle, rectangle, cross, diamond) and label (e.g., self-now, self-one, selfforty, stranger) above and below the fixation cross, respectively, for 100 ms. After each shape-label pairing was presented, the screen turned blank for a variable interval (i.e., 800 ms to 1,100 ms). Participants had to judge the accuracy of the pairings (i.e., whether they matched or mismatched the associations learned earlier) by pressing the corresponding button as quickly as possible within this time frame to encourage immediate responding (Sui et al., 2012). The meaning of the response buttons was counterbalanced across participants. Feedback (i.e., correct or incorrect response) was given on the screen for 500 ms at the end of each trial and participants were also informed of their overall accuracy at the end of each block of trials. Participants initially performed 18 practice trials, followed by seven blocks of 72 trials in which self-now, self-one, self-forty, stranger, and re-paired stimuli occurred equally often in a random order. In total, across all the blocks, there were 63 trials in each condition (i.e., self-now matching, self-now non-matching, self-one matching, self-one nonmatching, self-forty matching, self-forty nonmatching, stranger matching, stranger nonmatching). On completion of the task, participants were debriefed and dismissed.
Results and discussion
Perceptual matching Responses faster than 200 ms were excluded from the analysis, eliminating less than 1% of the overall number of trials. Table 1 shows the accuracy and response time (RT) data. Following Sui et al. (2012), a bootstrapping procedure was adopted to examine the distribution characteristic of perceptual-matching judgments in each condition, combining accuracy and RT performance (Davison & Hinkley, 1997). For each participant in each condition, accuracy and RT were paired as a single data point (x, y). A bootstrapped data set was then created by re-sampling the data with replacement, keeping the sample size as the number of participants. The mean of this bootstrapped data set was then calculated and plotted as a single data point in the distribution (x, y). This procedure was repeated 2,000 times to estimate the population mean and variation for each condition. The resultant distributions across the different shape-category judgments are displayed in Fig. 2. Whereas the bootstrapped sample mean observations for selfnow matching judgments fell in the lower right corner of the figure, all other matching judgments fell in an upper middle location (see Fig. 2a). In contrast, an overlapping distribution of observations emerged for responses to non-matching shape-category pairs (see Fig. 2b).
To explore the accuracy of participants' responses, a signal detection approach was adopted. For each shape-category, performance in the matching and non-matching conditions were combined to calculate a measure of sensitivity (d') and a single factor (shape category: self-now vs. self-one vs. self- (14) Self-one 749 (44) 72 (16) Self-forty 756 (35) 73 (14) Stranger 764 (51) 73 (11) Note. RT = reaction time. Standard deviations appear within parentheses Fig. 2 Distribution of bootstrapped sample means for matching (a) and nonmatching (b) trials (Experiment 1). The x-axis represents the accuracy rates and the y-axis the reaction times. c Shows d' as a function of shape category. Error bars represent +1 SEM forty vs. stranger) repeated-measures analysis of variance (ANOVA) was performed on these data (see Fig. 2c). This revealed an effect of shape category, F(3, 45) = 4.08, p = .012, η p 2 = .21, such that d' was larger for self-now than for self-one, t(15) = 3.23, p = .003, d = .81, and stranger, t(15) = 2.22, p =.02, d = .55. The difference between self-now and self-forty was not significant, t(15) = 1.62, p = .063.
Replicating Sui et al. (2012), these results demonstrate a self-prioritization effect on perceptual matching . Critically, however, performance was only enhanced when geometric shapes were associated with the current self. Future conceptions of the self produced patterns of performance equivalent to associating stimuli with a stranger, even when the self was construed only 1 year forwards in time (see Pronin et al., 2008;Pronin & Ross, 2006;Wakslak et al., 2008). This self-now prioritization effect confirms that temporal influences on self-construal impact perceptual matching.
Diffusion modeling
To identify the processes underlying task performance during perceptual matching, data were submitted to a HDDM analysis.
HDDM is an open-source software package written in Python for the hierarchical Bayesian estimation of drift diffusion model parameters (Wiecki et al., 2013). This approach assumes that the model parameters for individual participants are random samples drawn from group-level distributions and uses Bayesian statistical methods to estimate all parameters at both the group-and individual-participant level (Vandekerckhove, Tuerlinckx, & Lee, 2011). An advantage of this approach is that it is robust at recovering model parameters when less data (i.e., experimental trials) are available (Wiecki et al., 2013).
Models were response coded, such that the upper threshold corresponded to a matching response and the lower threshold to a nonmatching response. To test the hypothesis that selfprioritization is underpinned by a stimulus bias , separate drift rates (v) were estimated (i.e., allowed to vary) as a function of shape category and trial type (i.e., positive values = drift rate on matching trials, negative values = drift rate on nonmatching trials). A single starting value (z) was allowed to vary between the response thresholds, such that z = 0.5 indicates no bias (i.e., the starting point is located at the midpoint between the thresholds). Separate nondecisional processes (t 0 ) were estimated for matching and non-matching trials (Voss, Rothermund, Gast, & Wentura, 2013). Bayesian posterior distributions were modeled using a Markov Chain Monte Carlo (MCMC) with 10,000 bootstraps (following 1,000 burn in samples). Prior to analysis, trials with latencies faster than 200 ms were removed, and the HDDM software removed the 5% of trials with the longest response latencies (Ratcliff & Tuerlinckx, 2002). In this model, bias during decisional processing could be mapped on to the drift rate (v), indicating a stimulus bias; or the position of the starting point (z), indicating a response bias (White & Poldrack, 2014).
To determine the adequacy of this model, five additional models were tested for comparison. For the first two models, only the drift parameter (v) and starting point (z) were allowed to vary. For the other models, combinations of drift rate (v), starting point (z), and nondecision (t 0 ) processes were allowed to vary. As can be seen in Table 2, the model that included all three parameters yielded the best fit (i.e., smallest DIC value). Interrogation of the posterior distributions revealed evidence of both stimulus and response biases during decision-making (see Fig. 3). Specifically, on matching trials, drift rates (v) were higher (i.e., evidence accumulation was faster) for selfnow than all other shape-category pairings [p Bayes (self-now > self-one & self-forty & stranger) = 1.0]. This was not the case on nonmatching trials [p Bayes (self-now > self-one & self-forty & stranger) = .312]. In addition, comparison of the observed starting value (z = .62) with no bias (i.e., z = 0.5) revealed an a priori bias towards matching responses [p Bayes (bias > 0.5) = 1.0]. Finally, nondecisional processes (t 0 ) yielded no difference between matching and non-matching trials [p Bayes (matching < nonmatching) = .777].
These results explicate the basis of the self-prioritization effect on perceptual matching (Sui et al., 2012). Compared Note. v = drift rate, z = starting point, t 0 = nondecision processes. A DIC difference of 2 is positive evidence for a model, greater than 10 is strong evidence for a model (Kass & Raftery, 1995).
to other shape-category pairings (i.e., self-one, self-forty, stranger), self-now was characterized by a higher drift rate (v). This provides the first evidence that self-relevance enhances information uptake during perceptual matching . In addition, a bias in decisional processes relating to the position of the starting point (z) was also observed, indicating that participants favored matching over nonmatching responses.
Experiment 2
n Experiment 1, we expected self-prioritization effects to emerge when geometric shapes were associated with one's current (i.e., self-now) and near-future (i.e., self-one) self, but not with conceptions of the self in the distant future (i.e., self-forty). Interestingly, however, a different pattern of results was observed. Although stimulus prioritization was sensitive to temporal influences in self-construal (Pronin et al., 2008;Pronin & Ross, 2006;Trope & Liberman, 2003Wakslak et al., 2008), only shapes associated with the current self yielded enhanced performance, a self-now prioritization effect that was underpinned by a stimulus bias during decisional processing (Sui et al., 2012). It is conceivable, however, that for undergraduates in their early 20s even a single year into the future feels temporally distant (Pronin et al., 2008), thereby eliminating prioritized processing. Acknowledging this possibility, our next study comprised a replication of Experiment 1 but with an important modification. On this occasion, together with current self and stranger, geometric shapes were associated with future self in a day and in a month. Of interest was whether narrowing the future horizon would impact the emergence of the self-prioritization effect. As in Experiment 1, to identify the processes underlying task performance, the data were submitted to a HDDM analysis (Wiecki et al., 2013) Participants and design Sixteen undergraduates (three male, M age = 20.88 years, SD = 2.45) took part in the research, for which they received £5 (~$6.20). All participants had normal or corrected-to-normal visual acuity. Informed consent was obtained from participants prior to the commencement of the experiment, and the protocol was reviewed and approved by the Ethics Committee at the School of Psychology, University of Aberdeen. The experiment had a 4 (shape category: self-now vs. self-day vs. self-month vs. stranger) × 2 (trial type: matching vs. nonmatching) repeated-measures design.
Stimulus materials and procedure
Participants arrived at the laboratory individually, were greeted by an experimenter, and told they would be performing a perception task. The study closely followed Experiment 1 but with an important modification. On this occasion, during the learning phase, participants associated geometric shapes (i.e., circle, horizontal rectangle, cross, diamond) with self-now, self-in-a-day, self-in-a-month, and stranger. During the subsequent perceptual-matching task, participants had to report whether a series of shape-label pairings (e.g., circle & self-day, cross & self-month) were correct (or incorrect) on the basis of the associations learned previously. As in Experiment 1, participants initially performed 18 practice trials, followed by seven blocks of 72 trials in which self-now, self-day, self-month, stranger, and repaired stimuli occurred equally often in a random order. In total, across all the blocks, there were 63 trials in each condition. On completion of the task, participants were debriefed and dismissed.
Results and discussion
Perceptual matching Responses faster than 200 ms were excluded from the analysis, eliminating less than 1% of the overall number of trials. Table 3 shows the accuracy and response time (RT) data. As in Experiment 1, a bootstrapping procedure was adopted to examine the distribution characteristic of perceptual-matching judgments in each condition, combining accuracy and RT performance. The resultant distributions across the different shape-category judgments are displayed in Fig. 4. Whereas the bootstrapped sample mean observations for self-now matching judgments fell in the lower right corner of the figure, all other matching judgments fell in an upper middle location (see Fig. 4a). In contrast, an overlapping distribution of observations emerged for responses to non-matching shapecategory pairs (see Fig. 4b).
Replicating Experiment 1, these results demonstrate a selfprioritization effect on perceptual matching . As previously, however, prioritized processing only emerged when geometric shapes were associated with the current self (i.e., self-now prioritization effect). Even for characterizations of the self in the very near future (i.e., tomorrow, in a month) self-relevance did not facilitate performance (i.e., self = stranger). This self-now prioritization effect corroborates the contention that temporal influences on self-construal influence perceptual matching.
Diffusion modeling
To identify the processes underlying task performance, data were submitted to a HDDM analysis (Wiecki et al., 2013). As in Experiment 1, five additional models were tested for comparison. As can be seen in Table 2, the model that included all three parameters yielded the best fit (i.e., smallest DIC value). Interrogation of the posterior distributions revealed evidence of both stimulus and response biases during perceptual These results directly replicate the pattern of effects reported in Experiment 1. Compared to all other shape-category pairings (i.e., self-day, self-month, stranger), self-now was characterized by a larger drift rate (v), thus supporting the contention that self-relevance enhances information uptake during perceptual matching . As previously, a bias in decisional processes relating to the position of the starting point (z) was also observed. Specifically, participants favored matching over nonmatching responses. Corroborating Experiment 1, these findings confirm that, at least in the context of a shapelabel matching task, self-prioritization is underpinned by a bias in stimulus processing (Sui et al., 2012).
Experiment 3
Thus far, the results have revealed a self-now prioritization effect, such that perceptual-matching is enhanced when one's current self is contrasted with representations of the self in the distant (i.e., Expt. 1) and near (i.e., Expt. 2) future. What this demonstrates is that perceptual matching is influenced by temporally induced differences between one's current and future (i.e., hypothetical) selves (Trope & Liberman, 2003. It is worth noting, however, that the effects of temporal construal also extend to representations of the self in the past, representations grounded in actual experience (e.g., Frank & Gilovich, 1989;Libby & Eibach, 2002;Nigro & Neisser, 1983;Pronin & Ross, 2006;Robinson & Swanson, 1993). Echoing the principles of CLT (Trope & Liberman, 2003)-despite detailed knowledge of one's prior self (e.g., recollections of last week/month/ year)-self-relevant cognitions and memories increase in abstraction as one travels backwards in time (e.g., Broemer, Grabowski, Gebauer, Ermel, & Diehl, 2008;Conway & Pleydell-Pearce, 2000;D'Argembeau & Van der Linden, 2004;Johnson, Foley, Suengas, & Raye, 1988). This then raises an interesting question. Is the self-prioritization effect similarly sensitive to representations of the self in the past? We explored this issue in our final experiment. As previously, a HDDM analysis was used to interrogate the cognitive processes underpinning task performance (Wiecki et al., 2013).
Method Participants and design
Sixteen undergraduates (three male, M age = 20.44 years, SD = 1.67) took part in the research, for which they received £5 (~$6.20). All participants had normal or corrected-to-normal visual acuity. Informed consent was obtained from participants prior to the commencement of the experiment and the protocol was reviewed and approved by the Ethics Committee at the School of Psychology, University of Aberdeen. The experiment had a 4 (shape category: self-now vs. self-day vs. self-month vs. stranger) × 2 (trial type: matching vs. nonmatching) repeated-measures design.
Stimulus materials and procedure
Participants arrived at the laboratory individually, were greeted by a male experimenter, and told they would be performing a perception task. The study closely followed Experiment 2 but with an important modification. On this occasion, during the learning phase, participants associated geometric shapes (i.e., circle, horizontal rectangle, cross, diamond) with self-now, self-a-day-ago, self-a-month-ago, and stranger. During the subsequent perceptual-matching task, participants had to report whether a series of shape-label pairings (e.g., circle & self-day, cross & self-month) were correct (or incorrect) on the basis of the associations learned previously. As in Experiment 2, participants initially performed 18 practice trials, followed by seven blocks of 72 trials in which self-now, self-day, self-month, stranger, and repaired stimuli occurred equally often in a random order. In total, across all the blocks, there were 63 trials in each condition. On completion of the task, participants were debriefed and dismissed.
Perceptual matching
Responses faster than 200 ms were excluded from the analysis, eliminating less than 1% of the overall number of trials. One participant failed to follow the instructions, thus was excluded from the analysis. Table 4 shows the accuracy and response time (RT) data. As in Experiments 1 and 2, a bootstrapping procedure was adopted to examine the distribution characteristic of perceptual-matching judgments in each condition, combining accuracy and RT performance. The resultant distributions across the different shape-category judgments are displayed in Fig. 6. Whereas the bootstrapped sample mean observations for self-now matching judgments fell in the lower right corner of the figure, all other matching Note. RT = reaction time. Standard deviations appear within parentheses judgments fell in an upper middle location (see Fig. 6a). In contrast, an overlapping distribution of observations emerged for responses to nonmatching shape-category pairs (see Fig. 6b). As previously, a signal detection approach was adopted to explore the accuracy of participants' responses. For each shape, performance in the matching and nonmatching conditions were combined to calculate a measure of sensitivity (d') and submitted to a single factor (shape category: self-now vs. self-day vs. self-month vs. stranger) repeated-measures ANOVA (see Fig. 6c). This revealed an effect of shape category, F(3, 42) = 5.79, p = .002, η p 2 = .29, such that d' was larger for self-now than for self-day, t(14) = 3.87, p = .001, d = 1.03; self-month, t(14) = 2.98, p = .012, d = .87; but not for stranger, t(14) = 1.50, p =.21.
Replicating Experiments 1 and 2, these results demonstrate a self-prioritization effect on perceptual matching . As previously, however, prioritized processing only emerged when geometric shapes were associated with the current self (i.e., self-now prioritization effect). Even for characterizations of the self in the immediate past (i.e., yesterday) self-relevance did not facilitate performance. Thus, extended to representations in the past (Trope & Liberman, 2003, this selfnow prioritization effect corroborates the contention that temporal influences on self-construal influence perceptual matching.
Diffusion modeling
To identify the processes underlying task performance, data were submitted to a HDDM analysis (Wiecki et al., 2013). To determine the adequacy of this model, five additional models were tested for comparison. As can be seen in Table 2, the model that included all three parameters yielded the best fit (i.e., smallest DIC value). Interrogation of the posterior distributions revealed evidence of both stimulus and response biases during perceptual matching (see Fig. 7). Specifically, on matching trials, drift rates (v) were higher (i.e., evidence accumulation was faster) for self-now than all other shapecategory pairings [p Bayes (self-now > self-day & self-month & stranger) = 1.0]. This was not the case on nonmatching trials [p Bayes (self-now > self-day & self-month & stranger) = .202]. In addition, comparison of the observed starting value (z = .62) with no bias (i.e., z = 0.5) revealed an a priori bias towards matching responses [p Bayes (bias > 0.5) = 1.0]. Finally, nondecisional processes (t 0 ) yielded no difference between matching and non-matching trials [p Bayes (matching < nonmatching) = .790]. Extended to representations of the self in the past, these results further explicate the basis of the selfprioritization effect on perceptual matching (Sui et al., 2012). Compared to all other shape-category pairings (i.e., self-day, self-month, stranger), self-now was characterized by a higher drift rate (v). This supports the contention that self-relevance enhances information uptake during perceptual matching . In addition, a bias in decisional processes relating to the position of the starting point (z) was also observed, indicating that participants favored matching over non-matching responses. Thus, replicating Experiments 1 and 2, the self- Fig. 6 Distribution of bootstrapped sample means for matching (a) and nonmatching (b) trials (Expt. 3). The x-axis represents the accuracy rates and the y-axis the reaction times. c Shows d' as a function of shape category. Error bars represent +1 SEM prioritization effect during perceptual matching was underpinned by a bias in stimulus processing.
General discussion
A rapidly emerging literature has revealed a pervasive selfrelated bias in perceptual matching Sui et al., 2012;Sui, Liu, et al., 2013;Sui et al., 2014). Specifically, association with the self (vs. friend and stranger) facilitates the processing of otherwise meaningless geometric shapes. Developing this line of inquiry, here we considered the extent and basis of this selfprioritization effect. Across three experiments, a consistent pattern of results was observed. 5 First, prioritized processing was sensitive to temporal influences on self-construal (Pronin et al., 2008;Pronin & Ross, 2006;Trope & Liberman, 2003, such that self-prioritization only emerged when stimuli were associated with the current self (i.e., self-now prioritization effect). This effect, moreover, was insensitive to the direction of temporal construal. A prioritization effect was observed when current self was compared to representations of the self in both the future (i.e., Expts. 1 & 2) and past (i.e., Expt. 3). Second, a HDDM analysis revealed that task performance was underpinned by both stimulus and response biases. While participants favored matching over nonmatching responses in all three experiments-more critically, selfprioritization was underpinned by differences in the rate of information uptake (v). Specifically, self-now was characterized by a higher drift rate than all other shape-category pairings. These findings are noteworthy in the current academic climate as they demonstrate the replicability of the self-prioritization effect during perceptual matching across different temporal directions and timescales.
In showing that temporal influences on self-construal moderate the emergence of the self-prioritization effect , the current work resonates closely with almost 2 decades of research exploring the effects of psychological construal on socialcognitive functioning (Trope & Liberman, 2003. The gist of construal-level theory is quite straightforward. Scaled egocentrically, psychological distance is anchored in one's experience of the self in the here and now. What this means is that as objects and events-including the self-increase in distance (temporal & spatial) from this reference point, 5 It has been suggested that females report more vivid imaginary experiences than males (Richardson, 1991). As participants in the current research used imagery to associate shapes with the self at different points in time, it is possible that the effects observed were amplified by the preponderance of females in each experiment. Future research should consider this possibility. representations decrease in perceptual detail, specificity, and self-relevance. Indeed, on occasion, future selves are characterized and treated like other people (Hershfield, 2011;Mitchell et al., 2011;Parfit, 1971Parfit, , 1987Pronin et al., 2008;Pronin & Ross, 2006;Wakslak et al., 2008). At least in the context of perceptual matching, this self-becomes-other effect was observed only a single day into the future (and past), thereby revealing the primacy of the current self during sensorimotor processing (Conway, 2005).
That the self-prioritization effect was restricted to geometrical shapes associated with the current self underscores the nuanced character of self-referential processing (McConnell, 2011). A basic function of the self is to guide people's interactions with the world (Higgins, 1996). This can best be achieved not through the deployment of a rigid, unitary entity , but rather through the application of context-dependent representations (i.e., multiple selves) that are sensitive to the demands of the immediate situation (Ainslie, 1992;Kurzban & Aktipis, 2007;H. R. Markus & Nurius, 1986;McConnell, 2011;Parfit, 1971;Schelling, 1984;Thaler & Shefrin, 1981). From this perspective, it is unsurprising that temporal factors influence stimulus prioritization. By necessity, the version of the self that is called upon in immediate task contexts serves a quite distinct function to the one that simulates future events and experiences (Gilbert & Wilson, 2007Smallwood & Schooler, 2006). In particular, in the here and now, greater emphasis is placed on the construction of perceptually rich event (and self) representations (Trope & Liberman, 2003. Echoing this viewpoint, Conway and Pleydell-Pearce (2000) characterize the current self as a complex set of active goals and associated self-constructs in working memory that guide cognition flexibility and adaptively from one moment to the next (see also Conway, 2005). As demonstrated herein, the outputs of the current self also extend to perceptual processing. Consistent with the tenets of construal-level theory (Trope & Liberman, 2003, the self-prioritization effect in perceptual matching was confined to stimuli associated with the current self. In the available research to date, the self-prioritization effect is considered to be a perceptual phenomenon. Noting how perception can seemingly be modified by characteristics of the observer-including desires, values, and expectancies (e.g., Collins & Olson, 2014;Dunning & Balcetis, 2013;Lupyan, 2015;Vetter & Newen, 2014)-self-relevance is believed to exert a comparable influence on stimulus processing Sui et al., 2012;. A primary purpose of the current investigation was therefore to explore the processes underpinning task performance during perceptual matching using a HDDM approach (Wiecki et al., 2013). This confirmed that decisional evidence was accumulated most rapidly when geometric shapes were associated with self-now than all other shape-label pairings. What this reveals is that, at least in the context of perceptual matching, self-relevance influences stimulus processing (i.e., the rate of information uptake) during decision making . What remains to be seen, however, is whether self-relevance operates in a comparable way in other judgmental settings (e.g., Macrae, Visokomogilski, Golubickis, Cunningham, & Sahraie, 2017). Future research should explore this issue.
Here, we have shown when and how temporal influences on self-construal moderate the emergence of the selfprioritization effect during a shape-label matching task (Sui et al., 2012). Of course, the effects of self-relevance likely extend well beyond such a laboratory-based activity. Take, for example, object classification. Continual interaction with a complex and demanding environment necessitate that a fundamental function of the self is to classify objects based on their personal significance and meaning (e.g., mine vs. not mine, goal-relevant vs. goal-irrelevant; Berlad & Pratt, 1995;Constable, Kritilos, & Bayliss, 2011;Constable, Kritikos, Lipp, & Bayliss, 2014;Fischler, Jin, Boaz, Perry, & Childers, 1987;Folmer & Yingling, 1997;Gray, Ambady, Lowenthal, & Deldin, 2004;Miyakoshi, Nomura, & Ohira, 2007;Müller & Kutas, 1996). One's possessions are an obvious case in point, as evidenced by the fact that even young children understand the concept of ownership and afford it social significance (Fasig, 2000;Furby, 1980;Hay, 2006). When the perils of misappropriating other people's effects (e.g., pint of beer) can be substantial (e.g., an indecorous altercation in the pub), the ability to discriminate items that one owns from items that one does not is an invaluable skill (Constable et al., 2011;Constable et al., 2014). As such, one might expect ownership to enhance object classification.
In considering the wider implications of the current findings, other core facets of self-construal likely play a prominent role in determining the products of selfreferential processing. As noted, each person's self comprises a collection of multiple, context-dependent identities (e.g., humanitarian, golfer, curry lover) that vary in both centrality and significance (Higgins, 1987; H. R. Markus & Nurius, 1986;McConnell, 2011;Roberts & Donahue, 1994). Acknowledging this representational complexity, theories of identity-based motivation contend that when a specific component of the self is activated (i.e., a person's working self), processing resources are preferentially allocated to stimuli that bolster and enhance that identity if it is important to the individual (Oyserman, 2007(Oyserman, , 2009. Thus, attention is directed toward identity-consistent stimuli, while inconsistent information is neglected or downplayed (Berger & Heath, 2007;Coleman & Williams, 2015). The utility of this account lies in the flexibility it affords perceptual processing in complex settings. If stimulus relevance is identity-dependent, then a specific object (e.g., cheesecake) has the potential to modulate perceptual processing depending on which of an individual's multiple identities has been activated (e.g., dieter vs. dessert lover). A useful task for future research will therefore be to investigate the effects of identity activation and identity strength on the associations forged between the self and meaningful objects (cf. geometric shapes) in consequential task contexts. In addition, extending the age range of participants to include older adults may be revealing, especially for retrospective representations of the self that may be shaped by identity-relevant knowledge.
Beyond shape-label associations, recent work has demonstrated that the self-prioritization effect in perceptual matching extends to action planning. Modifying Sui et al.'s (2012) paradigm, Frings and Wentura (2014) initially required participants to associate labels (i.e., self, mother, stranger, no label) with arbitrarily assigned movements (i.e., up, down, left, right). During the subsequent testing phase, following execution of a cued movement, one of the four labels appeared and participants had to indicate whether the action and label matched their earlier learning experience. Extending Sui et al. (2012), a self-prioritization effect emerged such that performance was better for self-relevant actions than all other label-action pairings. As in the current investigation, it is possible that temporal influences on event construal may moderate the elicitation of this self-action prioritization effect (Frings & Wentura, 2014). Supported by an extensive literature, action planning is shaped by the timing of an event (Fujita, Henderson, Eng, Trope, & Liberman, 2006;Liberman & Trope, 1998;Liviatan, Trope, & Liberman, 2008;Trope & Liberman, 2003Vallacher & Wegner, 1989;Wakslak, Trope, Liberman, & Alony, 2006). Consider, for example, the act of traveling into town to purchase an expensive single malt whisky. If the event is about to happen in the here and now, low-level construal will generate details of how one's objective can best be accomplished (e.g., getting into the car, parking at the liquor store, locating malts from Speyside). In contrast, the same activity in the future will tend to be characterized in a decontextualized way (i.e., high-level construal) that emphasizes only the overall meaning of the episode (e.g., purchasing a birthday gift). By implication, one might expect these temporal differences in the specificity of action planning to influence the emergence of self-prioritization effects (Frings & Wentura, 2014)
Conclusion
In sum, here we demonstrated a critical determinant of the self-prioritization effect during perceptual matching (Sui et al., 2012) and identified the cognitive process through which this phenomenon arises. Specifically, stimulus prioritization was sensitive to temporal influences in selfconstrual (Trope & Liberman, 2003, such that only the association of geometric shapes with one's current (vs. future/past) self facilitated task performance. In addition, drift diffusion modeling revealed that self-prioritization was underpinned by a stimulus bias (White & Poldrack, 2014).
What has yet to be established, however, is whether comparable effects emerge in other task contexts in which stimuli vary in self-relevance.
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. | 2018-04-03T06:04:44.108Z | 2017-06-07T00:00:00.000 | {
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235359086 | pes2o/s2orc | v3-fos-license | Regularization in ResNet with Stochastic Depth
Regularization plays a major role in modern deep learning. From classic techniques such as L1,L2 penalties to other noise-based methods such as Dropout, regularization often yields better generalization properties by avoiding overfitting. Recently, Stochastic Depth (SD) has emerged as an alternative regularization technique for residual neural networks (ResNets) and has proven to boost the performance of ResNet on many tasks [Huang et al., 2016]. Despite the recent success of SD, little is known about this technique from a theoretical perspective. This paper provides a hybrid analysis combining perturbation analysis and signal propagation to shed light on different regularization effects of SD. Our analysis allows us to derive principled guidelines for choosing the survival rates used for training with SD.
Introduction
Stochastic Depth (SD) is a well-established regularization method that was first introduced by Huang et al. [2016]. It is similar in principle to Dropout [Hinton et al., 2012, Srivastava et al., 2014 and DropConnect [Wan et al., 2013]. It belongs to the family of noise-based regularization techniques, which includes other methods such as noise injection in data [Webb, 1994, Bishop, 1995 and noise injection throughout the network [Camuto et al., 2020]. While Dropout, resp. DropConnect consists of removing some neurons, resp. weights, at each iteration, SD randomly drops full layers, and only updates the weights of the resulting subnetwork at each training iteration. As a result of this mechanism, SD can be exclusively used with residual neural networks (ResNets).
There exists a stream of papers in the literature on the regularization effect of Dropout for linear models [Wager et al., 2013, Mianjy and Arora, 2019, Helmbold and Long, 2015, Cavazza et al., 2017. Recent work by Wei et al. [2020] extended this analysis to deep neural networks using second-order perturbation analysis. It disentangled the explicit regularization of Dropout on the loss function and the implicit regularization on the gradient. Similarly, Camuto et al. [2020] studied the explicit regularization effect induced by adding Gaussian Noise to the activations and empirically illustrated the benefits of this regularization scheme. However, to the best of our knowledge, no analytical study of SD exists in the literature. This paper aims to fill this gap by studying the regularization effect of SD from an analytical point of view; this allows us to derive principled guidelines on the choice of the survival probabilities for network layers. Concretely, our contributions are four-fold: • We show that SD acts as an explicit regularizer on the loss function by penalizing a notion of information discrepancy between keeping and removing certain layers.
• We prove that the uniform mode, defined as the choice of constant survival probabilities, is related to maximum regularization using SD.
• We study the large depth behaviour of SD and show that in this limit, SD mimics Gaussian Noise Injection by implicitly adding data-adaptive Gaussian noise to the pre-activations.
• By defining the training budgetL as the desired average depth, we show the existence of two different regimes: small budget and large budget regimes. We introduce a new algorithm called SenseMode to compute the survival rates under a fixed training budget and provide a series of experiments that validates our Budget hypothesis introduced in Section 5.
Stochastic Depth Neural Networks
Stochastic depth neural networks were first introduced by Huang et al. [2016]. They are standard residual neural networks with random depth. In practice, each block in the residual network is multiplied by a random Bernoulli variable δ l (l is the block's index) that is equal to 1 with some survival probability p l and 0 otherwise. The mask is re-sampled after each training iteration, making the gradient act solely on the subnetwork composed of blocks with δ l = 1.
We consider a slightly different version where we apply the binary mask to the pre-activations instead of the activations. We define a depth L stochastic depth ResNet by y 0 (x; δ) = Ψ 0 (x, W 0 ), y l (x; δ) = y l−1 (x; δ) + δ l Ψ l (y l−1 (x; δ), W l ), 1 ≤ l ≤ L, y out (x; δ) = Ψ out (y L (x; δ), W out ), where W l are the weights in the l th layer, Ψ is a mapping that defines the nature of the layer, y l are the pre-activations, and δ = (δ l ) 1≤l≤L is a vector of Bernoulli variables with survival parameters p = (p l ) 1≤l≤L . δ is re-sampled at each iteration. For the sake of simplification, we consider constant width ResNet and we further denote by N the width, i.e. for all l ∈ [L − 1], y l ∈ R N . The output function of the network is given by s(y out ) where s is some convenient mapping for the learning task, e.g. the Softmax mapping for classification tasks. We denote by o the dimension of the network output, i.e. s(y out ) ∈ R o which is also the dimension of y out . For our theoretical analysis, we consider a Vanilla model with residual blocks composed of a Fully Connected linear layer where φ(x) is the activation function. The weights are initialized with He init [He et al., 2015], e.g. for ReLU, W l ij ∼ N (0, 2/N ). There are no principled guidelines on choosing the survival probabilities. However, the original paper by Huang et al. [2016] proposes two alternatives that appear to make empirical consensus: the uniform and linear modes, described by Uniform: p l = p L , Linear: p l = 1 − l L (1 − p L ), where we conveniently parameterize both alternatives using p L .
Training budgetL. We define the training budgetL to be the desired average depth of the subnetworks with SD. The user typically fixes this budget, e.g., a small budget can be necessary when training is conducted on small capacity devices.
Depth of the subnetwork. Given the mode p = (p l ) 1≤l≤L , after each iteration, the subnetwork has a depth L δ = L l=1 δ l with an average L p := E δ [L δ ] = L l=1 p l . Given a budgetL, there is a infinite number of modes p such that L p =L. In the next lemma, we provide probabilistic bounds on L δ using standard concentration inequalities. We also show that with a fixed budgetL, the uniform mode is linked to maximal variability. Lemma 1 (Concentration of L δ ). For any β ∈ (0, 1), we have that with probability at least 1 − β, where L p = E[L δ ] = Lemma 1 shows that with high probability, the depth of the subnetwork that we obtain with SD is within an 1 error of v p u −1 log(2/β) vp from the average depth L p . Given a fixed budgetL, this segment is maximized for the uniform mode p = (L/L, . . . ,L/L). Fig. 1a highlights this result. This was expected since the variance of the depth L δ is also maximized by the uniform mode. The uniform mode corresponds to maximum entropy of the random depth, which would intuitively results in maximum regularization. We depict this behaviour in more details in Section 4. SD vs Dropout. From a computational point of view, SD has the advantage of reducing the effective depth during training. Depending on the chosen budget, the subnetworks might be significantly shallower than the entire network (Lemma 1). This depth compression can be effectively leveraged for computational training time gain (Fig. 1b). It is not the case with Dropout. Indeed, assuming that the choice of dropout probabilities is such that we keep the same number of parameters on average compared to SD, we still have to multiply matrices L times during the forward/backward propagation. It is not straightforward to leverage the sparsity obtained by Dropout for computational gain. In practice, Dropout requires an additional step of sampling the mask for every neuron, resulting in longer training times than without Dropout (Fig. 1b). However, there is a trade-off between how small the budget is and the performance of the trained model with SD (Section 6).
Effect of Stochastic Depth at initialization
Empirical evidence strongly suggests that Stochastic Depth allows training deeper models [Huang et al., 2016]. Intuitively, at each iteration, SD updates only the parameters of a subnetwork with average depth L p = l p l < L, which could potentially alleviate any exploding/vanishing gradient issue. This phenomenon is often faced when training ultra deep neural networks. To formalize this intuition, we consider the model's asymptotic properties at initialization in the infinite-width limit N → +∞. This regime has been the focus of several theoretical studies [Neal, 1995, Poole et al., 2016, Schoenholz et al., 2017, Yang, 2020, Xiao et al., 2018, Hayou et al., 2019, 2020, 2021a since it allows to derive analytically the distributions of different quantities of untrained neural networks. Specifically, randomly initialized ResNets, as well as other commonly-used architectures such as Fully connected Feedforward networks, convolutional networks and LSTMs, are equivalent to Gaussian Processes in the infinite-width limit. An important ingredient in this theory is the Gradient Independence assumption. Let us formally state this assumption first.
Assumption 1 (Gradient Independence). In the infinite width limit, we assume that the weights W used for back-propagation are an iid version of the weights W used for forward propagation.
Assumption 1 is ubiquitous in the literature on the signal propagation in deep neural networks. It has been used to derive theoretical results on signal propagation in randomly initialized deep neural network [Schoenholz et al., 2017, Poole et al., 2016, Yang and Schoenholz, 2017, Hayou et al., 2021b and is also a key tool in the derivation of the Neural Tangent Kernel [Jacot et al., 2018, Arora et al., 2019, Hayou et al., 2020. Recently, it has been shown by Yang [2020] that Assumption 1 yields the exact computation for the gradient covariance in the infinite width limit. See Appendix A0.3 for a detailed discussion about this assumption. Throughout the paper, we provide numerical results that substantiate the theoretical results that we derive using this assumption. We show that Assumption 1 yields an excellent match between theoretical results and numerical experiments.
Leveraging this assumption, Yang and Schoenholz [2017], Hayou et al. [2021a] proved that ResNet suffers from exploding gradient at initialization. We show in the next proposition that SD helps mitigate the exploding gradient behaviour at initialization in infinite width ResNets.
is some differentiable loss function. Letq l (x, z) = E W,δ ∇y l L 2 ∇y L L 2 , where the numerator and denominator are respectively the norms of the gradients with respect to the inputs of the l th and L th layers . Then, in the infinite width limit, under Assumption 1, for all l ∈ • Without Stochastic Depth (i.e. δ = 1),q l (x, z) = 2 L−l . Proposition 1 indicates that with or without SD, the gradient explodes exponentially at initialization as it backpropagates through the network. However, with SD, the exponential growth is characterized by the mode p. Intuitively, if we choose p l 1 for some layer l, then the contribution of this layer in the exponential growth is negligible since 1 + p l ≈ 1. From a practical point of view, the choice of p l 1 means that the l th layer is hardly present in any subnetwork during training, thus making its contribution to the gradient negligible on average (w.r.t δ). For a ResNet with L = 50 and uniform mode p = (1/2, . . . , 1/2), SD reduces the gradient exploding by six orders of magnitude. Fig. 2a and Fig. 2b illustrates the exponential growth of the gradient for the uniform and linear modes, as compared to the growth of the gradient without SD. We compare the empirical/theoretical growth rates of the magnitude of the gradient in Table 1; the results show a good match between our theoretical result (under Assumption 1) and the empirical ones. Further analysis can be found in Section A4.
Stable ResNet. Hayou et al. [2021a] have shown that introducing the scaling factor 1/ √ L in front of the residual blocks is sufficient to avoid the exploding gradient at initialization, as illustrated in Figure 2c. The hidden layers in Stable ResNet (with SD) are given by, The intuition behind the choice of the scaling factor 1/ √ L comes for the variance of y l . At initialization, with standard ResNet (Eq. (1) Hayou et al. [2021a] for more details). In the rest of the paper, we restrict our analysis to Stable ResNet; this will help isolate the regularization effect of SD in the limit of large depth without any variance/gradient exploding issue.
Nevertheless, the natural connection between SD and Dropout, coupled with the line of work on the regularization effect induced by the latter [Wager et al., 2013, Mianjy and Arora, 2019, Helmbold and Long, 2015, Cavazza et al., 2017, Wei et al., 2020, would indicate that the benefits of SD are not limited to controlling the magnitude of the gradient. Using a second order Taylor expansion, Wei et al. [2020] have shown that Dropout induces an explicit regularization on the loss function. Intuitively, one should expect a similar effect with SD. In the next section, we elucidate the explicit regularization effect of SD on the loss function, and we shed light on another regularization effect of SD that occurs in the large depth limit.
Explicit regularization on the loss function
be a smooth loss function, e.g. quadratic loss, crossentropy loss etc. Define the model loss for a single sample (x, t) ∈ D by where W = (W l ) 0≤l≤L . The empirical loss given by L To isolate the regularization effect of SD on the loss function, we use a second order approximation of the loss function around δ = 1, this allows us to marginalize out the mask δ. The full derivation is provided in Appendix A2. Let z l (x; δ) = Ψ l (W l , y l−1 (x; δ)) be the activations. For some pair (x, t) ∈ D, we obtain whereL(W , x) ≈ (y out (x; p), t) (more precisely,L(W , x) is the second order Taylor approximation of (y out (x; p), t) around p = 1 2 ), and g l (W , x) = z l (x; 1) T ∇ 2 y l [ • G l ](y l (x; 1))z l (x; 1) with G l is the function defined by y out (x; 1) = G l (y l−1 (x; 1) + 1 √ L z l (x; 1)). The first termL(W , x) in Eq. (4) is the loss function of the average network (i.e. replacing δ with its mean p). Thus, Eq. (4) shows that training with SD entails training the average network with an explicit regularization term that implicitly depends on the weights W .
SD enforces flatness. The presence of the hessian in the penalization term provides a geometric interpretation of the regularization induced by SD: it enforces a notion of flatness determined by the hessian of the loss function with respect to the hidden activations z l . This flatness is naturally inherited by the weights, thus leading to flatter minima. Recent works by [Keskar et al., 2016, Jastrzebski et al., 2018, Yao et al., 2018 showed empirically that flat minima yield better generalization compared to minima with large second derivatives of the loss. Wei et al. [2020] have shown that a similar behaviour occurs in networks with Dropout.
Let J l (x) = ∇ y l G l (y l (x; 1)) be the Jacobian of the output layer with respect to the hiden layer y l with δ = 1, and H (x) = ∇ 2 z (z) |z=yout(x;1) the hessian of the loss function . The hessian matrix inside the penalization terms g l (W , x) can be decomposed as in [LeCun et al., 2012, Sagun et al., 2017 ∇ 2 y l [ • G l ](y l (x; 1)) = J l (x) T H (x)J l (x) + Γ l (x), where Γ depends on the hessian of the network output. Γ is generally non-PSD, and therefore cannot be seen as a regularizer. Moreover, it has been shown empirically that the first term generally dominates and drives the regularization effect [Sagun et al., 2017, Wei et al., 2020, Camuto et al., 2020. Thus, we restrict our analysis to the regularization effect induced by the first term, and we consider the new version of g l defined by where ζ l (x, W ) = J l (x)z l (x; 1). The quality of this approximation is discussed in Appendix A4.
Information discrepancy. The vector ζ l represents a measure of the information discrepancy between keeping and removing the l th layer. Indeed, ζ l measures the sensitivity of the model output to the l th layer, where 1 l is the vector of 1 s everywhere with 0 in the l th coordinate.
With this in mind, the regularization term g l in Eq. (5) is most significant when the information discrepancy is well-aligned with the hessian of the loss function, i.e. SD penalizes mostly the layers with information discrepancy that violates the flatness, confirming our intuition above.
Quadratic loss. With the quadratic loss (z, z ) = z − z 2 2 , the hessian H (x) = 2I is isotropic, i.e. it does not favorite any direction over the others. Intuitively, we expect the penalization to be similar for all the layers. In this case, we have g l (W , x) = 2 ζ l (x) 2 2 , and the loss is given by where g l (W ) = 2 n n i=1 ζ l (x i , W ) 2 2 is the regularization term marginalized over inputs X . Eq. (6) shows that the mode p has a direct impact on the regularization term induced by SD. The latter tends to penalize mostly the layers with survival probability p l close to 50%. The mode p = (1/2, . . . , 1/2) is therefore a universal maximizer of the regularization term, given fixed weights W . However, given a training budgetL, the mode p that maximizes the regularization term 1 2L L l=1 p l (1 − p l )g l (W ) depends on the values of g l (W ). We show this in the next lemma. Lemma 2 (Max regularization). Consider the empirical loss L given by Eq. (6) for some fixed weights W (e.g. W could be the weights at any training step of SGD). Then, given a training budgetL, the regularization is maximal for p * l = min 1, max(0, 1 2 − Cg l (W ) −1 ) , where C is a normalizing constant, that has the same sign as L − 2L. The global maximum is obtained for p l = 1/2. Lemma 2 shows that under fixed budget, the mode p * that maximizes the regularization induced by SD is generally layerdependent ( = uniform). However, we show that at initialization, on average (w.r.t W ), p * is uniform. Theorem 1 (p * is uniform at initialization). Assume φ = ReLU and W are initialized with N (0, 2 N ). Then, in the infinite width limit, under Assumption 1, for all l ∈ [1 : L], we have As a result, given a budgetL, the average regularization term is maximal for the uniform mode p * = (L/L, . . . ,L/L).
The proof of Theorem 1 is based on some results from the signal propagation theory in deep neural network. We provide an overview of this theory in Appendix A0. Theorem 1 shows that, given a training budgetL and a randomly initialized ResNet with N (0, 2/N ) and N large, the average (w.r.t W ) maximal regularization at initialization is almost achieved by the uniform mode. This is because the coefficients E W [g l (W )] are equal under Assumption 1, which we highlight in Fig. 3. As a result, we would intuitively expect that the uniform mode performs best when the budgetL is large, e.g. L is large andL ≈ L, since in this case, at each iteration, we update the weights of an overparameterized subnetwork, which would require more regularization compared to the small budget regime. We formalize this intuition in Section 5.
In the next section, we show that SD is linked to another regularization effect that only occurs in the large depth limit; in this limit, we show that SD mimics Gaussian Noise Injection methods by adding Gaussian noise to the pre-activations.
Stochastic Depth mimics Gaussian noise injection
Recent work by Camuto et al. [2020] studied the regularization effect of Gaussian Noise Injection (GNI) on the loss function and showed that adding isotropic Gaussian noise to the activations z l improves generalization by acting as a regularizer on the loss. The authors suggested adding a zero mean Gaussian noise parameterized by its variance. At training time t, this translates to replacing z t l by z t l + N (0, σ 2 l I), where z t l is the value of the activations in the l th layer at training time t, and σ 2 l is a parameter that controls the noise level. Empirically, adding this noise tends to boost the performance by making the model robust to over-fitting. Using similar perturbation analysis as in the previous section, we show that when the depth is large, SD mimics GNI by implicitly adding a non-isotropic data-adaptive Gaussian noise to the pre-activations y l at each training iteration. We bring to the reader's attention that the following analysis holds throughout the training (it is not limited to the initialization), and does not require the infinite-width regime.
Consider an arbitrary neuron y i αL in the (αL) th layer for some fixed α ∈ (0, 1). y i αL (x, δ) can be approximated using a first order Taylor expansion around δ = 1. We obtain similarly, can therefore be seen as a perturbed version of y i αL (x; p) (the pre-activation of the average network) with noise γ α,L (x). The scaling factor 1/ √ L ensures that γ α,L remains bounded (in 2 norm) as L grows. Without this scaling, the variance of γ α,L will generally explode. The term γ α,L captures the randomness of the binary mask δ, which up to a factor α, resembles to the scaled mean in Central Limit Theorem(CLT) and can be written as γ α, ) . Ideally, we would like to apply CLT to conclude on the Gaussianity of γ α,L (x) in the large depth limit. However, the random variables X l are generally not i.i.d (they have different variances) and they also depend on L. Thus, standard CLT argument fails. Fortunately, there is a more general form of CLT known as Lindeberg's CLT which we use in the proof of the next theorem.
lim
exists and is finite. There is a clear downtrend as the depth increases; this trend is consistent throughout training, which supports the validity of the second condition in Theorem 2 at all training times. Theorem 2 shows that training a ResNet with SD involves implicitly adding the noise γ α,L (x) to y i αL . This noise becomes asymptotically normally distributed 3 , confirming that SD implicitly injects input-dependent Gaussian noise in this limit. Camuto et al. [2020] studied GNI in the context of input-independent noise and concluded on the benefit of such methods on the overall performance of the trained network. We empirically confirm the results of Theorem 2 in Section 6 using different statistical normality tests.
Similarly, we study the implicit regularization effect of SD induced on the gradient in Appendix A3, and show that under some assumptions, SD acts implicitly on the gradient by adding Gaussian noise in the large depth limit.
The Budget Hypothesis
We have seen in Section 4 that given a budgetL, the uniform mode is linked to maximal regularization with SD at initialization (Theorem 1). Intuitively, for fixed weights W , the magnitude of standard regularization methods such as . 1 or . 2 correlates with the number of parameters; the larger the model, the bigger the penalization term. Hence, in our case, we would require the regularization term to correlate (in magnitude) with the number of parameters, or equivalently, the number of trainable layers. Assuming L 1, and given a fixed budgetL, the number of trainable layers at each training iteration is close toL (Lemma 1). Hence, the magnitude of the regularization term should depend on how large/small the budgetL is, as compared to L. Small budget regime (L/L 1). In this regime, the effective depth L δ of the subnetwork is small compared to L. As the ratioL/L gets smaller, the sampled subnetworks become shallower, suggesting that the regularization need not be maximal in this case, and therefore p should not be uniform in accordance with Theorem 1. Another way to look at this is through the bias-variance trade-off principle. Indeed, asL/L → 0, the variance of the model decreases (and the bias increases), suggesting less regularization is needed. The increase in bias inevitably causes a deterioration of the performance; we call this the Budget-performance trade-off. To derive a more sensible choice of p for small budget regimes, we introduce a new Information Discrepancy based algorithm (Section 4). We call this algorithm Sensitivity Mode or briefly SenseMode. This algorithm works in two steps: 1. Compute the sensitivity (S) of the loss w.r.t the layer at initialization using the approximation, S l is a measure of the sensitivity of the loss to keeping/removing the l th layer. 2. Use a mapping ϕ to map S to the mode, p = ϕ(S), where ϕ is a linear mapping from the range of S to [p min , 1] and p min is the minimum survival rate (fixed by the user).
Large budget regime (L/L ∼ 1). In this regime, the effective depth L δ of the subnetworks is close to L, and thus, we are in the overparameterized regime where maximal regularization could boost the performance of the model by avoiding over-fitting. Thus, we anticipate the uniform mode to perform better than other alternatives in this case. We are now ready to formally state our hypothesis, Budget hypothesis. Assuming L 1, the uniform mode outperforms SenseMode in the large budget regime, while SenseMode outperforms the uniform mode in the small budget regime.
We empirically validate the Budget hypothesis and the Budget-performance trade-off in Section 6.
Experiments
The objective of this section is two-fold: we empirically verify the theoretical analysis developed in sections 3 and 4 with a Vanilla ResNet model on a toy regression task; we also empirically validate the Budget Hypothesis on the benchmark datasets CIFAR-10 and CIFAR-100 [Krizhevsky et al., 2009]. Notebooks and code to reproduce all experiments, plots and tables presented are available in the supplementary material. We perform comparisons at constant training budgets.
Implementation details: Vanilla Stable ResNet is composed of identical residual blocks each formed of a Linear layer followed by ReLU. Stable ResNet110 follows [He et al., 2016, Huang et al., 2016; it comprises three groups of residual blocks; each block consists of a sequence Convolution-BatchNorm-ReLU-Convolution-BatchNorm. We build on an open-source implementation of standard ResNet 4 . We scale the blocks using a factor 1/ √ L as described in Section 3. The toy regression task consists of estimating the function f β : x → sin(β T x), where the inputs x and parameter β are in R 256 , sampled from a standard Gaussian. CIFAR-10, CIFAR-100 contain 32-by-32 color images, representing respectively 10 and 100 classes of natural scene objects. We present here our main conclusions. Further implementation details and other insightful results are in the Appendix A4.
Gaussian Noise Injection: We proceed by empirically verifying the Gaussian behavior of the neurons as described in Theorem 4. For each input x, we sample 200 masks and the corresponding y(x; δ). We then compute the p-value pv x of the Shapiro-Wilk test of normality [Shapiro and Wilk, 1965]. In Fig. 5 we represent the distribution of the p-values {pv x | x ∈ X }. We can see that the Gaussian behavior holds throughout training (left). On the right part of the Figure, we can see that the Normal behavior becomes accurate after approximately 20 layers. In the Appendix we report further experiments with different modes, survival rates, and a different test of normality to verify both Theorem 2 and the critical assumption 2.
Empirical verification of the Budget Hypothesis: We compare the three modes: Uniform, Linear, and SenseMode on two benchmark datasets using a grid survival proportions. The values and standard deviations reported are obtained using four runs. For SenseMode, we use the simple rule p l ∝ |S l |, where S l is the sensitivity (see section 5). We report in Table 2 the results for Stable ResNet110. Results with Stable ResNet56 are reported in Appendix A4. The empirical results are coherent with the Budget Hypothesis. When the training budget is large, i.e.L/L ≥ 0.5, the Uniform mode outperforms the others. We note nevertheless that whenL/L ≥ 0.9, the Linear and Uniform models have similar performance. This seems reasonable as the Uniform and Linear probabilities become very close for such survival proportions. When the budget is low, i.e. L/L < 0.5, the SenseMode outperforms the uniform one (the linear mode cannot be used with budgetsL < L/2 when L 1, since p l /L > 1/2 − 1/(2L) ∼ 1/2), thus confirming the Budget hypothesis. Table 2 also shows a clear Budget-performance trade-off.
Related work
The regularization effect of Dropout in the context of linear models has been the topic of a stream of papers [Wager et al., 2013, Mianjy and Arora, 2019, Helmbold and Long, 2015, Cavazza et al., 2017. This analysis has been recently extended to neural networks by Wei et al. [2020] where authors used a similar approach to ours to depict the explicit and implicit regularization effects of Dropout. To the best of our knowledge, our paper is the first to provide analytical results for the regularization effect of SD, and study the large depth behaviour of SD, showing that the latter mimics Gaussian Noise Injection [Camuto et al., 2020]. A further analysis of the implicit regularization effect of SD is provided in Appendix A3.
Limitations and extensions
In this work, we provided an analytical study of the regularization effect of SD using a second order Taylor approximation of the loss function. Although the remaining higher order terms are usually dominated by the second order approximation (See the quality of the second order approximation in Appendix A4), they might also be responsible for other regularization effects. This is a common limitation in the literature on noise-based regularization in deep neural networks [Camuto et al., 2020, Wei et al., 2020. Further research is needed to isolate the effect of higher order terms. We also believe that SenseMode opens an exciting research direction knowing that the low budget regime has not been much explored yet in the literature. We believe that one can probably get even better results with more elaborate maps ϕ such that p = ϕ(S). Another interesting extension of an algorithmic nature is the dynamic use of SenseMode throughout training. We are currently investigating this topic which we leave for future work.
Appendix A0 An overview of signal propagation in wide neural networks
In section, we review some results and tools from the theory of signal of propagation in wide neural networks. This will prove valuable in the rest of the appendix.
A0.1 Neural Network Gaussian Process (NNGP)
Standard ResNet without SD. Consider a standard ResNet architecture with L layers. The forward propagation of some input x ∈ R d is given by where W l are the weights in the l th layer, Ψ is a mapping that defines the nature of the layer, and y l are the pre-activations. we consider constant width ResNet and we further denote by N the width, i.e. for all l ∈ [L − 1], y l ∈ R N . The output function of the network is given by s(y out ) where s is some convenient mapping for the learning task, e.g. the Softmax mapping for classification tasks. We denote by o the dimension of the network output, i.e. s(y out ) ∈ R o which is also the dimension of y out . For our theoretical analysis, we consider residual blocks composed of a Fully Connected linear layer where φ(x) is the activation function. The weights are initialized with He init He et al. [2015], e.g. for ReLU, W l ij ∼ N (0, 2/N ). The neurons {y i 0 (x)} i∈[1:N ] are iid normally distributed random variables since the weights connecting them to the inputs are iid normally distributed. Using the Central Limit Theorem, as N 0 → ∞, y i 1 (x) becomes a Gaussian variable for any input x and index i ∈ [1 : N ]. Additionally, the variables {y i 1 (x)} i∈[1:N ] are iid. Thus, the processes y i 1 (.) can be seen as independent (across i) centred Gaussian processes with covariance kernel Q 1 . This is an idealized version of the true process corresponding to letting width N 0 → ∞. Doing this recursively over l leads to similar results for y i l (.) where l ∈ [1 : L], and we write accordingly y i l ind ∼ GP(0, Q l ). The approximation of y i l (.) with a Gaussian process was first proposed by [Neal, 1995] for single layer FeedForward neural networks and was extended recently to multiple feedforward layers by [Lee et al., 2019] and [Matthews et al., 2018]. More recently, excellent work by [Yang, 2020] introduced a unifying framework named Tensor Programs, confirming the large-width Gaussian Process behaviour for nearly all neural network architectures.
For any input x ∈ R d , we have E[y i l (x)] = 0, so that the covariance kernel is given by Q l (x, x ) = E[y 1 l (x)y 1 l (x )]. It is possible to evaluate the covariance kernels layer by layer, recursively. More precisely, assume that y i l−1 is a Gaussian process for all i. Let x, x ∈ R d . We have that
Some terms vanish because E[W 1j
l ] = 0. Let Z j = N 2 W 1j l . The second term can be written as where we have used the Central Limit Theorem. Therefore, the kernel Q l satisfies for all . For the ReLU activation function φ : x → max(0, x), the recurrence can be written more explicitly as in [Hayou et al., 2019]. Let C l be the correlation kernel, defined as and let f : [−1, 1] → R be given by The recurrence relation reads Standard ResNet with SD. The introduction of the binary mask δ in front of the residual blocks slightly changes the recursive expression of the kernel Q l . It ii easy to see that with SD, the Q l follows where f is given by Eq. (A3).
We obtain similar formulas for Stable ResNet with and without SD.
Stable ResNet without SD.
(A6)
Stable ResNet with SD. • Standard ResNet without SD: Proof. Let us prove the result for Standard ResNet with SD. The proof is similar for the other cases. Let x ∈ R d . We know that where f is given by Eq. (A3). It is straightforward that f (1) = 1. This yields we conclude by telescopic product.
A0.3 Assumption 1 and gradient backpropagation
For gradient back-propagation, an essential assumption in the literature on signal propagation analysis in deep neural networks is that of the gradient independence which is similar in nature to the practice of feedback alignment [Lillicrap et al., 2016]. This assumption (Assumption 1) allows for derivation of recursive formulas for gradient back-propagation, and it has been extensively used in literature and empirically verified; see references below.
Gradient Covariance back-propagation. Assumption 1 was used to derive analytical formulas for gradient covariance back-propagation in a stream of papers; to cite a few, [Hayou et al., 2019, ?, Poole et al., 2016, Xiao et al., 2018, Yang and Schoenholz, 2017. It was validated empirically through extensive simulations that it is an excellent tool for FeedForward neural networks in Schoenholz et al. [2017], for ResNets in Yang and Schoenholz [2017] and for CNN in Xiao et al. [2018].
Neural Tangent Kernel (NTK When used for the computation of gradient covariance and Neural Tangent Kernel, [Yang, 2020] proved that Assumption 1 yields the exact computation of the gradient covariance and the NTK in the limit of infinite width. We state the result for the gradient covariance formally. Lemma A2 (Corollary of Theorem D.1. in [Yang, 2020]). Consider a ResNet of the form (1) or (3) with weights W . In the limit of infinite width, we can assume that W T used in back-propagation is independent from W used for forward propagation, for the calculation of Gradient Covariance. Lemma A3 (Gradient Second moment). Consider a ResNet of type Eq. (3) without SD. Let (x, t) ∈ D be a sample from the dataset, and define the second of the gradient withq l (x, t) = E W ∂L(x,W ) ∂y l . Then, in the limit of infinite width, we have that As a result, for all l ∈ [1 : L], we have that Proof. It is straighforward that Using lemma A2 and the Central Limit Theorem, we obtaiñ We conclude by observing that E[φ (y l i (x)) 2 ] = P(N (0, 1) > 0) = 1 2 .
A1 Proofs
A1.1 Proof of Lemma 1 Lemma 1 (Concentration of L δ ). For any β ∈ (0, 1), we have that with probability at least 1 − β, Proof. The concentration inequality is a simple application of Bennett's inequality: Let X 1 , ..., X n be a sequence of independent random variables with finite variance and zero mean. Assume that there exists a ∈ R + such that X i ≤ a almost surely for all i. Define S n = n i=1 X i and σ 2 n = n i=1 E[X 2 i ]. Then, for any t > 0, we have that Now let us prove the second result. Fix someL ∈ (0, L). We start by proving that the function ζ(z) = z u −1 α z is increasing for any fixed α > 0. Observe that u (t) = log(1 + t), so that This yields For z > 0, the numerator is positive if and only if α z > u α z , which is always true using the inequality log(1 + t) < t for all t > 0. Now let α = log(2/β). Without restrictions on L p , it is straightforward that v p is maximized by p = (1/2, ..., 1/2). With the restriction L p =L, the minimizer is the orthogonal projection of p onto the convex set {p : L p =L}. This projection inherits the symmetry of p , which concludes the proof.
∇y L L 2 , where the numerator and denominator are respectively the norms of the gradients with respect to the inputs of the l th and L th layers . Then, in the infinite width limit, under Assumption 1, for all l ∈ [L] and (x, z) ∈ R d × R o , we have Denote for clarity for any neuron i and layer l, The equation can be written We notice that for any k ≥ l + 1 and any i, d i,k on depends on W l+1 through the forward pass, and that the terms W ji l+1 in equation (A9) come from the backward pass. Therefore, Lemma A2 entails where 2 N is the variance of W ji l+1 (N is the width of the network). Again using Lemma A2 and taking the expectation with respect to the remaining weights and mask concludes the proof.
A1.3 Proof of Lemma 2
Lemma 2 (Maximal regularization). Consider the empirical loss L given by Eq. (6) for some fixed weights W (e.g. W could be the weights at any training step of SGD).Then, for a fixed training budgetL, the regularization is maximal for p * l = min 1, max(0, where C is a normalizing constant, that has the same sign as L − 2L. The global maximum is obtained for p l = 1/2. Proof. Let a l = g l (W ). Noticing that p l (1 − p l ) = 1/4 − (p l − 1/2) 2 , it comes that with the abuse of notations √ a = ( √ a 1 , ..., √ a L ) and where stands for the element-wise product.
We deduce from this expression that p = 1 2 is the global maximizer of the regularization term. With a fixed training budget, notice that the expression is maximal for p * √ a the orthogonal projection of , where λ 0,l , λ 1,l ≥ 0. Taking p = p * , β = C, λ 0,l = 1/2 + βa −1 l and λ 1,l = 1/2 − βa −1 l . Since the program is quadratic, the KKT conditions are necessary and sufficient conditions, which concludes the proof.
A1.4 Proof of Theorem 1
Theorem 1 (p * is uniform at initialization). Assume φ = ReLU and W are initialized with N (0, 2 N ). Then, in the infinite width limit, under Assumption 1, for all l ∈ [1 : L], we have As a result, given a budgetL, the average regularization term 1 2L L l=1 p l (1 − p l )E W [g l (W )] is maximal for the uniform mode p * = (L/L, . . . ,L/L).
Proof. Using the expression of g l , we have that Thus, to conclude, it is sufficient to show that for some arbitrary i ∈ [1 : n], we have for all l ∈ [1, L]. Fix i ∈ [1 : n] and l ∈ [1 : L]. For the sake of simplicity, let z l = z l (x; 1) and y l = y l (x; 1). We have that Using Assumption 1, in the limit N → ∞, we obtain Let us deal first with the term E W [(z k l ) 2 ]. Let k ∈ N. Recall that for finite N , we have that z k l = N m=1 W k,m l φ(y m l−1 ) and W k,m l ∼ N (0, 2 N ). Thus, using the Central Limit Theorem in the limit N → ∞, we obtain where Q l−1 (x, x) is given by Lemma A1. We obtain The termq j l = E W This yields and we conclude that The latter is independent of l, which concludes the proof.
Let γ α,L (x) = 1 √ L αL l=1 η l z l , ∇ y l G i l (y l (x; 1)) . The term γ α,L captures the randomness of the binary mask δ, which up to a factor α, resembles to the scaled mean in Central Limit Theorem(CLT) and can be written as where X l,L (x) = η l z l , ∇ y l G i l (y l (x; 1)) . We use a Lindeberg's CLT to prove the following result.
lim L→∞
exists and is finite.
Theorem 3 (Lindeberg's Central Limit Theorem for Triangular arrays). Let (X n,1 , . . . , X n,n ) n≥1 be a triangular array of independent random variables, each with finite mean µ n,i and finite variance σ 2 n,i . Define s 2 n = n i=1 σ 2 n,i . Assume that for all > 0, we have that Given an input x ∈ R d , the next lemma provides sufficient conditions for the Lindeberg's condition to hold for the triangular array of random variables X l,L (x) = η l µ l,L (x). In this context, a scaled version of γ L (x) converges to a standard normal variable in the limit of infinite depth. Lemma A4. Let x ∈ R d , and define X l,L (x) = η l µ l,L (x) where µ l,L is a deterministic mapping from R d to R, and let σ 2 l,L (x) = Var δ [X l,L (x)] for l ∈ [L]. Assume that 1. There exists a ∈ (0, 1/2) such that for all L, and l ∈ [L], p l ∈ (a, 1 − a).
lim L→∞
Then for all > 0, we have that The proof of Lemma A4 is provide in Section A1.6. Corollary A1. Under the same assumptions of Lemma A4, we have that The proof of Theorem 2 follows from Corollary A1.
A1.6 Proof of Lemma A4
Lemma A4. Let x ∈ R d , and define X l,L (x) = η l µ l,L (x) where µ l,L is a deterministic mapping from R d to R, and let σ 2 l,L (x) = Var δ [X l,L (x)] for l ∈ [L]. Assume that 1. There exists a ∈ (0, 1/2) such that for all L, and l ∈ [L], p l ∈ (a, 1 − a).
lim
Then for all > 0, we have that . For l ∈ [L], we have that σ 2 l,L = p l (1 − p l )µ 2 l,L . Therefore, Under conditions 1 and 2, it is straightforward that s 2 L = Θ(L). To simplify our notation in the rest of the proof, we fix x ∈ R d and denote by X l := X l,L (x) and µ l := µ l,L (x). Now let us prove the result. Fix > 0. We have that where the lower bound diverges to ∞ as L increases by assumption. We conclude that . where W = (W l ) 0≤l≤L . With SD, we optimize the average empirical loss given by
A2 Full derivation of explicit regularization with SD
To isolate the regularization effect of SD on the loss function, we use a second order approximation of the loss function of the model, this will allow us to marginalize out the mask δ. Let z l (x; δ) = Ψ l (W l , y l−1 (x; δ)) be the activations. For some pair (x, t) ∈ D, the second order Taylor approximation of (y L (x), t) around δ = 1 = (1, . . . , 1) is given by where G l is the function defined by y out (x; 1) = G l (y l−1 (x; 1) + 1 √ L z l (x; 1)). Taking the expectation with respect to δ, we obtain whereL(W , x) ≈ (y out (x; p), t) (more precisely,L(W , x) is the second order Taylor approximation of (y out (x; p), t) around p = 1 5 ), and g l (W , x) = z l (x; 1) T ∇ 2 y l [ • G l ](y l (x; 1))z l (x; 1). The first termL(W , x) in Eq. (4) is the loss function of the average network (i.e. replacing δ with its mean p). Thus, Eq. (4) shows that training with SD entails training the average network with an explicit regularization term that implicitly depends on the weights W .
A3 Implicit regularization and gradient noise
The results of Theorem 2 can be generalized to the gradient noise. Adding noise to the gradient is a well-known technique to improve generalization. It acts as an implicit regularization on the loss function. Neelakantan et al. [2015] suggested adding a zero mean Gaussian noise parameterized by its variance. At training time t, this translates to replacing ∂L ∂wt by ∂L ∂wt + N (0, σ 2 t ), where w t is the value of some arbitrary weight in the network at training time t, and σ 2 t = a(1 + t) −b for some constants a, b > 0. As t grows, the noise converge to 0 (in 2 norm), letting the model stabilize in a local minimum. Empirically, adding this noise tends to boost the performance by making the model robust to over-fitting. Using similar perturbation analysis as in the previous section, we show that when the depth is large, SDmimics this behaviour by implicitly adding a Gaussian noise to the gradient at each training step.
Consider an arbitrary weight w in the network, and let h(x; δ) = ∂L(x,W ;δ) ∂w be the gradient of the model loss w.r.t w. h(x; δ) can be approximated using a first order Taylor expansion of the loss around δ = 1. We obtain, Let γ L (x) = 1 √ L L l=1 η l ∂ ∂w z l , ∇ y l [ •G l ](y l (x; 1)) . With SD, the gradient h(x; δ) can therefore be seen as a perturbation of the gradient of the average network h(x; p) with a noise encoded by γ L (x). The scaling factor 1/ √ L ensures that γ L remains bounded (in 2 norm) as L grows. Without this scaling, the variance of γ L generally explodes. The term γ L captures the randomness of the binary mask δ, which resembles to the scaled mean in Central Limit Theorem and can be written as (x; 1)) . Ideally, we would like to apply Central Limit Theorem (CLT) to conclude on the Gaussianity of γ L (x) in the large depth limit. However, the random variables X l are generally not i.i.d (they have different variances) and they also depend on L. Thus, standard CLT argument fails. Fortunately, there is a more general form of CLT known as Lindeberg's CLT which we use in the proof of the next theorem. Theorem 4 (Asymptotic normality of gradient noise). Let x ∈ R d , and define X l,L (x) = η l µ l,L (x) where µ l,L (x) = ∂ ∂w z l , ∇ y l [ •G l ](y l (x; 1) , and let σ 2 l,L (x) = Var δ [X l,L (x)] = p l (1−p l )µ l,L (x) 2 for l ∈ [L]. Assume that 1. There exists a ∈ (0, 1/2) such that for all L, and l ∈ [L], p l ∈ (a, 1 − a).
lim
exists and is finite.
As a result, SD implicitly mimics regularization techniques that adds Gaussian noise to the gradient.
The proof of Theorem 4 follows from Lemma A4 in a similar fashion to the proof of Theorem 2. Under the assumptions of Theorem 4, training a ResNet with SD entails adding a gradient noise γ L (x) that becomes asymptotically close (in distribution) to a Gaussian random variable. The limiting variance of this noise, given by v ∞ (x), depends on the input x, which arises the question of the nature of the noise process γ L (.). It turns out that under some assumptions, γ L (.) converges to a Gaussian process in the limit of large depth. We show this in the next proposition
A4 Further experimental results
Implementation details: Vanilla Stable ResNet is composed of identical residual blocks each formed of a Linear and a ReLu layer. Stable ResNet110 follows [He et al., 2016, Huang et al., 2016; it comprises three groups of residual blocks; each block consists of a sequence of layers Convolution-BatchNorm-ReLU-Convolution-BatchNorm. We build on an open-source implementation of standard ResNets 6 . We scale the blocks using a factor 1/ √ L as described in Section 3. We use the adjective non-stable to qualify models where the scaling is not performed. The toy regression task consists of estimating the function where the inputs x and parameter β are in R 256 , sampled from a standard Gaussian. The output is unidimensional. CIFAR-10, CIFAR-100 contain 32-by-32 color images, representing respectively 10 and 100 classes of natural scene objects. The models are learned in 164 epochs. The Stable ResNet56 and ResNet110 use an initial learning rate of 0.01, divided by 10 at epochs 80 and 120. Parameter optimization is conducted with SGD with a momentum of 0.9 and a batch size of 128. The Vanilla ResNet models have an initial learning rate of 0.05, and a batch size of 256. We use 4 GPUs V100 to conduct the experiments. In the results of Section 3 and 4, the expectations are empirically evaluated using 500 Monte-Carlo (MC) samples. The boxplots are also obtained using 500 MC samples.
The exploding gradient of Non-Stable Vanilla ResNet: In Table 6 and Table 5, we empirically validate Proposition 1. We compare the empirical values of and compare it to the theoretical value (in parenthesis in the tables). We consider two different survival proportions. We see an excellent match between the theoretical value and the empirical one.
Proposition 1 coupled to the concavity of log(1 + x) implies that at a constant budget, the uniform rate is the mode that suffers the most from gradient explosion. Figures 2a and 2b illustrate this phenomenon. We can see that the gradient magnitude of the uniform mode can be two orders of magnitude larger than in the linear case. However, the Stable scaling alleviates this effect; In Figure 2c we can see that none of the modes suffers from the gradient explosion anymore. Second order approximation of the loss: In Table 3 we empirically verify the approximation accuracy of the loss (equation (6)). L is the loss that is minimized when learning with SD.L is the loss of the average model y out (x; p). The penalization term is 1 2L L l=1 p l (1 − p l )g l (W ) (more details in Section 4). At initialization, the loss of the average model accurately represents the SD loss; the penalization term only brings a marginal correction. As the training goes, the penalization term becomes crucial;L only represents 12% of the loss after convergence. We can interpret this in the light of the fact thatL converges to zero, whereas the penalization term does not necessarily do. We note that the second-order approximation does not capture up to 25% of the loss. We believe that this is partly related to the non-PSD term Γ l that we discarded for the analysis.
Further empirical verification of assumption 2 of Theorem 2: Under some assumptions, Theorem 2 guarantees the asymptotic normality of the noise γ. Further empirical verifications of assumption 2 are shown in Fig. 6 and Fig. 7. The downtrend is consistent throughout training and modes, suggesting that assumption 2 is realistic. In Fig. 8 we plot the distributions of the pvalues of two normality tests: the Shapiro-Wilk (Shapiro and Wilk [1965]) test and the the D'Agostino's K 2 -tests (D'Agostino [1970]).
Further empirical verification of the Budget Hypothesis:
We also compare the three modes (Uniform, Linear and SenseMode) in CIFAR10 and CIFAR100 for Stable Resnet56. The results are reported in Table 6. These results confirm the observations discussed in the main text. | 2021-06-08T01:16:31.807Z | 2021-06-06T00:00:00.000 | {
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186380995 | pes2o/s2orc | v3-fos-license | Field observations and finite element 3-D analysis of soil displacements close to unsupported excavation
. Soil displacements due to unsupported deep excavation may cause severe damages to the nearby structures and foundations systems. Such excavations affect the state of stresses and displacements field of the surrounding soil. In this study, the soil displacements at five observation points were continuously monitored for23 days, which was the time period of excavation of about 7 m deep open tunnel. The reference points were installed on the ground surface at horizontal distance ranging from 1.25 to 3.25 m from tunnel excavation edge. The construction work was related to the project of developing the Army Channel/ Zeyouna section in the Eastern part of Baghdad City. The field observations indicate upward vertical displacement and outward lateral displacements during the first 10 days when the excavation depth was within 4 m. After that, the displacement trend was generally reversed. The displacement values were within 12 mm and vary from one observation point to another depending on the lateral distance of each point from excavation edge. The finite element package PLAXIS 3D was used to simulate the problem after obtaining the required soil parameters by an extensive site investigation. The analysis results in general, compare well with the field observations in terms of soil displacements at the reference points, especially during the first 10 days. This finding may reveal the reliability of the analysis results at other locations in the surrounding soil.
Introduction
Open excavations for constructing high rise buildings in crowded inhabited areas have become increasingly a common practice in civil engineering. Such excavations are often deeper than the foundations level of the adjacent buildings. Unless there is a well-designed shoring and sound support, these excavations will necessarily cause lateral and vertical movements of the soil mass surrounding the excavated pits, [1][2][3][4][5][6][7][8]. Such movements may cause severe damages to the adjacent structures. The design of these excavations should therefore include an estimation of the ground movement as well as a stability check of the adjacent buildings, [2][3][4][5][6]. For example, a deep foundation pit nearby a subway line in Taipei was excavated and caused the line tunnel damages and great economic losses, [2]. Fig. 1 displays a case study of a collapsed 13-floor building in Shanghai, China due to an adjacent deep excavation and a heavy rainfall, [2& 9]. The patterns of movement of a soil mass surrounding an open excavation can be explained as follows: Removing the excavated soil mass is eventually unloading the soil from its geostatic stress state. The resulting displacements due to this unloading are related to the soil elastic rebound. The surrounding soil will, generally, be displaced upward while the pit's sides tilt outside the excavation center in the horizontal direction.
The other pattern of soil mass movement is related to the loss of lateral support upon excavation. The pit sides will consequently be displaced laterally towards the pit center as the lateral soil pressure will change from its at rest (k o ) condition to zero, for the case of unsupported excavation.
Field work
The project of developing the Army Canal in the Eastern part of Baghdad city comprises of excavation of a number of open tunnels. Zayona tunnel, which is taken as the case study in this paper, is a part of this project. The tunnel occupies an approximate area of about (45x28) m 2 . The proposed unsupported excavation depth is about 7 m. The soil profile at the tunnel location consists of about 20 m deep clayey silt to silty clay layer of low plasticity (CL). The water content is ranging from 15 to 18%, the liquid limit is about 33 and plasticity index of 18. Five observation points were installed, before starting the excavation process, close to two perpendicular sides of the tunnel boundaries as shown in
Results of field work
The field observations of soil displacements are measured by means of surface trigonometry using the surveying instruments such as Theodolite and Levels. Fig. 3. displays the variation of vertical displacement with time at each of the five observation points. It can be noticed that the vertical displacement is gradually increased in the upward direction (positive) until it reached its maximum value after about 10 days when the excavation depth became 4m. After that, the vertical displacement tends to decrease and after about 15 days, downward movement (negative) is detected. In general, the vertical displacements of the points (1 and 3) whether it is positive or negative are greater than that of the other points because these points are closer to the excavation edge. Fig. 4. and Fig. 5. show the variation of the lateral displacements in X and Y directions at each of the observation points. It is important to note that the tunnel local coordinates axes make an angle of about 45 o with the global coordinate axes. In order to figure out the lateral displacements whether they are outward or towards the excavation center, the following should be considered; for points 1 and 2, the resultant of positive X and positive Y displacements indicates outward lateral movement. For points 3, 4, and 5, the resultant of positive X and negative Y displacements indicates the outward movement. In general, the trend of variation of lateral displacement is almost similar to that of vertical displacement especially for observation point 1.
Finite element modelling
A series of 3D finite element analyses are implemented, by using PLAXIS 3D Foundation program [12], to analyze the ground movement at the location of observation points. A 10 nodded tetrahedron element is used in the analyses. The Silty Clay layer is modeled as a Hardening soil model and the relevant soil properties are listed in Table 2.The excavation process is modeled as five stages. The relevant excavation depths of these stages are; 1, 2, 3, 5 and 7 m, respectively. Fig. 8 illustrates the distribution of lateral ground movement at each of the excavation depths. For excavation depths less than 3 m, the lateral grounds surface movements is in the outward direction and increase with increasing excavation depth. After that depth of excavation, the lateral grounds surface movements indicate a reverse direction and the negative lateral grounds movements (towards the excavation center) are developed below the level of ground surface. The measured displacements at the observation points are compared with those obtained by the finite element analysis. Fig. 9 displays the measured and calculated vertical displacements at four of the observation points. In general, the trend of variation is almost similar. Good agreements are noticed particularly during the first 10 days when the excavation depth less than 5 m. The comparison between measured and calculated lateral displacements at two of the observation points is displayed in the Fig 10. It can be noticed that all the measured and calculated values of lateral displacements are within the range of +8 mm to -17 mm with almost similar trend of variation.
Conclusions
All the observation points exhibit upward (positive) vertical displacement with increasing depth of excavation down to a depth of 4.0 m (at the tenth day of the excavation process). After that, downward vertical (negative) displacements are monitored. In general, the values of vertical displacement at the observation points whether they are positive or negative are greater for the points that are closer to excavation boundaries. The values of displacements obtained by the finite element analysis reveal good agreements in terms of variation trend and magnitudes with the measured ones especially for excavation depth about 5 m (less than the 10 days). The differences between measured and calculated values become more pronounced for excavation depths greater than 5 m. | 2019-06-13T13:16:37.760Z | 2018-01-01T00:00:00.000 | {
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258907401 | pes2o/s2orc | v3-fos-license | Consequences of China’s special send-down movement on infectious disease control in rural areas: a natural experiment
Background China's send-down movement in the 1960s and 1970s, as a natural experiment, provides a unique opportunity to investigate the relationship between peers' dissemination of health literacy, community health workers, and infectious disease control in areas with weak health systems and inadequate human resources. To address the lack of studies on the health effects of the send-down movement, this study examined the associations between prenatal exposure to the send-down movement and infectious diseases in China. Methods We analyzed 188,253 adults born in 1956–1977 with rural hukou who participated in the Second National Sample Survey on Disability in 2006 across 734 counties of China. Difference-in-difference models were used to detect the effect of the send-down movement on infectious diseases. Infectious diseases were ascertained by using the combination of self- or family members’ reports and on-site medical diagnosis of disabilities attributed to infectious disease by experienced specialists. The density of the relocated urban sent-down youth or “sent-down youths” (SDYs) in each county defined the intensity variable of the send-down movement. Results Individuals in SDY-receiving areas with increased intensity of prenatal exposure to the send-down movement had a decreased probability of infectious diseases (β = −0.0362, 95% CI: 0.0591, −0.0133) after controlling for a set of regional and cohort characteristics. This association was stronger in counties with more prevalent infectious diseases prior to the send-down movement (β = −0.0466, 95% CI: 0.0884, −0.0048) than in those with less prevalence (β = −0.0265, 95% CI: 0.0429, −0.010). No substantial differences were found across sex-specific groups or by strictness of send-down movement implementation. On average, prenatal exposure to the send-down movement corresponded to a decrease in the probability of infectious diseases in rural areas by 19.70%. Conclusions For areas with weak health systems, strengthening community health workers and promoting health literacy may be two key points to address the burden of infectious diseases. Increasing education and primary health care through peer-to-peer dissemination may contribute to the reduction of infectious disease prevalence.
Introduction
In addition to COVID-19, other infectious diseases, such as tuberculosis and malaria, still remain global health challenges, with 8 million new cases of tuberculosis and 300-500 million new cases of malaria each year. Although some developing countries have eliminated a number of infectious diseases, the continued prevalence of infectious diseases in regions, such as Sub-Saharan Africa, challenges the achievement of the United Nation's 2030 Sustainable Development Goal target on communicable diseases. Since 1949, China has made large gains in infectious disease control, and was awarded polio-free and malaria-free certifications by the WHO in 2000 and 2021, respectively. The rapid progress in strengthening the dissemination of health literacy dissemination of health literacy and the primary health care system are key points for China's ability to combat infectious diseases (Jiang et al., 2020;Yu et al., 2018;L. Zhang & Wilson, 2012), which may provide lessons for other regions working towards eliminating communicable diseases.
In the late 1960s, China's government launched the "send-down movement," also known as the "rustication movement" or "going up to the mountains and down to the villages." While this policy was in effect, over 16 million sent-down youth (middle and high school graduates) were mandated to move from urban to rural areas (Cao, Chen, Pang, Zheng, & Nilsson, 2012;Y. Chen, Fan, Gu, & Zhou, 2020;Xie, Jiang, & Greenman, 2008). Prior research has reported both harmful and beneficial long-term effects of this movement on the sent-down youth who were sent, but there is evidence that, from the rural perspective, the arrival of those sent-down youth may have improved community health literacy in less developed regions and relieved shortages of healthcare workers (Cao et al., 2012;Y. Chen et al., 2020;Xie et al., 2008). This mass movement could have affected the incidence of infectious diseases in rural areas in the following ways: 1) Through community-based peer-to-peer education, the urban sent-down youth increased rural residents' knowledge of and ability to gather and process information on disease transmission mechanisms and vector control strategies; 2) Large proportions of sent-down youth were assigned to be counselors and teachers in countryside, introducing new values and healthy behaviors in school settings; 3) Some sent-down youth with basic medical and preventive techniques were assigned to be community health workers ("barefoot doctors"), guiding vaccination and prenatal health care programs and contributing to the construction of the Rural Cooperative Medical System (Yanhong, 2008.1.23. ;Yu et al., 2018).
The send-down movement provides a unique opportunity to investigate how such an unprecedented scale of resettlement of urban sentdown youth affected infectious diseases in rural areas of China with less education levels and poor health systems. This movement was launched in 1968 and declined after Mao Zedong's death in 1976. In 1977, these "sent-down youths" began leaving the countryside for urban areas, ending with a sharp decline in the number of sent-down youths still residing in rural areas in 1978 (Y. Chen et al., 2020). Specific cohorts born in rural areas during the sent-down movement may have had lasting health values, knowledge, and behaviors imparted to them by the sent-down youth, but those health effects are not well-studied.
To date, no studies have examined the effect of such a massive movement of sent-down youth on infectious disease control in rural areas. Treating the send-down movement of 1968-1976 as a natural experiment, this study used difference-in-difference models to capture the send-down movement's effect on the risk of infectious diseases, which may further elucidate the contribution of community health workers and peers' dissemination of health literacy to infectious disease control in poor areas and provide lessons for countries with increased burden of infectious diseases exacerbated by weak health systems and inadequate human resources. sent-down youth.
Our research revealed that prenatal exposure to the send-down movement corresponded to a decreased probability of infectious diseases. This association was particularly pronounced in counties with a higher prevalence of infectious diseases before the send-down movement, as evidenced by our heterogeneity analysis. On average, prenatal exposure to the send-down movement resulted in a 19.70% reduction in the probability of infectious diseases in rural areas. This study is structured as follows: Section 2 outlines the data and sample selection process. Section 4 presents the empirical findings, while Section 5 offers a discussion and conclusions.
Study design and data sources
This study used data from the Second National Sample Survey on Disability (SNSSD), which was a nationally representative investigation conducted from 1 April to 31 May 2006, covering 31 provinces, 734 counties or districts, 2980 towns or streets and 5964 communities or villages in China. The SNSSD collected information to characterize disabilities in China, including prevalence rates, causes and severities of disability, rehabilitation demands and medical needs, and living conditions of disability. This survey was derived by four-stage stratified random cluster sampling (counties, towns, interview sites, and households) with probability proportional to size. The SNSSD involved 2,526,145 participants with a 99.87% response rate, thus making up 0.19% of noninstitutionalized residents in China. Additional details about the SNSSD can be found elsewhere (Zheng et al., 2011).
We defined the treatment group as individuals born in 1968-1977 whose prenatal period overlapped with the arrival of sent-down youth. Thus, prenatal exposure was defined as maternal exposure (approximately 300 days from conception to delivery) for pregnancies during the send-down movement in 1968-1976. The control group was comprised of individuals born in 1956-1967, i.e., born prior to the send-down movement. We first excluded counties or districts in areas that were more likely to be districts that sent rather than received urban sent-down youth (including Beijing, Tianjin, Shanghai, and all districts in prefecture-level cities), and then excluded counties with missing information on the density of sent-down youth. Our final analytic sample was restricted to 188,253 adults with rural hukou born in 1956-1977, which the hukou refers to a household register. The flowchart of sample selection can be found in Fig. 1.
Infectious diseases
The SNSSD identified individuals with disabilities attributed to infectious diseases through self-reports by participants or their family members and on-site diagnosis by experienced medical specialists. Participants with functional limitations were identified by trained field interviewers with a structured five-item screening questionnaire developed according to the 'Guidelines and Principles for the Development of Disability Statistics.' This questionnaire was collected by face-to-face interviewing and was demonstrated to have high reliability (J. Zhang, 2010). Individuals who answered a positive response to the questionnaire were determined as likely to meet the criteria for functional limitations. Then, persons with a World Health Organization Disability Assessment Schedule, Version II (WHO DAS II) score ≥52 were diagnosed with functional limitations evaluated by experienced specialists (Field et al., 2000). Finally, experienced specialists diagnosed infectious diseases among persons who had disabilities attributed to any of the following diseases: poliomyelitis, tuberculosis, trachoma, maternal infection-related diseases (including infections with viral hepatitis, sexually transmitted diseases, etc.) and other infectious diseases related to hearing impairment (including rubella, measles, and epidemic cerebrospinal meningitis, etc.). The prevalence of disabilities attributed to infectious disease was used as a proxy for prevalence of infectious disease in the county.
Send-down intensity
As an obligatory and temporary policy, the send-down movement can be treated as a natural experiment to study how the arrival of new health knowledge and behaviors from urban areas influence infectious disease control in rural areas. During the send-down period, sent-down youth could not migrate or return to their sending areas because of stringent hukou restrictions and the mandatory send-down policy. At the end of the send-down movement, about 95% of sent-down youth and their offspring left the receiving countryside, which reduces the possibility that sent-down youth (and their offspring), with better health literacy and behaviors, were counted as rural residents. Additionally, the send-down movement ended in 1978, which excludes the effects on infectious disease control by national immunization programs that were not initiated until 1978.
Send-down movement intensity was measured by the density of sentdown youth in each county obtained from Chen et al. (2020) (Y. Chen et al., 2020). The density of sent-down youth is proxied through the total number of sent-down youths during the send-down movement divided by the total number of persons in each county in 1964. Chen et al. (2020) collected these data from over 3000 local gazetteers and the 1964 census. A higher density of sent-down youth represents a higher magnitude of the send-down movement, which we hypothesized to have a greater effect on infectious disease control. We obtained the density of sent-down youth of 557 counties ranging from 0.10% to 18.43% with a mean of 2.34%, i.e., the average density of sent-down youth was 23.4 sent-down youth per 1000 locals during the send-down movement.
Control variables
Sex (male or female) and ethnicity (Han ethnicity or minority) were included as covariates. We also adjusted for the fixed effects of each county, interaction terms between province and cohort, and the interaction terms between infectious disease prevalence before the start of the send-down movement for each county and birth cohort to control for time-invariant traits of counties and unobservable differences in cohort trends. The prevalence of infectious diseases at the county-level was calculated as the prevalence of infectious diseases of the pre-send-down movement control group using the 2006 SNSSD database.
Statistical analysis
We used the cohort difference-in-differences (DID) method (Esther, 2001) to estimate the impact of the send-down movement on the probability of having a disability attributed to infectious diseases. The cohort difference-in-differences (DID) method is a statistical technique used to evaluate the causal effect of a treatment or intervention on an outcome variable, which is particularly useful when dealing with observational data. Two variations were estimated: variation in intensities of send-down movement exposure across regions and variation in intensities of send-down movement across birth cohorts. The effects of the send-down movement on each cohort could be separately estimated. The equation of linear probability models with the DID estimator is as follows: where Y ijk is the probability of infectious diseases for person i born in county k and year j. sent-down youth k is the density of sent-down youth in county k. Additionally, γ j represents the cohort fixed effect, θ k is the cohort fixed effect of the density of sent-down youth, and β jk is the estimator of the send-down movement effect on the probability of infectious diseases. X ijk denotes a series of control variables, including sex and ethnicity, county k is the country fixed effects, prov m × Cohort j represents the province-cohort fixed effects, Λ k × Cohort j is the interaction term between county-level infectious disease prevalence prior to the arrival of sent-down youth and cohort dummy variables, and ε ijk denotes the standard error.
To test the robustness of our findings, we performed a heterogeneous analysis of prenatal exposure to send-down movement on the probability of infectious diseases by the subgroup of males vs. females and the subgroup of individuals in counties with more vs. less prevalent infectious diseases. Counties with "less prevalent" infectious diseases were defined as areas with county-level infectious disease prevalence (before the sent-down youth's arrival) at or below the median prevalence of 0.36%; counties with "more prevalent" infectious disease had countylevel prevalence above that median. Additionally, we analyzed the effect of prenatal exposure to send-down movement and infectious diseases by the strictness of send-down movement, i.e., the degree to which the send-down policy was implemented. More strictly implemented send-down cohorts were the individuals born in 1968-1972, and less strictly implemented send-down cohorts were born in 1973-1977, following similar methods of a previous study (Ye, Zhu, & He, 2021).
This study used Stata 15 (Stata Corp, College Station, TX, USA) for data analysis.
Results
The characteristics of participants born in rural areas from 1956 to 1977 are presented in Table 1. The average (±SD) age of the 1956-1977 birth cohorts at the time of the survey was 39.05 (±6.03) years. A total of 49.69% were males, and 17.88% were of minority ethnicity. A total of 0.14% of the control group experienced disabilities attributed to infectious diseases, with an average age of 33.85 (±2.79) years, whereas 0.36% of the 1968-1977 treatment group experienced disabilities attributed to infectious diseases, with an average age of 44.01 (±3.61) years. Among the control group, 49.33% were males, and among the treatment group 50.06% were males. The proportions of minority ethnicity in the control and treatment groups were 19.25% and 16.58%, respectively. Although a higher prevalence of infectious diseases was initially observed in the treatment group as compared to the control group, this unadjusted calculation cannot distinguish whether the difference is attributed to effects of sent-down youth or of other unobserved regional or cohort characteristics. Table 2 presents the adjusted DID regression coefficients of senddown movement on the probability of having a disability attributed to infectious diseases. Individuals who were born in areas that received sent-down youth and with greater intensity of the send-down movement had a lower probability of infectious diseases (β = − 0.0362, 95% CI: 0.0591, − 0.0133). Given that the average density of sent-down youth was 2.34% and the prevalence of infectious diseases in rural areas before the send-down movement was 0.43%, exposure to the send-down movement during the prenatal period decreased the prevalence of infectious diseases in rural areas by 19.70% (=(2.34% × 0.0362)/0.43% × 100).
Results from heterogeneous analyses showed that the send-down movement substantially decreased the probability of infectious diseases in both male and female subgroups, with coefficients of − 0.0409 (95% CI: 0.0811, − 0.0006) and − 0.0315 (95% CI: 0.0562, − 0.0067), respectively. However, the interaction terms of sex and the association of prenatal exposure to the send-down movement with infectious diseases were not substantial (see Table 3 and eTable 4). There also appeared to be a heterogeneous effect of the send-down movement on infectious disease by prevalence of county-level infectious diseases: effects of send-down movement were larger for individuals in counties with more prevalent infectious disease (β = − 0.0466, 95% CI: 0.0884, − 0.0048) as compared to counties with less prevalent infectious disease (β = − 0.0265, 95% CI: 0.0429, − 0.0100), and this difference was substantial (see Table 3 and eTable 4). Additionally, the effects of the senddown movement were observed in cohorts with more strictly implemented send-down movement (β = − 0.0372, 95% CI: 0.0626, − 0.0119) and less strictly implemented send-down movement (β = − 0.0311, 95% CI: 0.0581, − 0.0034), but the difference between these two groups was not substantial (see Table 3 and eTable 4).
Discussion
By considering the mandatory send-down movement of 1967-1978 in China as a natural experiment, we studied the effects of sent-down youth on infectious diseases in rural counties. To our knowledge, this study is the first to examine the health consequences of the send-down movement in the rural areas receiving urban sent-down youth. We estimated that prenatal exposure to the send-down movement corresponded to a decrease in the probability of infectious diseases in rural areas on average by 19.70%, and this send-down movement effect on infectious disease control was stronger in counties with more prevalent infectious diseases than in counties with less prevalence. While the senddown movement had wide-ranging consequences on the sent-down youth who were moved from urban to rural areas, these sent-down youth may have served as an important bridge connecting urban and rural areas, leading to improvements in the rural areas receiving them. The sent-down youth could have contributed to health improvements in their receiving countryside counties by introducing urban techniques, values, and behaviors, while also helping rural residents understand, apply, analyze, and disseminate health information or health services. This historical event provides a unique opportunity to investigate the contribution of human capital accumulation and health literacy transmission to infectious disease control in rural areas.
Two mechanisms may explain the effect of prenatal exposure to the send-down movement on infectious disease control. First, sent-down youth were important in helping rural women ensure adequate nutrition intake and learn coping strategies for stressful relationships during pregnancy, which are crucial to the prevention of infectious diseases and subsequent congenital complications that could lead to disabilities (J. Notes: All models adjusted for the county fix effect, province-cohort fix effect, base infectious diseases prevalence × cohort fix effect. care conditions. Healthy pregnancies, especially in addressing malnutrition and avoiding prenatal stress, lead to healthy development and growth, which may further decrease the risk of infectious diseases in offspring (Marques et al., 2013;Nielsen et al., 2011). Second, sent-down youth assigned as community health workers ("barefoot doctors") played an important role in health system development and infectious disease prevention. Generally, these community health workers undertook various tasks, including case management of maternal and childhood illnesses, promotion of healthy behaviors, and delivery of preventive interventions of communities (A et al.). In China, "barefoot doctors" reached almost every village, devoting much time to preventive medicine, and eventually became the backbone of rural cooperative medical services (C; L & H). They largely alleviated vaccine shortage conditions prior to national immunization campaigns and enhanced access to health care in rural areas (Y. Chen et al., 2020;Gu, 2009;Rosenthal & Greiner, 1982). While China experienced an acute shortage of health workers and vaccines during the 1960s and 1970s, "barefoot doctors" were responsible for transporting vaccines from hospitals in urban areas to their respective villages, which promoted infectious disease control (Li, 2015). As additional sensitivity analyses, we compared the impacts of the send-down movement on infectious diseases across exposures during different developmental stages ranging from prenatal to early adulthood (see eTable 3). We found that the send-down movement exposure effect was decreased in the infant stage, and no substantial associations were found between send-down movement exposure during preschool, teenage, or early adulthood stages and infectious diseases. Although early childhood is a crucial period for the prevention of infectious diseases (Gurgel & Correia, 2007;Jung, Min, Kim, & Min, 2021), the prenatal stage, including prenatal care and health education, may be the most important stage for the prevention of disabilities caused by infectious diseases. However, these findings may have been biased in that most of the infectious diseases contributing to the disabilities we studied (e.g., poliomyelitis, cerebrospinal meningitis) were infections that present in newborn or early childhood stages (Khandaker, Stochl, Zammit, Lewis, & Jones, 2015;Schwoebel et al., 1992).
As another check for robustness, we included the additional years of 1979-1988 and used DID models to separately estimate the effects of send-down movement prenatal exposure on each year's cohort compared with the control group of 1951-1955 birth cohorts (see eFig. 1). Our findings showed that no heterogeneous effect existed in cohorts prior to the send-down movement relative to the density of sentdown youth, except for the rising trend in the 1961 birth cohort, which was possibly impacted by the Great Famine of 1959-1961. The Great Famine could have increased risk of infectious diseases (Cheng et al., 2020). Our analysis with the exclusion of the 1962 cohort indicated that the findings in this study are robust after the exclusion of potential immediate impacts of the Great Famine (see eTable 1). Furthermore, we found that the coefficients gradually decreased after the start of the send-down movement in general, with later cohorts having a lower probability of infectious diseases with increasing density of sent-down youth. The lasting effects of the send-down movement or the later impacts of expanded immunization programs may have contributed to the continuous decline of infectious diseases after the movement ended in 1978.
Several limitations exist in this study. First, this study could not separate the confounding age effect from the cohort effect in a crosssectional dataset based on cohort DID strategy identification. Although most of the infectious diseases involved in this study presented through infection in early childhood and the confounding age effect can be alleviated to some extent, our results should be interpreted with caution. Second, other historical events may have influenced the distribution of our outcome. For instance, as previously mentioned, the Great Famine prior to the send-down movement may have created conditions that increased the risk of infectious diseases, thus leading to an overestimation of the effect of sent-down youth on infectious disease control. The Great Famine also resulted in a tragic loss of life, and mortality rates could have varied by county, resulting in potential differential censoring rates before individuals could be surveyed. Further studies with more granular and longitudinal data are warranted to confirm our results. Third, while we restricted our study sample to individuals with rural hukou, unobserved migration may affect our results. Given hukou system restrictions and the strict regulations on rural-to-urban migration, the living place at the time of survey was used to identify an individual's birthplace at the county-level. However, the estimated density of sentdown youth for an individual's prenatal exposure may not reflect the true density if the individual moved, or if the density was not uniform within a county. Forth, the infectious diseases examined in this study were identified based solely on functional impairments associated with those diseases due to no exclusion for people with disability, which could result in an underestimation of both the prevalence of infectious diseases and the impact of sent-down youth on infectious disease control.
Conclusions
In conclusion, our study found a beneficial effect of prenatal exposure to the send-down movement on infectious disease control in rural Chinese counties. While this study does not suggest mandated relocation per se, community health activities and education from sent-down youth may have led to improved infectious disease prevention in rural areas. Future studies could evaluate other community health outcomes from the send-down movement and examine potential mechanisms to health promotion in rural areas. Our findings also highlight the potential of Table 3 Heterogeneous effect of prenatal exposure to send-down movement on the probability of infectious diseases, based on linear probability regression models with difference-in-difference estimators, the Second National Sample Survey on Disability in 2006. Notes: All models adjusted for the county fix effect, province-cohort fix effect, base infectious diseases prevalence × cohort fix effect.
factors such as human resource accumulation, community health workers, and peers' dissemination of health literacy to improve infectious disease control in poor areas. These factors may be helpful to improve the ability of countries with weak health systems and an inadequate supply of health workers to combat the burden of infectious disease.
Ethics approval and consent to participate
This survey was conducted in all provinces by the Leading Group of the National Sample Survey on Disability and the National Bureau of Statistics with approval by the State Council of China. The study protocol of SNSSD was approved by the China State Council (No. 20051104) and conducted in accordance with the legal framework of the Statistical Law of the People's Republic (version 1996) of China. All participants provided informed consent to the government of China.
Funding
This work was supported by the Major Project of the National Social Science Fund of China (21&ZD107; 21&ZD187) , National Science and Technology Major Project (SQ2022YFC3600291), and and Promotion of Initiative and Empowerment for Women (ANSO-SBA-2020-02), and Young Elite Scientists Sponsorship Program by CAST (2022QNRC001).
eTable 3
The effect of prenatal exposure to send-down movement on the probability of infectious diseases by overlap with development stages, based on linear probability regression with difference-in-difference estimators, the Second National Sample Survey on Disability in 2006 Notes: All models adjusted for the sex, ethnicity, county fix effect, province-cohort fix effect, base infectious diseases prevalence × cohort fix effect. | 2023-05-27T05:05:40.822Z | 2023-05-11T00:00:00.000 | {
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244949820 | pes2o/s2orc | v3-fos-license | Identification and Validation of N6-Methyladenosine-Related Biomarkers for Bladder Cancer: Implications for Immunotherapy
N6-methyladenosine (m6A) has emerged as one of the most important modifications of RNA. Based on the expression of 23 different modes of m6A regulatory factors, we identified three different m6A modification patterns in bladder cancer. The effects of the three different modes of m6A modification on clinicopathological characteristics, immune cell infiltration levels and expression levels of immune checkpoint genes were comprehensively analyzed. In addition, the effects of different modes of m6A modification on the therapeutic efficacy of anti-PD-L1 immunotherapy (atezolizumab) are also discussed. Our results confirm that m6A methylation plays an important role in immune cell recruitment in the tumor microenvironment of bladder cancer, which influences the efficacy of anti-PD-L1 therapy for bladder cancer. We further confirmed the important role of FTO protein in the biological function of bladder cancer cells by performing in vitro experiments. FTO functions as an oncogene in bladder cancer cells, and upon FTO knockdown, the level of m6A enzyme activity in bladder cancer cells was significantly increased, apoptosis was increased, and cell proliferation and cell invasion were reduced. In addition, our study also confirmed that K216H and K216E are probably important targets for regulating FTO. We provide new insights into the regulatory pathways of the immune microenvironment and the methylation function of m6A in bladder cancer, which will help in designing novel diagnostic methods, prognostic tools, and therapeutic targets.
INTRODUCTION
Bladder cancer is the most common cancer of the urinary system and is characterized by a difficult early diagnosis, rapid metastasis, and resistance to treatment. Immune checkpoint therapy (ICT) targets immune pathway effectors such as cytotoxic lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1) (1). Anti PD-L1 immunotherapy has been approved by the Food and Drug Administration (FDA) for the treatment of metastatic bladder cancer (2,3). Immunotherapeutics that target PD-1 or PD-L1 have significantly improved the survival outcomes for some patients, showing an astonishing effect in a small number of patients with sustained efficacy. The improved clinical efficacy for the detection of cancer of the urinary system including advanced-stage bladder cancer has changed the intervention measures used (4); however, some patients with bladder cancer undergoing immunotherapy show no response to ICT or display resistance to drugs, and this scenario does not meet the clinical needs of these patients (5,6). At present, multiple studies have confirmed that the immune response resulting due to many tumors, including those found in bladder cancer, is related to the level of immune cell infiltration in the tumor microenvironment, the expression of PD-1/PD-L1, and the tumor mutation burden (TMB) (7,8).
The microenvironment in which tumor cells grow and survive is called the tumor microenvironment (TME), which includes stromal cells such as cancer-related fibroblasts and macrophages, immune infiltrating cells (myeloid cells and lymphocytes), bone marrow-derived cells (BMDCs) such as endothelial progenitor cells and hematopoietic progenitor cells, and secretory factors such as cytokines (9). There is mounting evidence that TME plays a crucial role in tumor progression, immune escape, and has an impact on the response to immunotherapy (10,11). A comprehensive analysis of the heterogeneity and complexity of the TME is a key step in improving the success rate of existing ICTs and the development of new immunotherapy strategies. Further exploration of the regulatory mechanisms underlying TME cell infiltration will improve the ability to guide and predict the response to immunotherapy, and may help identify new therapeutic targets.
N6-methyladenosine (m 6 A) is the most common and abundant RNA modification in eukaryotes. As a reversible epigenetic modification, m 6 A is found in almost all types of RNAs, including mRNAs, ribosomal RNAs (rRNAs), long noncoding RNAs (lncRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), and circular RNAs (circRNAs), and are dynamically regulated in many physiological processes (12,13). The m 6 A modification is a dynamic and reversible process in mammalian cells and is regulated by various methyltransferases, demethylases, and binding proteins, also known as writers, erasers, and readers, respectively. The process of m 6 A methylation is catalyzed by methyltransferases such as RBM15, ZC3H13, METTL3, METTL14, WTAP, and KIAA1429, and the removal process is mediated by demethylases such as FTO and ALKBH5 (14). Further, a group of specific RNA binding proteins, such as YTHDF1/2/3, YTHDC1/2, HNRNPA2B1, and IGF2BP1/2/3 can recognize the m 6 A motif, thus affecting the function of m 6 A (15). Results from various studies have shown that the abnormal expression and changes to gene expression for m 6 A regulatory factors are related to the dysregulation of multiple biological processes essential for the initiation, progression, metastasis, drug resistance, recurrence, and immune regulation of cancer (16).
Many studies have confirmed the special correlation between m 6 A modification and changes in the TME. Li et al. (17) observed that when METTL3 was depleted, the steady-state growth of T cells was blocked and was maintained at a slow rate. Further, their research showed that METTL3 deletion resulted in a decrease in the level of m 6 A, and an increase in the mRNA stability and protein levels of SOCS1, SOCS3, and CISH, thus negatively regulating the IL-7 signal production in CD4 + T cells. Results from another study (18) revealed the role of m 6 A in dendritic cell activation, in which METTL3 mediated the m 6 A modification of the transcripts for the adaptor Tirap for CD40, CD80, and TLR4. Signal adaptors enhanced the translation of METTL3 in dendritic cells, thereby enhancing TLR4/NF-kB signal transduction, producing cytokines, and stimulating T cell activation. From the perspective of immune checkpoint therapy, the m 6 A binding protein YTHDF1 can be used to modulate antitumor immunity and improve immunotherapy, as it can regulate the expression of lysosomal proteinase in an m 6 A dependent manner. YTHDF1 can recognize the transcription of m 6 A modified transcripts and encodes a lysosomal protease to increase translation in dendritic cells. Moreover, the deletion of YTHDF1 enhances the cross-presentation of tumor antigens and the cross initiation of CD8 + T cells in vivo. Further, Wang et al. confirmed that the deletion of YTHDF1 enhances the therapeutic effect of the PD-L1 checkpoint blockade (19).In melanoma, elevated FTO expression levels promote tumor growth by reducing the methylatin of m 6 A in the mRNA for PD-1 (PDCD1), CXCR4, and SOX10 and preventing their RNA decay mediated by YTHDF2. Knockout of FTO in melanoma cells induces tumor cells to become sensitive to interferon-gamma (IFNg) in vitro and promotes a sensitizing response of melanoma cells to anti-PD-1 antibody in mice (20). Together, these data prove that the m 6 A modification plays an important role in regulating the tumor immune microenvironment, and the combined use of m 6 A modulators and PD-1/PD-L1 drugs may prove to be a new strategy to enhance the clinical efficacy of tumor immunotherapy.
In this study, we examined the effect of the m 6 A methylation modification on the invasion characteristics of immune microenvironment cells and the effect of anti-PD-L1 therapy on bladder cancer. Our study helps elucidate the molecular mechanism underlying the regulation of the immune microenvironment in bladder cancer and provides new predictors, potential auxiliary targets, and directions for more effective immunotherapy strategies for combating bladder cancer.
(TCGA) genome characterization center. Level 3 data were downloaded from the TCGA data coordination center. This dataset shows the gene-level transcription estimates, i.e., the log 2 (x+1) transformed RSEM normalized counts, and somatic mutation data was downloaded from the site https://portal.gdc. cancer.gov/. IMvigor 210 cohort is a multicenter, single-arm phase II clinical study for evaluating the safety and efficacy of Tecentriq, a PD-L1 inhibitor, in patients with advanced urothelial carcinoma (21,22). The complete processed expression data, detailed clinical annotations, and somatic mutation data were obtained from IMvigor210CoreBiologies, which is a complete documentation software and data package for the R statistical computing environment. The software package is available free of charge under the Creative Commons 3.0 License.
Common Molecular Typing of Bladder Cancer
We used several published subtype classification systems for bladder cancer, including the Baylor subtype described by Mo et al. (26), who defined urothelial differentiation based on 18 gene markers, thus dividing the muscle-invasive bladder cancers (MIBC) and non-MIBCs (NMIBC) into basal and differentiated subgroups. Damrauer et al. (27) identified basal and luminal cancer subtypes by consensus cluster analysis and further identified 47 genes as predictors of these subtypes. The MDA subtype (28) was analyzed by using 2252 genes (2697 probes) from 73 freshly frozen primary MIBC samples, including p53 like, luminal, and basal cancer. The Lund group (29) presented a six-class system based on global mRNA expression, including the urothelial-like, genomically unstable, epithelial infiltrated, SCClike/Mes-like, SCC-like/UroB, and Sc/NE-like cancer. TCGA genotyping (30) used 2707 genes to conduct unsupervised cluster analysis on 129 patients with MIBC. The patients were classified into four subtypes (I, II, III, and IV), using the Cartes d'Identitedes Tumeurs (CIT)-Curie subtypes (31). These classifiers were combined into an R package (BLCA subtyping) (32), which can be found at https://github.com/cit-BioInfo/ BLCAsubtype. We applied these classifiers independently to the TCGA-BLCA cohort and the IMvigor210 cohort to analyze the relationship between the subtypes of different classification methods and three different m 6 A modification patterns. Further, we interpreted the differences among the three different m 6 A modified subtypes.
Estimation of TME Cell Infiltration
We used the single-sample gene set enrichment analysis (ssGSEA) algorithm to quantify the relative abundance of cell infiltration in TME. We obtained gene sets from the data of Qingzhu Jia et al. (33) to label the different types of TME infiltrating immune cell types. As demonstrated by Jia et al. (33), we calculated the antitumor-immunity-score as the sum of the immune cell scores of various antitumor cells. Further, the pro-tumor-immunity-score is the sum of the scores of tumorpromoting immune cells, and we define the tumor immunity score as the difference between the pro-tumor-immunity score and the antitumor-immunity-score for unified analysis. In addition, we also used the ESTIMATE algorithm (34) to estimate the ratio of the immune matrix components of each sample in the TME to further explore the differences of TME components including immune cells and stromal cells scores among different m 6 A modification patterns. The results were presented in three forms: immune score, stromal score, and the ESTIMATE score; the higher the score from the immune score or stromal score, the more immune or matrix components in TME. The ESTIMATE score is the sum of the immune score and stromal score, which represents the level of the two components in TME.
Identification of Differentially Expressed Genes (DEGs) Among Three Types of m 6 A Modified Patterns
DEGs were identified via the Bayesian method between different m 6 A modification patterns using the limma R package (35), the absolute value of logFC > 1 as the significance criteria. We used the ssGSEA method to score the samples based on the 23 m 6 A regulatory factors. Based on the median value of the m 6 A score, the patients were classified into a high m 6 A score group or a low m 6 A score group. The difference between the two groups was analyzed by the empirical Bayes method using the limma R package, taking the corrected p-value < 0.05, and the absolute value of logFC > 1 as the significant criteria and 647 DEGs were obtained for subsequent signal pathway analysis.
Differential Signaling Pathway Enrichment Among the Three m 6 A Modified Modes
The enrichment of signaling pathways for the DEGs expressed relative to the different phenotypes of m 6 A were analyzed using the metascape website (36) http://metascape.org. We determined the statistically rich terms, accumulated hypergeometric p values, and enrichment factors, and used these in a filtering step. Next, according to the Kappa statistical similarity between the gene members, the remaining important terms were clustered in a hierarchical structure, and the kappa score of 0.3 was used as a threshold to convert the terms to clusters (36). We selected the genes with the best p-value in each cluster as its representative term and displayed them in the heatmap. The databases for the KEGG pathway, GO biological processes, Reactor gene sets, Canonical pathways, and the CORE resources were used for pathway and process enrichment analysis applying a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 (the enrichment factor is the ratio between the observed count and the chance expected count). The GSEA analysis (37) evaluates the skewness of the two distributions for the selected gene set in the gene list sorted by a specific phenotype. The analysis gene set was obtained from the hallmark gene sets provided by the Molecular Signatures Database (https://www.gsea-msigdb.org/). In this study, we used the clusterProfiler R package to implement the GSEA analysis (38).
Construction of a Scoring System to Evaluate the Level of m 6 A Modification in Individual Samples
To quantify the m 6 A modification pattern in a single patient. Based on the mRNA expression level of the 23 identified m 6 A regulatory molecules in each sample, we constructed a scoring system called m 6 Ascore by single sample Gene Set Enrichment Analysis (ssGSEA, based on GSVA R package).
Analysis of Copy Number Variation in Different Subtypes of Somatic Cells
For copy number variation analysis, we used the GISTIC.2 to identify significantly amplified or missing parts of the genome. The burden of copy number loss or gain was calculated as the total number of genes with copy number changes at the focal and arm level.
The differential gene expression of the core biological pathway genes between the different m 6 A phenotypes of the
Cell Lines and Cell Culture
The human bladder cancer cell lines were obtained from the Oncology Institute of Central South University. The cell lines were incubated at 37°C under a humidified atmosphere with 5% CO2 and cultured in DMEM (Invitrogen, CA), supplemented with 10% fetal bovine serum (FBS) (GIBCO, NY), 1 mmol/L glutamine, and 1% penicillin/streptomycin.
Cell Viability, Apoptosis, and Invasion Assays
Cell proliferation was analyzed using a commercial CCK-8 assay kit (#C0038, Beyotime). We employed fluorescence-activated Cell Sorting (FACS) to assess apoptosis with the Annexin V-FITC/PI staining kit (Mbchem). According to the manufacturer, the transwell assay was used to measure cell invasion with the 6well insert devices (8 mm pore size; Corning Life Sciences, Bedford, MA) and Biocoat Matrigel (BD Biosciences). We counted the cells from the middle and surrounding 5 fields of view and averaged the counts.
Western Blotting
Western blotting was performed as described previously (39). Briefly, total protein was extracted with RIPA lysis buffer and estimated using the bicinchoninic acid (BCA) Protein Assay Reagent Kit. The proteins were separated using 10% SDS-PAGE and electrophoretically transferred on a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk and incubated overnight at 4°C with primary antibodies targeting FTO (27226-1-AP) and b-actin (1:2000, Ptgcn, 66009-1-Ig) and anti-GAPDH (Abcam, ab125247), followed by incubation with the appropriate secondary antibodies for one hour. Positive bands were visualized using an enhanced chemiluminescence system.
Measurement of Total m 6 A
Total m 6 A content was measured in the aliquots of 200-ng of total RNA extracted from cells using an m 6 A RNA methylation quantification kit (cat. no. P-9005; Epigentek) according to the manufacturer's instructions.
Statistical Analysis
The non-paired t-test was used to compare the normally distributed variables between the two groups; the Mann-Whitney U test (also known as the Wilcoxon rank-sum test) was used to estimate the statistical significance of the nonnormal distribution variables. The Kruskal-Wallis test and a one-way ANOVA were used as nonparametric and parametric analysis methods (40). The correlation coefficient between TME infiltrating immune cells and the expression of m 6 A regulatory genes was calculated using the Pearson correlation method. We used the univariate Cox regression model to calculate the risk ratio (HR) for the m 6 A regulatory genes. The survminer R software package was used to determine the cut-off point for the correlation between the m 6 A scores and patient survival in each data set subgroup. The "surv-cut point" function of the maximum rank statistic was used to double score the m 6 A score by repeatedly testing all possible cut-off points, and further, the patients were classified into "high" and "low" subgroups, according to the maximum selected log-rank statistic, to reduce the batch effect in the calculations. The Kaplan-Meier method was used to plot the survival curve for prognosis analysis, and a log-rank test was used to determine the significance of the difference. The waterfall diagram of the maftools software package was used to show the mutations in the 23 m 6 A regulatory factors in the TCGA-BLCA cohort. The thermograms of the 23 mutations for the m 6 A regulatory factors were plotted. All statistical P-values were bilateral, and P < 0.05 was considered to be statistically significant.
Survey of 23 m 6 A Regulatory Factors in Bladder Cancer
In this study, we analyzed 23 m 6 A regulatory factors related to bladder cancer, including 2 erasers (ALKBH5, FTO), 13 readers (FMR1, HNRNPA2B1, HNRNPC, IGF2BP1, IGF2BP2, IGF2BP3, YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, ELAVL1, LRPPRC), and 8 writers (KIAA1429, METTL14, METTL3, RBM15, RBM15B, WTAP, ZC3H13, CBLL1). We first mapped the network for the 23 m 6 A regulatory factors in bladder cancer ( Figure 1A), and examined the interactions and their significance for the prognosis of bladder cancer patients. We found that there were significant correlations between the expression of m 6 A regulatory factors in writers, erasers, and readers, and most of the correlations were positive (Supplementary Table 1). Based on these correlations, the 23 m 6 A regulatory factors were divided into four categories (Clusters A-D). Figure 1B summarizes the somatic mutation frequency of the 23 m 6 A regulators. In 116 out of 412 patient samples, a mutation in m 6 A regulators was observed, with a frequency of 28.16%. Most of the mutations were missense mutations, and the regulators KIAA1429, METTL3, and ZC3H13 had the highest mutation frequency (4%), while HNRNPC, YTHDF3, and FMR1 did not harbor any mutations. Subsequently, we found that FMR1 and YTHDF2, YTHDF1 and KIAA1429, YTHDF2 and KIAA1429, WTAP, and METTL3, ZC3H13 and LRPPRC, and ZC3H13 and YTHDC2 had significant symbiotic relationships ( Figure 1C, P < 0.05). The differential expression analysis for the 23 m 6 A regulatory factors in bladder cancer and its adjacent tissues is shown in Figure 1D. The expression of ALKBH5, FTO, METTL14, WTAP, YTHDC1, YTHDF3, and ZC3H13 in adjacent cancer tissues was significantly higher than that in cancer tissues, while the expression of METTL3, YTHDF1, YTHDF2, ELAVL1, IGF2BP1, IGF2BP3, and HNRNPA2B1 in cancer tissues were significantly higher than those in non-cancerous tissues. However, due to the small sample size of the paracancerous tissues, this result needs further verification. A univariate Cox regression analysis was performed to study the effect of the 23 m 6 A regulatory factors on the overall survival (OS) of bladder cancer patients ( Figure 1E). It was observed that six m 6 A regulatory factors had a significant effect on the prognosis for bladder cancer patients. YTHDC1 and WTAP may be protective factors (HR < 1, P < 0.05), while IGF2BP2, ALKBH5, IGF2BP3, and FTO are risk factors for bladder cancer (HR > 1, P < 0.05).
Three Different m 6 A Modification Patterns Defined by the 23 m 6 A Regulatory Factors
Based on the gene expression of the 23 m 6 A regulatory factors, 407 patients with bladder cancer were classified using the Rpackage ConsensusClusterPlus. Our analysis showed that 3 clusters were optimal (Figures 2A, B and Supplementary Figure 1A-D) and the principal component analysis showed that all patients could be well classified into three subtypes ( Figure 2C), and we identified three different m 6 A modification patterns in 407 patients with bladder cancer, which we named Cluster 1-3. We plotted a heatmap ( Figure 2D) and boxplot ( Figure 3A) for the expression of the 23 m 6 A regulatory factors in Cluster 1-3 and found that the expression of IGF2 BP1, IGF2BP2, IGF2BP3, KIAA1429, and RBM15 in Cluster 2 was lower than in the other clusters (P < 0.01), while the expression of METTL3, RBM15B, YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, and ELAVL1 was higher in Cluster 2 than the other clusters. Significantly higher overall survival was observed in the patients who had m 6 A modifications from cluster 2 ( Figure 3B). To better understand the potential differences between the three m 6 A modification patterns, we compared them with the published molecular typing of bladder cancers, including Baylor, MDA, CIT-Curie, and UNC ( Figure 3C). Using the CIT classification, Cluster 1 and Cluster 3 showed more of MC7 subtypes; whereas, more MC1 subtypes were seen in Cluster 2; Using the Baylor classification, Cluster 1 and Cluster 3 had more basal subtypes, and Cluster 2 contained more differentiated subtypes; based on the UNC classification, the basal subtype was more frequent in Cluster 1 and Cluster 3 than the luminal subtype, while Cluster 2 showed the opposite result. To further characterize the molecular differences between the RNA based subtypes, we analyzed the expression of 23 regulatory genes associated with bladder cancer significantly higher than that in Cluster 1 and Cluster 3, while the opposite relation was found for STAT3, FOXM1, EGFR, HIF1A, GATA6, FGFR1, and RARB ( Figure 3D). Further, we identified differences in the mutation of 20 genes in the three m 6 A modification patterns as shown in the waterfall diagram (Supplementary Figure 1E). and we analyzed the difference in Copy number alterations across three m 6 A modification patterns. (Supplementary Figures 2, 3).
Difference in Immune Cell Infiltration Characteristics in the Tumor Immune Microenvironment for Different m 6 A Modification Patterns
To reveal the role of m 6 A modification patterns in TME immunoregulation, we calculated the abundance of 28 cellular subsets in each patient sample. The heatmap shows the average difference in the infiltration level of 28 types of immune cells in the three different m 6 A modification patterns (Figure 4A, Supplementary Tables 2, 3), We found significant differences in the characteristics of cellular infiltration in the TME; Cluster 2 is characterized by immunosuppression, and most of the infiltrating cells are immune cells. The infiltration level was significantly lower than that of cluster1 and cluster3 (Supplementary Figure 4A). In addition, the anti-tumor immunity score and the pro-tumor immunity score of Cluster 2 were significantly lower than that of Cluster 1 and Cluster 3 (Supplementary Figures 4B, C), and the pro-tumor score of Cluster 2 is significantly higher than in Cluster 1 and Cluster 3 ( Figure 4B). The ESTIMATE algorithm can estimate the ratio of the immune and matrix components of each sample in the TME, which is presented in the form of three scores: the immune score, stromal score, and the ESTIMATE score. We found that the three scores in Cluster 2 were significantly lower than those in Cluster 1 and Cluster 3 ( Figure 4C), suggesting that m 6 A methylation may be involved in regulating the type of TME infiltrating cells and play a central role in immune regulation. However, the mechanism for how m 6 A modification affects the immunophenotype is unclear. Therefore, we analyzed the correlation between the 23 m 6 A regulatory factors and the 28 types of immune cells ( Figure 4D). A heatmap of the correlation showed that the m 6 A regulators were highly correlated with most of the immune cells, and WTAP, IGF2BP2, and IGF2BP3 showed the most significant correlation with most immune cells. Further, YTHDF2, YTHDC1, METTL3, and ELAVL1 showed a high negative correlation with most immune cells, indicating that these regulators may play an important role in the differentiation and recruitment of immune cells. To further explore the possible role of m 6 A related phenotypes in immunotherapy, we studied the difference in the expression of the immune checkpoint related genes CD274, PDCD1LG2, CTLA4, LAG3, PDCD1, HAVCR2, TIGIT, and the CD8+T cell marker genes including CD8A, GZMB, CXCL9, CXCL10, PRF1, TBX21, and CD8B in the three different m 6 A modification patterns ( Figure 4E, F). Our results showed that the expression of these genes in Cluster 2 was significantly lower than that in Cluster 1 and Cluster 3 (P < 0.0001). The results suggest that m 6 A methylation affects the type of TME infiltrating cells, and Cluster 2 had a significantly lower level of immune cell infiltration related to the immune checkpoint than the other two modification patterns. The gene expression in Cluster 2 is significantly low, and we speculated that the response to immunotherapy in patients showing Cluster 2 type m 6 A modifications may be worse than that of Cluster 1 and Cluster 3.
Modes of m 6 A Modification
To reveal the biological differences among the three different m 6 A modification patterns, we undertook expression profiling of the patient tissue samples and identified 403 DEGs ( Figure 5A). The Circos diagram shows the overlap of the DEGs obtained by pairwise comparison of the three different m 6 A modification patterns. Cluster 2 had more DEGs than did Cluster 1, and there are more common genes in the list of DEGs obtained for Cluster 1 than that obtained for Cluster 2, and between Cluster 3 and Cluster 2 ( Figure 5B). In Figure 5B, the blue lines connect genes belonging to the same ontology term showing the number of functional overlaps between different groups. We used Metascape (36) to analyze the enrichment of signaling pathways between the different m 6 A modification classes. The results are shown in the heatmap which is colored by p-values, and the white cells indicate a lack of enrichment of the term in the corresponding gene list (Figures 5C, D, and Supplementary Figures 5A, B). Additionally, a GSEA was performed for further signaling pathway enrichment analysis ( Figure 5E), IL2/STAT5 signaling, EMT, inflammatory response, interferon-gamma response, and TNFa signaling via NFkB were enriched in Cluster 1. Allograft rejection, complement, EMT transition, inflammatory response, kras signaling are enriched in Cluster 3. These signaling pathways involve different core biological processes, most of which are related to the regulation of immunity, and some of them have been confirmed to have a correlation in immunotherapy (48)(49)(50), which provides a basis for further exploration of the effect of m 6 A modifications on the immunophenotype.
The m 6 A Score Is an Important Prognostic Biomarker and Predictor of Bladder Cancer
We constructed and evaluated a scoring system called the m 6 Ascore to quantify the level of m 6 A modification in each patient sample by ssGSEA, taking into account the individual differences and complexity of the m 6 A modification. First, we conducted a survival analysis using the m 6 Ascore and found that patients with high m 6 Ascore had a poor prognosis (p = 0.036, Figure 6A). There were also significant differences in the m 6 Ascore among the three different m 6 A modification patterns, with the lowest and highest m 6 Ascores being assigned to Cluster 3 and Cluster 2, respectively ( Figure 6B). Next, we analyzed the differences in m 6 Ascore in the MDA cancer subtype and found the highest score for the basal subtype, followed by the luminal subtype, with the lowest expression level observed in the p53-like subtype ( Figure 6C). To explore the potential biological relevance of m 6 Ascore, we classified 407 patients into a high m 6 Ascore group and a low m 6 Ascore group based on the expression level of the 23 m 6 A regulators. We first examined the difference in the composition of immune cells between the two patient groups ( Figure 6D). Our results showed that CD56 dim natural killer cells, central memory CD4T cells, eosinophils, mast cells, monocytes, and type 17 T helper cells showed a significantly low enrichment in the high m 6 Ascore group. Further, the activated CD4 + T cells, memory B cells, natural killer T cells, neutrophils, and type 2 T helper cells showed highly significant enrichment in the low m 6 Ascore group. We analyzed the DEGs, and additionally examined the signaling pathways showing enrichment via GO and KEGG pathway analysis between the two groups. The pathways showing enrichment for molecular function (MF), cellular component (CC), and biological process (BP) via GO analysis were examined. Signaling pathways enriched for BP include the xenobiotic metabolic process, hormone metabolic process, epidermis development, etc.; pathways enriched for CC included the apical part of the cell, apical plasma membrane, cornified envelope, etc.; and pathways enriched for MF include monooxygenase activity, serine-type endopeptidase activity, and other signaling pathways (Supplementary Figures 5C-E). A KEGG pathway analysis showed that the DEGs were mainly enriched in glutathione metabolism, ether lipid metabolism, and other signaling pathways (Supplementary Figure 5F). A GSEA analysis ( Figures 6E, F) showed that pathways for the inflammatory response, interferon-gamma response, myc targets V1, TNFa signaling via NFkB, and other genes were concentrated in the high m 6 Ascore group. These results strongly suggest a significant correlation between a high m 6 Ascore and immune activation. The results also demonstrate the central role of m 6 A methylation in different biological processes in bladder cancer, including different aspects of immune regulation. The m 6 Ascore may be used to evaluate the characteristics of immune cell infiltration in bladder cancer tissue to predict the clinical response of patients to immunotherapy. Thus, the m 6 Ascore may have the potential to be used as an independent prognostic biomarker to predict the OS rate of patients, guiding more effective clinical practice.
Effect of m 6 A Methylation Modification on Anti-PD-L1 Immunotherapy for Bladder Cancer
To explore the potential effect and mechanism of m 6 A methylation on immunotherapy for bladder cancer, we obtained data for the IMvigor210 cohort, which is an openlabel, multicenter, single-arm phase II clinical study to evaluate the safety and efficacy of Tecentriq, an anti-PD-L1 agent, in patients with advanced urothelial cancer (Supplementary Table 4). Patients who responded in whole or in part were classified as responders and were compared with non-responders who showed the incidence of stable or progressive disease. We performed an unsupervised clustering using data for mRNA expression for the 23 m 6 A regulatory factors from all patients. We identified three different m 6 A modification patterns and calculate the m 6 Ascore for subsequent analysis. Additionally, we performed a combined heatmap-based analysis (Figure 7) to find the correlation between the different m 6 A methylation modes and the Lund and TCGA molecular typing for the anti-PD-L1 based immunotherapy. Further, for the three groups of m6A modification patterns, we also analyzed the differences in PD-L1 expression in immune cells (IC) and tumor cells (TC), patients' response to anti-PDL1 efficacy, TMB, m 6 Ascore, and the important gene mutations prevalent in bladder cancer. We also analyzed the differences in the expression of genes in Each signal path is represented by a circular node, whose size is directly proportional to the number of input genes in the term. The color represents the cluster identity, i.e., nodes with the same color belong to the same cluster. Terms with similarity score > 0.3 are linked by edges (the thickness of the edge represents the similarity score). The network was visualized using Cytoscape (v3.1.2).
(E) GSEA of the marker gene set downloaded from the MSigDB database. All transcripts were sequenced according to the multiple variations (log 2 ) obtained using the different analyses among three different m 6 A modification patterns. GSEA analysis evaluated the skewness of the two distributions of the selected gene set in the sequenced gene list. (51), including the FGFR3 gene signature; CD8 Teff signature; antigenprocessing machinery; immune checkpoint signature; MKI67 and cell cycle genes; DNA replication-dependent histones; DNA damage-repair genes; TGFb receptor and ligand; F-TBRS genes; EMT markers; and the angiogenesis signature. In the comparison between the different m 6 A modification patterns and the TCGA and Lund classification using a heat map-based analysis, we found that most genes in Cluster 1 were classified as subtype I and the least as subtype IV. For the Lund classification, we observed that the UroA subtype had the highest proportion in Cluster 1, the Inf type has the highest proportion in Cluster 2, and the SCCL subtype was more enriched in Cluster 1. Additionally, we found that the Cluster 3 modification mode had higher levels of PD-L1 expression in immune cells and tumor cells, a higher proportion of CR/PR (Figure 7 and Figure 8A). These results suggest that m 6 A methylation may be involved in the response to anti-PDL1 therapy. Additionally, among the three different m 6 A methylation patterns in bladder cancer, there were differences in gene mutations (51) for TP53, RB1, FGFR3, CDKN2A, ERBB2, and PIK3CA. Patients had a greater number of FGFR3 and CDKN2A mutations in Cluster 1 than in Cluster 2 or Cluster 3, while the converse was true for TP53 mutations. There were significant differences among the Figure 8A). We found differences in m 6 Ascore levels among the three m 6 A modification patterns, with the lowest m 6 Ascore level found in Cluster 2, while the m 6 Ascore of Cluster 3 and Cluster 1 were the same, with no significant difference ( Figure 8B). Differences in the m 6 Ascore were also observed in immunophenotyping, with the excluded subtype having the highest m 6 Ascore, and no significant difference in m 6 Ascore found between the inflamed and desert subtypes ( Figure 8C). Next, we analyzed the correlation between m 6 Ascore and biological pathway gene scores (39) in bladder cancer ( Figure 8D). The m 6 Ascore was positively correlated with the scores for DNA replication, mismatch repair, Fanconi, base excision repair, nucleotide excision repair, homologous recombination, cell cycle, and DDR, and negatively correlated with EMT2, EMT1, immune checkpoint, CD8 T effector, APM, etc. The effect of the m 6 Ascore on OS was observed by plotting the KM curve, which indicated that a higher m 6 Ascore had a poor prognosis (P = 0.032, Figure 8E). Using the SubMAP algorithm, we predicted response to the anti-PD1 and anticytotoxic T-lymphocyte-associated protein 4 (CTLA4) immunotherapy in the groups with high and low m 6 A score. High m 6 Ascore group may respond better to the PD-1 treatment (Bonferroni-corrected P = 0.03)
FTO Might Play an Important Role in Anti-PD-L1 Immune Checkpoint Therapy
The results from our data analysis showed that patients with high expression of FTO had a poor prognosis in the IMvigor210 cohort (p = 0.0035; Figure 9A) and there were differences in FTO expression in the three groups ( Figure 9B). We also queried the predictive value of FTO expression in the cohort for anti-PD-L1 immunotherapy and found that there was a significant difference in FTO expression between non-responders and objective remission groups. Non-responsive patients showed significantly higher FTO expression ( Figure 9C), suggesting that FTO may play an important role in the anti-PD-L1 treatment of bladder cancer. Upon further examination, FTO expression was positively correlated with the EMT signaling pathway and negatively correlated with the DDR/cell cycle, nucleotide excision repair, Fanconi anemia pathway, and other signaling pathways ( Figure 9D), and additionally, was negatively correlated with the protein level of PD-L1 in immune cells ( Figure 9E). FTO had no significant correlation with the total PD-L1 level in tumor cells ( Figure 9F). The analysis of data from the TCGA database showed that the prognosis of patients with a high expression of FTO was worse (p = 0.0015; Figure 9G), and FTO expression was higher in patients with a high stage of bladder cancer ( Figure 9H). Additionally, the enrichment results of FTO-related gene pathways showed that FTO may be involved in the regulation of focal adhesion, the Hippo signaling pathway, the TGF-b signaling pathway, and other signal pathways ( Figure 9I). Previous studies have confirmed that the activation of EMT and TGF-b related pathways reduces T cell migration to tumors and weakens their tumor-killing effect (5,51). Therefore, FTO may play an important role in the occurrence and development of bladder cancer, the efficacy of anti-PD-L1 immune checkpoint therapy, and might be useful for predicting the prognosis. (H) Box plot showing the difference in FTO expression between stage I and II and stage III and IV; the asterisk represents the statistical p-value (**P < 0.01; ****P < 0.0001). (I) Pearson correlation was used to calculate the correlation between FTO and all other protein-coding genes, and genes with a correlation coefficient R >= 0.03, and p < 0.05 were selected as FTO-related genes. These genes were enriched by KEGG signaling pathway analysis to study FTO function.
FTO Could Be Involved in the Occurrence and Development of Bladder Cancer
We examined the nuclear, cytoplasmic, and overall expression levels of FTO in nine human bladder cancer cell lines: BIU87 cells, 5637 cells, T24 cells, EJ cells, RT4 cells, J82 cells, UM-UC-3 cells, TCCSUP cells, and human bladder epithelial immortalized SV-huv-1 cells ( Figure 10A). Using immunofluorescence two cell lines with high FTO expression (EJ and T24 cells) and two cell lines with low FTO expression (BIU87 and RT4) were identified. We found that FTO was expressed in both cytoplasm and nucleus of these cell lines ( Figure 10B). Subsequently, we knocked down the FTO in EJ and RT4 using siRNA. CCK-8 examination revealed that cell proliferation was reduced in the si-FTO group ( Figure 10C), and transwell assays showed reduced cell invasion in the si-FTO group ( Figure 10D).
Flow cytometry revealed increased apoptosis after the knockdown of FTO ( Figure 10E). The level of total cellular FTO-m 6 A enzyme activity was significantly increased ( Figure 10F), and total FTO protein was decreased in the si-FTO group ( Figure 10G). These results confirm the critical role of FTO in bladder cancer. According to our previous study (53), we confirmed the existence of important modification sites K216 for FTO protein, including K216R, K216H, K216S, and K216E. We constructed plasmids and transfected them into BIU87 and RT4 cells, as well as detected the expression of FTO protein in each group by WB (Western blot) ( Figure 11A). The apoptosis was increased in the FTO-MT (K216H) and MT (K216E) groups ( Figure 11B transwell assay ( Figure 11C), and the total FTO-m 6 A enzyme activity level was significantly increased in these two groups ( Figure 11D). The above results suggest that K216H and K216E are the important targets for regulating FTO in the future. The histogram data for each group is the average of three independent replicates; bars represent mean ± SD; *P < 0.05, ***P < 0.001, ****P < 0.0001, ns P > 0.05. maladjusted m 6 A regulatory factors alone (or editing targeted mutant or dysfunctional m 6 A sites through targeted transcriptomics), or in combination with other therapies. Targeting the m 6 A modification may have a strong therapeutic potential for the treatment of all types of cancers, especially those that are resistant to existing treatments (56).
DISCUSSION
Due to technical and resource constraints, most studies have focused on a single TME immune cell type or a single m 6 A. The overall infiltration characteristics of the TME mediated by the combined effects of multiple m 6 A regulators have not been widely analyzed (57,58). The modification of m 6 A is a reversible process that is affected by various writers, erasers, and readers. In this study, we explored the effects of multiple m 6 A regulators on the infiltration of immune cells in the TME of bladder cancer, the molecular mechanisms underlying m 6 A modification in bladder carcinogenesis, the immune response, and the drug resistance of immune checkpoint inhibitors. We also provide new insights into the role of m 6 A modification patterns in TME cell infiltration and developing more effective immunotherapy strategies.
We classified patients with bladder cancer based on the expression of 23 m 6 A regulatory molecules and identified three different m 6 A modification patterns. We found that Cluster 2 showed unique specificity compared with the other two types. A quantification of 28 immune cells in the immune microenvironment of bladder cancer using the ssGSEA algorithm showed that Cluster 2 had a low level of immune cell infiltration characterized by immune inhibition, which corresponds to the immune desert phenotype. Furthermore, an analysis performed using the ESTIMATE algorithm showed that the stromal and immune scores in Cluster 2 were lower than the other two subtypes, which corroborates our previous results. The expression of immune checkpoint and CD8 + T cell markers were lower in Cluster 2 than in the other two groups, and studies have shown that the expression of these markers is highly sensitive to immune checkpoint inhibitors (52). Thus, different m 6 A modification patterns are significantly related to immune activation, and a comprehensive evaluation of the m 6 A modification patterns will enable the understanding of the characteristics of TME cell infiltration. An analysis of DEGs between the different m 6 A modification patterns uncovered m 6 A-related signaling pathway genes. These were mainly enriched in the biological pathways related to the matrix and immune activation. In the anti-PD-L1 treatment cohort, we identified three different m 6 A modification patterns and found significant differences in gene expression and mutations in biological pathway genes associated with the three subtypes of bladder cancer. We constructed a scoring system to quantify and evaluate the m 6 A modification level in a single tumor and found that a high m 6 Ascore was related to poor prognosis in the TCGA and anti-PD-L1 cohorts. Patients with a high or low m 6 Ascore showed differences in immune cell infiltration in the tumor immune microenvironment. A GSEA signaling pathway analysis indicated that the high and low m 6 Ascore are mainly enriched in the matrix and immunerelated signaling pathways. Together, these results indicate that the m 6 A score is a reliable tool for the comprehensive evaluation of m 6 A modification patterns in tumors in individuals, and to determine the mode of infiltration in the TME. The expression and gene changes for m6A regulatory factors are related to a variety of biological processes, especially the matrix and immune activation. Our findings may provide new impetus for improving the clinical response of patients with bladder cancer to immunotherapy, the identification of different immunophenotypes in bladder cancer, and promoting individualized immunotherapy.
The role of FTO in cancer has recently garnered increasing attention. Previous studies conducted by our team have shown that FTO-mediated m 6 A modification plays an important role in hepatocellular carcinoma, and the SIRT1 deacetylase can play a carcinogenic role by down-regulating FTO. FTO is an RNA demethylase that can remove the methylation of m 6 A in mRNA both in vitro and in vivo (59,60). FTO has catalytic demethylation activity for both cap-m 6 Am and internal m 6 A. As the abundance of m 6 A in mRNA is much higher than that of its preferred binding target m 6 Am, the main target of FTO is m 6 A (61). Reports indicate that the FTO-mediated demethylation of cap-m 6 Am leads to mRNA degradation (62), but the evidence for this is inconsistent. PCIF1 is the cap-m 6 Am methyltransferase, which processes cap-m 6 A alone but not internal m 6 A. Reports indicate that the cap-m 6 Am added by PCIF1 does not change the level of gene expression or the stability of transcripts (63)(64)(65). Additionally, studies (66) have found that the spatial distribution of FTO can also play a regulatory role. The N-terminus of FTO has an NLS, which can be partially distributed in the nucleus and the cytoplasm, and the distribution of FTO is different in different cell lines, its role and regulation being affected by the environment. Yang et al. (20) have shown that the induction of FTO can be used as an adaptive mechanism to combat metabolic stress in melanoma cells, thus increasing their proliferation, invasion, and migratio n, an d p ro mo ting the tumorigen esis an d development of melanoma in mice. Further, the authors showed that an FTO knockout can increase m 6 A methylation in key oncogenic melanoma cells, including the loci for PD-1 (PDCD1), CXCR4, and SOX10. The inhibition of FTO makes melanoma cells sensitive to interferon-g (IFN-g) and anti-PD-1 therapy in mice, indicating that FTO plays an important role in promoting the occurrence of melanoma and anti-PD-1 drug resistance. The role of m 6 A involvement with FTO in bladder cancer had not been reported, and we found that in the TCGA and IMvigor210 cohorts, patients with high FTO expression had a poor prognosis, and the expression of FTO was higher in patients with a higher stage of bladder cancer.
In addition, we analyzed the predictive value of FTO expression in the cohort subjected to anti-PD-L1 immunotherapy, and found that there was a significant difference in FTO expression between the non-responders and the objective remission group. The level of FTO expression in the patients non-responsive to anti-PD-L1 therapy was significantly higher than in patients in remission. The data showed that FTO expression was negatively correlated with the protein level of PD-L1 in immune cells ( Figure 9E), was positively correlated with the EMT signaling pathway, and negatively correlated with DDR, cell cycle, nucleoside exercise repair, Fanconi anemia pathway, and other signaling pathways. The enrichment of FTO-related gene pathways indicated that FTO might be involved in the regulation of focal adhesion/Hippo signaling pathway/TGF-b signaling, these results suggest that FTO plays an important role in bladder cancer. The combination of FTO targeted regulation and anti-PD-L1 blockers may have great therapeutic potential for reducing the resistance of bladder cancer to immunotherapy. Thus, this study furthers the understanding of the regulation of m 6 A modification in the tumor immune microenvironment of the bladder cancer and contributes to the development of new predictive indicators, drug combination strategies, and new immunotherapeutic strategies for cancer immunotherapy. However, its specific role and mechanism need further experimental study.
In this study, we systematically analyzed the mutation and correlation of 23 kinds of m 6 A regulatory factors in bladder cancer, and their effects on OS and immune invasion, We found three different patterns of m 6 A modification and compared them with other important molecular types of bladder cancer, such as MDA, Lund, and Baylor, the effects of the three kinds of m 6 A modification patterns on the mutation characteristics, clinicopathological characteristics, gene expression, immune cell infiltration level, and gene expression level of immune checkpoint regulators were comprehensively analyzed. Further, we investigated the effect of m 6 A modification mode on the therapeutic efficacy of bladder cancer immune checkpoint inhibitor anti-PD-L1. Our results confirmed that m 6 A methylation is involved in the process of immune cell recruitment in the TME of bladder cancer, and may affect the efficacy of anti-PD-L1 therapy. In addition, we established a method to quantify the level of m 6 A modification (m6Ascore), which we found to be an important and powerful prognostic biomarker and predictor for bladder cancer. Thus, this study furthers the understanding of the regulation of m 6 A modification in the tumor immune microenvironment of bladder cancer and contributes to the potential development of new predictive indicators, drug combination strategies, and new immunotherapeutic strategies for cancer immunotherapy.
CONCLUSION
This study comprehensively recognized the role of m 6 A methylation modification on the invasion characteristics of bladder cancer immune microenvironment cells and the effect on the anti-PD-L1 treatment for bladder cancer. The difference in m 6 A modification mode is an important factor indicating the heterogeneity and complexity of the tumor microenvironment and the immunotherapy impact. The m 6 A modification mode helps decipher the molecular mechanisms underlying the immune microenvironment regulation in bladder cancer and provides new predictive indicators, possible auxiliary targets, and directions for guiding more effective immunotherapy strategies in the future.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
ETHICS STATEMENT
The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Ethical Review Committee of the Third Xiangya Hospital of Central South University. Written informed consent was obtained from individuals or guardians of the participants.
AUTHOR CONTRIBUTIONS
XC and KC designed the study. XC and HD analyzed and interpreted the data. HD and XC wrote this manuscript. HD, FT, MZ, and DS conducted the experiments and edited and revised the manuscript. All authors have seen and approved the final version of the manuscript. The box plot shows the difference for 28 immune cell infiltration levels in the three m 6 A modification patterns; the asterisk represents the statistical p-value ( * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001). (B, C) The differences in the antitumor immunity score and pro-tumor immunity score among three kinds of m 6 A modification patterns; the asterisk represents the statistical p-value.
Supplementary Figure 5 | Signaling pathway enrichment analysis. (A, B) In the TCGA-BLCA cohort, there are three different m 6 A modification patterns, and different gene signaling pathways are enriched; the same enrichment network has its nodes displayed as pies. Each pie sector is proportional to the number of hits that originated from a gene list. Color code for the pie sector represents a gene list and is consistent with the colors used for the table rows in the slide. The same enrichment network has its nodes colored by p-value, as shown in the legend. Darker color indicates more statistically significant nodes (see legend for p-value ranges).
(C-E) The enrichment analysis of Gene Ontology (GO) signaling pathways in the high and low m 6 A groups was divided into three parts: C, cellular component (CC); D, biological process (BP); E, molecular function (MF). (F) Enrichment analysis of KEGG signaling pathways of DEGs in high and low m 6 A groups. | 2021-12-08T16:20:44.588Z | 2021-12-06T00:00:00.000 | {
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2969269 | pes2o/s2orc | v3-fos-license | Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis
Background α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.
Introduction
Targeting cell surface death receptors, especially tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) binding receptors, holds promise for cancer treatment [1,2]. TRAIL selectively induces apoptosis in a wide variety of cancer cells with little or no toxicity towards normal cells [1,2]. Thus, agents that can enhance TRAIL death receptor (TRAIL-R/DR4 or TRAIL-R2/DR5) signaling or sensitize TRAIL resistant cells to TRAIL induced apoptosis are of interest [2,3]. TRAIL/DR4/DR5 apoptotic signaling includes: interaction of TRAIL with DR4 or DR5, receptor clustering, recruitment of the adaptor molecule FADD, and activation of initiator caspases-8 or -10, leading to cleavage of downstream effector caspases (mitochondrial-independent apoptosis) or cleavage of Bid, a pro-apoptotic Bcl-2 family member, leading to mitochondrial-dependent apoptosis [4].
Evading apoptosis is a hallmark of cancer [5]. One way tumor cells can escape death signals is by expression of anti-apoptotic pro-survival proteins [6]. Therefore, targeting anti-apoptotic proteins also holds promise for killing cancer cells and sensitizing them to different therapeutics [7]. c-FLIP (cellular FADD-like IL-1a-converting enzyme inhibitory protein), is a death effector domain containing protein that regulates extrinsic death receptor signaling from the tumor necrosis factor-a (TNF-a) family of cell surface death receptors, including DR4/DR5, Fas (CD95/APO-1), and TNFR [8]. c-FLIP is a catalytically inactive caspase-8/10 homolog and typically functions as a caspase-8 inhibitor resulting in chemotherapeutic drug resistance [8].
These data are the first to show that a-TEA induces ER stress dependent increases in death mediators JNK/CHOP/DR5 and decreases in survival mediators c-FLIP-L and Bcl-2 in human breast cancer cells. These ER stress mediated events function downstream of a-TEA triggered TRAIL/DR5/caspase-8 signaling, leading to up-regulation of JNK, CHOP and DR5 and downregulation of c-FLIP and Bcl-2.
Materials and Methods
Chemicals a-TEA was made in house as previously described [9]. ER stress inhibitor salubrinal was purchased from Calbiochem (La Jolla, CA). Caspase-8 inhibitor Z-IETD-FMK was purchased from BioVision (Mountain View, CA).
Cell Culture
MDA-MB-231 estrogen-receptor negative human breast cancer cells were purchased from the American Type Culture Collection (Manassas, VA). MCF-7 estrogen-responsive human breast cancer cells were originally provided by Dr. Suzanne Fuqua (Baylor College of Medicine, Houston, TX). Both cell lines were cultured in MEM media with 10% FBS. For experiments, FBS was reduced to 2% and cells were allowed to attach overnight before treatments. a-TEA (40 mM) was dissolved in ethanol as stock solution. Equivalent level of ethanol (0.1%) was used as vehicle control (VEH) for a-TEA treatment (40 mM).
Quantification of apoptosis
Apoptosis was quantified by Annexin V-FITC/PI assay following the manufacturer's instructions (Invitrogen).
RT-PCR detection of DR5, Bcl-2 and XBP-1 mRNA expression
Total RNA was extracted using RNA isolation kit (Qiagen Inc. Valencia, CA). Semi-quantitative analyses were conducted to detect DR5, Bcl-2 and XBP-1mRNA form by reverse transcriptase -polymerase chain reaction (RT-PCR) using the housekeeping gene b-actin as control. 5 mg total RNA was reverse transcribed to cDNA using 1 ml Superscript RTase (250 U, Invitrogen) following the manufacture's instructions. 1 ml cDNA was used per PCR reaction with 15 ml Taq PCR Master Mix Kit (Qiagen Inc) plus 10 mM oligonucleotide primer pairs (Invitrogen). Primer sequences are available upon request.
RNA Interference
A scrambled RNA duplex purchased from Ambion (Austin, TX) that does not target any of the known genes was used as the nonspecific negative control for RNAi (referred to as control siRNA). Transfection of MCF-7 or MDA-MB-231 cells with siRNA to DR5, TRAIL, CHOP, JNK, Itch or control (Ambion, Austin, TX) was performed in 100 mm cell culture dishes at a density of 2610 6 cells/dish using Lipofectamine 2000 (Invitrogen) and siRNA duplex, resulting in a final siRNA concentration of 30 nM following the company's instructions. After one day exposure to transfection conditions, the cells were re-cultured in 100 mm dish at 2610 6 cells/dish and incubated for one day followed by treatments.
Chromatin immunoprecipiation (ChIP) assay
MDA-MB-231 cells were treated with 40 mM of a-TEA or vehicle (0.1% ethanol) for 15 hours. Protein to DNA cross-linking was conducted by adding 1% formaldehyde to the cell culture medium for 12 min followed by the addition of glycine (0.125M) to stop the cross-linking. Cells were collected for ChIP assay as described by Nelson et al. [25]. The CHOP antibody used for the Western blot analysis was used for immunoprecipitation. Normal mouse IgG 1 purchased from Santa Cruz Biotechnology was used as an isotype control. Polymerase chain reaction (PCR) was conducted using the primers as described by Adbelrahim et al. [26] to detect CHOP binding sites in the DR5 promoter region.
Ectopic Expression of c-FLIP (L)
Transient transfection of MCF-7 cells with wildtype His-tagged FLIP expression construct, pcDNA3.1-His-c-FLIP (L), which was kindly provided by Dr. John C. Reed (The Burnham Inst. La Jolla, CA) [27] was performed following the procedure published before [23]. Transfected cells were cultured overnight before a-TEA treatment.
Statistical Analyses
Apoptosis data were analyzed using a two-tailed student t-test to determine statistical differences among treatments. Differences were considered statistically significant at p,0.05.
Results
a-TEA induces TRAIL/DR5 and CHOP dependent apoptosis a-TEA treatment of MDA-MB-231 and MCF-7 breast cancer cells up-regulates DR5 and CHOP protein levels in a time dependent manner (Fig 1 A). siRNA knockdown of TRAIL, DR5 or CHOP significantly reduces the ability of a-TEA to induce apoptosis in both cell lines as detected by FACS analyses of expression of the apoptotic biomarker, Annexin V (Fig 1 B), suggesting that both TRAIL/DR5 and CHOP are involved in a-TEA induced apoptosis. Since CHOP has been shown to act as a positive transcriptional factor for DR5 [28], siRNA to CHOP was used to examine if CHOP regulate a-TEA induced upregulation of DR5. Data show that siRNA to CHOP reduced the ability of a-TEA to increase CHOP and DR5 (L/S) levels, as well as to cleave PARP, a biomarker of apoptosis, in MCF-7 cells (Fig 1C). To further confirm that DR5 is regulated by CHOP in a-TEA induced apoptosis RT-PCR to detect mRNA levels of DR5 and ChIP analyses to assess CHOP binding activity on the DR5 promoter were conducted in MDA-MB-231 cells. Data show that a-TEA induces increased levels of DR5 mRNA ( Fig 1D) and increased CHOP binding activity on DR5 promoter ( Fig 1E) in comparison to VEH treated control cells. These data indicate that CHOP contributes to a-TEA mediated increases in DR5 protein levels and induction of apoptosis.
a-TEA mediated increases in DR5 and CHOP protein levels are JNK dependent and CHOP influences JNK phosphorylation status Since prior studies showed that JNK activation is a critical component in a-TEA induced apoptosis and other studies have shown that JNK is involved in upregulation of CHOP protein expression [29] the possible involvement of JNK in a-TEA mediated increases in CHOP/DR5 protein expression was investigated in MCF-7 cells. siRNA to JNK reduced total JNK and reduced the ability of a-TEA to increase phosphorylated JNK2/1 (pJNK), increase CHOP and DR5 (L/S) protein levels and to induce apoptosis as measured by PARP cleavage (Fig 2A), suggesting that JNK is a critical contributor to increases in CHOP and DR5 (L/S) protein level. Interestingly, siRNA to CHOP reduced a-TEA mediated increases in Figure 1. a-TEA induces TRAIL/DR5 and CHOP dependent apoptosis. A. MDA-MB-231 and MCF-7 cells were treated with 40 mM a-TEA for 9, 15, and 24 hrs. Western blot analyses were performed to evaluate DR5 (L/S) and CHOP protein levels using GAPDH as loading control. B. MDA-MB-231 and MCF-7 cells were transiently transfected with siRNAs to DR5, TRAIL or CHOP using non-specific siRNA as negative control followed by treatment with 40 mM a-TEA for 18 hrs. Apoptosis was determined by Annexin V/FACS. C. Samples (from B) were analyzed by western blot for PARP cleavage, CHOP and DR5 (L/S) protein levels using GAPDH as loading control. D. mRNA levels of DR5 were determined by RT-PCR. E. The binding activity of CHOP on DR5 promoter was determined by CHIP assay. Data from A, C, D and E are representative of two or more independent experiments. Data from B are the mean 6 S.D. of three independent experiments. * p,0.05 = significantly different from control siRNA determined by t-test. doi:10.1371/journal.pone.0011865.g001 Figure 2. a-TEA induced increased CHOP and DR5 (L/S) protein levels are JNK dependent. In turn, CHOP regulates pJNK2/1 protein levels. A. MCF-7 cells were transiently transfected with siRNA to JNK using non-specific siRNA as negative control followed by treatment with 40 mM a-TEA for 18 hrs. Western blot analyses were performed to evaluate PARP cleavage, pJNK2/1, total JNK2/1, CHOP, and DR5 (L/S) protein levels using GAPDH as loading control. B. MCF-7 cells were transiently transfected with siRNA to CHOP using non-specific siRNA as negative control followed by treatment with 40 mM a-TEA for 18 hrs. Western blot analyses were performed to evaluate pJNK2/1 protein levels using GAPDH as loading control. Data from A and B are representative of two or more independent experiments. doi:10.1371/journal.pone.0011865.g002 pJNK2/1 protein levels (Fig 2B), suggesting that a-TEA mediated increases in JNK phosphorylation are dependent on CHOP expression.
Endoplasmic reticulum stress involvement in a-TEA induced apoptosis contributes to JNK/CHOP/DR5 upregulation a-TEA treatment of MDA-MB-231 and MCF-7 cells increased levels of endoplasmic reticulum stress (ER-stress) indicators: phosphorylated eukaryotic initiation factor 2-alpha (peIF-2a) and glucose-regulated protein of 78kDA (GRP78) also known as BiP (Fig 3A), as well as spliced mRNA forms of X-box binding protein-1 (XBP-1) (Fig 3B) in a time-dependent manner, suggesting that a-TEA treatment induced ER stress in both cell lines. Furthermore, ER-stress inhibitor salubrinal significantly reduced the ability of a-TEA to induce apoptosis detected by Annexin V/FACS (Fig 3C). Since JNK/CHOP/DR5 pathway can be activated in response to ER stress [30], studies were conducted to examine if a-TEA mediated JNK/CHOP/DR5 (L/S) signaling events via ER stress. Data show that ER-stress inhibitor salubrinal significantly reduced the ability of a-TEA to upregulate pJNK2/1, CHOP, and DR5 (L/S), as well as to induce PARP cleavage in both cell lines (Fig. 3D). Salubrinal significantly reduced the ability of a-TEA to upregulate ER stress markers GRP78 and peIF2a, confirming the efficiency of ER-stress inhibitor. These data demonstrate that a-TEA induction of JNK/CHOP/DR5 and apoptosis is mediated via ER stress.
a-TEA induction of ER stress-dependent JNK/CHOP/DR5 signaling is mediated by TRAIL/DR5 pathway Since both JNK and DR5 can be regulated by TRAIL/DR5 signaling [30,31], studies were conducted to determine if a-TEA mediated ER stress events are downstream events of TRAIL/DR5 using siRNA knockdown procedures. MCF-7 and MDA-MB-231 cells transiently transfected with siRNAs to DR5 or TRAIL exhibited reduced levels of DR5 and TRAIL, respectively, and showed blockage of a-TEA's ability to produce PARP cleavage and to increase levels of pJNK2/1, CHOP, DR5 (L/S), as well as blocked a-TEA's ability to upregulate ER stress markers GRP78 and peIF-2a (Fig 4 A, B & C), demonstrating that a-TEA induction of ER stress-dependent JNK/CHOP/DR5 signaling is mediated by TRAIL/DR5 pathway.
ER stress dependent JNK/CHOP/DR5 upregulation occurs both upstream and downstream of caspase-8 activation
In an effort to determine if ER stress proceeds or follows a-TEA activation of DR5 signaling, caspase-8 inhibitor (Z-IETD-FMK) was employed. Data show that caspase-8 inhibitor significantly reduced a-TEA abilities to induce apoptosis (Fig 5A), and cleavage of Bid, caspase-9 and PARP, as well as increase protein levels of pJNK2/1, CHOP, DR5, and ER stress markers GRP78 and peIF-2a in both cell types (Fig 5B). These data suggest that a-TEA activation of caspase-8 is upstream of ER-stress mediated JNK/ CHOP/DR5 signaling. siRNA knockdown of JNK, CHOP and DR5 in MCF-7 cells reduced the ability of a-TEA to cleave caspases 8 and 9 (Fig 5 C), suggesting that not only DR5 but also JNK and CHOP are involved in activation of caspases-8 and -9. Furthermore, treatment of MDA-MB-231 and MCF-7 cells with ER inhibitor salubrinal blocked a-TEA's ability to induce caspases-8 and -9 cleavage (Fig 5 D), providing additional evidence for caspase 8 acting prior to and after a-TEA induced ER stress. (Fig 6A). MCF-7 cells transiently transfected with siRNA to CHOP, JNK or DR5, and both MDA-MB-231 and MCF-7 cells cultured with ER-stress inhibitor salubrinal or caspase 8 inhibitor Z-IETD-FMK prevented the ability of a-TEA to decrease c-FLIP (L) protein levels (Fig 6B and C), providing evidence that a-TEA downregulation of c-FLIP is mediated by caspase-8/ER stress/ JNK/CHOP/DR5 signaling. Since c-FLIP has been reported to be degraded via JNK-dependent E3 ubiquitin ligase Itch [32], studies were conducted to determine if Itch E3 ligase mediated ubiquitination was involved in a-TEA down-regulation of c-FLIP. Knockdown of Itch E3 ligase with siRNA to Itch reduced a-TEA's ability to downregulate c-FLIP (L) protein levels in MCF-7 cells (Fig. 6D). siRNA to Itch significantly reduced the ability of a-TEA to induce apoptosis in MDA-MB-231 and MCF-7 cells (Fig 6E). These data suggest that a-TEA induced downregulation of c-FLIP (L) is mediated by a caspase-8/ER stress/JNK/CHOP/DR5 pathway via Itch ligase ubiquitination. Over-expression of c-FLIP enhanced c-FLIP (L) levels ( Fig 6G) and siRNA to itch blocked a-TEA's ability to reduce c-FLIP protein expression (Fig 6D). Both overexpression of c-FLIP (L) protein and restoration of c-FLIP protein expression by silencing itch in a-TEA treatment reduced the ability of a-TEA to cleave PARP and caspase-8, and to increase protein levels of pJNK2/1, CHOP, DR5, and GRP 78 (Fig 6G and H). These data suggest that a-TEA's ability to downregulate c-FLIP (L) is important for caspase-8 activation and subsequent activation of the ER stress/ JNK/CHOP/DR5 signaling.
a-TEA downregulation of Bcl-2 protein and mRNA expression is mediated by ER stress a-TEA treatment of MDA-MB-231 and MCF-7 cells reduced both Bcl-2 protein and mRNA levels in a time dependent manner (Fig 6A and F). siRNA to JNK, CHOP, and DR5 (Fig 6B) as well as caspase-8 and ER stress inhibitors (Fig 6C) blocked the ability of a-TEA to decrease Bcl-2 protein expression. These data suggest that a-TEA induced down-regulation of Bcl-2 is mediated by caspase-8, ER stress, JNK, CHOP and DR5.
Discussion
The underlying mechanisms of how a-TEA, a potential chemotherapeutic drug, enhances DR5 death signaling pathways were investigated. For the first time, studies demonstrate that: (i) a-TEA induces ER stress dependent apoptosis, (ii) a-TEA induced ER stress regulates JNK/CHOP/DR5 signaling and is mediated by TRAIL/DR5/caspase-8, forming a positive loop, (iii) a-TEA downregulation of c-FLIP is mediated by ER stress-dependent JNK/CHOP/DR5 signaling via JNK activation of Itch E3 ligase ubiquitination and involved in activation of the ER-stress-dependent events via reducing the inhibitory effect of c-FLIP on caspase-8, and (iv) a-TEA downregulation of Bcl-2 protein levels is mediated by the ER stress-dependent events. Taken together, these data show that a-TEA induces TRAIL/DR5/caspase-8 dependent ER stress that triggers JNK/CHOP/DR5 signaling enhancing DR5/caspase-8 mediated mitochondria-dependent apoptotic signaling and produces a downregulation of two key anti-apoptotic factors; c-FLIP and Bcl-2. Based on these data, we provide a schematic diagram depicting a series of events including a positive-acting feedback loop, which is operative in a-TEA induced DR5 dependent apoptosis of human breast cancer cells in culture (Fig 7). DR5 signaling triggers caspase-8 activation, leading to two a-TEA initiated pathways: (i) a classic mitochondria-dependent apoptotic cascade initiated by cleavage of Bid and (ii) an ER-stress dependent activation of JNK/CHOP/DR5 sequence leading to further activation of caspase-8 and downregulation of antiapoptotic factors; c-FLIP and Bcl-2. Importantly, downregulation of c-FLIP serves to further enhance caspase-8 activation, which sustains the ER-stress/JNK/CHOP/DR5 amplifying loop.
TRAIL has emerged as a potent anticancer agent based on its ability to induce apoptosis in tumor cells but not in most normal cells [1,2,3]. Thus, targeting TRAIL/DR4/DR5 signaling pathways holds promise for inducing pro-apoptotic signaling in many cancer types while sparing normal cells and tissues [33,34]. Although considerable effort has been made in investigating the biological ramifications of TRAIL death receptor signaling, a complete understanding of the multiple pathways and events involved remain unclear. Typically, signaling pathways that initiate apoptosis have been broadly classified as (i) extrinsic death receptor initiated pathways or (ii) intrinsic pathways initiated by mitochondrial events [35]. These two pathways are not mutually exclusive, and JNK and caspase-8 have been observed to play central roles in both [4,36]. Our findings that TRAIL/DR5/casapse-8 triggers an ER stress dependent JNK/CHOP/DR5 amplifying loop and a JNK/c-FLIP/caspase-8 amplifying loop in a-TEA induced apoptosis provides new insights into TRAIL/DR5-mediated apoptotic signaling.
DR5 upregulation has been reported to be mediated by NF-kB in TRAIL-induced apoptosis in MCF-7 and MDA-MB-231 cells [37]. However, inhibition of NF-kB using IkB phosphorylation inhibitor BAY-11-7085 [38] enhances a-TEA induced upregulation of DR5 and apoptosis in MCF-7 cells (data not shown), suggesting that NF-kB plays an anti-apoptotic role in a-TEAinduced apoptosis. Data reported here showing that a-TEA upregulates DR5 via CHOP binding to the DR5 promoter is supported by published reports showing that CHOP can directly regulate DR5 expression through a CHOP binding site in the 59flanking region of the DR5 promoter [28,39].
JNK is a stress responsive kinase that has been reported to induce CHOP expression via an AP-1 binding site in the CHOP promoter [40,41]. JNK has been shown to be activated and is required for a-TEA-induced apoptosis of human breast, ovarian and prostate cancer cells [17,18,19,20]. Data presented here show that siRNA to JNK blocks a-TEA induced CHOP as well as DR5, indicating that CHOP/DR5 upregulation is mediated via JNK in a-TEA induced apoptosis. These data are in agreement with other studies showing that CHOP is an ER stress-regulated protein [42], JNK activation can contribute to CHOP expression during ER stress [43], and coupling of JNK/CHOP/DR5 are involved in ER stress-mediated apoptosis [29].
Present study show that a-TEA induces increased levels of peIF-2a and GRP78 proteins and spliced XBP-1, all of which are recognized as ER stress markers [44,45] and that salubrinal, a selective inhibitor of eIF-2a dephosphorylation (protects cells from ER stress) [46] blocked a-TEA induced apoptosis and the ability of a-TEA to activate JNK/CHOP/DR5 and upregulate ER stress markers, confirming that ER stress is induced by a-TEA and is involved in both apoptosis and activation of JNK/ CHOP/DR5. Present data show that a-TEA induction of the ER stress dependent JNK/CHOP/DR5 pathway is a downstream consequence of TRAIL/DR5 signaling. Furthermore, a caspase-8 inhibitor not only blocks a-TEA induced mitochondria-dependent apoptotic cascade as indicated by blockage of Bid and procaspase-9 cleavage, but also blocks ER stress signaling events, indicating that TRAIL/DR5 downstream effector caspase-8 contributes to a-TEA induced ER stress-dependent events. To our knowledge, this is the first report that ER stress can be triggered by a TRAIL/DR5/caspase-8 pathway and that ER stress induces a JNK/CHOP/DR5 amplifying loop that can further enhance TRAIL/DR5-mediated apoptosis. The mechanism(s) whereby activated caspase-8 induces ER stress were not investigated in this study. However, studies using hepatoblastoma HepG2 cells suggest that activated caspase-8 may promote and amplify the ER stress response by cleaving BAP31, an integral membrane protein of the ER, in glycochenodeoxycholic acidinduced apoptosis [47].
As a caspase-8 inhibitor, c-FLIP plays an important role in death receptor dependent apoptosis. Published data showed that down-regulation of c-FLIP is involved in a-TEA induced apoptosis in human ovarian and prostate cancer cells [18,22,23]. However, how a-TEA regulates c-FLIP is not known. Data reported here show that down-regulation of c-FLIP is mediated by the caspase-8 dependent ER stress JNK/CHOP/ DR5 loop via JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch. JNK-mediated protein degradation of c-FLIP has been reported for TNF-induced cell death [48]. However, it is the first report that JNK-mediated c-FLIP degradation is ER stress dependent.
Bcl-2 is a well established anti-apoptotic factor, which inhibits mitochondrial dependent apoptosis. It has been reported that ER stress induces downregulation of Bcl-2 via CHOP [49]. Although CHOP is a transcription factor, a CHOP responsive site in the Bcl-2 promoter has not been reported; suggesting that if CHOP mediates transcriptional regulation of Bcl-2 expression it must form a complex with other proteins [49]. Here, for the first time, we demonstrate that a-TEA downregulates Bcl-2 at both protein and mRNA levels, which is regulated by ER-stress mediated JNK/CHOP/ DR5 signaling.
In conclusion, data show that a-TEA induces a TRAIL/DR5/ caspase-8 dependent ER stress-mediated JNK/CHOP/DR5 loop, which amplifies caspase-8-mediated mitochondrial-dependent apoptosis via downregulation of c-FLIP and Bcl-2. These data are significant in that they provide a better understanding of the pro-apoptotic actions of the anti-cancer agent a-TEA, and provide new insights into DR5-mediated apoptotic signaling in human breast cancer cells. treatment with 40 mM a-TEA for 18 hrs. Western blot analyses were performed to evaluate over expression of c-FLIP on ability of a-TEA to cleave PARP and caspase-8, and down-regulate c-FLIP (L), pJNK2/1, CHOP, DR5 (L/S), peIF-2a and GRP78 protein expression using GAPDH as loading control. H. MCF-7 cells were transiently transfected with siRNA to Itch, using non-specific siRNA as negative control followed by treatment with 40 mM a-TEA for 18 hrs. Western blot analyses were performed to evaluate elevated levels of c-FLIP (L) on ability of a-TEA to cleave PARP and caspase-8 and to down-regulate pJNK2/1, CHOP, DR5 (L/S) and GRP78 protein levels using GAPDH as loading control. Data for A, B, C, D, F, G and H are representative of two or more individual experiments. Data from E are the mean 6 S.D. of three independent experiments. * p,0.05 = significantly different from control siRNA determined by t-test. doi:10.1371/journal.pone.0011865.g006 | 2014-10-01T00:00:00.000Z | 2010-07-29T00:00:00.000 | {
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30008608 | pes2o/s2orc | v3-fos-license | Chromosomal control of early embryonic development in mice. II. Experiments on embryos with structural aberrations of autosomes 7, 9, 14 and 17.
Peculiarities of preimplantation and early postimplantation development were studied in embryos with partial deletions and duplications of chromosomes 7, 9, 14 and 17, in the progeny of mice heterozygous for the unequal reciprocal translocations T(7; 14)2Iem, T(16; 17)43H and T(9; 17)138Ca. Deficiencies for any part of autosomes 9 or 14 combined with duplications of the corresponding segments of autosomes 7 or 17 do not affect preimplantation development, though they are lethal soon after implantation. Deficiency for the distal part of chromosome 7 (Df 7F4) induces embryonic death by the early blastocyst stage. Deficiencies for the distal part of chromosome 17 (Dfl7El-E5), as well as for its proximal region (Dfl7AB) carrying all genes of the T-t complex, have no detrimental effects on cleavage, blastulation and implantation, but are lethal after implantation, mostly during early neurulation. Deficiency for the middle part of chromosome 17 (Dfl7CD) is expressed just after a few cleavage divisions, and these embryos all die by the morula stage. It is suggested that the genes of the CD region of chromosome 17 and of the F4 region of chromosome 7 are of major significance for genetic control of early development in the laboratory mouse.
It was recently found that chromosome 17, though comparatively small in size, plays a major role in genetic control of the initial stages of embryogenesis in mice. Trisomy of 17 is lethal during major organogenesis (Baranov & Dyban, 1970;Baranov & Udalova, 1975;Gropp, Putz & Zimmerman, 1976), while monosomy of 17 affects cleavage and blocks embryonic development by the morula stage (Baranov, Dyban & Chetobar, 1980). However, it is not known what genes in chromosome 17 produce these affects. Mice heterozygous for the reciprocal translations T(16; 17)43H (hereafter T43) and T(9; 17)138Ca (hereafter T138), which divide chromosome 17 into three almost equal parts (see Text- fig. 1), were used to study this problem. One breakpoint in translocation T43 disrupts the centromeric heterochromatin of chromosome 16 while the other break is situated in the distal end of band 17B (Searle et al. 1978). Breakpoints in translocation T138 are located in the D segment of chromosome 17 and B segment of chromosome 9 (Miller & Miller, 1975).
In contrast to chromosome 17, our knowledge on developmental effects of aneuploidy for chromosome 7 is very scarce. Some preliminary observations indicate early death after implantation of embryos with trisomy 7 (Gropp, 1981 a, b), but nothing is known about the expression of monosomy 7. Some data on paternal gene Gpi-I expression (Brinster, 1973) and the effects of the lethal albino deletion mutations (Gluecksohn-Waelsch, 1979) suggest that chromosome 7 plays a major role in the control of morphogenetic and biochemical differentiation in early development. This hypothesis can be tested on mice with the unequal reciprocal translocation T(7; 14)2Iem (hereafter T2), which has one of its breakpoints in the distal part of chromosome 7 (presumably in bands 7F3-4) and the other in the centromeric region of chromosome 14 (band 14B). Both translocation products are easily identified in metaphase plates without differential staining (Baranov, 1979).
Neither trisomy nor monosomy for chromosomes 9, 14 or 16, involved in translocations T138, T2 and T43, respectively, interferes with preimplantation development (Dyban & Baranov, 1978;Baranov, 1980). Therefore it was of special interest to study early developmental profiles of mouse embryos with partial deletions of chromosomes 7 and 17. The results of the present communication indicate that both the middle part of chromosome 17, primarily the CD region, and the distal part of chromosome 7 are involved in the genetic control of preimplantation development.
(i) Mice
Breeding nuclei of mice homozygous for translocation T43 and for translocation T138 were obtained through the courtesy of Dr J. Forejt, Institute of Molecular Genetics, Prague, CSSR, and Dr A. Malaschenko, Laboratory of Biological Models, Yurlovo, Moscow, USSR. Translocation T2 arose spontaneously in an outbred stock of mice kept in the Institute for Experimental Medicine, Leningrad, USSR (Baranov, 1979). Translocation heterozygotes T43/ + , T138/+ and T 2 / + were obtained from the mating of corresponding homozygotes to CBA/Lac mice of normal karyotype.
(ii) Embryological methods
Mice, heterozygous for the translocations, were crossed either to F x (CBA x C57BL) (hereafter F a ) or to mice with the Robertsonian translocation Rb(2.6)4Iem (Baranov, 1981). Embryos were examined before implantation (days 230 V. S. BARANOV 1978), were stained with Giemsa solution and used for exact counts of total chromosome number in all available metaphase plates. At the next step the preparations were destained with pure methyl alcohol and processed for G-bands according to the conventional trypsin-Giemsa method (Wurster, 1972) with some modifications. Chromosome preparations from postimplantation embryos were made according to the modified Wroblewska-Dyban technique (Dyban & Baranov, 1978). Chromosome counts were made on 10-15 metaphase plates, stained for G bands. Translocation products and normal homologues were identified in each embryo. Karyo types were constructed according to the Committee on Standardized Genetic Nomenclature for Mice (Committee, 1972). A total of 568 embryos (343 before and 225 after implantation) were analysed both morphologically and cy togenetically.
RESULTS
(i) Translocation T2Iem Table 1 shows that the overwhelming majority of all the embryos with known karyotype in the progeny of T2/ + mice on the 3rd day (100 out of 109) and on the 4th day (90 out of 97) had a normal diploid chromosome number. However, in at least half of them only one translocation product can be identified -either a large marker, 7 14 , or a small one, 14'. So these embryos had structural aberrations of the translocated chromosome. It should be pointed out that the small chromosome 14' always combined with two chromosomes 7 and one chromosome 14. Vice versa, a large marker 7 14 combined with two chromosomes 14 and only one chromosome 7. Taking into account the positions of the exchange points, this means that the embryos of the first group had duplication for a small distal part of chromosome 7 and deletion of practically the whole chromosome 14 except for its small centromere region (Dp7F4; Dfl4BCDE), while the embryos of the second group The frequencies of chromosomally unbalanced embryos of both groups were similar on the 3rd day (29 and 20, respectively, P > 005) (Text- fig. 2); while on the 4th day the number of embryos with a small marker (group 1) was at least three times the number with a large marker (group 2) (31-9% and 10-3%, respectively, P<001). Not a single embryo of group 2 was recovered after implantation (9-10th days), but there were 19 embryos of group 1 among 78 embryos with known karyotype. A few of the embryos studied before and after implantation were aneuploids (see Table 1). Aneuploidy was due to the imbalance of non-translocated chromosomes in 14 of 21 embryos, while 7 embryos had an excess or deficiency of translocated marker chromosomes.
Morphologically, all the chromosomally unbalanced embryos resembled quite normal morulae in the process of compaction on the 3rd day (mean cell count 102 + 2-3 compared to 13-1 + 1-8 in the control litter-mates) and normal early blastocysts on the 4th day (mean cell count 341 + 29 and 395 + 4-3, respectively). It is worth mentioning, however, that numerous pyknoses and micronuclei, indicating progressive degeneration, were present in 6 out of 10 embryos of group 2. Taking into account the greatly reduced proportion of group 2 embryos on the 4th day, these observations indicate that chromosomal unbalance due to Df 7F4; Dpl4BCDE affects blastulation and is always lethal by the early blastocyst stage. Group 1 embryos (Dp7F4; Dfl4BCDE) were recovered both before and after implantation in about equal proportions, though at later stages they looked abnormal.
Of the embryos of group 1, 10 of 19 were represented by small empty yolk sacs without traces of embryoblast derivatives, and 9 had remnants of neural folds. Maternal blood in the implantation chamber and the absence of allantois proved progressive resorption of these embryos. Two aneuploid embryos with partial trisomy of a small marker chromosome (Tsl4 7 T2Iem) looked morphologically quite normal on the 10th day.
(ii) Translocation T43H Table 2 shows structural chromosomal aberrations in almost half of all karyotyped embryos (75 out of 163) in the progeny of T43/ + female mice. The number of chromosomally abnormal embryos was somewhat higher before than after implantation (47% and 44%, respectively). All four main groups of chromosomal aberrations expected from non-disjunction of translocated products were registered before implantation and only three of them were found after it ( Table 2). The proportion of the embryos for each unbalanced group varied. Before implantation embryos with Dfl7AB prevailed, but at later stages embryos with Dpl7AB represented the most numerous group. Deletion of the CDE region of chromosome Chromosomal control of early development in mice 233 per embryo with Dfl7CDE was 2-3 times less than in the embryos of genetically balanced karyotype of the same cross (12-1 ±2-6 and 31-2 + 1-8, respectively). Numerous micronuclei, pyknoses, unusually large and light nuclei, combined with metaphase chromosomes separated prematurely into paired chromatids, indicated advanced degeneration and the death of these embryos by the morula stage. The deficiency for the proximal part of autosome 17 (Dfl7AB) or the duplication of any region of chromosome 17 (Dpl7AB or Dpl7CDE) had no harmful effect on preimplantation development. Mean cell count per embryo in these groups was 28-32 (Plate 1 b) and was equal to that in the control group. Some embryos with Dfl7AB remained alive after implantation, though all of them looked abnormal. Nine of these were represented by very small trophoblastic vesicles without embryoblast derivatives visible under the stereomicroscope, and three looked like an abnormal neurula (Plate 2a). Meanwhile, the complete absence of the embryo remnants in at least half of all implantation sites on the 9-10th days proved that embryonic death occured soon after implantation.
Out of 14 embryos with Dpl7AB, 11 looked quite normal and were registered only at chromosomal preparation, 2 embryos were slightly retarded and 1 was malformed.
(iii) Translocation T138Ca
A total 216 viable embryos were obtained from 38 females, and 125 embryos were successfully karyotyped: 55 before and 70 after implantation (Table 3). The frequency of chromosomally unbalanced embryos on the 4th day was almost twice that on days 8-10 (47-3% and 25-7%, respectively), indicating that embryonic selection was operative by implantation. Actually, at least one main group of chromosomally unbalanced embryos deficient for the proximal segment of chromosome 17 (Dfl7ABCD + Dp9AB) was registered exclusively before implantation. All 6 embryos of that group on the 4th day were composed of 6-12 blastomeres (mean cell count 8-7 + 3-6 compared to 315+1-8 in controls) some of which were pyknotic or abnormally large with very light transparent nucleoplasm (Plate 2b). These embryos are dead by the morula stage. Out of 18 chromosomally unbalanced embryos on the 8-10th days, 9 were deficient for the distal part of chromosome 17 (Df 17E) and had in excess most of chromosome 9 (Dp9CDEF). These aberrations did not interfere with cleavage, blastulation and implantation but were selected soon thereafter. The development of chromosomally unbalanced embryos of two other groups (see Table 3) Dpl7E; Df9CDEF and Dpl7ABCD; Df9AB, was morphologically similar to that of the former group (Dfl7E; Dp9CDEF). It should be pointed out first that structural aberrations of chromosomes 7 and 17 in the present work always combined with genetic unbalance of the other chromosomes involved in the reciprocal translocations, namely no. 16 (T43), no. 9 (T138) or no. 14 (T2). It should also be remembered that monosomy for any of the latter autosomes does not affect cleavage or blastulation and is often compatible with implantation (Dyban & Baranov, 1978;Baranov et al. 1980;Baranov, 1981) while their trisomy is lethal during organogenesis (11-12 days for Ts9), or in the foetal period (16-18 days for Tsl6 and 14) (Baranov & Udalova, 1975;Gropp et al. 1976). Therefore the genetic unbalance of these chromosomes is morphologically evident at relatively late stages of embryogenesis, and so the detrimental effects of structural aberrations of these chromosomes might be at least partly neglected when the preimplantation development is considered. This aberration though rather common on the 3rd day, was registered in quite a few degenerating early blastocysts. Such an early lethal effect of a very small deletion requires special consideration.
Chromosome 7 is one of the most thoroughly studied genetically, with at least 30 structural genes mapped on it (Eicher & Washburn, 1978). Some of these genes are known to be expressed as early as the 8-blastomere stage (McLaren, 1976). c 2bH deletion in the middle of chromosome 7 (Miller et al. 1974 (Gluecksohn-Waelsch, 1979). The breakpoint T2Iem in chromosome 7 is located in the most distal region, probably in segments 7F3-F4, so it presumably shares no common region with the c 2bH deletion. The genetic map of segment 7F4 is almost completely unknown (Eicher & Washburn, 1978;Womack, 1978). Meanwhile, the results of the present work indicate the great significance of the genes in the distal segment of chromosome 7 for the control of early development in mice. Whether that effect is due to the expression of certain structural genes or is caused by the disbalance of genes with regulatory functions is still unknown.
(ii) Deletions for the different parts of chromosome 17
The present study of T43 and T138 heterozygotes provided evidence that deficiencies for different parts of chromosome 17 impaired development potentials of mouse embryos to different extents. The absence of the distal part (17E-1 to E-5) or of the proximal region (Al-A3; B) of this chromosome (Fig. 1) does not affect cleavage, blastulation or implantation, and some of these embryos might be recovered from implants during neurulation, though all of them look abnormal. Contrary to these observations the effects of deletions 17ABCD and 17CDE are already evident during cleavage and they completely block embryonic survival by the morula stage. So the damaging effect of these deletions is almost identical and is expressed at almost the same stage of development as in monosomy for the whole autosome 17 . Both deletions studied (17ABCD and 17CDE) have in common the region of chromosome 17 enclosed between the breaks T43 and T138 (Text- fig. 1). As it may be inferred from the present data the deletions for the proximal (AB) or the distal (E) segments of chromosome 17 do not affect preimplantation development. There is, therefore, sufficient evidence for the assumption that the absence of the middle segment encompassed between exchange points of T43 and T138 is actually responsible for the early lethal effects of both partial deletions 17CDE and 17ABCD as well as monosomy of chromosome 17. If this suggestion is really true it might be inferred that region CD of this chromosome contains the genes controlling the initial stages of mouse embryogenesis. Therefore, the genetic content of the middle part of chromosome 17 is of special interest and should be discussed in more detail.
The genetic map, related more or less precisely to the cytological map, of chromosome 17 is drawn schematically in Text- fig. 1. The breakpoint of translocation T43H is located to band 17B just distally to tf (Lyon et al. 1979) and the following linkage relationship of three gene markers to the breakpoint was established T-tf-T4SH.-H2 (Forejt, Capkova & Gregorova, 1980). It means that the T43 breakpoint separates physically the two major genetic complexes T-t and H2, with the T-t located in the large translocated product 17 16 , and H-2 in 16 17 . Genes of the complex T-t system are known to be intimately involved in embryonic development and spermatogenic differentiation (Bennett, 1975(Bennett, , 1981Sherman & Wudl, 1977;Lyon et al. 1979). Nevertheless, duplication of the AB segment, e.g. trisomy for the whole set of T-t genes, does not interfere with embryonic survival Baranov, Gregorva & Forejt, 1981). Moreover, this trisomy is compatible with normal postnatal development and normal 236 V. S. BARANOV fertility in both sexes (Forejt et al. 1980). Even more unexpected are the data of the present work which demonstrate normal preimplantation development of embryos with deficiency for all maternal genes of the T-t locus though they die during neurulation. Embryos deficient for a large distal segment, E1-E5, show normal development before implantation and perish soon after it. Unfortunately, the genetic content of this segment is still almost unknown.
The genetic map of the central part of chromosome 17 encompassed by the breakpoints T43 and T138 is known in more detail. Besides the genes of the complex H-2 locus it carries DNA for a number of other structural genes such as catalase (Ce-2), phosphoglycerate kinase (Pgk-2), glyoxalase (Glo-1), etc. (Womack, 1978). Expression of some H-2 genes from the early blastocyst stage can be reliably demonstrated by immunofluorescent methods (Searle et al. 1976;. Therefore the disbalance for these genes does not seem to be responsible for the death of CD-deficient embryos by the morula stage. On the other hand, according to more recent data at least some H-2 associated genes, named Ped (preimplantation embryonic development) do affect the time of the first cleavage division and the rate of subsequent development (Goldbard, Verbanac & Warner, 1982a, b). Though the real nature of Ped genes as well as their gene products remains unknown they might be considered as main candidates for so-called 'early genes' (Dyban, 1974) controlling initial stages of embryogenesis in mammals.
It should be mentioned, however, that the lethal effect of the CD deletion is already expressed in heterozygotes and this differs from the other early lethal mutations such as t li or c 2bH (Bennett, 1975(Bennett, , 1981Gluecksohn-Waelsch, 1979) which are lethal only in homozygotes. One might suggest therefore that deficiency 17CD in some still unknown way influences the function of the relevant segment of the normal homologous chromosome 17. So at the cellular level deficiency CD expresses as a nullisomy of corresponding segment 17CD. These results therefore might be considered as additional arguments in favour of a recent suggestion on mutual activation of homologous chromosomes and their segments at initial stages of embryogenesis (Baranov, 1982). The latter suggestion is also supported in part by the remarkable phenomenon discovered by D. R. Johnson (1974Johnson ( , 1975) that postulates the existence of a special activation centre in the segment of chromosome 17, proximal to the T138 breakpoint, which is probably necessary for activation of the normal homologous chromosome 17 during embryogenesis (Lyon & Glenister, 1977).
Whatever the extact mechanism of early embryo lethality caused by the partial deletions studied in the present work, it seems clear that chromosomes 7 and 17 actually control initial embryogenesis in mice, and that most probably the regions 17CD and 7E4 are especially concerned with early genetic activity of these chromosomes. | 2018-04-03T01:34:58.303Z | 1983-06-01T00:00:00.000 | {
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220714065 | pes2o/s2orc | v3-fos-license | Use of outpatient mental healthcare services and upper-secondary school completion in young women with migrant background – A population-based study
Mental disorders typically develop during adolescence, with young women being particularly at risk. Mental disorders during this period can negatively affect both current and future life prospects such as school completion. Migrants are at increased risk of developing mental disorders as a result of their experiences prior to, during and after migration. Additionally, they are less likely to complete upper-secondary school when compared to the majority population. Thus, being a young migrant woman with a mental disorder may have adverse consequences for school completion, which in turn can affect socioeconomic status later in life. In this study, we aimed to investigate the association between mental disorders, defined as having used outpatient mental healthcare services (OPMH), and completion of upper-secondary school among young women living in Norway, using national registry data. Additionally, we examined differences in probability of school completion between Norwegian majority, migrants and migrant descendants between those who used and did not use OPMH. The sample consisted of women born between 1990 and 1993 (N = 122,777). We conducted hierarchical, multivariable logistic regression analysis. In unadjusted analysis, we found that young women who used OPMH services had lower odds of school completion than those who did not, even after adjustment for migrant background and parental education. However, by calculating predictive margins, we found that descendant women, who had used OPMH services, had significantly higher probability of completing upper-secondary education than Norwegian majority women who had used services. None of the four migrant groups differed significantly from majority women. Use of OPMH services, had most adverse effect on majority, migrants from Nordic and Western countries and descendants, when compared to non-users. Future interventions should aim to increase school completion among young women with mental disorders.
Introduction
Mental disorders are a public health challenge. Globally about 16% were affected by a mental or addictive disorder in 2016 (Rehm & Shield, 2019). In Norway, almost a quarter of young women aged 16-24 years report significant mental problems (Nes & Clench-Aas, 2011;Norhealth, n.d.), making them the most vulnerable group. Several mental disorders often have an onset during the teenage years (Jones, 2013), due to biological, cognitive and emotional changes during adolescence (Lee et al., 2014). The research focus on mental disorders in young women is important, not only due to the impact on current life but also in the long term. Poor mental health negatively influences other health outcomes and increases the risk of school noncompletion (Bowman, McKinstry, & McGorry, 2017). In turn, this can further result in lower salary, poorer workforce attachment and increased dependence on welfare services in adulthood (Ngui, Khasakhala, Ndetei, & Roberts, 2010;Trautmann, Rehm, & Wittchen, 2016).
As well as young women, migrants are at increased risk of mental disorders as a result of migration-related experiences (Delara, 2016). Worldwide, the number of international migrants is rapidly growing (United Nations, 2017) and Norway is no exception. The migrant population in Norway has increased significantly over the past decades (Sandnes, 2017). The combination of being young, a woman and a migrant may therefore have adverse effects on mental health. Cultural differences regarding gender roles, lack of social support and discrimination may not only increase the risk of mental disorders (Abebe, Lien, & Hjelde, 2014;Ll� acer, Zunzunegui, Del Amo, Mazarrasa, & Bolůmar, 2007), but may also have a negative impact on socioeconomic outcomes, such as school completion. In Norway, young migrant women have lower upper-secondary school completion rates compared to both majority and descendant women (Steinkellner, 2017). However, we lack information on the impact of mental disorders on school completion among this group. In this study, we aim to investigate whether there is an association between mental disorders, as measured by the use of outpatient mental healthcare services (OPMH), and upper-secondary school completion.
Previous research
Research on the association between mental disorders and school completion or other school-related outcomes is not new (Kessler, Foster, Saunders, & Stang, 1995). Two systematic reviews summarizing research on this topic concluded that mental disorders were negatively associated with school completion and educational attainment and positively associated with early school leaving (Esch et al., 2014;Melkevik, Nilsen, Evensen, Reneflot, & Mykletun, 2016). However, authors point to the potential mediating effect of socioeconomic background and family support on these associations (Esch et al., 2014). Despite focusing on different types and severity of mental disorders, the majority of studies indicate that mental disorders have a negative impact on educational attainment such as school completion, college enrolment and academic performance (Br€ annlund, Strandh, & Nilsson, 2017;Breslau, Lane, Sampson, & Kessler, 2008;Leach & Butterworth, 2012;McLeod, Uemura, & Rohrman, 2012;Mojtabai et al., 2015;Myer et al., 2009;Needham, 2009;Van der Ende, Verhulst, & Tiemeier, 2016). It is important to stress that few of the above studies stratified findings by gender and most relied on self-reported data. Studies that did investigate gender differences found poor mental health to have a stronger negative effect on school completion among young women than young men (Br€ annlund et al., 2017;Fletcher, 2008;Needham, 2009).
The above studies can be placed under the social selection perspective, which assumes that mental disorders have a detrimental effect on later life achievements and maintenance of social status (Mossakowski, 2014). Previous research pinpointed that the presence of social selection is strongest for disorders that are serious or socially disruptive, such as schizophrenia, antisocial personality disorder and conduct disorders (McLeod & Kaiser, 2004;Mossakowski, 2008). The relationship between mental disorder and school completion may alternatively be confounded by other factors associated with both mental disorder and school completion. Lynch and von Hippel (2016) investigated these explanations for self-rated health and education. They concluded that good self-rated health in adolescents predicted higher educational attainment (i.e., the social selection mechanism) but that family background and college aspirations partly confounded this association. Similarly, the association between mental disorder and educational attainment may be confounded by family background variables. Family socioeconomic status (SES), such as parental educational attainment for instance, is associated with both risk of mental disorder in (Reiss, 2013), and educational attainment of children (Davis-Kean, 2005). Parental country of birth has also been used as an indicator for family SES and is a potential confounder in the association between mental disorder and school completion (Br€ annlund et al., 2017). Studies investigating the association between use of OPMH services, or mental disorders, and school completion should control for confounding factors in order to measure the possible direct effect of mental disorders on school completion.
Norwegian research on the association between mental disorders and school completion mainly relies on self-reported mental disorders. Selfreported mental disorders are associated with non-completion of uppersecondary school (Sagatun, Heyerdahl, Wentzel-Larsen, & Lien, 2014). Furthermore, adolescent mental disorders reduce years of schooling, and the probability of upper-secondary school completion and college enrolment (Evensen, Lyngstad, Melkevik, & Mykletun, 2016). These studies have not focused on young women in particular nor considered differences between majority populations and migrants. To the best of our knowledge, there is a lack of research looking at the relationship between mental disorders, treated at outpatient level, and school completion among women with migrant background. Our study will contribute to filling the gap in the existing literature by looking at OPMH service use and upper-secondary school completion among young majority, migrant and descendant women.
Migrant women in Norway
Norway, like several other European countries, has experienced an increase in the number of migrants during recent decades. Between 1970 and 2017, the percentage of migrants rose from about one percent to more than 14% of the total Norwegian population (Dzamarija, 2017). Together with their descendants, migrants made up almost 18% of the population in 2019 (Statistics Norway, 2019b). Depending on country of origin and reason for migration, migrants may have a different risk of mental disorder and use of mental healthcare services. Migrants, particularly those from low-and middle-income countries, may be at greater risk of mental health problems compared to the majority population (Abebe et al., 2014). There are also differences in use of primary healthcare services (PHC) for mental health problems where, for instance, refugee women have higher use than non-refugee women (Straiton, Reneflot, & Diaz, 2017). Use of PHC also varies by country of origin, though migrant women in general have lower usage compared to their Norwegian counterparts (Straiton, Reneflot, & Diaz, 2014). Use of OPMH services is also lower for both migrant and descendant women compared to their Norwegian counterparts, although again there is considerable variation by country of origin (Straiton, Corbett, Hollander, & Hauge, 2019).
Regarding school completion, young migrant women are more likely to drop out of upper-secondary school and less likely to complete school within the normative time frame of five years, compared to descendants and the Norwegian majority (Statistics Norway, 2019a). Among descendant women, the dropout percentage is lower compared to majority women, though, a lower percentage complete upper-secondary school within the normative time frame (Statistics Norway, 2019a). Among those who do complete upper-secondary school education, a higher percentage of descendant women enrol in higher education compared to majority women (Steinkellner, 2017).
Current study
Previous research on the association between mental disorders and school completion is mainly based on survey studies, which are known for being prone to selection bias due to low response rates among migrants and minority populations (Ll� acer et al., 2007). Furthermore, none of the above studies investigated whether the effect of OPMH service use on school completion varies between majority, migrant or descendant young women. In the current study, we use register data with national level coverage to look at upper-secondary school completion among young women who have used OPMH services, as an indicator of mental disorders, and those who have not. We also control for migrant background and parental education to look at whether these factors confound the association.
Additionally, we examine whether the probability of completing upper-secondary school among those using and not using OPMH services, varies between young majority, migrant and descendant women.
Based on the available evidence on migrant women's lower use of healthcare services, despite increased risk of mental disorders, and lower rates of school completion compared to both descendants and Norwegian majority, we hypothesize that young migrant women will have lower probability of completing upper-secondary school when using OPMH services than majority and descendant women. We also expect probabilities to vary among migrants, depending on their region of origin and when compared to non-users within the same group.
Study design and data sources
In this cohort study of young women in Norway, we used longitudinal population-based data from several Norwegian registries, namely the Central Population Registry, the National Database for the Reimbursement of Health Expenses (KUHR) and the National Education Database. The Central Population Registry provided demographic information such as year of birth, sex and country of origin. Additionally, it contains information on time of immigration, emigration or death, and thus, whether an individual resided in Norway during respective years of study. This was used to define the eligible study sample. The KUHR database contains data on healthcare professionals' reimbursement claims of fee-for-service in Norway, such as claims from OPMH services as used in this study. Each contact with healthcare providers is registered with a date and type of contact. OPMH services are located throughout the country and available for all Norwegian residents. After referral from a general practitioner (GP) or psychologist, individuals with acute problems or those with a need for long-term follow-up care can receive help. We use information on contact with OPMH services for years 2006-2011. From the National Education Database, we used information on the highest obtained education level and any initiated education for years 2011-2014 in order to identify those who did or did not complete upper-secondary education. Additionally, we extracted information on parental education when the individual was aged 16.
A unique personal identification number (PIN) is assigned to all Norwegian citizens at birth, as well as all individuals registered as residents in Norway for at least six months. We used a pseudonymised version of a PIN to combine variables across the registries necessary to examine the objectives of interest. We also used family number to extract the information on parental education.
Study sample
The study population consisted of 149,271 young women born between 1990 and 1993. We excluded individuals who died (N ¼ 993) or emigrated permanently (N ¼ 5,319) at the age of 18 or earlier, as they did not meet inclusion criterion. We also excluded those who immigrated to Norway at the age of 18 or later (N ¼ 17,043). Furthermore, we included only young women who started upper-secondary education and resided in Norway during the exposure period (excluded N ¼ 2,879). We excluded women from South America due to a small number of individuals in this group (N ¼ 260). The eligible study sample for the aims of this study constituted of 122,777 young women. We followed each of them for five years, between the age of 16 and 21.
Outcome variable
Upper-secondary education was used as outcome variable and was defined as completion of upper-secondary education by the age of 21 and treated as a binary variable in the analyses. In Norway, all youths between 16 and 19 have the right to start upper-secondary education after completing compulsory education, with the majority of students starting upper-secondary education the year they turn 16. The Norwegian school system is largely public and free of charge. Compulsory education lasts for 10 years, when aged 6-16 years. All children who have stayed in Norway for at least three months have the right and duty to attend compulsory education until summer the year they turn 16. After completing compulsory education, all students have the right to continue into upper-secondary education, which is free of charge when public. Upper-secondary education is divided into general and vocational tracks. The general track lasts for three years and, after passing all required exams, is a qualification for tertiary education. The vocational track has three pathways lasting for three or four years. Two of them give qualifications for practical occupations, without qualification for tertiary education, while the third path leads to a tertiary education qualification, if specific required courses are taken. Due to variations in the length of study programs, the normative time frame for uppersecondary school completion is set to five years i.e. by the age of 21 (Statistics Norway, 2019a).
Exposure variable
OPMH service use was utilized as our exposure variable and an indicator of mental disorders. We employed a dichotomous indicator (yes/ no) to identify individuals with at least one contact with OPMH services during late adolescence (aged between 16 and 19), with a start point on 1 st of July and end point on 30 th of June, three years later, as shown in Fig. 1.
Migrant background
We divided our sample into three main groups: migrant (born abroad to two foreign-born parents), descendant (Norwegian-born to two foreign-born parents) and majority women (Norwegian-born with at least one Norwegian-born parent, and foreign-born with at least one Norwegian-born parent). We further divided migrants into four categories as 1) Nordics and Western countries (including Nordic countries, Western Europe, USA, Canada, Australia and New Zealand), 2) Eastern Europe, 3) Africa and 4) Asia.
Parental education
Parental education refers to the mother or father's highest attained level of education by the time the individual was aged 16. This variable had three possible values: upper-secondary school or lower, tertiary education, and unknown parental education.
Analyses
By using chi-square tests and post-hoc Bonferroni tests for analysis of variance, we compared the characteristics of descendants and each migrant group to majority women as a reference group. To investigate the association between use of OPMH services and upper-secondary school completion among all young women we used a hierarchical, multivariable logistic regression model. First, we applied univariate logistic regression model, and thereafter adjusted for migrant background and parental education to control for confounding, in model 2 and 3 accordingly. Furthermore, in model 4, we tested for statistical interactions between use of OPMH services and migrant background in order to examine whether the effect of use of OPMH services on school completion varies with migrant background. We compared young women who did and did not use OPMH services with different migrant backgrounds by using predictive margins. Results are presented as predicted probabilities (Pr) with 95% confidence intervals (95% CI). This eased the interpretation of results as predictive margins compare the predicted probabilities of school completion for all groups (Williams, 2012). The analyses were conducted with inclusion of individuals with unknown parental education, as treating this category as missing would make the groups of migrants smaller, resulting in more imprecise and wider confidence intervals. An additional analysis with complete cases did not change the direction of the results or show visual changes in the main outcome. Hence the risk of bias due to missingness seems to be limited. Stata 15.0 was used to execute the analyses.
Ethics
This project has been granted approval by the Regional Committee for Medical and Health Research Ethics, South East Norway (REK 2014(REK / 1970) and all registry owners approved the use of their data. Table 1 shows that of the 122,777 young women, 91.6% were classified as Norwegian majority, 5.1% as migrants divided into four groups, and 3.3% as descendants. There were significant differences in use of OPMH services; with a higher percentage of Norwegian majority using services compared to descendants, and migrant women from Eastern Europe, Asia and Africa but not migrant women from Nordic and Western countries (X 2 ¼ 595.0, df ¼ 5, p < 0.001). We also found significant differences between majority women and all groups of migrants, but not descendants, regarding school completion (X 2 ¼ 244.3, df ¼ 5, p < 0.001). Parental tertiary education was most common among migrant women from Nordic and Western countries and the Norwegian majority, while parental upper-secondary education or lower was most common among descendants and migrants from Africa. All groups differed significantly from the majority (X 2 ¼ 2300.0, df ¼ 5, p < 0.001).
Results
In Table 2, we display the results of the hierarchical, multivariable logistic regression analysis conducted to investigate the association between use of OPMH services and upper-secondary school completion. In model 1, the results show the unadjusted analysis, with a clear negative association between use of OPMH services and school completion. Young women who had contact with OPMH services had significantly lower odds of completing upper-secondary school within five years compared to those who did not use OPMH services (OR ¼ 0.20, CI 95% 0.20-0.21). In the adjusted analysis, all control variables were significantly associated with school completion, although they had modest influence on the OR for OPMH services. All four migrant groups and descendants had significantly lower odds of school completion compared to the Norwegian majority (Model 2). However, after controlling for parental education, descendants did not differ significantly from the Norwegian majority, while all migrant groups had significantly lower odds than the Norwegian majority (Model 3). The results showed that having parents with tertiary education increased the odds of school completion compared to having parents with upper-secondary education or lower. In model 4 (results not shown), we conducted interaction analysis between use of OPMH services and migrant background to examine whether the effect of use of OPMH services on school completion differed between groups. The effect of moderation was significant for all groups, indicating the need for additional analyses. To investigate whether the effect of use of OPMH services on uppersecondary school completion varied between the Norwegian majority and the four migrant groups and descendants, we compared predicted probabilities of completing upper-secondary education among the groups by using predictive margins (results are presented in Fig. 2).
In Fig. 2, results are presented as predicted probabilities with 95% confidence intervals to demonstrate the probability of completing upper-secondary education among young women who did and did not use OPMH services. We present predicted probabilities for the Norwegian majority, descendants and four groups of migrants. Regarding Fig. 1. Timeline for the study, with point of measurement of exposure, OPMH service use, and outcome, upper-secondary school completion, for each of age cohorts.
Discussion
In this study, we investigated the association between use of OPMH services, an indicator of mental disorders, and upper-secondary school completion among young women in Norway. Furthermore, we aimed to examine whether the probability of completing upper-secondary education when using and not using OPMH services differs for the Norwegian majority, migrants and descendants. We hypothesized that use of OPMH services would have more adverse effect on the probability of completing upper-secondary for migrant women, compared to descendants and the Norwegian majority. We also expected probabilities to vary among migrants, depending on their region of origin and when compared to non-users within the same group.
The results showed that being in contact with OPMH services during adolescence reduced the odds of upper-secondary school completion for all young women in our study sample. The negative association remained after adjustment for migrant background and parental education. By use of predictive margins, we compared the predicted probabilities of school completion when using and not using OPMH services for all groups of migrants, descendants and the majority. Descendants Table 2 Crude and adjusted odds ratios (OR) with 95% confidence intervals (95% CI) and significance level for the association between use of OPMH services and uppersecondary school completion. All models were conducted on the sample of 122,777 individuals. Estimates are odds ratios (OR), with 95% confidence intervals (95% CI) in brackets.
Fig. 2.
Predicted probabilities with 95% confidence intervals of school completion for women who had contact with OPMH services and those who did not, by migrant background.
who had used OPMH services appeared to fare significantly better than their Norwegian majority counterparts, but also migrant women from Nordic and Western countries in terms of school completion. Contrary to our expectations, none of the four migrant groups differed significantly from the Norwegian majority when comparing OPMH service users. We also found that within groups, use of OPMH services had the most adverse effects on school completion among majority, migrants from Nordic and Western countries and descendants, when compared to nonusers.
Our main findings are in accordance with earlier research on the field, claiming that young women experiencing mental disorders are at greater risk of not completing upper-secondary education compared with their healthy counterparts (e.g. Fletcher, 2008;Needham, 2009). This supports the social selection perspective. The previous studies we refer to relied on self-reported mental health measures collected through surveys. The results are therefore prone to both recall bias and selection effects, since those who choose to participate may differ from those who choose not to. Br€ annlund et al. (2017), however, based their study on objective data from Swedish registries but with drug prescription as a proxy for mental disorders and also found a negative association between mental disorders and educational achievement. Yet, none of these studies considered differences between adolescents or young women with different migrant background. Previously, it has been pointed out that surveys often suffer from bias due to lower response rates among minority populations, mainly due to low language proficiency (Ll� acer et al., 2007). In our study, the data has national level coverage and uses objective sources, thus excluding recall and selection bias. Nevertheless, it is important to stress that contact with OPMH services only detects those who sought help there and not all young women experiencing mental disorders. We also lack information about those who used a private psychologist, psychiatrist or those who get inpatient treatment. However, inpatient services constitute only five percent of contacts with mental healthcare services (Norwegian Directorate of Health, 2019). According to Ngui et al. (2010) over two thirds of all people affected by mental disorders do not receive the care they need, mainly due to not contacting healthcare services. Thus, even possessing information about the whole population, we were unable to determine the real scope of the problem and the impact mental disorders may have on school completion.
Furthermore, our findings did not support the confounding hypothesis, as the association between use of OPMH services and school completion remained virtually unchanged when controlling for migrant background and parental education. This was unexpected, as previous studies found that several family background variables such as parental education and migrant background had an influence on both mental health and educational outcomes among young people (George, 2013;Reiss, 2013). Nonetheless, results from a Swedish register-based study found the effect of mental disorders on school completion was robust, despite adjustment for several family background variables (Br€ annlund et al., 2017), as in our study.
Our study aimed to fill the gap on the lack of research looking at differences between young women with migrant background in the association between use of OPMH services and school completion. As migrants and descendants make up a growing part of the society in many parts of the world, their mental health and its impact on other aspects of life is important to map. Based on previous research pinpointing migrants' increased risk of development of mental disorders and lower school completion rates than the majority population, we hypothesized that the effect of use of OPMH services on school completion would be even stronger for migrants compared to descendants and the Norwegian majority. Additionally, we expected the effect to vary among migrants. The interaction analysis showed that the effect of use of OPMH services on school completion varied between migrants, descendants and the Norwegian majority. We, unexpectedly, found that descendant women had higher probability of completing school when using OPMH services compared to the Norwegian majority and migrant women from Nordic and Western countries. We also found use of OPMH services to have less adverse effect on school completion among migrant women from Eastern Europe, Asia and Africa. The strongest effect was found for majority women, migrant women from Nordic and Western countries and descendant women. This might be due to the greater social mobility and higher educational aspirations among descendants, and some migrant groups in the Norwegian society. A previous study suggested that descendants and several groups of migrants seem to have similar or higher educational attainment than the Norwegian majority when comparing groups with similar socioeconomic background (Hermansen, 2016). Furthermore, descendants, but also migrants, might have higher educational aspirations and expectations of life, as they report their parents having higher educational ambitions on their behalf compared with the Norwegian majority (Friberg, 2016). Also contrary to our expectations, the effects of OPMH service use on school completion was not stronger for migrants than for the Norwegian majority. While the above argumentation about educational aspirations may also apply, the unexpected findings could relate to help seeking. We know that migrants seek healthcare services to a lesser extent than the Norwegian majority (Abebe, Lien, & Elstad, 2017;Straiton et al., 2014Straiton et al., , 2019. Additionally, we might expect migrants to experience greater barriers to seeking help for mental disorders than non-migrants. Thus, those who are in contact with OPMH services may be in a better position in terms of SES than those who do not seek help. This could mean that a larger proportion of resourceful young women with migrant background use OPMH services than resourceful majority women, resulting in the unexpected finding that descendants fare better and migrants do no worse regarding school completion, when using OPMH services than the majority population. However, it is important to emphasize that this does not mean descendants or migrants are not at a disadvantage having had a mental disorder.
Limitations
Our study has several other limitations. Firstly, the age period for the main exposure is measured when individuals are aged 16-19 years. This age period is slightly overlapping with the period of the outcome, as the majority of students complete upper-secondary education when aged between 19 and 21. Ideally, the exposure should be measured prior to enrolment in upper-secondary education. However, due to the limited number of years the registry data was available, and in order to use the same number of exposure years for each cohort while simultaneously maximising the use of the available information, we had to observe individuals when aged 16-19 years.
Secondly, we do not differentiate between different mental disorders in this study. Previous studies found that internalizing and externalizing disorders have a different impact on educational attainment. The effect of externalizing problems, such as conduct and attention problems, on educational attainment seems to be stronger than for internalizing problems such as depression and anxiety (e.g. Evensen et al., 2016;McLeod et al., 2012). However, research comparing those two categories is inconsistent.
A third limitation of this study is our operationalization of migrant background. To ensure the statistical power of our analysis and the anonymity of individuals included in our study, we were only able to divide migrants into four groups. However, we are aware of that the variety within the groups may influence our results and that these broad categories will mask important differences. More details about migrant background are needed to really understand how and why these groups may be different from each other.
Finally, we lack information on several other variables that could potentially confound or contribute to a better understanding of the association between use of OPMH services and school completion. Unmeasured factors such as one's own educational aspirations or parental expectations regarding one's educational attainment could contribute to understanding the underlying mechanisms of the association between mental disorder and school completion. For instance, Reisel, Hermansen, and Kindt (2019) suggested that research based on registry data could benefit from including survey-based data on ambitions, motivations and mechanisms that drive adolescents when it comes to educational choices and academic achievements. Other research also pinpointed such factors as possible confounders in the association between health and educational outcomes (Lynch & von Hippel, 2016). We agree and suggest that future research should incorporate personal perspectives as well as parental aspirations, since these could contribute to explaining the differences between majority, descendant and migrant women regarding OPMH service use and educational attainment. We also recommend future research to address the limitations of our study.
Conclusion
In summary, use of OPMH services is negatively associated with upper-secondary school completion among young women, and is robust to adjustments for potential confounding variables. Furthermore, our hypothesis that migrant women who use OPMH services would have a lower probability of completing upper-secondary education than their Norwegian and descendant counterparts was partly rejected. We found that young descendant women who use OPMH services had significantly higher probability of completing upper-secondary school compared to their Norwegian counterparts and migrant women from Nordic and Western countries. Migrant women from Eastern Europe, Asia and Africa did not significantly differ neither from the majority nor from descendants. However, this does not mean that having had a mental disorder is not a disadvantage for descendants. Migrants and descendants are less likely to use healthcare services than majority women, thus those who use OPMH services may be more resourceful than both migrants who do not seek help, as well as the majority in contact with OPMH services.
Our findings are of great importance as they show the negative impact of mental disorders on school completion in young women, which can subsequently influence other aspects of life and result in lower salary, limited workforce participation and have adverse effects on health. Such limitations have costs for the whole society, due to reduced ability to gain social capital, and increased dependency on welfare services of people affected by mental disorders.
Financial disclosure statement
This study was funded by the Research Council of Norway through the 'Women's Health programme' project number 273262.
Declaration of competing interest
There is no conflict of interest. | 2020-07-16T09:04:52.439Z | 2020-07-15T00:00:00.000 | {
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49864758 | pes2o/s2orc | v3-fos-license | Phaseolus vulgaris Exerts an Inhibitory Effect on Platelet Aggregation through AKT Dependent Way
The Phaseolus vulgaris (common bean), a worldwide vegetable of high consumption, can act as a nutritional supplement in the diet of oversized individuals to reduce weight. Studies have demonstrated the existence of molecules capable of inhibiting the breakdown of carbohydrates via inhibition of both α-amylases and glycosidases. Here, we describe a novel property of the Phaseolus vulgaris: inhibition of thrombotic cardiovascular events. Using assays to test platelet aggregation and secretion, and flow cytometry against the surface expression of P-Selectin. We show that bean extracts significantly reduced adenosine 5′-diphosphate and arachidonic acid induced-platelet aggregation. The mechanism underlying such effect appears to be mediated by AKT, since AKT hypo-phosphorylation decreases the “inside out” activation of platelets. In sum, our results support the hypothesis that common beans are nutritional ingredients that help reduce the risk of cardiovascular diseases associated with platelet hyper-reactivity.
INTRODUCTION
Cardiovascular diseases (CVD) are the leading cause of death in the world, ∼30% of all deaths are due to vascular affections (1) (17.3 million/year), a number expected to increase by 2030 (2). Atherosclerosis is among the most frequent causes of CVD. In the arterial walls, the accumulation of lipids (atheroma) that contain cholesterol, inflammatory cells, collagen, and elastin (3) stimulate the proliferation and inflammation of the endothelium. Over time, the atheroma, via interactions with extracellular matrix factors, promotes the recruitment of new platelets. The accumulation of disc-shaped fragments at the site of arterial damage further increases the inflammatory process that consequently interferes with blood flow (4). Thus, aggregation of platelets represents the cellular bridge between inflammation and thrombosis (4). Indeed, pharmacological agents that block platelet activity significantly reduce the formation of thrombotic and inflammatory events (5), key steps in the progression of CVDs. As a consequence, platelet aggregation inhibitors are widely used as primary and secondary prevention methods against ischemic insults (6,7).
There are lifestyle factors that help reduce the risk of CVDs. For example, regular exercise and avoiding the consumption of tobacco are two factors that significantly reduce the formation of atheromas (8,9). Other lifestyle factor considered to decrease CVD risk is the consumption of fruits and vegetables (10). In fact, there is consensus that the beneficial effect of a balanced diet is not limited to the nutritional content, but it provides factors that protect arteries against toxic agents that increase the risk of CVDs (11). In here, we examine the group of leguminous plants that have edible seeds and are consumed in five continents as essential components of the diet. Particularly, we studied the common bean (Phaseolus vulgaris) that is consumed either as dry or fresh grain (12).
In Chile, this legume (locally called poroto) is consumed in three vegetative stages. The first is when the grain is not yet possible to separate from the pod, its color is green (green beans) and is consumed mainly in salads and typical dishes; the second form is the fresh grain, this is harvested when the grain has reached its final maturity, is separated from the pod and is consumed mainly in traditional cooking, this grain still conserves the water it obtained from the plant (fresh beans); the third presen- tation is stored grain, the grain is harvested at the end of their maturation and drying to be stored without risk of contamination, this type of grain is consumed in the winter season. Previous studies have shown the beneficial properties of bean consumption as antioxidant (13,14), appetite inhibitor (15,16), and carbohydrate metabolism (16,17). Here, we provide evidence indicating that Phaseolus vulgaris inhibits platelet aggregation. Overall, our aim is to transform diet guides in detailed food catalogues that help people choose the appropriate types and amounts of foods that favor a healthy condition.
Plant samples
Phaseolus vulgaris varieties at different growing seasons (green beans, with green pods and green grain; fresh beans, shelled) were collected and processed as indicated below. All beans were obtained from the Regional Supply Center of Talca (CREA).
Aqueous extract
The aqueous beans extract was prepared as follows: beans (1 kg) were washed with pure water, macerated with a blender (Oster) and dissolved in water. The homogenate was filtered with gauze, then cotton wool, and Whatman paper (pore size 0.45 μm). The extract was lyophilized (FreeZone 6, Labconco, Kansas City, MO, USA) and stored (−70 o C).
The chemical composition of the bean extracts was analyzed by the Institute of Chemistry at Universidad of Talca according to standardized methods (Association of Official Analytical Chemists) (18).
Determination of total phenols concentration
The total amount of phenols in each bean extract was determined by a modified version of the Folin-Ciocalteu method (19). Briefly, 0.1 mL of a given extract was mixed with 0.5 mL of the Folin-Ciocalteu phenol reagent (Merck, Darmstadt, Germany). After 5 min of incubation, 0.5 mL of sodium carbonate (Na2CO3; 10% w/v) was added. Then, after 15 min at room temperature (RT), the absorbance (A) of the homogenate was measured at 640 nm (SPECTRO-UV11, mrclab, Haifa, Israel). The total concentration of phenols in the extract was expressed as mg of gallic acid equivalents/g dry weight.
Determination of antioxidant activity Quantification of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Fluka Chemie GmbH, Buchs, Switzerland) was measured using the method of von Gadow et al. (20). Briefly, 0.1 mL of the bean extracts were mixed with 2 mL of DPPH (20 mg/L) and incubated for 8 min (RT). The absorbance of the gallic acid at different concentrations was used as a standard, and the DPPH capture expressed as mg of chromogenic acid equivalents/g fresh weight ( Table 1).
Preparation of platelets
Platelets were prepared as previously described (21). Blood samples were obtained from 3 healthy adult volunteers (BD Vacutainer Ⓡ , BD Biosciences, Franklin Lakes, NJ, USA), none of which took non-steroidal anti-inflammatory drugs for at least 8 days prior to the study (informed consent was obtained from participants). Samples were collected in falcon tubes containing sodium citrate 3.2% (ratio blood/anticoagulant=9/1 v/v). After centrifugation of the citrated blood, at 250 g for 10 min, a platelet rich plasma (PRP) was obtained. After counting the platelets in a hematological counter (Bayer Advia 60, Diamond Diagnostics, Holliston, MA, USA), PRPs were washed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-Tyrode buffer plus PGE1 (120 Determination of platelet aggregation and adenosine triphosphate (ATP) secretion Platelet aggregation and ATP secretion were monitored by a turbidometric method using light transmission in an aggregometer (22) To determine P-selectin expression, 50 μL of the platelet sample was incubated with anti-CD62-PE and anti-CD61-FITC, and maintained for 25 min in the dark. After a quench-dilution step (<4-fold), samples were analyzed in an Accuri C6 flow cytometer (BD Biosciences). Distinct platelet populations were gated on cell size using forward vs. side scatter, the CD61 positivity separated to distinguish it from the electronic noise. Light scatter and fluorescence channels were set at logarithmic gain, and 5,000 events per sample were analyzed. Fluorescence intensities of differentially stained populations were expressed as a mean channel value using the BD Accuri C6 Software (BD Biosciences).
Western blotting
Platelets (200×10 6 platelets/mL) were pre-incubated with either the vehicle (0.9% NaCl) or bean extracts (1 mg/ mL) for 10 min. Then platelets were lysed with 0.2 mL of lysis buffer in ice (30 min), and heated at 95 o C (10 min). Equal quantities of total protein (30 mg) were subjected to SDS-PAGE, and transferred under reduced conditions to a nitrocellulose membrane. To detect AKT1 phosphorylation, a polyclonal antibody against anti-phospho-AKT1 (AKT1, phosphor S473, Abnova, Taipei, Taiwan) was used. A monoclonal antibody against anti-γ-tubulin [γ-tubulin monoclonal antibody (4D11), Thermo Scientific, Waltham, MA, USA] was used as the control. After incubation with a second antibody conjugated to horseradish peroxidase [peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA], samples were developed via chemiluminescence (western blotting substrate, Thermo Scientific) under a radiographic film. Bands were analyzed using ImageJ plugins. All measurements were performed from 6 separate platelet donors.
Statistical analysis
Data were analyzed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) and expressed as mean±standard error of mean (SEM). Results were expressed as percent of aggregation over the vehicle (considered as 100%). Statistically significant differences were analyzed by paired or unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. P-values <0.05 were considered significant.
RESULTS
Inhibitory effect of bean extracts on platelet aggregation activity Platelet aggregation was achieved by the use of two agonists, ADP or AA. As shown in Table 2, both the aqueous and methanolic bean extracts obtained from fresh beans showed anti-platelet action. In the presence of ADP 8 μM, the aqueous and methanolic bean extracts inhibited platelet aggregation by 28±3% and 49±5%, respectively (P<0.01). A similar effect was observed when 1 mM AA was used as the activator of platelet aggregation. The % of inhibition were 28±4% (P<0.05) and 35±3% (P< 0.01) for the aqueous and methanolic bean extracts respectively. When aqueous and methanolic extracts obtained from green beans were studied, similar anti-platelet aggregation effects were observed. Overall, the results indicate that both fresh and green varieties have equivalent activity.
Effects of bean extracts on platelet secretion
Platelets contain important inflammatory/hemostatic mediators within their granules. As a result of the activation, platelets release the granules to the extracellular space.
To determine whether aqueous and methanolic bean extracts modulate platelet secretion, we measured the levels of ATP in the extracellular media. Our results (Table 3) indicate that the methanolic extract obtained from fresh beans (1 mg/mL) inhibited the secretion of ATP mediated by ADP (8 μM) and AA (1 mM). The maximum blockades of ATP secretion were 34±11% and 29±13% (P<0.05) for ADP and AA, respectively. Similar results were obtained when methanolic extracts obtained from green beans were used. The maximum inhibitions of ATP secretion were 24±9% and 28±14% for ADP and AA, respectively. Interestingly, the aqueous extracts of both varieties, fresh and green beans, showed no significant inhibitory effects on platelet secretion.
Effects of bean extracts on platelet P-selectin expression
Expression of P-selectin on the membrane of activated platelets is one of the most widely used markers to evidence the degree of platelet activation. This same parameter determined in vitro in human platelets of healthy volunteers is useful to determine the degree of reactivity of the platelets on exposure to an agonist in the presence or absence of the different extracts and thus determine if the extracts are able to reduce the activation of platelets. Because most favorable results were presented to the methanol extract of bean stew, this was subjected to tests to determine its effect on the expression of the P-selectin molecule actively involved in platelet activity. As shown in Fig. 1, the shelled bean methanol extract at a concentration of 1 mg/mL induced a decrease in the expression of P-selectin mediated ADP 8 μM almost 15% (P<0.05). Thus, the shelled bean methanolic extract inhibits platelet activation, decreasing the expression of Pselectin.
Methanolic bean extracts alter the phosphorylation state of AKT-1 To investigate the plausible mechanism by which bean extracts inhibited platelet activation and aggregation, we evaluated the phosphorylation of AKT1, a key intermediary in intracellular platelet signaling. Fig. 2 shows a decrease in phosphorylation of AKT (S473) using methanolic fresh bean extract to 1 mg/mL, using it as agonist ADP 8 μM aggregation. This result by western blot shows that the methanol extract of Phaseolus vulgaris is capable of inhibiting AKT phosphorylation, blocking subsequent platelet activation and aggregation.
DISCUSSION
The mortality associated with CVD directly relates to the prevalence in the population (24). To promote a healthy lifestyle, and an appropriate diet in the population, it is essential to encourage consumption of foods rich in fruits and vegetables. It is necessary to create an educated "nutritional catalog" that relates the benefits of a healthy diet with factors that reduce CVD risks. One of the main pathophysiological events involved in CVDs is the aggregation of platelets, a central phenomenon in thrombosis and atherogenesis (25,26).
In the case of Phaseolus vulgaris, with the results presented in Table 1 and 2, it is shown that Phaseolus vulgaris has an antiplatelet effect and that this effect is dependent upon the state of maturation of the grain. Although Phaseolus vulgaris has multiple effects on human health, there are no reports about its specific effect on platelets. To our knowledge, this study is the first to describe this direct and effect dependent on the state of maturation of the beans on the activity of platelets.
In order to partially characterize the mechanism by which Phaseolus vulgaris exerts its anti-platelet effect, it was necessary to study the methanol extracts, and the ability of these to suppress platelet secretion. P-selectin plays an essential role in platelet function, also considered important in thrombotic events (27)(28)(29). P-selectin is a cell adhesion glycoprotein in resting platelets found in the alpha granules (30). When platelets are activated, due to secretory mechanisms of surrounding cells as histamine and thrombin, P-selectin is translocated to the membrane and binds to its ligand, promoting aggregation (30). The effects against expression of this adhesion protein were evident in the platelets administered prior to the methanol extracts.
Similar results on the activity of platelets and platelet P-selectin expression versus the presence of ADP were obtained using other vegetables and fruits, such as tomatos (31,32). The proposed reduction in P-selectin on the platelet membrane relaxation mechanism is the cAMP levels in platelets by modulating the activity of PKA (27).
The AKT/PKB signaling protein is fundamental in signaling from the inside out and from the outside in, triggered by the activation of platelets (33,34). Our results demonstrate that the mechanism of action of shelled bean extracts involves the inhibition of AKT phosphorylation at residue (S473). This may explain the effect on activation, aggregation, and secretion in treated platelets. This effect takes on a central importance in the context of developing new strategies that aim to lower the high rates of death from ischemic CVD.
A study by Vaiyapuri et al. (35) showed that nobiletin, a flavonoid, has the ability to inhibit platelet function. There is a growing interest in the search for extracts or compounds of natural origin that are capable of reducing the risk factors of CVD, this is mainly due to the low adherence of pharmacological therapies and a large number of undesirable side effects presented by groups pharmacological agents such as statins. Current pharmacotherapy, although it has managed to counteract the formation of atheromatous plaques in individuals at risk, has increased thrombotic events due, among other aspects, to the endothelial erosion that they produce. These clinical data motivate the search for clinical therapies of natural origin that together with reducing risk factors, do not increase thrombotic events secondarily. It should be noted that extracts of Phaseolus vulgaris are used today in the reduction of body weight in overweight and obese individuals. These extracts could be an alternative to reduce the side effects of pharmacological therapies or be alone therapies tending to reduce the risk factors. | 2018-08-01T20:48:44.203Z | 2018-06-01T00:00:00.000 | {
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244352895 | pes2o/s2orc | v3-fos-license | The Aftermath of the COVID-19 Crisis in Saudi Arabia: Respiratory Rehabilitation Recommendations by Physical Therapists
Since late 2019, the number of COVID-19 patients has gradually increased in certain regions as consecutive waves of infections hit countries. Whenever this wave hits the corresponding areas, the entire healthcare system must respond quickly to curb the diseases, morbidities, and mortalities in intensive care settings. The healthcare team involved in COVID-19 patients’ care must work tirelessly without having breaks. Our understanding of COVID-19 is limited as new challenges emerge with new COVID-19 variants appearing in different world regions. Though medical therapies are finding solutions to deal with the disease, there are few recommendations for respiratory rehabilitation therapies. A group of respiratory rehabilitation care professionals in Saudi Arabia and international experts have agreed with the World Health bodies such as the World Health Organization (WHO) on the treatment and rehabilitation of patients with COVID-19. Professionals participating in COVID-19 patient treatment, rehabilitation, and recovery formulated respiratory rehabilitation guidelines based on the DELPHI Method, combining scientific research and personal practical experience. As a result, it is envisaged that the number of individuals in the region suffering from respiratory ailments due to post-COVID-19 will decrease. This narrative review and clinical expertise guidelines may give physiotherapists acceptable and standard clinical guideline protocols for treating COVID-19 patients.
Introduction
The COVID-19 disease appeared in China as acute pneumonia in December 2019 [1]. COVID-19-affected patients presented with fever, breathing difficulty, cough, and a few patients presented with severe pneumonia symptoms [2]. People with immunodeficiency or chronic diseases (e.g., cancer, diabetes, lung diseases) or the elderly are prone to infections and more severe complications [3]. Individual care and well-being may also impact the likelihood of the new coronavirus attachment and its severity [2]. In this pandemic, those aged between 45 and 50 are more likely to be afflicted by the virus [4].
Respiratory rehabilitation is a comprehensive therapy approach focused on individualized assessment and treatment to improve or maintain people with pulmonary diseases' physical, social, and mental health [8,9]. It includes fitness training, education, and lifestyle modification, and aims to improve pulmonary disease patients' overall health [8,10]. Physical therapy techniques are particularly relevant in the rehabilitation of patients with COVID-19 [9,11].
International organizations have attempted to recommend respiratory rehabilitation in their own countries [9][10][11]. Therefore, there was a need to establish standard respiratory rehabilitation guidelines for COVID-19 care in Saudi Arabia. Based on our clinical experience after dealing with several COVID-19 patients and through a literature search on respiratory rehabilitation, we propose these guidelines for physiotherapists involved in treating and managing COVID-19 patients in Saudi Arabia. These are the opinions of respiratory rehabilitation experts and experts from other disciplines summoned to deal with the crisis.
Methodology
This narrative review focuses on respiratory rehabilitation guidelines for treating COVID-19 patients based on experts' personal experiences and available scientific literature. The flow diagram shows the review process and the study synthesis by the experts (Figure 1). The studies included for clinical recommendations for COVID-19 physical therapy care by experts are presented in Table 1. A group of international cardiorespiratory physical therapy experts (n = 12) compiled the clinical recommendations. The expert group included seven clinical cardiopulmonary physical therapists and five academic physical therapy professionals. The first author organized a virtual forum to discuss the necessity for consensus for physical therapists to produce respiratory rehabilitation recommendations for COVID-19 patients. The author group first met virtually on April 30 2020, to discuss the immediate need for global acute care to manage COVID-19 patients and the need to develop respiratory rehabilitation practice guidelines. As a result, the task of developing scientific instructions for physical therapists in critical care settings was promptly prioritized.
Literature Search
From creating databases from 30 April 2020, through 30 June 2021, we searched the published literature in Scopus, PubMed (Medline), Embase, Ovid databases, and other related scientific websites regarding respiratory rehabilitation and COVID-19 for any randomized clinical trials (RCT), systematic reviews, meta-analyses, or international recommendations and guidelines. The inclusion criteria included only articles that were written in English, and the articles' full text could be retrieved and read. The exclusion criteria included articles that do not fall under the scope of rehabilitation or physical therapy and articles that are not related to patients (such as animal experiments or viral studies). The search terms included are: "COVID-19", "new coronavirus pneumonia", "acute respiratory distress syndrome (ARDS)", "severe acute respiratory syndrome (SARS)", "acute hypoxemic respiratory failure", "influenza", "Middle East respiratory syndrome (MERS)", "pulmonary/respiratory rehabilitation or physical therapy and infectious diseases", "noninvasive ventilation and acute hypoxemic respiratory failure", "acute respiratory failure (ARDS)", "mechanical ventilation", and "weaning from mechanical ventilation." The flow diagram shows the review process and the study synthesis by the experts (Figure 1). The recommendations are based on the published literature, and additional literature was consulted until all panel members agreed on the final draft of the respiratory rehabilitation guidelines. The panel virtually met on several occasions to formulate the recommendations, and the DELPHI method was followed to reach a common consensus ( Figure 2).
In the absence of dedicated staff, healthcare professionals must clear the training test based on clear principles of behavior and action. In addition, respiratory rehabilitation providers must be appropriately skilled and experienced with respiratory physical therapy. All preventive measures must be implemented, and both operators and patients must wear all protective equipment stated in the relevant papers. RR is a three-phased systematic nonpharmacological therapy that includes assessment, treatment, and re-assessment (the evaluation is primarily functional, focusing on consciousness, pulmonary, cardiovascular, physical functions, and quality of life) [15]. The risk of droplet formation in the airways of a patient is very high. According to the WHO, to avoid or limit the possibility of this, all interventions and activities must be carried out-particularly concerning airway clearance interventions-with utmost care [16].
Whatever intervention mode, intensity, and timing are used, it must be tailored to each patient's specific needs, especially for individuals with severe or critical conditions, the elderly, morbidly obese patients, comorbidities, and other complications [16]. Throughout the recovery phase, assessment and monitoring of COVID-19 patients should be continued [17]. Respiratory rehabilitation providers can also help patients cope with delirium, rage, fear, sleep disturbances, panic attacks, or a sensation of loneliness during isolation, and rigorous therapy and those at risk of non-compliance with treatment [18].
COVID-19 Respiratory Signs and Symptoms Are Discussed, as well as Possible Physical Therapy Referrals and Interventions
Mild symptoms without severe respiratory impairment, such as a fever and a dry cough, but no changes in a chest X-ray [19]. Treatment considerations: There will be no physical therapy contact with the patient, but telerehabilitation can be given for respiratory care advice [19]. Low oxygen demand (e.g., O 2 flow of 5 liters/minute for maintaining SpO 2 > 90%), cough with or without sputum production, and the ability to remove secretions autonomously are all characteristics of pneumonia [20]. Treatment considerations: There will be no physical therapy direct contact with the patient, but telerehabilitation can be given for respiratory care advice.
Mild symptoms with or without pneumonia characteristics such as comorbidity in the respiratory or neuromuscular systems (e.g., chronic obstructive pulmonary disease, myopathies or neuropathies, cystic fibrosis, bronchiectasis, spinal cord injury) [20]. Treatment considerations: Referral to physical therapy for airway clearing, and physical therapists must use airborne precautions. For non-ventilated patients, wearing a surgical mask during respiratory therapy sessions should be mandatory.
Mild disease with or without pneumonia symptoms, with signs of exudative secretions and ineffective self-secretion clearance capacity [20]. Treatment considerations: Referral to physical therapy with the intent of clearing the airway, and physical therapists must use airborne precautions. For non-ventilated patients, wearing a surgical mask during respiratory therapy sessions should be mandatory.
Severe disease suggests infections of the lower respiratory tract (e.g., increased O 2 requirements; mild to high-grade fever; difficulty breathing or shortness of breath; severe, frequent, or productive or non-productive coughing episodes; radiological changes consistent with secretion accumulation) [20]. Treatment considerations: Referral to physical therapy for airway clearing, especially if the cough is ineffective and productive. There is radiological evidence of secretion accumulation. Physical therapists must take airborne precautions, and for the non-ventilated patient wearing a surgical mask during respiratory therapy sessions should be mandatory. It is recommended to optimize care as soon as possible and involve respiratory rehabilitation in the intensive care unit (ICU).
Mobilization, exercises, and rehabilitation [21]: If the patient is or with critical functional constraints, there is a high risk of developing problems such as frail or multiple comorbidity impairment of independence, for instance, in patients with ICU who experience a considerable decrease in functionality or at risk of ICU diseases, for example, mobilization, exercise, and rehabilitation. Treatment considerations for ICU-acquired patients: Referring to physical therapy, droplet precautions and precautions should be taken against airborne diseases when close contact is needed, or aerosol generation procedures may be necessary. For the non-ventilated patient, the patient must wear an operational mask throughout respiratory therapy sessions.
Physical Therapy for COVID-19 Individuals Who Are in the Critical Phase of the Disease
In conscious or unconscious patients, frequent posture changes, passive or active mobilization, and electrical stimulation (neuromuscular) are used to prevent disability [22]. Positional therapy with proper supervision is advised to maximize the V/Q ratio and avoid injury from immobility [22,23]. The prone position is suggested for several hours, but it must be discontinued if oxygenation deteriorates or if any significant problem arises. The trend in peripheral muscular strength changes should be assessed as soon as possible using appropriate tools.
The RR techniques in the critical phase must begin after the patient has attained some clinical stability to deal with ventilation and weaning. Treatments should be discontinued in the event of a high fever, increasing dyspnea, tachypnea >30 breaths/min, SpO 2 <90% on O 2 therapy, systemic arterial hypertension, respiratory distress, bradycardia, or tachycardia.
In individuals with minor bronchial obstruction, airway clearing procedures are not suggested during the acute phase [24]. Indeed, the possible benefits for operators do not justify the pollution concerns [24]. In patients with bronchiectasis or obvious bronchial burden, the risk/benefit ratio should be assessed on an individual case-by-case basis, employing techniques that ensure a safe distance from the patients [24].
Standard respiratory physical therapy protocols for dyspnea reduction, skeletal muscle training, tracheobronchial clearance, and activities of daily living training are not recommended since they can overburden the patient's respiratory system and create distress [24]. It is recommended that ventilatory clinical parameters (cough, dyspnea, temperature, respiratory rate, SpO 2 , SpO 2 /FiO 2 , and thoracoabdominal dynamics) be evaluated twice a day.
COVID-19 Patients Receiving Physical Therapy Rehabilitation in the Acute Phase
Mobilization such as getting out of bed, making frequent postural changes, including continuous rotational therapy, asking patients to perform active upper and lower limb exercises, implementing muscle conditioning exercises, and strengthening upper and lower limb muscles are all recommended to prevent disability during this acute phase [24,25]. If patients have inspiratory muscular weakness, therapy includes activities to strengthen the muscles of the respiratory system [24]. Sedation may be used to relieve fatigue and dyspnea in patients with a non-productive dry cough [26]. Airway clearance procedures, ideally using single-use devices with self-management, are appropriate in excessive secreting patients with chronic pulmonary illnesses [26].
COVID-19 Patients Receiving Physical Therapy Rehabilitation in the Post-Acute Phase
For assessing peripheral muscle strength, manual or instrumental (e.g., handgrip dynamometer) muscle testing, isokinetic testing, and joint range assessments are recommended [27,28]. Reconditioning procedures are recommended for weaned patients and those on mechanical ventilation and oxygen for a long time to improve their physical functions and correct the motor and psychological effects of extended immobilization in the ICU [28]. Because the impact of viral infections on muscle activity is unknown, exercises are aimed at gradually increasing the load based on physical symptoms to maintain normal function [29]. Low-intensity exercises (≈3.0 MET) along with regular patient counseling and education are recommended [30]. A telehealth system can eventually perform rehabilitation programs for isolated individuals (i.e., educational videos, teleconsultation).
Patients who are discharged from hospitals should be given instructions on how to cope with physical activity and should be evaluated regularly in terms of function, capability, and involvement once they have been cured and no longer pose a risk of infection [30]. Balance evaluation is recommended for long-term bedridden patients [25]. A schedule should be established at the earliest possible time for evaluating the exercise capacity and O 2 saturation response during efforts using walking tests and nighttime performance [25]. Patients who have recovered from severe COVID-19 frequently have physical and functional disabilities (such as changes in respiratory function, musculoskeletal and neurological function, limited participation in activities of daily living, and a decline in quality of life [31,32]. The severity of the normocapnic respiratory failure and the presence of physical and psychological dysfunction determine the length of time it takes to recover (anxiety, abandonment, depression, posttraumatic stress syndrome) [33]. It may take longer for patients with comorbidities to return to their previous status. Daily checks should be made for temperature, respiratory rate, cough, dyspnea, SpO 2 , SpO 2 /FiO 2 , and thoracoabdominal dynamics [33]. Regular and straightforward treatment regimens should be used for weaning oxygen therapy.
Specific Respiratory Physical Therapy Interventions
Bronchoscopy, invasive or non-invasive mechanical ventilation, intubations, and cardiopulmonary resuscitation are some of the procedures that produce aerosols when dealing with critical COVID-19 patients (in cardiac or respiratory failure) [34]. Treatment goals for oxygen therapy may differ based on the patient's condition. Oxygen saturation, i.e., SpO 2 > 92 to 96%, is the goal for severe hypoxia, respiratory distress, or shock patients [34,35]. When a patient is stable, the goal for SpO 2 maintenance is > 92% in adults and approximately between 92-95% in pregnant females. The SpO 2 aim should not be maintained over 96% in persons with COVID-19 and acute hypoxemic respiratory failure [35,36].
If treating therapists wear appropriate personal protective equipment (PPE), highflow nasal oxygen (HFNO) and non-invasive ventilation is recommended for treating hypoxemic respiratory failure-related hypoxia because it has a limited risk of producing aerosols at flow rates of 40 to 60 L/min or with helmet and filters during non-invasive ventilation [37]. All these cases are required to be used in negative pressure rooms. When proper COVID-19 prevention measures are implemented, the risk of airborne transmission to employees is minimal [37,38]. Patients who require HFNO should use negative pressure chambers [39]. Only patients in airborne isolation rooms should be supplemented with HFNO. The flow rate should be kept at less than 30 liters/minute, which may help prevent viral transmission [39]. Despite the accumulated data in favor of the use of different non-invasive respiratory therapies for acute respiratory failure in COVID-19, it is not fully understood when to start, escalate, and de-escalate the best respiratory supportive option for the different timing of the disease. For many countries, non-invasive mechanical ventilation (NIMV) is not advised for routine use due to the significant hypoxic respiratory failure rate associated with COVID-19-related hypoxia [37,40,41]. However, evidence has gradually been consolidating in favor of non-invasive ventilatory strategies in the management of hypoxemic respiratory failure caused by COVID-19 [37].
Nebulized drugs such as salbutamol or similar types are not recommended for treating COVID-19 in non-intubated patients because they increase the risk of aerosol generation and infection spread to nearby health care professionals [42,43]. Wherever possible, metered-dose inhalers or spacers should be used. If a nebulizer is required, consult local standards to keep aerosolization to a minimum (e.g., using a Pari Sprint with an inline viral filter). Nebulizers, NIMV, HFNO, and spirometry should be avoided, and permis-sion from senior medical professionals should be requested before using them. Airborne precautions should be used if they are deemed necessary. Although there is evidence favoring the prescription of some physical therapy techniques, some of these approaches are still recommended, such as postural drainage positioning, percussion, and vibrations, breathing techniques such as autogenic drainage, or active cycle breathing. Furthermore, several of these procedures, such as manual or mechanical hyperinflation, positive expiratory pressure (PEP) therapy, and mechanical insufflation-exsufflation, appear to be more effective and physiologically plausible for treating these patients [26]. Respiratory muscle training must be incorporated in various phases of rehabilitation to improve their strength and endurance [44]. Physiotherapists can also use inspiratory positive pressure breathing techniques in some instances, such as patients with rib fractures. NIMV can also be utilized for respiratory failure management or during exercise as part of airway clearance methods [45]. Airway suctioning and assisted or encouraged cough motions are two techniques that can help with secretion clearing [45][46][47].
Conclusions
Respiratory rehabilitation professionals in Saudi Arabia have been motivated to respond to the current COVID-19 pandemic. A group of physical therapy professionals involved in COVID-19 patient treatment, rehabilitation, and recovery developed respiratory rehabilitation guidelines based on the DELPHI Method, which combined scientific research with personal practical experience. To improve cardio-respiratory status, various physical therapy interventions have been found to be effective in different phases of COVID-19 patients' care. For instance, in the acute phase/ICU, airway clearance and positioning are critical, whereas ambulation and respiratory muscle training are beneficial in the subacute phase. It is anticipated that these guidelines may help physiotherapists to effectively treat and manage COVID-19 patients with respiratory symptoms. | 2021-11-19T16:19:06.263Z | 2021-11-01T00:00:00.000 | {
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237502962 | pes2o/s2orc | v3-fos-license | Propagation of smallness and spectral estimates
The purpose of this article is to show that the spectral projector estimates for Laplace operators can be deduced from Logunov-Malinnikova's Propagation of smallness estimates for harmonic functions [11, 10, 9]. The main point is to pass from the local estimates obtained in [4] (on a compact manifold) to global estimates. We also state classical consequences in terms of observability and control for heat equations, which are direct consequences of these spectral projector estimates..
Introduction
In this article, we continue our investigation of Logunov and Malinnikova's results on the propagation of smallness for elliptic equations and their consequences on the propagation of smallness, observation and control for heat equations. Here our purpose is to extend our previous results [4] on compact manifolds (with or without boundaries) to the non compact setting. We consider on R d the following Laplace operator where we assume that the coefficients κ, g are Lipschitz and that g is uniformly elliptic: Recall that the n-Hausdorff content (or measure) of a set E ⊂ R d is and (for a bounded set) the Hausdorff dimension of E is defined as dim H (E) = inf{n; C n H (E) = 0}. Let ω ⊂ R d satisfying the following condition (see [3,8]) (1.3) ∃R, δ > 0; ∀x ∈ R d , meas(ω ∩ B(x, R)) ≥ δ, or (1.4) ∃R, δ > 0; ∀x ∈ R d , C n H (ω ∩ B(x, R)) ≥ δ. The first purpose of this note is to show that Jerison-Lebeau's spectral estimate from [7] still holds under these very weak assumptions (assuming in (1.4) that n < d is sufficiently close to d i.e. the set ω is not too thin). Let us recall that the operator −∆ on L 2 (R d , κ(x)dx), with domain H 2 (R d ) is self adjoint with non negative (continuous) spectrum. As a consequence, using the spectral theorem, it is possible to write (here dm λ is the spectral measure of the operator √ −∆): and consequently Assume that ω satisfies the assumption (1.3). Then there exists C > 0 such that for any u ∈ L 2 (R d ), Assume now that ω satisfies the assumption (1.4) for some n < d sufficiently close to d. Then there exists C > 0 such that for anu u ∈ L 2 (R d ), Let us now give three applications to the observation and control of the heat equation. The first application is very standard and follows from the works by Miller [12], Phung-Wang [13] and Apraiz, Escauriaza, Wang, Zhang [1,2]. We also refer to [4,Section 5].
Theorem 2 (Null controllability from sets of positive measure). Let F ⊂ (0, T ) a set of positive measure. Let E ⊂ R d satisfying (1.3). Then, there exists C > 0 such that for any u 0 ∈ L 2 (M ) the solution u = e t∆ u 0 to the heat equation As a consequence, for all u 0 , v 0 ∈ L 2 (M ) there exists f ∈ L 2 (F × E) such that the solution to The second application is a control and observability result from sets of positive Hausdorff content. It follows from (1.7) following the proof of [4,Theorem 3] given in [4,Section 5]. The only difference is that in [4] we use the duality between continuous functions and Radon measures while here we use the duality between the spaces G, G defined below.
Theorem 3 (Null controllability from sets of positive Hausdorff content). Let F ⊂ (0, T ) be a set of positive measure. Let E ⊂ R d satisfying (1.4). Then, there exists C > 0 such that for any u 0 ∈ L 2 (M ) the solution u = e t∆ u 0 to the heat equation Let us denote by G the set of functions on F with values locally bounded Radon measures on E, such that Remark that G is the dual space of G, defined as the space of functions g on F with values continuous functions on E such that We endow G with its natural norm. We deduce from (1.9) that for all is well defined. We deduce by duality that if M is the set or Radon measures on E, the map v ∈ M → χv ∈ D(−∆) −σ is also well defined and continuous. As a consequence, G is continously embeddeed in and equation (1.10) is just solved by The third application is the observation and control for discrete times from [4,Theorem 4].
Theorem 4 (Observability and exact controllability using controls localised at fixed times). Let m > 0, τ ∈ (0, 1) and D > 0. There exists C > 0, such that if E ⊂ R d satisfies (1.3) then for any sequence (s n ) n∈N , J = {0 < · · · < s n < · · · < s 0 < T } converging not too fast to 0, i.e., we have that for any u 0 ∈ L 2 (M ), the solution u = e t∆ u 0 to the heat equation As a consequence, given any sequence (t n ) n∈N , converging not too fast to T , and the solution to Remark 1.4. In [4], the spectral estimates are obtained in manifolds with boundaries under Dirichlet or Neumann boundary conditions. It would also be possible to include such boundaries in a non compact setting provided we still control the geometry at infinity. For example, we could include holes as long as their diameters remain in a compact set of (0, +∞) and they remain disjoint from each other (with a fixed lower bound on their distances).
proof of Theorem 1
We start with the first part in Theorem 1. Let us define Recall that As a consequence, we have Without loss of generality we can assume that in (1.3) and (1.4), the constant R is chosen large enough so that Let 1 = p χ(x − p) be a partition of unity associated. Consider, for T 2 > T 1 > 0 the sets which by construction satisfy the inclusions E p ⊂ K p ⊂ Ω p . Now assumption (1.3) ensures that |F p | ≥ δ. Remark that in (1.2), since F p has a diameter at most 2R, when computing the Hausdorff content of F p , we can assume that ∀j, r j ≤ 4R. As a consequence, For sufficiently small ǫ > 0 we can now apply [11, Theorem 5.1] and get where the constant is uniform with respect to p because the metric and κ are bounded on B(p, 2R) (uniformly with respect to p while the metric is uniformly elliptic).
We first replace L ∞ norms by L 2 norms. We can assume v µ ≡ 0 Let 2) would hold with E p replaced by E ′ p (and the same constants), leading to As a consequence, We now need a variant of Sobolev embeddings, which we prove for the reader's convenience: Let us first, assuming Lemma 2.1, finish the proof of (the first part of) Theorem 1. Observe that Young's inequality yields Hence, fom (2.4) we deduce for all D > 0, (E∩B(p,R)) . Now, from Lemma 2.1 we deduce , and the proof of Theorem 1 for µ ≥ µ 0 follows from taking D > 2K.
Let us now prove Theorem 1 in the second case, f µ ≥ µ 0 . From assumption 1.4, we have and we deduce from [11,Theorem 5.1] that if n is sufficiently close to d that where the constant is uniform with respect to p because the metric and κ are bounded on B(p, 2R) (uniformly with respect to p while the metric is uniformly elliptic). We deduce we deduce for all D > 0, 2α Dµ u 2 L ∞ (E∩B(p,R)) . Now, from Lemma 2.1 we deduce and the proof of the second part in Theorem 1 for µ ≥ µ 0 follows from taking D > 2K. Finally, for µ < µ 0 , apply Theorem 1 for µ = µ 0 to u = Π µ (u).
Proof of Lemma 2.1 for smooth metrics. We start with an elementary proof (which works only for smooth metrics) relying only on Sobolev embeddings. Later on we give the general proof for Lipschitz metrics. Let χ ∈ C ∞ (R d+1 ) non negative be equal to 1 on (−T 2 , T 2 ) × B(0, R). Let s > d+1 2 , and χ p (x) = χ(x − p).
By Sobolev embeddings, for all p ∈ Z d , and we deduce .
We now use the elliptic regularity result (here we have to assume that κ, g ∈ C k , k ≥ s), and we get (recal that −∆ acts on the spectral measure as the multiplication by λ 2 ): where in the last estimate we used (1.5).
15) and considering the function we get from (2.13) (with f = 0): As a consequence, summing with respect to p, using quasi-orthogonality for the H 1 norm, we get On the other hand, using (1.5) we have x ≤ Ce 2T µ u 2 L 2 (κdx) .
Next, using that the metric g is uniformly elliptic according to (1.1), for any t ∈ (−τ, τ ), we have Using again (1.5) we deduce ≤ Ce 2τ µ u 2 L 2 (κ(x) dx) . This concludes the proof of Proof of Lemma 2.1 for Lipschitz metrics. | 2021-09-15T01:16:05.682Z | 2021-09-14T00:00:00.000 | {
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231750715 | pes2o/s2orc | v3-fos-license | The complete chloroplast genome of Euphorbia ebracteolata Hayata (Euphorbiaceae)
Abstract Euphorbia ebracteolata is a perennial medicinal plant and widely used in China for thousands of years. The complete chloroplast genome reported here is 163,090 bp in length, including two inverted repeats (IRs) of 26,699 bp, which are separated by a large single-copy (LSC) and a small single-copy (SSC) of 91,943 and 17,749 bp, respectively. The whole chloroplast genome of E. ebracteolata contains 112 genes, including 78 protein-coding genes, 30 transfer RNA, and 4 ribosome RNA. Phylogenetic analysis result strongly indicated that E. ebracteolata is closely related to E. helioscopia.
The genus Euphorbia is the largest in the spurge family, some of which have been used as medicinal plants for a long time. Euphorbia ebracteolata Hayata (Euphorbiaceae), a perennial herb distributed in the central and eastern of China. The root of this plant is poisonous, named 'LangDu' which is used as a traditional herbal medicine to treat edema, asthma, tuberculosis, and tumor diseases for thousands of years (Fu et al. 2006). Because 'LangDu' is widely used and extremely important, it is necessary to understand the background of the evolutionary biology of E. ebracteolata. Chloroplast genome is used as a source of valuable data for phylogenetic analysis, genetic diversity evaluation, and plant molecular identification (Dong et al. 2018;Sun et al. 2020). In this study, the completed chloroplast genome sequence of E. ebracteolata is determined which provides valuable genetic information for the phylogenetic studies.
The fresh samples of E. ebracteolata were collected from Huaining county Anhui province, China (30 44 0 1 00 N, 116 49 0 47 00 E). Voucher specimen was stored at the herbarium of Institute of Chinese Materia Medica (CMMI), China Academy of Chinese Medical Sciences with the specimen voucher number is 340822LY0035. Total genomic DNA from fresh leaves of a single individual using the method of Li et al. (2013). And the sequencing library was constructed and quantified following the methods introduced by Dong et al. (2017).
The whole-genome sequencing was conducted with 150 bp paired-end reads on the Illumina HiSeq X Ten platform. Contigs were assembled from the high-quality paired-end reads by using the SPAdes version 3.6.1 program (Kmer ¼ 95) (Bankevich et al. 2012) after low-quality reads were filtered. The chloroplast genome contigs selected by the Blast program (Altschul et al. 1990), taken Malva parviflora (GenBank: MK860036) as the reference. The selected contigs were assembled using Sequencher version 4.10 (Gene Codes Corporation, Ann Arbor, MI http://www.genecodes. com). Gene annotation was performed with Plann (Daisie et al. 2015), taken the cp genome of E. esula (GenBank: KY000001) as the reference and manually corrected for codons and gene boundaries using BLAST searches. The annotated E. ebracteolata genomic sequence has been deposited into GenBank with the accession number MT830860.
The complete chloroplast genome reported here is 1,63,090 bp in length, including two inverted repeats (IRs) of 26,699 bp, which are separated by a large single-copy (LSC) and a small single-copy (SSC) of 91,943 and 18,089 bp, respectively. The overall GC-content of the chloroplast genome was 35.5%. The chloroplast DNA of E. ebracteolata comprised 112 distinct genes, including 78 protein-coding genes, 4 transfer RNA, and 30 ribosome RNA, do not contain ycf15. In these genes, 17 harbored a single intron, whereas two (ycf3 and clpP) contained double introns. The rps12 gene is a trans-spliced gene with 5 0 end located in the LSC region and the 3 0 end located in the IR region. The gene trnK-UUU has the largest intron, which contains the matK gene.
To confirm the phylogenetic location of E. ebracteolata within the genus Euphorbia, total 11 complete cp genomes were downloaded from Genbank and the genus Daphniphyllum was taken as an outgroup. All chloroplast genome sequences were aligned using MAFFT (Katoh et al. 2019). Phylogenetic analysis was conducted based on IQ-tree using PhyloSuite under the TVM þ FþI þ G4 model with 1000 bootstrap replicates (Nguyen et al. 2015;Zhang et al. 2020). The phylogenetic tree showed that all species of Euphorbia form a monophyletic group with 100% support, and E. ebracteolata was closely related to E. helioscopia (Figure 1). The chloroplast genome of E. ebracteolata provided a lot of genetic information for species conservation and taxonomy of genus Euphorbia.
Disclosure statement
No potential conflict of interest was reported by the author(s). | 2021-02-03T05:07:15.149Z | 2021-01-02T00:00:00.000 | {
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216079336 | pes2o/s2orc | v3-fos-license | Virtual and augmented reality in finance: State visibility of events and risk
The recent financial crisis and its aftermath motivate our re-thinking of the role of Information and Communication Technologies (ICT) as a driver for change in global finance and a critical factor for success and sustainability. We attribute the recent financial crisis that hit the global market, causing a drastic economic slowdown and recession, to a lack of state visibility of risk, inadequate response to events, and a slow dynamic system adaptation to events. There is evidence that ICT is not yet appropriately developed to create business value and business intelligence capable of counteracting devastating events. The aim of this chapter is to assess the potential of Virtual Reality and Augmented Reality (VR / AR) technologies in supporting the dynamics of global financial systems and in addressing the grand challenges posed by unexpected events and crises. We overview, firstly, in this chapter traditional AR/VR uses. Secondly, we describe early attempts to use 3D/ VR / AR technologies in Finance. Thirdly, we consider the case study of mediating the visibility of the financial state and we explore the various dimensions of the problem. Fourthly, we assess the potential of AR / VR technologies in raising the perception of the financial state (including financial risk). We conclude the chapter with a summary and a research agenda to develop technologies capable of increasing the perception of the financial state and risk and counteracting devastating events.
Introduction
The recent financial crisis and its aftermath motivate our re-thinking of the role of Information and Communication Technologies (ICT) as a driver for change in global finance and a critical factor for success and sustainability. We attribute the recent financial crisis that hit the global market, causing a drastic economic slowdown and recession, to a lack of state visibility of risk, inadequate response to events, and a slow dynamic system adaptation to events. There is evidence that ICT is not yet appropriately developed to create business value and business intelligence capable of counteracting devastating events. The aim of this chapter is to assess the potential of Virtual Reality and Augmented Reality (VR / AR) technologies in supporting the dynamics of global financial systems and in addressing the grand challenges posed by unexpected events and crises. We overview, firstly, in this chapter traditional AR/VR uses. Secondly, we describe early attempts to use 3D/ VR / AR technologies in Finance. Thirdly, we consider the case study of mediating the visibility of the financial state and we explore the various dimensions of the problem. Fourthly, we assess the potential of AR / VR technologies in raising the perception of the financial state (including financial risk). We conclude the chapter with a summary and a research agenda to develop technologies capable of increasing the perception of the financial state and risk and counteracting devastating events.
Traditional use of VR / AR
Virtual Reality and Augmented Reality tools and technologies supply virtual environments that have key characteristics in common with our physical environment. Viewing and interacting with 3D objects is closer to reality than abstract mathematical and 2D representations of the real world. In that respect Virtual and Augmented reality can potentially serve two objectives (Maad et al, 2001): (a) reflecting realism through a closer correspondence with real experience, and (b) extending the power of computer-based technology to better reflect "abstract" experience (interactions concerned with interpretation and manipulation of symbols that have no obvious embodiment e.g. share prices, as contrasted to interaction with physical objects). The main motivation for using VR / AR to achieve objective (a) is cost reduction (e.g. it is cheaper to navigate a virtual environment depicting a physical location such as a theatre, a road, or a market, than to be in the physical location itself), and more scope for flexible interaction (e.g. interacting with a virtual object depicting a car allows more scope for viewing it from different locations and angles). Objective (b) can be better targeted because the available metaphors embrace representations in 3D-space (c.f. visualization of the genome). VR and AR technologies are currently widely used in the exploration of real physical objects (e.g. car, cube, molecule, etc.) or a physical location (e.g. shop, theatre, house, forest, etc..). In the course of exploration the user is immersed in the virtual scene, and can walkthrough or fly through the scene. The user's body and mind integrate with this scene. This frees the intuition, curiosity and intelligence of the user in exploring the state of the scene. In a real context, agents intervene to change the state of current objects/situations (e.g. heat acts as an agent in expanding metallic objects, a dealer acts as an agent in changing bid/ask quotes and so affects the flow of buyers and sellers). Introducing agency into a VR or an AR scene demands abstractions to distinguish user and non-user actions especially when these go beyond simple manipulation of objects by the user hand, or walking through and flying physical locations (Maad et al, 2001).
Uses of AR / VR in finance
This section considers three case studies of the use of 3D, VR and AR in Finance. The first case study was developed by the author at Fraunhofer Institute of Media Communication in Germany. It involves the development of a live edutainment financial content MARILYN (Chakaveh, 2005;Maad, 2003a;Maad, 2003b). MARILYN consists of a 3D virtual human avatar presenting in an entertaining, informative, and interactive way multilingual live financial data (news and tickers) taken from various international stock exchanges. The second case study is a virtual reality application in financial trading previously developed by the author at the University of Warwick in UK in collaboration with Laboratoire de Robotique de Paris (Maad et al, 2001). The third case study describes a novel application drawing expertise from two important fields of study, namely digital image processing and augmented reality. The application is developed within the context of the CYBERII project 1 (Maad et al, 2008). It features a human (the user) immersion in a virtual world of financial indicators for exploration and apprehension. This immersion aims at augmenting the user's perception of the financial market activity and at equipping him/her with concrete information for analysis and decision making. The user immersion in a virtual world of financial indicators serves in reflecting realism through a closer correspondence with real world experience, and in extending the power of computer-based technology to better reflect abstract experience.
A comparison is drawn to highlight an added value in the shift from the use of 3D to Virtual Reality and Augmented Reality in finance.
Use of 3D / VR in finance
This section describes MARILYN Marilyn (Multimodal Avatar Responsive Live Newscaster) a system for interactive television, where a virtual reality three-dimensional facial avatar responds to the remote control in real time, speaking to the viewer and providing the requested information (Chakaveh, 2005;Maad, 2003a;Maad, 2003b). Marilyn informs the viewer with a click of a button on daily financial news. The focus is on the provision of choice as well as personalization of information in an entertaining manner. As well as offering live financial data from leading stock exchanges such as New York, London, Frankfurt and Tokyo, multilingual aspects of the information are also catered for. Traditionally, financial news has been regarded as content-based and rather rigid in format. In contrast, the edutainment aspects of Marilyn can make such a program entertaining as well as informative. MARILYN consists of a 3D virtual human avatar presenting in an entertaining, informative, and interactive way multilingual live financial data (news and tickers) taken from various international stock exchanges. The use of the 3D virtual character aims at reducing the rigidity of delivering purely structured financial content consisting solely of indicators and charts. The voice replies, facial expressions, and hands and body gestures of the virtual human avatar provide a rather entertaining and informative medium to convey financial knowledge to the target audience. Five stages are adopted in the development of this live edutainment financial content (Maad, 2003a;Maad, 2003b): Stage 1 involves the development of a template for an animated virtual human avatar enriched with facial expressions, voice replies, and hand gestures. The template gives the option to choose a desired virtual human character. Stage 2 involves developing several multilingual templates for live financial news content (tickers, and news) taken from various international stock exchanges. Stage 3 involves establishing a bi-directional communication medium between the virtual human avatar and the financial content. Stage 4 involves establishing an interactive communication medium between the animated virtual human avatar and the target audience. Stage 5 involves checking the compliance of the used technology with the prevailing standards for the target media delivery platform. The first prototype of the live edutainment financial news content, depicted in Figure 1 above, was developed for an internet platform and was called MARLYN (Multimodal Avatar Responsive LIve News caster). The prototype for the Internet platform consists of: A facial animation player applet 2 that is MPEG-4 FBA 3 (facial and body animation) compatible: The facial animation player decodes an MPEG-4 facial animation content generated from a given source and apply the results to a face model developed using 3D face modelling tools. The MPEG-4 FBA decoding process is based on integer arithmetic and adopts the interpolation from key position method (the same as the morph target approach widely used in computer animation and the MPEG-4 Facial Animation Table (FAT) approach).
A facial and body animation generator tool, Fbagen 4 , for dynamic generation of speech and lip sync bit stream files:
For generating speech bit stream file, the Fbagen tool uses Microsoft SAPI 5 (Speech Application Programming Interface) as an interface for the SVOX 6 text to speech synthesiser (TTS) that uses SVOX voices. Fbagen also uses a lip sync encoder for generating the lip sync files. The lip sync file is encoded as an MPEG-4 FBA bit stream using high-level viseme parameters for lip movement encoding and viseme blend with linear interpolation for co-articulation. The Fbagen tool takes as input a text string and generates the corresponding FBA streams (.fba files) and speech files (.wav files). The generated FBA streams and speech files constitute the facial animation content that is input to the facial animation player.
JAVA programs for extracting live financial contents (news and tickers) by parsing web financial sites:
The extracted data (put in the form of a String) is passed as a command line argument to the Fbagen tool in order to generate the corresponding FBA streams and speech files.
A Flash 7 interface for menu-like interaction with the 3D virtual character:
The interface provides 2-dimensional navigation of various selection options of the multilingual financial content.
A Flash display of the live financial news content:
This content is basically the one extracted using the JAVA programs. A web page framing the whole work: This web page consists of 4 frames. These frames are for the applet, the flash interface, the flash financial content display, and the application's header respectively. This web page is preceded by an introductory web page motivating the subsequent content. The Internet prototype of the live edutainment content is implemented in a client/server architecture. At the server-side, the financial content is extracted and the MPEG4 FBA and speech files are generated. At the client-side, the play-out out of the facial animation, the interaction, and the display of the live financial content takes place, and are framed in a web page.
The portability of the live edutainment financial content to a mobile Personal Digital Assistant (PDA) platform involves the customisation of the user interaction interface to a mobile device style of interaction, and the in-house development of new versions of the facial animation player and the facial and body animation generator. The new design would ultimately use less system resources and is rather compatible with the uses of high quality voices produced by various text to speech synthesisers (TTS) and speech application programming interfaces (SAPI).
Use of VR in finance
Over recent years it has become increasingly obvious that text-based communication poses significant stumbling blocks to unambiguous communication. Virtual reality showed its benefit for the transfer of information and knowledge through the concept learning from the interaction with immersive 3D environment. Virtual reality and related technologies allows creating virtual environments with key characteristics that represent real world situations. The visualization and interaction with virtual objects is closer to reality than abstract mathematical and 2d representations of complex scenarios. In this respect virtual reality can potentially serve two objectives: (a) reflecting realism through a closer correspondence with real experience, and (b) extending the power of computer-based technology to better reflect "abstract" experience such as interactions concerned with interpretation and manipulation of symbols that does not have obvious embodiment. The main motivation for using VR in financial trading is to enhance the user perception and cognition about the complexity of the financial market mechanisms and manifestations. The added value of using virtual reality in modeling financial market is its advanced visualization technique and metaphoric representation that mimic real world situations. Immersive multisensory interaction that includes vision, spatialized audio and haptic sensation provides the user with the illusion of being in real financial market. The user is confronted with nested scenarios and has to generate solutions by taking into account multicriteria affects. He can develop various strategies in virtual environment to support decision making that reduces the risk of making wrong decision in real financial trading conditions. This approach improves learning, allows generating empirically driven decision and gaining experience from a synthetic world close to reality. Being immersed in a virtual environment the user acquires the feeling that he is interacting with real financial trading market. He interacts with the virtual agents and metaphors representing trading operators, he optimizes his actions using the procedure of trial and error. The user can define strategies, he takes actions, and perceives the consequence of his decisions with high degree of visualization that enhances his awareness about the complexity and unpredictable events that could happen in real financial market. This section describes a virtual reality application in financial trading developed by the author at the University of Warwick in UK in collaboration with Laboratoire de Robotique de Paris (Maad et al, 2001). The virtual reality application, depicted in Figure 2 below, involves a user playing the role of a dealer who sets his/her bid/ask spread to attract buyers and sellers. The presence of the dealer in the rendered virtual reality scene is a simulated presence. As such the virtual reality simulation is fully detached from the real world. The virtual environment is represented to the user through a head mounted display. This peripheral provides the sensation of being immersed in the virtual scene. The actions of the user are communicated to the virtual environment using a 3D interaction tool: a pair of a. The virtual financial environment b. The interaction mode Pinch Gloves. Figure 4 shows a screenshot of the virtual financial environment. The dealer manipulates his bid and ask prices (red and blue cylinders) to attract buyers and sellers (informed and uninformed traders).
Despite the added value of virtual reality approach in financial trading, in terms of the improved user perception of the financial market activities and of the role of the dealer in manipulating the true price of a security, the isolation of the user from the real world and the constrained interaction (the user has to wear a head mounted display and a pair of Pinch Gloves to acquire the feeling of immersion in the virtual world) makes the experience lived by the user (dealer) not sufficiently realistic (Maad et al., 2001).
Use of AR in finance
In this section we overview the CYBERII project (Hasenfratz et al 2004;Hasenfratz et al, 2003) and present the case study of the use of CYBER II technology in Finance (Maad et al, 2008). The CYBERII project (Hasenfratz et al 2004;Hasenfratz et al, 2003) aims at simulating, in real-time, the presence of a person (e.g. a TV presenter or a teacher) in a virtual environment. Novel paradigms of interaction are proposed within the context of this project. These paradigms involve full body interaction with the virtual world in real time. This goes beyond traditional modes of interaction involving the use of mouse, remote control, pinch Gloves, or human sensor equipments. The CYBERII project adopts a five steps technique to insert one or several humans in a virtual interaction space. It uses the notion of "Active Regions" to initiate the interaction with the virtual world. Actions are triggered upon body interaction with "Active Regions". This interaction may take the form of touching the "Active Region" and may be technically sensed using image analysis techniques that identify a body presence in the "Active Region". As described in (Hazenfratz et al., 2004;Hazenfratz et al., 2003), the CYBERII project adopts the following five steps: Multi-camera image acquisition, from different view points, of real moving bodies (see Figure 3 -left image). Reconstruction, modelling, and motion tracking of the acquired bodies and the surrounding environment (see Figure 3 -right image).
Rendering of the reconstructed bodies and their surrounding. The creation of patterns of interaction in the rendered world using "Active Regions" as shown in Figure 2. Examples of "Active Regions" in a virtual interaction space include: the On/Off Button "Active Region" (see Figure 4 -left image) and the moving slider (see Figure 4 -right image). Data management and distributed parallel computing to meet the real time and realistic rendering constraints. The CYBERII project targets various industries including: (1) the TV industry (e.g. virtual sets and online presentations); (2) the game industry (e.g. inserting real persons in the game world); and education and entertainment (e.g. allowing visits and presentation of remote places). The proposed novel paradigm of interaction developed within the scope of the CYBERII project, namely full body interaction with active regions, promises universal accessibility to media content. For instance, physically impaired users may find a greater convenience in full body interaction with a virtual scene instead of interaction with a mouse, a keyboard, or a remote control. The CYBERII augmented reality financial application, depicted in Figure 5 below, involves the insertion of a user (playing the role of a financial dealer) in a virtual world, and the use of the technique of sliders "Active Regions" to enable the body interaction of the user (dealer) to set his/her bid/ask prices for attracting buyers and sellers (Maad et al, 2008). The flow of buyers and sellers is abstractly represented by synthetic objects generated in the augmented scene following the user interaction with active regions. Figure 5 shows how the technique of slider "Active Regions" can help the dealer adjust his quotes to attract buyers and sellers. The flow of buyers and sellers is represented by red balls (for sellers flow) and yellow balls (for buyers flow). This flow is trigerred through body interaction with the sliders "Active Regions". This illustrates how the CYBERII concept of full body interaction with a virtual environment and the technique of slider "Active Regions" could be used for greater engagement and unconstrained immersion in a virtual world of financial indicators. The simulation augments the real world with perceptual knowledge about bid-ask spread variation and about the flow of traders (buyers and sellers) as a result of this variation.
Comparative analysis of the use of AR and VR in finance
MARILYN aims at substituting reality (use of 3D avatar instead of real human to present TV programs). The use of augmented reality instead of virtual reality aims at breaking the separation of the financial application from the real world and at augmenting its realism. Further reality augmentation to the rendered scene can be gained by adding, as a background, a synthetic depiction of the activity of the financial market. This is shown in Figure 6 below. Reality augmentation can be also gained by adding a synthesized speech that tells us about the current actions taken by the dealer (the incrusted human), the impact of the dealer's action on the flow of traders (buyers and sellers), as well as on the whole financial market activity. Many challenges may face the use of the application 3D/AR/VR in finance. These include: The maturity of the technology: Earlier empirical studies conducted in (Maad, 2003a) reveals the limitation of 3D and audio technologies in meeting standards for authoring interactive television content. This is attributed to the lack of realism of the media content authored using the prevalent technologies. For instance in the CYBERII application, we can see that the rendered human(s) lack realism as shown in the figures. This may have an impact on the appeal of the current technology to the financial community. The choice of the medium of delivery: Four potential medium of delivery can be considered, these include: the PC, the Internet, the interactive television (ITV), or other home/office devices. A closer look at the hardware setup of the CYBERII project, described in (Hazenfratz et al., 2004;Hazenfratz et al., 2003) as a networked set of 4 cameras, 4 PC, and one supercomputer, reveals great challenges in the portability and interoperability of the CYBERII technology. Users interest: The widespread penetration of 3D/AR/VR technologies among the financial community depends on the current demand for applications such as the one described above. If this demand ever exists then it would be limited among a few novice community who fancy new technologies. In face of these challenges, more research work, beyond the development of the technology, need to be undertaken to justify the usability and utility of 3D/AR/ VR technologies in meeting various industries' needs.
Financial state visibility case study
The urgent economic problem, linked to the financial crisis, challenges current research and technological development. The scale of the fiscal crisis that undermined the credibility of the financial system motivates the consideration of "Global Financial State Visibility" as a key global challenge that validates research and technological development activities to support the engineering dynamics of automatically adaptable software services along the "global financial supply chain" (Maad et al, 2009). Financial state could be conveyed in various ways: perception the state of financial risk perception of financial events percent of the financial activity perception of the financial system and regulatory framework Our aim is to align the prevalent thinking in terms of mediating the financial state using reports or static models to mediating the financial state using advanced visualisation and interaction technique. Key issues to consider are who will access, manipulate, and govern the financial state. Various entities (policy makers, regulators, auditors, accountants, investors, consumers, suppliers, producers, individuals) need to access /govern / adapt Financial state Visibility depending on service level agreements SLA (see Figure 7 below).
Fig. 7. Actors that need to access /govern / adapt Financial state Visibility
The Financial state visibility challenge has various dimensions: Domain knowledge dimension: this involves building the financial state knowledge base (the development of techniques to store and manipulate the semantic representation of the financial state by various stakeholders including regulators, investors, and central banks worldwide); and the visual rendering (using techniques such AR/VR) of the financial state knowledge base. Converged ICT and media dimension: this involves the development of an interoperability layer at the service infrastructure and interface levels to guarantee instant access to financial state via various converged ICT and media devices. Global factors dimension: financial state knowledge is stored and manipulated in different languages and different structures and formats. This raises geographical cultural, and accessibility technical challenges. Rendering of the financial state needs to adapt to "global context on demand". Governance dimension: There is an urgent need to support the governance of the financial state with greater perception and manipulation capability tools. Various financial state visibility services, depicted in Figure 8 below, could be developed taking into account the above considered dimensions. These services can be grouped into various levels and categories: Mediate Integrative services to various users at various levels according to Service Governance Level Agreement Mediator Service Exchange Adaptation Service Mediators The design and engineering of these state visibility services involves: composition, workflow, adaptation at design time and run time consideration, agency interaction, verification and validation. Key issue is the delivery of AR / VR along the chain of services workflow. Users of the services are conceived at two levels: level 1 end users are developers of internet of things; and level 2 end users are end users of internet of things Users at both level govern access, discovery, and manipulation of state visibility services according to Governance Service Level Agreements GSLA.
Use of AR / VR for greater financial state visibility
This section proposes a blend of approaches to mediate financial state visibility as a service using VR / AR technologies. The 3 applications overviewed in section 3 above highlight a limitation of existing 3D/VR / AR technologies in wide industry penetration. This is mainly attributed to portability and utility of the technology. We suggest below service oriented software engineering and Empirical Modelling as a blend of approaches for greater portability and usability of 3D / VR/ AR technologies
Service software engineering
Given the need of massive computational power and distributed access by various actors of the financial state, 3D / VR / AR technologies can largely benefit from service oriented computing to mediate, using high end visualization, the financial state and risk to various actors. According to (Di Nitto et al, 2009), service-oriented computing (SOC) represents one of the most challenging promises to support the development of adaptive distributed systems. Applications can open themselves to the use of services offered by third parties that can be accessed through standard, well defined interfaces. The binding between applications and the corresponding services can be extremely loose in this context, thus making it possible to compose new services on the fly whenever a new need arises. Around this idea a number of initiatives and standards have grown up, some of them focusing on how such roles need to interact with each other, and some others on how to engineer systems based on such a model and on how to provide foundational support to their development. In particular, socalled service-oriented applications and architectures (SOAs) have captured the interest of industry, which is trying to exploit them as the reference architecture to support Business to Business B2B interaction. According to Forrester Research, the SOA service and market had grown by $U.S. 4.9 billion in 2005, and it is forecasted to have an interesting growth rate until 2010, with a compound annual growth rate of 88 percent between 2004 and 2009. The SOC road map, figure 9 below extracted from (Di Nitto et al, 2009), separates basic service capabilities provided by a services middleware infrastructure and conventional SOA from more advanced service functionalities that are needed for dynamically composing (integrating) services. The SOC road map highlights the importance of service modeling and service-oriented engineering (i.e., service-oriented analysis, design, and development techniques and methodologies that are crucial elements for the development of meaningful services and business process specifications).
Empirical modelling
The challenges faced by the use of 3D/ VR / AR for constructing virtual environments for finance are best revealed by drawing a comparison with its use in computer-aided assembly (Garbaya et al, 2000). This comparison reveals a difference in the objective, considerations, approaches, and user role in constructing visually rich virtual environment for different contexts. The main objective in using 3D/ VR / AR in finance is enhanced cognition of state; in the case of virtual assembly the main objective is to minimise the need for building physical prototypes. The issues to be considered in applying 3D/ VR / AR for financial state visibility and in virtual assembly differ in nature and importance. In virtual assembly, the major concerns are proper 3D picture capturing, conversion, and adding behaviour to objects; in 3D/ VR / AR for financial state visibility, they are geometric abstraction of financial concepts, integration with financial database, and distributed interaction. The steps followed to create a virtual scene for virtual assembly and for financial state are different. A linear, preconceived, set of processes can be followed to develop a scene for virtual assembly. These can be framed in three stages: defining objects to be assembled, preparing the assembly geometry for visualisation, and adding behaviour to visualised objects. Creating a virtual scene depicting financial state is more complicated and cannot be framed adequately in a pre-conceived way. However, a broad outline can be traced to guide the construction process. This involves: identifying entities (both those that admit geometric abstraction and those that have already a well recognised geometric representation) to be included in the virtual scene; choosing an appropriate geometric representation for these entities; adding a situated behaviour and visualisation to entities; identifying the external resources (such as databases, files, data feeds, etc.) to be interfaced to the virtual scene; and framing the role of the user intervention in the simulation. Where human intervention is concerned, the user's role in the 3D/VR/AR scene is more open-ended in a financial context than in an assembly context. In a 3D/ VR / AR scene for assembly the immersion of the user is very important: the user can manipulate virtual objects with his hands. The user's hands, guided by the user's brain, interact directly with virtual objects. This makes virtual reality approach appropriate for modelling the process of assembly task planning (see Figure 10). a. Virtual assembly environment b. Assembly planning and operation with haptic interface Fig. 10. Example of VR modelling for areas such as assembly planning Construing financial phenomena is a function performed by the human brain. The mental model of the designer can be abstracted in a static diagram, a 2D computer artefact, or a VR / AR scene. Geometric objects in the virtual scene might admit no counterpart in the real world -they are purely geometric metaphors. This makes a virtual scene just one of several possible representations. It also motivates a prior situated analysis exploring possible construals pertaining to the social context. The above comparison highlights the need to support 3D/ VR / AR technologies with principles and techniques to analyse and construe social contexts and to adopt appropriate visualisations for abstract entities (such as financial indicators) that have no real geometric counterpart. Current technologies for Empirical Modelling (Beynon, 1999) can help in construing financial situations and in representing state and the analysis of agency in state change, whilst 3D / VR / AR technologies offer enhanced visualisation and scope for user immersion and experience of state.
Conclusion
This chapter reflected on the potential of Virtual Reality and Augmented Reality (VR / AR) technologies in supporting the dynamics of global financial systems and in addressing the grand challenges posed by unexpected events and crisis. The chapter briefly overviewed traditional VR/AR uses and described three early attempts to use 3D/ VR / AR technologies in Finance. In light of the recent financial crisis, there is a potential added valued in harnessing the use of VR/AR technologies to convey a greater visibility of the financial state (including visibility of financial risk). Various dimensions of the problem are considered. The chapter suggested a blend of service oriented computing SOC and empirical modelling technologies to support the use of VR / AR technologies in raising the perception of financial state and risk.
There is a very significant distinction between VR modelling for areas such as assembly planning as represented in papers such as (Garbaya et al, 2000), and its application for greater visibility of the financial state and risk. Whilst we can reasonably speak of "using VR to model the reality of the process of assembly planning", the reality of the financial state is an altogether more elusive concept. Where manufacturing assembly deals with objects and actions whose objectivity and real-world authenticity is uncontroversial, financial state visibility is a prime example of an activity in which the impact of technology upon human cognition is prominent, and character of its agencies and observables is accordingly hard to capture in objective terms. Empirical Modelling supplies an appropriate framework within which to address the ontological issues raised by such applications of VR / AR (Beynon, 1999). This chapter points to the following conclusions: 3D/ VR/ AR technologies can help in exploring a particular state in a social context. The pre-construction phase of the virtual world can benefit greatly from concepts drawn from the Empirical Modelling literature such as modelling state, state change, and the initiators of state change. 3D/ VR / AR technologies needs to be better adapted for the representation of multiple agents acting to change the state and corresponding visualisation. The successful application of 3D / VR /AR technologies in modelling social and data intensive environment relies upon integrating these technologies with other programming paradigms and architectures such as service oriented computing and architecture (SOC and SOA). Future research agenda involves the development of quantitative and qualitative metrics to assess the potential benefits of 3D / VR / AR in modelling a state in a social context. Two dimensions are appropriate: the visibility and the viscosity. | 2017-05-02T05:24:55.172Z | 2010-01-01T00:00:00.000 | {
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16090759 | pes2o/s2orc | v3-fos-license | Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study
Introduction We compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions. Methods Altogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases. Results CLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue. Conclusions The significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.
Introduction
In epithelial cells, tight junctions (TJs) are the most apical intercellular junctions, and function as selective barriers to macromolecules and prevent diffusion of lipids and proteins between apical and basolateral membrane domains. TJs appear as a network of continuous and anastomosing filaments on the protoplasmic face of the plasma membrane [1]. The molecular architecture of TJ strands has been described in recent years. The proteins involved in the formation of TJs have been divided into two categories: integral membrane proteins, such as claudins (CLDNs), occludins and JAM (junctional adhesion molecule) [2]; and peripheral membrane proteins, such as zonulae occludens (ZO)-1, ZO-2 and ZO-3, cingulin, symplekin, pilt, MAGI-1 (MAGUK [membrane associated guanylate kinase] inverted protein 1) and AF-6 (afadin) [3].
CLDNs, a family comprising 24 members, are the main family of proteins that make up the TJs [2,4]. The tightness of individual TJ strands in situ is determined by the various combinations of CLDN family members. CLDNs have a membrane topology similar to that of occludin, even though they are smaller proteins of about 22 kDa. Their protein structure, which is uniform among family members, contains four transmembrane domains with amino-and carboxyl-termini in the cytoplasm and two extracellular loops [5].
Until now only few data have been reported regarding the role played by CLDNs in breast lesions, and findings on their function remain controversial [6,7]. There is 38% homology between human CLDN1 and CLDN2 at the amino acid sequence level [8,9]. Expression of the fourexon CLDN1 gene, formerly named senescence associated epithelial membrane protein, was identified in testis [10], colorectal carcinoma [11] and lung [12]. The direct involvement of CLDN1 in the barrier function of TJs has been demonstrated in CLDN1-over-expressing MDCK cells [13].
In a study conducted in breast carcinomas by Kramer and coworkers in 2000 [7] it was shown that there are no genetic alterations in the promoter or coding sequences in the CLDN1 gene that could account for the loss of CLDN1 protein expression in tumour cells. Of the relatively few publications concerning CLDN expression in the breast, one study [14] found that CLDN7 protein and mRNA are downregulated in primary breast cancers as compared with normal breast epithelium. Taken together, these observations indicate that there is a need to study the possible role played by CLDNs in carcinogenesis and tumour differentiation.
CLDN3 and CLDN4 can function as receptors for Clostridium perfringens enterotoxin [15]. The one-exon genes of CLDN3 and CLDN4 are closely linked (CLDN3 7q11 and CLDN4 7q11.23) and share extended homology of approximately 80% at the DNA level. SAGE (serial analysis of gene expression) demonstrated that, of differentially upregulated mRNAs, CLDN3 and CLDN4 were among the six most upregulated in primary ovarian carcinoma cells [16]. Van Itallie and coworkers [17] demonstrated the close correlation between the level of CLDN4 expression and change in both conductance and ionic selectivity.
Thus far there is no evidence on how CLDN3 and CLDN4 are expressed in breast carcinomas. The present study was designed to determine whether human CLDN1, CLDN3 and CLDN4 are expressed in different types of breast lesions, what differences there are between different CLDNs in breast, and how the protein expression correlates with CLDN mRNA expression.
Materials
For immunohistochemistry, paraffin-embedded sections were used. For immunofluorescence, paraffin-embedded sections and frozen resected breast tissue samples were analyzed. For real-time PCR RNA isolated from frozen surgical breast tissues were analyzed.
Materials used for immunohistochemistry
Altogether, 56 paraffin-embedded sections from 52 surgically resected breast specimens were analyzed for expression of CLDN1, CLDN3 and CLDN4 using immunohistochemistry. Tissue samples were collected with the patient's written consent and conforming with national law (23/2002.V.9 EüM) regarding human studies. The age of the 52 female patients ranged between 31 and 80 years. Carcinomas were graded according to the Elston-Bloom and Richardson system [18].
Materials used for immunofluorescene
Immunofluorescence detection of CLDN1, CLDN3 and CLDN4 was performed on frozen sections (IDC and surrounding breast) as well as on tissue microarray of IDCs obtained from paraffin-embedded blocks.
Materials used for CLDN1, CLDN3, CLDN4 and GAPDH mRNA expression with real-time PCR In 17 out of the 52 cases analyzed by immunohistochemistry, the level of mRNA expression of CLDN1, CLDN3 and CLDN4 were also analyzed using real-time PCR. The histopathological diagnoses of these 17 breast lesions are as follows: fibrocystic changes/breast lesions (n = 3), IDC ± DCIS (n = 12), ILC (n = 1) and intraductal papilloma (n = 1). In the following text only the real-time PCR results for the 13 invasive carcinomas (12 IDC ± DCIS and 1 ILC) are discussed in detail.
Because relative quantification of the real-time PCR results was used for data analyses in all 17 breast samples, the normal component of the breast tissue was simultaneously investigated for mRNA expression of CLDN1, CLDN3, CLDN4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. GAPDH was used as the reference gene, and so data are normalized to GAPDH.
Breast tissue samples, obtained in accordance with current local and ethical recommendations, were snap frozen in liquid nitrogen within 30 min of surgical removal and stored at -80°C.
Immunohistochemistry for CLDN1, CLDN3 and CLDN4 detection
Immunohistochemical detection of CLDNs was performed in formalin-fixed, paraffin-embedded sections. Sections (5 µm thick) were cut from each paraffin block, deparaffinized and rehydrated. Immunohistochemical staining for CLDN1, CLDN3 and CLDN4 was performed using the streptavidinperoxidase procedure.
For CLDN1 and CLDN3 proteins, antigen retrieval was performed with two different antigen retrieval solutions in parallel sections: 20 min in Vector (Burlingame, CA, USA) antigen retrieval solution in a microwave oven; and 40 min in DAKO (Carpinteria, CA, USA) antigen retrieval solution in a microwave oven. For CLDN4, antigen retrieval was performed for 20 min in Vector antigen retrieval solution in a microwave oven. Endogenous peroxidase was blocked with 1% H 2 O 2 for 10 min at room temperature.
Antigen-bound primary antibody was detected using standard avidin-biotin immunoperoxidase complex (DAKO LSAB2 Kit). The chromogen substrate was aminoethylcarbazol. In each case, for counter-staining Mayer's haemalaun was used. For each CLDN a negative control with omission of the primary antibody was included. Colon carcinoma was used as positive control. In each case, two independent observers (A-MT and JK) recorded the distribution of staining, intensity and localization. Staining of CLDNs was expressed relative to adjacent normal mammary epithelium.
Immunofluorescence microscopy for CLDN1, CLDN3 and CLDN4 detection
Sections (10 µm thick) were cut from frozen tissue and from breast tissue array paraffin blocks. The tissue arrays were constructed in our laboratory. The selected breast tumour samples were obtained from paraffin blocks from our files. In brief, the study involved breast tumours from 30 T1N0 and 30 T1N1 cases. Histopathological data included tumour stage according to the UICC TNM system. With the guidance of an experienced pathologist (JK), representative core tissue biopsies (2 mm in diameter) were taken from archival paraffin-embedded primary breast carcinomas and seeded in new paraffin blocks using an instrument provided by Histopathology Company (Pécs, Hungary). Two biopsies were taken from each paraffin block. For paraffin-embedded sections antigen retrieval was performed as described above (under Immunohistochemistry for CLDN1, CLDN3 and CLDN4 detection).
Sections were incubated with goat antiserum for 1 hour at room temperature and then incubated with the primary antibodies (Zymed) to CLDN1, CLDN3 and CLDN4 (1:50) overnight. CLDN1 and CLDN3 antigen-bound primary antibody was detected using anti-rabbit IgG conjugated to Alexafluor 488 (Molecular Probes, Eugene, OR, USA) and CLDN4 stained sections was detected using anti-mouse IgG conjugated to Alexafluor 488 (Molecular Probes) for 30 min at room temperature. Cell nuclei were counterstained with propidium iodide (Molecular Probes) for 10 min at room temperature. For each CLDN a negative control with omission of the primary antibody was included. Coverslips were mounted with fluorescent mounting medium (DAKO). Stained specimens were analyzed by laser scanning confocal microscopy (MRC 1024; Bio-Rad, Hercules, CA, USA).
Western blotting
The monospecificity of CLDN1, CLDN3 and CLDN4 antibodies was tested by our group in whole tumour cell lysate by Western blot analyses, as described previously [14]. In brief, from snap frozen tissues of IDC of the breast and normal tissue surrounding the tumour, total protein was extracted using lysis buffer (100 mmol/l NaCl, 1% NP-40, 2 mmol/l EDTA, 50 mmol/l TRIS, 20 µg/ml aprotinin, 20 µg/ ml leupeptin, 1 mmol/l PMSF, pH 7.5). Subsequently, the samples were mixed with 2× Laemmli sample buffer and boiled for 10 min, followed by centrifugation at 13,000 rpm for 15 min. Similar amounts of protein were loaded in each lane and run on 10% SDS-PAGE under reducing conditions. Then, gel electrophoresis proteins were transferred to nitrocellulose membranes. For immunodetection, blots were incubated with CLDN1 polyclonal antibody (1:400), CLDN3 polyclonal antibody (1:800) and CLDN4 monoclonal antibody (1:200) overnight. The biotinylated secondary antibody, goat anti-mouse IgG (diluted 1:2000, E433; DAKO), was applied for 1 hour at room temperature. Antibody detection was conducted using an enhanced chemiluminescent reaction system.
RNA isolation
Breast tissue (50-100 mg) was homogenized and RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA) [19], in accordance with the manufacturer's instructions, using a Polytron homogenizer (Kinematica AG, Littau/ Lucerne, Switzerland). Briefly, the RNA was precipitated with 0.5 ml isopropyl alcohol in the aqueous phase. The RNA pellet was washed once in 70% ethanol, dried and resuspended in 50 µl of RNAse-free water and kept at -80°C until use. Total RNA integrity was verified by electrophoresis using ethidium bromide staining as well as by measuring optical density (OD). OD 260 nm/OD 280 nm absorption ratio was accepted between 1.70 and 2.8.
Reverse transcription of RNA
Total RNA (700 ng) was reverse transcribed for 40 min at 48°C in 30 µl with 2.5 unit M-MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA) in the presence of RNAse inhibitor (Applied Biosystems) using Random Hexamers (Applied Biosystems).
Real-time PCR
Conditions for real-time PCRs were optimized in a gradient cycler (Mastercycler Gradient, Eppendorf, Hamburg, Germany). Optimized conditions were transferred to the Bio-Rad real time PCR detection system as follows. Typical real-time PCR reaction was conducted with 2 µl cDNA template in a total volume of 25 µl, using the Bio-Rad iCycler Real Time PCR detection system (BioRad 1708740). For CLDNs and GAPDH the SYBR Green based real-time PCR method was used. Each PCR was conducted in 25 µl volume of 1 × PCR puffer (BioRad 1708882) with 300 nmol/l of each primer (Table 1) for 2 min at 95°C for initial denaturing, then 40 cycles of 95°C for 20 s, 63°C for 30 s, and 72°C for 30 s, followed by melting analyses from 55 to 95°C. Melting curve analyses resulted in single product specific melting temperatures for the analyzed CLDN1, CLDN3, CLDN4 and GAPDH genes. No primer-dimer formations were observed during the 40 real-time PCR amplification cycles. PCR reaction for each sample was done in duplicate in 96-well plates. In addition, the resulting realtime PCR product (10 µl) was loaded onto 2% agarose gel prepared in Tris-borate EDTA puffer and stained with ethidium bromide (Bio-Rad). Each PCR was optimized to ensure that no bands corresponding to genomic DNA amplification or primer-dimer pairs were present. The relative quantification method (described in detail by Pfaffl and coworkers [20] from the Technical University of Munich) was used for data analysis of real-time PCR. The program included a statistical evaluation. Data were analyzed for significant differences by analysis of variance using approxi-mate tests [20]. The relative expression is based on the expression ratio of a target gene versus a reference gene (GAPDH).
Electron microscopy
In two cases both the normal breast tissue and the tumour sample were investigated by transmission electron microscopy in order to analyze the presence of TJs in tumours compared with nontumourous tissue, as described previously [21].
Results
Immunohistochemical localization of CLDNs indicated that CLDNs were expressed along the entire length of the lateral plasma membranes between epithelial cells, including apical areas containing TJ structures. Using electron microscopy, TJ structures were identified in normal breast as well as in breast tumour tissue. Cell types in breast tissue are heterogeneous, and it is therefore noteworthy that CLDNs were expressed only in epithelial cells. Surrounding fibroblasts and adipocytes served as negative controls because these cells were negative for CLDN antibodies.
Immunohistochemistry of CLDN1
The two antigen retrieval methods used yielded similar results. The monospecificity of the CLDN antibodies was tested by Western blot analyses, and a single band was found at approximately 20 kDa for CLDN1, CLDN3 and CLDN4 (Fig. 1).
Immunohistochemical localization of CLDN1 indicated that CLDN1 was present in the membranes of normal duct cells in the majority of cases (Fig. 2a). CLDN1 positivity was also observed in some cell membranes in pure DCIS cases or in the DCIS component of invasive carcinomas. On the other hand, major differences in CLDN1 expression were observed among tumours. Two major differences were noticed. First, in 36 of the 39 carcinomas examined, invasive tumour cells were either negative for CLDN1 or it was present in a scattered distri- Table 1 The CLDN1, CLDN3, CLDN4 and GAPDH primer sequences, position and product size bution among such tumour cells (Fig. 2b). These findings were confirmed by confocal microscopy images (Fig. 2c). There was no difference immunohistochemically in CLDN1 expression between different types of invasive carcinomas or between ductal carcinomas of different grade. The second major difference from normal tissue was that CLDN1 was frequently present (eight out of nine samples) in areas of apocrine metaplasia (Fig. 2d). In these cases positivity was observed only in cells of apocrine metaplasia, and the neighbouring tumour tissue was negative for CLDN1.
Immunohistochemistry of CLDN3 and CLDN4
CLDN3 positivity was observed in the majority (49/56) of cases examined (12 fibrocystic breasts, 5 DCIS, 23 IDC, 4 ILC, 2 mucinous, 2 tubular and 1 papillary breast carcinomas) and in all cases analyzed by confocal microscopy. There were no remarkable differences in CLDN3 expression in different types of breast lesions or between normal and tumour components (Fig. 3a,b). CLDN3 expression is evaluable by standard immunohistochemistry and by comparing two antigen retrieval methods; in the majority of cases CLDN3, similar to CLDN1, was observed along the entire length of the epithelial cell membrane, but in a few cases, especially in normal tissues and in tissue with fibro-cystic changes, CLDN3 appeared to be localized close to the apical end of the cells or at the basolateral membrane of the epithelial cells. These findings were seen on both confocal and light microscopy (Fig. 3c,d).
CLDN4 positivity was present in all 56 tissue sections in epithelial cell membranes as well as in the frozen section and breast tumour array analyzed by confocal microscopy.
Intense positivity was observed in normal epithelial cells (Fig. 4a) and in tumours of grade 2 and grade 3 (Fig. 4b), and was greatly reduced in the majority of tumours of grade 1.
CLDN4 was absent or its expression was greatly reduced in tumour cells of 17 out of 21 grade 1 tumours (Fig. 4c), but it was present in the normal epithelial components within the same blocks (14 IDC NST [no special type], 5 special types, 2 ILC). Positivity was seen in 8 out of 11 grade 2 tumours and in 5 out of 7 grade 3 invasive carcinomas. In the normal tissue surrounding grade 2 and 3 tumours, the CLDNs were consistently present. CLDN4 was absent or its expression was decreased in the seven special-type breast carcinomas (mucinous, tubular, papillary) but it was present in the normal tissue surrounding these tumours. In contrast to CLDN1, CLDN4 was consistently absent in regions of apocrine metaplasia (Fig. 4d).
Real time-PCR results
Real-time PCR using CLDN1, CLDN3, CLDN4 and GAPDH specific primers was performed in the 17 samples detailed above. mRNA was expressed in all of the analyzed cases. Relative quantification using GAPDH as the reference gene was applied [20]. mRNA expression in 13 invasive tumours out of the 17 samples was analyzed as the sample group, and the surrounding breast tissue was used as the control group. During the real-time PCR reaction, CLDN1 mRNA was detected in 12 out of 13 analyzed tumours and in 12 out of 13 corresponding surrounding nontumourous breast tissue samples. In normal, surrounding breast tissue, the mean value of crossing points (cycle threshold) was 34,02 whereas in the tumour tissue samples it was 36,22 (indicating low levels of CLDN1 mRNA). CLDN1 was downregulated by 12-fold in the sample (tumour) group in comparison with the control group using GAPDH as the reference gene. CLDN3 mRNA was present in all tumours and surrounding tissues. CLDN3 exhibited no expressional difference between invasive tumours and nontumourous tissue samples.
CLDN4 mRNA was also found to be present in these 13 invasive tumours and surrounding mammary tissues using real time-PCR. According to relative quantification, CLDN4 mRNA expression did not appear to be changed in the invasive tumours in comparison with the surrounding normal tissue. It is important to emphasize that there were only two The monospecificity of claudin (CLDN)4 antibody using Western blot analysis The monospecificity of claudin (CLDN)4 antibody using Western blot analysis. CLDN4 protein expression in invasive breast carcinoma, surrounding normal breast and human liver. Western blot analysis was performed on equal amounts of protein from total cell lysates using CLDN4 monoclonal antibody. A single band was shown at approximately 22 kDa for CLDN4. IDC, invasive ductal carcinoma. grade 1 invasive tumour samples among the 13 investigated invasive tumours.
The program used by us and described by Pfaffl and coworkers [20] (Relative Expression Software Tool [REST] for group-wise comparison and statistical analysis of relative expression results in real-time PCR) includes a statistical analysis (analysis of variance). Using this program, we found no statistically significant differences in the distribution of data between the sample and control groups in terms of CLDN expression (the P values for CLDN1, CLDN3 and CLDN 4 were 0.166, 0.928 and 0.996, respectively). However, only 13 tumours were analyzed for RNA expression using real-time PCR because fresh tumour tissue and corresponding normal tissue surrounding the tumour were available in only 13 cases. In these cases the results of immunohistochemistry and real-time PCR appeared to correlate; specifically, CLDN1 appeared to be downregulated in tumours as compared with normal breast, whereas CLDN3 was expressed at similar levels in tumours and normal breast.
In the case of CLDN4 expression the differences observed by immunohistochemistry between tumours of different grade were not observed by real-time PCR. CLDN4 mRNA expression did not appear to be changed in the invasive tumours in comparison with the surrounding normal tissue. Real-time PCR remains to be performed in a greater number of cases, including tumours of different grade.
Apart from melting analysis, the specificity of real-time PCR results was confirmed on 2% agarose gel electrophoresis. The PCR product was seen at the appropriate size and nonspecific bands were not visualized. The specificity of real-time PCR of CLDN4 was also analyzed by sequencing (data not shown).
Discussion
In the present study we examined the expression in breast lesions of three members of the CLDN family, namely CLDN1, CLDN3 and CLDN4. The majority of studies published to date have described roles for CLDNs in forming TJs [22], conferring ionic selectivity [17] and functioning as a barrier [23], but only few data have been published on the expression of CLDNs in the breast [14]. CLDN1 and CLDN2 were the first described CLDN components of TJs, and so the majority of data are related to these proteins. It was demonstrated that CLDN1 and CLDN2 function as major structural components of TJ strands, and that occludin is an accessory protein within this structure [8,24].
Well developed TJs found in the lung contain CLDN3, CLDN4 and CLDN5, which are abundant, whereas levels of expression of CLDN1 and CLDN2 are reduced [12]. These findings suggest that different members of the CLDN family are involved in the formation of TJ strands in different tissues. The extent to which CLDN expression is tissue dependent remains a subject of research interest, as does the physiological relevance of the existence of multiple CLDN species. In CLDN1-, CLDN2-and CLDN3-transfected mouse L-cells, Kubota and coworkers [25] showed that CLDNs may act as calcium-independent adhesion molecules, although the adhesion strength is low. It was hypothesized by Rangel and coworkers [26] that the precise ratio of different CLDNs determined the permeability of TJs. In ovarian tumours, the same group suggested that CLDN3 and CLDN4 are required for signalling through survival or proliferative pathways.
In the present study, immunohistochemistry revealed expression of CLDNs in TJs along the lateral plasma membranes of epithelial cells and along the basal plasma membrane of the epithelium, suggesting that CLDNs are not exclusively localized to TJs. Localization of these three CLDNs together in breast tissue sections has not previously been reported, but based on the observations presented here CLDN1, CLDN3 and CLDN4 are not exclusively localized to areas of TJs. Gregory and coworkers [27] described similar observations for CLDN1 in the rat epididymis.
We demonstrated that immunohistochemically detectable CLDN1 protein is absent or its expression is markedly decreased in the majority of different types of invasive breast carcinomas as compared with normal ducts. Of 39 malignant tumours examined by immunohistochemistry, 36 were negative for CLDN1 or it was present in a scattered distribution among the tumour cells. CLDN1 mRNA expression investigated by real-time PCR confirmed this finding. The mRNA level in tumour is considerably lower than that in normal breast tissue. Theoretically, several regulatory pathways could influence CLDN1 protein synthesis and expression. How these regulatory pathways are involved in cellular transformation and oncogenic progression in the breast remains to be elucidated. Previous studies reported that CLDN1 mRNA expression is absent or downregulated in the majority of breast cancer cell lines [28]. Kramer and coworkers [7] found no evidence for alterations in the CLDN1 gene being responsible for the loss of expression of CLDN1 protein. The same group found a similar loss of CLDN1 expression in five primary breast tumours. This finding is in contrast to the broad spectrum of CLDN1 expression found in other human tissues, such as liver and pancreas (our unpublished data). This may suggest that CLDN1 might be involved in the development of breast cancer or other epithelial tumours [7]. Whether TJs are simply lost in breast neoplasia and whether downregulation of CLDN1 is among the causative factors in this process are unknown. Alterations in the number, appearance and permeability of TJs have been described in various tumour types [21,29]. Abnormalities in TJ permeability and number greatly influence the transepithelial flux of growth factors [30] and hence the development of epithelial tumours [31]. In two cases, both normal breast tissue and tumour sample were investigated by transmission electron microscopy. Membrane densities corresponding to TJ structures were identified in both normal and neoplastic epithelial cells. The eventual abnormalities of TJs in breast tumours must be analyzed in a large series of breast tumours. Although the similar CLDN3 and CLDN4 TJ proteins are highly expressed in breast neoplasia, we hypothesize that mechanisms other than alteration in TJs are involved in the loss of CLDN1 expression in breast tumours. The role played by CLDN1 positivity in apocrine cells observed in this study requires further investigation. In the present study, CLDN3 appeared to be expressed in a similar manner in primary breast cancers as in normal epithelium. Immunohistochemistry and real-time PCR yielded similar findings without notable upregulation or downregulation in different groups of tumours or compared with normal epithelium. In the majority of cases, CLDN3 was found to be localized along the entire membrane of epithelial cells, but in some cases it was only detected close to the apical end or at the basolateral membrane of the epithelial cells.
Although this observation was confirmed by both light and confocal microscopy, in the breast this staining pattern has not previously been reported. Interestingly, Rangel and coworkers [26] did not find CLDN3 exclusively at the membrane in any of the samples studied; the majority of samples of primary ovarian tissue exhibited a combination of cytoplasmic and membrane staining. In gut, Rahner and coworkers [32] found CLDN3 to be strongly expressed in the surface epithelial cells of the stomach fundus, and have found it to be distributed predominantly along the basolateral membrane and not at the TJ. That group concluded that CLDNs have different patterns of expression in different gastrointestinal tissues -patterns that should account for differences in paracellular permeability.
We found that CLDN4 protein expression was downregulated in grade 1 IDCs. Further sample collection and analysis are required to confirm this result at the mRNA level, because only two grade 1 IDCs were available for PCR examination in our series. However, mRNA and protein were expressed in grade 2 and 3 IDCs to degrees similar to expression in surrounding breast tissue. In a study conducted in ovarian tissue, Hough and coworkers [16] found that expression of CLDN3 and CLDN4 protein was undetectable in normal ovarian cells and highly expressed on the membranes of ovarian carcinoma cells. Contrary to the findings in ovarian tumours reported by Rangel and coworkers [26] that CLDN3 and CLDN4 exhibited cytoplasmic staining and that over-expression of these proteins were associated with malignancy, in our study in breast we did not observe clear positivity of CLDNs in cytoplasm. A close relation between CLDN4 expression and changes in both conductance and ionic selectivity has been described. Further important observations were reported by van Itallie and coworkers [17]; they found that the number of junction strands was increased by over-expression of CLDN4, and that the levels of several other strand proteins were unaffected. Loss of CLDN4 from the cell surface coincides with dramatic changes in the morphology of TJ fibrils. Removal of CLDN4 disrupts fibril organization and increases junc-tion permeability [15]. A recent study [33] identified CLDN4 as a potent inhibitor of the invasiveness and metastatic phenotype of pancreatic carcinoma cells by playing role in the transforming growth factor-β and Ras/Raf signal regulated kinase pathway. No studies to date have reported decreased expression of CLDN4 in grade 1 breast carcinomas, in special types of breast tumour, or in apocrine metaplasia compared with normal epithelium, as were observed in our study. The mRNA expression of different CLDNs in a large number of special-type breast carcinomas and tumours of different grades requires investigation so that the decrease in CLDN4 expression in special-type breast carcinomas and IDCs of grade 1 can be unequivocally confirmed.
Conclusion
Our observations suggest that, in breast tissue, CLDN3 expression is similar in tumours and surrounding normal tissue, as demonstrated by immunohistochemistry and realtime PCR. In contrast, absent or decreased expression of CLDN1 in invasive breast carcinomas was demonstrated at both mRNA and protein levels. These data suggest that CLDN1 is involved in invasion and metastasis, and in mammary carcinogenesis. Furthermore, loss of or decrease in CLDN4 expression in grade 1 IDCs and the absence of CLDN4 in special-type breast carcinomas and in areas of apocrine metaplasia in benign breast tissue, as compared with normal epithelium, suggest a role for CLDN4 in cellular differentiation. The implications of this unique feature require further investigation. The important issue is whether loss of CLDN1 and CLDN4 plays a significant role in cellcell adhesion and tumour differentiation. | 2014-10-01T00:00:00.000Z | 2005-01-31T00:00:00.000 | {
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262081391 | pes2o/s2orc | v3-fos-license | Fault Diagnosis Method for High Voltage Power Metering System Based on the PCA-ELM Algorithm
The current fault diagnosis of high-voltage power metering systems lacks the processing of information dimensions, which leads to the low correctness of fault diagnosis results. To this end, a fault diagnosis method based on the PCA-ELM algorithm for high-voltage power metering systems is proposed. Eliminate data information noise. Extract fault information features. Based on PCA to reduce the information dimension, the ELM algorithm is used to analyze the fault information characteristics. The fault information characteristics correspond to the fault types to complete the fault diagnosis of the high-voltage power metering system. Comparative experiments are designed for verification. The measurement results show that the accuracy rate of fault diagnosis for this method is 99.89%, and the average accuracy rate of fault type diagnosis is 98.87%, which substantially improves the correct rate of diagnosis results.
Introduction
A high-voltage power metering system is a very important part of basic power facilities.The main working equipment of high-voltage power metering systems are voltage transformers, voltage transformers, and energy meters.In the process of operation of these devices, if a failure occurs, it will have a more serious impact on the whole system.In high-voltage power metering systems, load currents are transmitted, and as demand changes, so does the size of the load, which leads to faults such as short circuits in high-voltage power metering systems, which need to be diagnosed promptly and accurately [1].PCA can process a large amount of data information and reduce the dimensionality of the information feature space.PCA reduces the loss of the information contained in the original indicators and enables a comprehensive analysis of the collected data, reducing the workload of data analysis and increasing the accuracy of data analysis.ELM is a feedforward neural network, which reduces the training error through the setting of weights and the formulation of hidden layers.ELM has been widely used in many fields [2].Combining PCA with ELM to form an extreme learning machine neural network based on principal component analysis can reduce the workload of data analysis by reducing unnecessary analysis indexes in the case of a large amount of data information, while ELM is used to analyze the extracted data features and complete the analysis of data information features in the process of multiple training iterations.In the high-voltage power metering system, the algorithm of PCA-ELM can accurately analyze the large amount of data information generated in the operation of the system, extract the fault characteristics in a large amount of data information, and complete a higher quality fault diagnosis of the high-voltage power metering system [3].
Eliminate Data Information Noise
High-voltage power metering systems generate a lot of data information during operation.This data information is not directly usable because of the presence of a lot of interfering information, i.e., noise.Noise is a useless signal generated by an independent random process that has nothing to do with the content of the information.The noise will be eliminated and the real data information can be obtained.We design an active filter circuit in a high-voltage power system.The active filter circuit can suppress data information outside the specified frequency range by setting the frequency at which the signal is allowed to pass [4].We set the cutoff frequency f and determine the proportionality between the damping R and the cutoff frequency as shown in the following table : Table 1 Proportional relationship between cutoff frequency and damping F(Hz) R (μF) 1-10 20-1 10-100 1-0.1 100-1000 0.1-0.01 The active filter circuit is designed according to the proportional relationship required in the noise elimination process.The active filter circuit designed in this paper is shown in the following figure: In Equation (1), b0, b1, and b2 are the frequencies set to allow passage, λ is the damping factor in this circuit, ω0 is the intrinsic frequency, and P is the frequency of the data information in the highvoltage power metering system.In this function expression, when the values of b0 and b1 are both 0, the data information allowed to pass is low-frequency information.
Extract Fault Information Features
The implied layer is the data information processing module.Let the real data information be Xi, i=1,2...n, and the desired output fault signal be Yi∈{-1,1}, where -1 is characteristic of normal operation data information and 1 is characteristic of fault data information.Then the classification hyperplane equation is: In Equation ( 2), (•) denotes the vector dot product, and η is the parameter of the constraint condition.The constraint condition is the parameter that separates the normal data information from the faulty data information.To make the extraction of fault information features more adequate, the classification interval is designed to satisfy the following constraints.
After the classification of the real data information is completed in the implicit layer, the extracted fault data information is passed to the undertaking layer.The bearer layer is responsible for filtering and classifying the features of the fault data information [5][6][7].As shown in the above figure, because of the different magnitudes of the fault information features, the influence of the magnitudes needs to be eliminated before the dimensionality reduction process.Normalize the principal component features of fault information.Then the individual principal components of the fault data information are arranged in the order from largest to smallest according to the correlation between the fault information features.The variance share S of each principal component is calculated as:
Analyzing Features Based on PCA-ELM Algorithm
In Equation ( 4), a1, a2, ..., ai are the eigenvectors corresponding to the fault information eigenvalues, and ZX1, ZX2, ..., and ZXi are the raw variable values after normalization.Among all the results, the one with the largest variance is the first principal component.ELM will continue to analyze and process the fault information features after the dimensionality reduction process, and the process of analyzing and processing the fault information features after the PCA process is shown in the following figure.The number of rows is the number of input fault information features.Columns are the number of nodes.The optimal number of nodes is obtained, and the number of nodes in the implied layer should meet the contribution rate of more than 99.5% to the ELM output matrix.In the ELM algorithm, there is no error node in the output layer, so the output weight can be solved to minimize the error function to complete the analysis of fault information characteristics.The error function F of the ELM algorithm is calculated as: In Equation ( 5), G is the output matrix of the hidden layer, T is the fault information feature after PCA processing, and ‖ is the Frobenius parametric number of the matrix elements.Through iterative calculation, the matrix weights of the ELM algorithm are continuously reduced to avoid redundancy and improve the stability and generalization ability of the ELM algorithm.Finally, according to the calculated matrix weights, the fault information features after PCA processing are analyzed and the analysis results are output to complete the analysis of fault information features of high-voltage power metering system based on the PCA-ELM algorithm.
Diagnose Faults in the High-Voltage Power Metering System
The fault type of each fault message is diagnosed based on the results of the fault message characteristics analyzed by the PCA-ELM algorithm.The fault diagnosis is divided into its types according to the fault location and the cause of the fault, as shown in the following table: According to the correspondence in the above table, the fault information characteristics processed by the PCA-ELM algorithm correspond with the fault location and fault phenomenon to obtain the detailed fault information of the high-voltage power metering system and complete the diagnosis of system faults.
Experimental preparation
To verify the feasibility of the fault diagnosis method of a high-voltage power metering system based on the PCA-ELM algorithm proposed in this paper, a fault diagnosis comparison experiment is designed.All the data information generated by the operation of a high-voltage power metering system in a city is collected, which contains 105, 326 items.Among them, it contains 1630 IA fault information, 3604 Ia fault information, 586 Ib fault information, 423 Ua fault information, 899 Ub fault information, 2547 ua fault information, 1368 ub fault information, a total of 11,057 pieces of all fault information, and 94, 269 pieces of normal operation data information.The above data information is aggregated into an experimental dataset, and 70% of the data information of each category in this dataset is extracted for algorithm training.The remaining 30% of the data information in this dataset is used for the data of the fault diagnosis experiment.Finally, there are 28280 normal data, 489 IA fault messages, 1081 Ia fault messages, 176 Ib fault messages, 127 Ua fault messages, 270 Ub fault messages, 764 ua fault messages, and 410 ub fault messages in the diagnosis experiment, totaling 31597.
To verify the feasibility of the proposed method, comparison object 1 is the fault diagnosis method of a high-voltage power metering system based on the Elman network.Comparison object 2 is a fault diagnosis method of a high-voltage power metering system based on momentum BP neural network.In this fault diagnosis test experiment, the fault sign parameter of the Elman network is set to 8, the input vector dimension to 8, the output vector dimension is set to 4, and the number of hidden layers is 17.The algorithm is trained by the gradient descent learning method.The learning rate of the momentum BP neural network learning algorithm is set to 0.1.logsig function is used as the transfer function of BP neurons.The momentum factor is set to 0.8.The training times of all three fault diagnosis methods for high-voltage power metering systems are set to 1000 times.
Analysis of experimental results
The 31597 data information in the experimental dataset is applied to three fault diagnosis methods respectively to test the correct rate of fault diagnosis of different diagnosis methods under the same training times.The fault diagnosis method of a high-voltage power metering system based on the PCA-ELM algorithm proposed in this paper is named Method 1.The Elman network-based fault diagnosis method for high-voltage power metering systems is named Method 2. The momentum BP neural network-based fault diagnosis method for high-voltage power metering systems is named Method 3. The experimental results are shown in the following table: According to the above table, the correct rate of determining fault data information for three types of high-voltage power metering systems can be derived, and the number of information determined as fault information is compared with the number of real fault information to derive the correct rate results shown in the following table: We can see from Table 4 that method 1 proposed in this paper has a high correct rate for fault determination of data information of high-voltage power metering systems, which is 3.95% and 4.58% higher than the other two fault diagnosis methods, respectively.
According to the experimental results in Table 3, the correct diagnosis rate of the three diagnostic methods for each fault type was calculated and the results are shown in the following table: We can see from Table 5 that method 1 proposed in this paper has a high correct diagnosis rate for various fault types, which is above 97%, and the average correct diagnosis rate is 98.87%.In the other two diagnosis methods, the highest correct rates are 77.91% and 90.24%, respectively.The highest correct rates are 22.09% and 9.76% lower than those of the fault diagnosis methods proposed in this paper, with a large gap.In addition, the average correct rates of the other two diagnostic methods were 63.98% and 54.67%, respectively.The average correct rates are 34.89% and 44.20% lower than those of the diagnosis methods proposed in this paper, which has a large gap.Thus, the fault diagnosis method of the high-voltage power metering system based on the PCA-ELM algorithm proposed in this paper can significantly improve the correct diagnosis rate of high-voltage power metering system faults, which has high practical significance.
Conclusion
In this paper, a fault diagnosis method based on the PCA-ELM algorithm for high-voltage power metering systems is proposed for the problem that the correct rate of diagnosis results is not high in fault diagnosis of high-voltage power metering systems.The fault information of high-voltage power metering systems is diagnosed by PCA descending fault information features and ELM algorithm analyzing fault information features.The diagnosis method effectively avoids the influence of highdimensional information on the diagnosis results and greatly improves the accuracy of fault diagnosis, which has a greater application value in the operation and maintenance of high-voltage power metering systems.
Figure 1
Figure 1 Active Filter Circuit Diagram We create an active filter circuit as shown above.Establish the transfer function of the data signal according to this circuit diagram.The transfer function expression is K(P), and its calculated expression is: 2 0 1 2 2 2 0 b 2 P b P b K P P P λ ω 0 Write the PCA-ELM-based algorithm to analyze the features of fault information.PCA is used to analyze the principal components of the fault data information.The operation flow is shown in the following figure.
Figure 2 PCA
Figure 2 PCA Analysis of Fault Information Principal Component Process
Figure 3
Figure 3 ELM Algorithm Process
Table 4
Accuracy of Fault Diagnosis by Different Fault Diagnosis Methods
Table 5
Diagnostic accuracy of different fault types using different methods | 2023-09-21T20:03:34.166Z | 2023-09-01T00:00:00.000 | {
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157273800 | pes2o/s2orc | v3-fos-license | Factors Affecting Demand Planning in the South African Clothing Industry
: The decline in the number of clothing manufacturers and the effect of globalisation have contributed to complexities in estimations and the scheduling of demand, as well as lead time management in the South African clothing industry. This article explores demand planning factors affecting the South African clothing industry, with specific reference to Gauteng. The study was necessitated due to demand planning challenges facing the South African clothing industry as well as economic factors which contribute to inaccuracies in clothing demand planning. The study makes an impact in the garment production factories of Gauteng in South Africa and adds to the philosophy of demand planning practices. It uncovers key factors affecting demand planning practices in the Gauteng clothing industry, South Africa. The study is explorative and descriptive in nature and it uses SPSS to analyse data. The findings revealed that there were factors affecting how demand planning practices were conducted in the clothing industry. The factors that have a significant influence on clothing demand planning include the scheduling of the manufacturing of customers’ orders, planning for fashion clothes, the use of the POS system, clothing imports, estimating future clothing requirements, recession and the effect of the late arrival of clothes. Therefore, clothing industry stakeholders should take these factors into consideration when planning for their demand to ensure customer needs can be fully met, thus improving the performance of the clothing industry.
Introduction
Since 2004, when the South African government lowered tax duties which are paid when goods/services are imported into the country, the South African clothing industry has been declining (Nattrass & Seekings, 2012:10). This decline had a negative effect on the level of sales, employment, gross domestic product (GDP), as well as on the accuracy of demand planning (Kim, 2012(Kim, :1285Rotunno, Vѐzina & Wang, 2013:2). In SA the fashion industry is one of the fifth major employment segments in the country. The SA clothing industry also makes a significant contribution to the country's growth in terms of sales (Gauteng Growth and Development Agency (GGDA), 2014). Hence, demand planning is critical in the SA clothing industry. Demand planning is critical in the clothing industry. Globally, the clothing industry is associated with a minimum period of garment existence, unstable customer needs and a variety of fashion styles with extensive lead times (Wong & Guo, 2010:614). According to Jonson and Tolstoy (2012:62), proper application of demand planning activities is required for organisations to achieve a competitive advantage. Demand planning, however, is subject to discrepancies and complexities, and this is one of the greatest challenges faced by the SA clothing industry (Aksoy et al., 2012:222;Bhardwaj & Fairhurst, 2010:165). The decline in the figure of garment production factories in the country and the effect of globalisation have contributed to complexities in estimations, scheduling, as well as lead time management (Sekerden, 2011:7). In addition, the influx of Chinese products into the country has affected the accuracy of demand planning in the clothing industry of the country (Rotunno et al., 2013:2).
The clothing industry has also experienced a reduction in the quantity of clothing organisations, a reduction in manufacturing output figure as well as fluctuating employment as reflected in section 1.2.3 (GGDA, 2014:3; IQ Business, 2014:3; Nattrass & Seekings, 2012:2). Numerous studies have been conducted on demand planning in the clothing industry. Authors such as Priest (2005), Sen (2008), Wark (2006) and Steytler and Powell (2010:6) explored selected components of demand planning such as fashion, clothing estimations and recession. In South Africa, authors such as Volk (2006), Nattrass and Seekings (2012) and Oberhofer (2012) low income payer with fewer possibilities for research innovations and it is associated with strong workforce legislations (Nattrass & Seekings, 2012:8). According to Vlok (2006:227), the industry remains a critical division with regards to employment development. Having realised the importance of demand planning, in 1994 the city of Johannesburg established centres where various clothing styles are produced with the aim of improving clothing demand (Oberhofer, 2012:7). The SA clothing industry consists of three key stakeholders, namely fabric suppliers, fashion designers and clothing manufacturers (GGDA, 2014:2;Oberhofer, 2012:65;Staritz & Morris, 2013:9). It is critical to note that, in the process of satisfying customers' clothing demands, the SA clothing industry is faced with opportunities as well as challenges (Nattrass & Seekings, 2012:6). Due to the lack of proper demand planning, there are inconsistencies that have affected the industry in the following ways: Changes in the number of clothing production organisations in South Africa: There have been changing: trends in the number of clothing organisations in South Africa over the years. In 1998, there were 784 clothing manufacturers. By 2000, there was a decline to 672, while there was an increase to 1008 clothing production organisations in 2006 (GGDA, 2014:3). These trends may have emanated from the effect of inconsistencies in demand planning, as well as other factors, such as clothing imports and the recession (Gereffi & Lee, 2012:28;Steytler & Powell, 2010:2).This clearly indicates that there are changing trends in the number of clothing production organisations in South Africa. Decline in manufacturing output: The clothing industry makes a significant contribution to the manufacturing sector of a country. In 2005, the clothing industry in SA grew by over R12 500 million and then there was a sharp decline to less than R11 500 million in 2006 (IQ Business, 2014:3). From 2007 to 2012, the industry was stable, and in 2013 there was a slight growth to almost R13 500 million (IQ Business, 2014:3). The decline in the contribution of the manufacturing sector in this industry from 2006 to 2012 may be the reason for a decline in employment in the clothing industry (IQ Business, 2014:3). Fluctuation in employment: Over the years, the clothing industry has also experienced fluctuations with regard to employment (GGDA, 2014:3). According to Nattrass and Seekings (2012:2), there is evidence of fluctuations in the number of employees within the SA clothing industry. In 1950, the level of employment in the industry was at 50 000 workers, in 2005 the employment figure was at 142 865 workers, while in 2013, the figure was at 80 000. This decline indicates that the employment level may be the result of material shortages, as well as a rise in the import of clothing from China.
As indicated in the discussion above, the productivity of the SA clothing industry can be affected by changing trends in the number of clothing production organisations, the decline in manufacturing output, as well as fluctuations in employment. This indicates that better approaches are required to improve planning for clothing demands.
Factors affecting demand planning in the clothing industry: There are challenges that have an effect on demand planning in the clothing industry in South Africa. These challenges stem from the incorrect scheduling of clothing material, planning for fashion clothes, incorrect use of the point of sale (POS) system, an influx of clothing imports, incorrect estimations of clothes, the recession as an unfavourable economic condition, and the late arrival of clothing material (Aksoy et al., 2012:223;Nattrass & Seekings, 2012:17). These challenges have an effect on demand planning in the SA clothing industry. The challenges are briefly explained below.
• Incorrect scheduling of clothing material: The scheduling of clothing material is one of the challenging aspects that affect proper demand planning (Annadurai & Uthayakumar, 2010:3465). Scheduling is defined as the utilisation of correct and timely resources in manufacturing, fabric acquisition and production capabilities (Maravelias, 2012(Maravelias, :1815. The scheduling of material can become multifaceted in the clothing industry due to the various stages involved in the production process, as well as the variation in clothing and the types of customers involved (Aksoy et al., 2012:222). Gereffi and Frederick (2010:17) mentioned that in South Africa, the complexity of clothing production is increasing as garments produced require more detailed trimming, especially with fashion clothing. Planning for fashion clothes: The fashion industry is a fast-moving industry with customers who are fashion-driven and who are not willing to compromise for the styles they demand (Wang, Gou, Sun & Yue, 2012:462). Customers' hesitation regarding fashion clothes has increased (Shen, Choi, Wang & Lo, 2013:266). This causes demand planning and prediction regarding fashion to become challenging. According to Wang et al. (2012:462), fashion clothes last for a short period. Bhardwaj and Fairhurst (2010:166) confirmed that fashion clothes change constantly and require constant follow-ups. Hence, demand planning in the SA clothing industry becomes difficult. Alack of skills to analyse fashion trends is also a challenge, which affects demand planning in the Gauteng clothing industry. Barnes and Lea-Greenwood (2010:762) argued that fashion customers consult garment style magazines and watch garment style shows on television and search for new fashion designs when shopping for clothes. Hence, it becomes challenging to implement demand planning for fashion clothes with accuracy. Incorrect use of the point of sale (POS) system: POS is an automated system used by retail outlets to process the sale of items bought by customers (Karnin & Walach, 2015:1). According to Folk, Bohen, Sanders and Johnson (2011:5), the POS system reflects the history of sold inventories. This service technology, called the POS system, also assists SA clothing manufacturers to obtain sales data from retail clothing stores. This prevents human errors from happening when manual sales are recorded in retail stores (Pretorius, 2013:1). Hamister (2011:432) said that information distortion is a challenge when using the POS system. According to Chaudhry and Hodge (2012:70), it is questionable whether the POS systems of clothing retailers are linked to the demand planning system of clothing manufacturers. When the clothing industry's system is not linked to the customers' systems, errors are bound to occur in demand planning (Ni & Fan, 2011:1531. Influx of clothing imports: The influx of imports has reduced employment globally, as well as in South Africa (Ramdass, 2007:1). Imports are the merchandise or goods that enter a country (Ramdass, 2007:4). Laudal (2010:72) alluded that imports emanate from international trade, which is now affecting the economy of growing countries. According to GGDA (2014:4), the increase of imports in the Gauteng clothing industry affects the proper implementation of demand planning processes in the industry. This results in discrepancies in demand planning for clothes as additional clothing supplies in the country make it difficult to predict the market for clothes and to deal with competition in the clothing industry. Incorrect estimations of clothes: Estimating with accuracy affects the complexity of manufacturing processes (Thomassey, 2010:471;Aksoy et al., 2012:222). In order to know the quantities of clothes that will be required in future, estimations have to be implemented. The correct and accurate estimation of clothing requirements is crucial in the clothing industry. According to Bhardwaj and Fairhurst (2010:169), failure to estimate future clothing styles will affect demand planning as the industry will not be able to attract fashion-sensible customers.
Research Design and Methodology
This article explores factors affecting demand planning in the clothing industry in Gauteng, South Africa. An exploratory and descriptive study was conducted. A mixed research approach (qualitative and quantitative) was used. A survey was conducted among clothing industry stakeholders in Gauteng. A survey involves obtaining data on situations, occurrences or events (Leedy & Ormrod, 2014:195).In South Africa, the clothing industries are found in the Western Cape, KwaZulu-Natal, the Free State and Gauteng. Gauteng was chosen because it was convenient and accessible to the researcher. A non-probability sampling technique was used. This means the researcher chose a sample size that was easily available and accessible. Convenient sampling was employed in this study. Population of study constituted 306 key stakeholders in the clothing industry: fabrics suppliers, fashion designers and clothing manufacturers. A total sample of 56 key clothing industries was chosen Managers, supervisors and specialists constituted the sample of the study based on convenience. A semi-structured questionnaire was mailed and hand-delivered to the respondents, who were based in Pretoria and Johannesburg. The questionnaire was measured using a Likert scale format. The structured questions were analysed descriptively using the statistical package for the social sciences (SPSS) and are presented in this article by means of tables and figures.
Results
This section of the article presents the findings on the factors affecting demand planning in the Gauteng clothing industry. Respondents were asked to indicate their level of agreement on the following factors that affect demand planning in the clothing industry: scheduling, planning for fashion, point of sale (POS) systems, imports of clothes, estimating future clothing requirements, the influence of a recession and the effect of the late arrival of clothes.
Scheduling the manufacturing of customers' orders:
Respondents were asked to indicate their level of agreement on fourteen (14) statements relating to scheduling the manufacturing of customers' orders. Figure 1 below shows the percentage of respondents of the clothing industry stakeholders who strongly disagreed or disagreed, who neither agreed nor disagreed and who agreed or strongly agreed with statements regarding the scheduling of the manufacturing of customers' orders. Table 1 presents the description of the statement numbers in Figure 1. * Note: some of the percentages do not add up to 100% on account of missing values, because some of the respondents did not answer specific statements relating to factors affecting demand planning.
As is shown in the graph (see Figure 1), a large majority (above 70%) of the clothing industry stakeholders agreed or strongly agreed with 11 of the 14 procedures that need to be followed when scheduling the
SCHEDULING THE MANUFACTURING OF CUSTOMER ORDERS
manufacturing of customers' orders. However, with regard to the statement whether retailers' demands regarding low cost charges on orders should be accommodated, only 48,2% of the respondents agreed or strongly agreed with the statement, indicating that more than half of the responses were neutral or in disagreement with the statement. In addition, only 60,7% of the respondents agreed or strongly agreed with the statements about clothing materials or fabrics needed in the manufacturing process that may arrive late, also indicating that a high 40% of the respondents were neutral or in disagreement with this statement. There was strong agreement (85,7%) that communicating customers' orders in terms of size, colour and clothing styles is crucial and that follow-ups should be conducted on material shortages with suppliers (80,3%). A very high level of agreement (85,7%) was also found with regard to the importance of manufacturers informing retail stores about stock shortages or any production problems, as well as the continual reviewing of the product process of the supply of clothes (82,2%). The following statements had very high levels of agreement: Communicating customers' orders in terms of size, colour (85,7%) Follow-ups on material shortages with suppliers (80,3%) Manufacturers informing retail stores about stock shortages or any production problems (85,7%) Continual reviewing of the product process of the supply of clothes (82,2%) The development of a team that plans for customer needs is crucial. A2.3 Continual reviewing of the product process of the supply of clothes is important. A2.4 Manufacturing processes that are not properly managed will result in continuous rework.
A2.5
It is important for manufacturers to inform retail stores about stock shortages or any production problems. A2.6 It is necessary to conduct follow-up on material shortages with suppliers.
A2.7
Manufacturing processes can only be finalised when customers' orders and specifications are confirmed. A2.8 Fabric orders must be placed before the sales season starts. A2.9 The manufacturing of clothes must be completed before the sales season starts. A2.10 Retailers' demands regarding low cost charges on orders should be accommodated. A2.11 Clothing materials or fabrics needed in the manufacturing process may arrive late.
A2.12
Meeting customers' orders on time is affected by the late arrival of clothing materials or fabrics.
A.2.13
Communicating customers' orders in terms of size, colour and clothing styles is crucial. A2.14 Manufacturers should communicate orders with the retail stores before manufacturing begins. Source: Questionnaire Planning for fashion clothes: Respondents were asked to indicate their level of agreement on fifteen (15) statements relating to planning for fashion clothes. Figure 2 below shows the percentage of respondents of the clothing industry stakeholders who strongly disagreed or disagreed, who neither agreed nor disagreed and who agreed or strongly agreed with the statements regarding planning for fashion clothes. Table 2 presents the description of the statement numbers in Figure 2. Monitoring fashion movements is important.
A3.4 Historical sales information should be updated continuously for timely and accurate planning. A3.5 It is not easy to plan for fashion clothes, because their lifespan is short. A3.6 It is easy to plan for basic clothes, because their lifespan is long. A3.7 It is important to have short manufacturing plans for fashion clothes. A3.8 Long manufacturing plans may exist for basic clothes. A3.9 Having manufacturers who think upfront and react to market changes, is crucial.
A3.10
Recognising fashion trends has never been easy due to multiple styling levels being available. A3.11 It is not easy to plan for fashion clothes due to constant changes in fashion. A3.12 Fashion styles may change 810 times a year.
A3.13
Manufacturers take lots of pressure from retail stores demanding more fashion variety. A3.14 Tracking and recording of customer changes regarding clothing needs are crucial. A3.15 It is not easy to keep track of fashion trends due to constant changes in fashion. Source: Questionnaire The level of agreement varied with 10 of the statements where respondents indicated agreement of more than 50%. Statements with a low level of agreement were: It is not easy to plan for fashion clothes due to constant changes in fashion (44,7%). Fashion styles may change 810 times a year (19,7%). It is not easy to keep track of fashion trends due to constant changes in fashion (37,5%). Recognising fashion trends has never been easy due to multiple styling levels available (48,2%). It is not easy to plan for fashion clothes because their lifespan is short (48,2%).
The following statements had very high levels of agreement:
PLANNING FOR FASHION CLOTHES
Customers' changing needs regarding fashion clothes should be considered when planning and estimating future clothing needs (78,6%). Having manufacturers who think upfront and react to market changes is crucial (78,6%). Monitoring fashion movements is important (78,5%). Historical sales information should be updated continuously for timely and accurate planning (76,8%). Tracking and recording customer changes regarding clothing needs are crucial (71,5%).
The point of sale (POS) system usage:
Respondents were asked to indicate their level of agreement on eleven (11) statements relating to the POS system. Figure 3 below shows the percentage of respondents of the clothing industry stakeholders who strongly disagreed or disagreed, who neither agreed nor disagreed and who agreed or strongly agreed with statements on the POS system usage. Table 3 presents the description of statements in Figure 3.
In terms of the perception of the respondents on the POS system in Figure 4.6, on average half (50%) of the respondents were in agreement with seven of the 11 statements relating to the usage of the POS system. These statements were: Having a system that communicates clothing information among participants in the clothing industry faster is crucial (57,2%). Incorrect sales information captured in the POS system will result in incorrect manufacturing plans (53,5%). A clothing manufacturer should store historical sales information of clothes on the mainframe computer (53,7). Manufacturers can use data on the POS system of retail stores to plan future clothing needs (51,8%). The POS system information of retail/customers provides a history about clothing sales (57,1%). Access to the POS system information of retail stores/customers is crucial for manufacturers (50%).
The importance of sharing sales information among suppliers, manufacturers and the end user customers (48,2%). Figure 4.
B1
Manufacturers can use data on the POS system of retail stores to plan future clothing needs.
B2
The POS system information of retail/customers provides history about clothing sales.
B3
Access to the POS system information of retail stores/customers is crucial for manufacturers.
B4
Incorrect sales information captured in the POS system will result in incorrect manufacturing plans.
B5
A clothing manufacturer should store historical sales information of clothes on the mainframe computer. B6 Storing garment details according to size, colour and design is crucial.
B7
Historical sales information used by manufacturers to plan future clothing needs can be purchased from marketing or advertising firms.
B8
Manufacturing factories can send company representatives to gather or collect historical sales information from retail stores manually.
B9
Having a system that communicates clothing information among participants in the clothing industry faster is crucial.
B10
The sharing of sales information among suppliers, manufacturers and the end user customers is crucial.
B11
Test the accuracy of historical sales information on a monthly basis before planning for future clothing production begins. Source: Questionnaire Storing garment details according to size, colour and design is crucial, and this statement had a slightly higher level of agreement (62,5%). A low level of agreement was found for testing the accuracy of historical sales information on a monthly basis before planning for future clothing production begins (41,1%). An alarming response (over 70%) of the clothing industry stakeholders revealed a neutral reaction or disagreement with regard to the following statements: Historical sales information used by manufacturers to plan future clothing needs can be purchased from marketing or advertising firms (only 21,4% agreement). Manufacturing factories can send company representatives to gather or collect historical sales information from retail stores manually (only 25% agreement).
Imports of clothes:
When it comes to the imports of clothes, respondents were asked to indicate their level of agreement on twelve (12) statements relating to clothing imports. Figure 4 below shows the percentage of respondents who strongly disagreed or disagreed, who neither agreed nor disagreed and who agreed or strongly agreed with the statements on the importing of clothes. Table 4 presents the description of statements in Figure 4.
C1
The influx of illegal and legal imports into South Africa results in inaccurate planning in manufacturing processes.
C2
The implementation of low tariff agreements on import rates by the South African government has contributed to an oversupply of clothes in the country. C3 Chinese imports contribute to an oversupply of clothes in the clothing manufacturing firms.
C4
Manufacturing firms do not have enough resources to supply large volumes of orders to the clothing retail stores in South Africa.
C5
The oversupply of clothes has resulted in high levels of unsold stock for manufacturers and wholesalers and in retail stores.
C6
There is a lack of demand for SA clothes because of a high number of clothing imports from China.
C7
Manufacturing processes need to be reduced to avoid the piling up of clothes in manufacturing factories. C8 There is a need for government subsidies in the SA clothing manufacturing industry.
C9
There is a need for SA clothing retailers and wholesalers to support local clothing manufacturers. C10 Currently, the SA government is not financing the clothing manufacturing industry. C11 There is a lack of capital equipment in SA clothing manufacturing industries. C12 Currently, SA retailers are not supporting local clothing manufacturers. Source: Questionnaire The statement with the highest level of agreement was that regarding the need for SA clothing retailers and wholesalers to support local clothing manufacturers (82,1%). In addition, the majority (67,8%) of the clothing industry stakeholders were in agreement that the SA government should support local clothing manufacturers by subsidising the clothing manufacturing industry. However, less than half of the respondents (48,2%) were in agreement that the SA government is currently not financing the clothing manufacturing industry, while only (41,1%) of the clothing industry stakeholders were in agreement that manufacturing firms do not have enough resources to supply large volumes of orders to the clothing retail stores. In addition, only 42,9% were in agreement that manufacturing processes need to be reduced to avoid the piling up of clothes in manufacturing factories. In terms of the influx of clothing imports, more than half of the respondents were in agreement about the following:
IMPORTS OF CLOTHES
The influx of illegal and legal imports into South Africa results in inaccurate planning in manufacturing processes (62,5%). The implementation of low tariff agreements on import rates by the SA government has contributed to an oversupply of clothes in the country (58,9%). Chinese imports contribute to the oversupply of clothes in the clothing manufacturing industry (60,7%). There is a lack of demand for SA clothes because of the high number of clothing imports from China (53,6%).
In addition, the responses of the clothing industry stakeholders on the above statements relating to the influx of clothing imports match the arguments indicated in the study literature (Chapter 2) pertaining to the influx of clothing imports.
Estimating future clothing requirements: The estimation of future clothing requirements was one of the factors that were perceived to have an effect on demand planning. Respondents were asked to indicate their level of agreement on ten (10) statements relating the estimating of future clothing requirements ranging from 1 = strongly agree to 5 = strongly disagree. Figure 4.8 below shows the percentage of respondents who strongly disagreed or disagreed, neither agreed nor disagreed and who agreed or strongly agreed with the clothing industry stakeholders regarding estimating future clothing requirements. Table 5 presents the description of statements in Figure 5. The implementing of different methods of estimation for basic and fashion clothing styles is required. D2
ESTIMATING FUTURE CLOTHING REQUIREMENTS
Attendance of international trade exhibitions in order to plan ahead for the season. D3 Estimators of clothes should undergo formal training to improve their skills. D4 The tool used for estimating future clothing needs can lose track of clothing sales information. D5 Fashion clothes change constantly, which results in errors when estimating trends. D6 Basic clothes are stable and result in low variances when predicting the future demand. D7 Manufacturers should conduct estimations of future customer needs according to the number of sizes sold previously. D8 Variance that exists regarding future clothing estimations should be investigated. D9 Manufacturers should conduct estimations of future customer needs on each clothing style. D10 Balancing the demand and supply of clothes is crucial to avoid high or low estimations. Source: Questionnaire Figure 5 indicates that, on average, half (50%) of the respondents were in agreement with seven (7) of the ten (10) steps listed on the estimation of future clothing requirements. The highest level of agreement (58,9%) was on the statement relating to the importance of balancing the demand and supply of clothes to avoid high or low estimations. Furthermore, 51,8% were in agreement, which means that basic clothes are stable and result in low variances when estimating clothes, and with the statement indicating that manufacturing firms should conduct estimations on future customer needs according to the number of sizes sold previously (51,8%). In addition, clothing estimators should undergo formal training to improve their skills. Surprisingly, only 28,2% of the clothing industry stakeholders were in agreement that fashion clothes change constantly and result in errors when estimating trends, and that the tool used for estimating future clothing needs can lose track of clothing sales information.
Recession as an unfavourable economic condition:
Respondents were asked to indicate their level of agreement on six (6) statements relating to recession. Figure 6 below shows the percentage of respondents who strongly disagreed or disagreed, who neither agreed nor disagreed and who agreed or strongly agreed with the clothing industry stakeholders regarding the recession as an unfavourable economic condition. Table 6 presents the description of the statements in Figure 6. Figure 4.9 regarding recession Statements Description of statements E1 When the SA economy is stable, clothes will sell in large numbers. E2
RECESSION AS AN UNFAVOURABLE ECONOMIC CONDITION
The use of historical clothing sales data does not assist in estimating accurate future clothing needs. E3 Before a recession, the use of historical sales data contributes to the level of accuracy when estimating future clothing needs. E4 The purchase of non-essential items, such as clothing, fell as a result of the recession. E5 During the period of a recession, it is crucial to reduce future customers' orders as customers' needs decline. E6 During the period of a recession, it is crucial to manufacture clothes in small batches. Source: Questionnaire Figure 6 reflects that the majority (over 70%) of the clothing industry respondents were in agreement (agreed or strongly agreed) with three of the statements relating to the recession. The highest levels of agreement were on the following statements: When the SA economy is stable, clothes will sell in large numbers (75%). The purchase of non-essential items, such as clothing, fell as a result of the recession (73,2%). During the period of a recession, it is crucial to manufacture clothes in small batches (71,4%).
For the other three statements, half or more than half of the respondents were in agreement as shown below: During the period of a recession, it is crucial to reduce future customers' orders as customers' needs decline (64,3%). Before a recession, the use of historical sales data contributes to the level of accuracy when estimating future clothing needs (55,3%). The use of historical clothing sales data does not assist in estimating accurate future clothing needs (50%).
The effect of the late arrival of clothes:
Respondents were asked to indicate their level of agreement on twelve (12) statements relating to the effect of the late arrival of clothes. Figure 7 below shows the percentage of respondents who strongly disagreed or disagreed, neither agreed nor disagreed and who agreed or strongly agreed with the clothing industry stakeholders regarding the effect of the late arrival of clothes. Table 7 presents the description of the statements in Figure 7. It is not possible to have control over the timely arrival of fabrics.
F3
Fabrics and clothing material used in clothing manufacturing can only be purchased overseas.
F4
Fabrics and clothing material used in clothing manufacturing can be purchased from local suppliers. F5 Orders placed with overseas suppliers may arrive too late for manufacturing.
F6.
Late arrival of clothing materials or fabrics should be considered when estimating future clothing needs.
F7
The timely arrival of clothing material or fabrics may reduce the level of errors when estimations regarding future customer needs are implemented.
F8
Operating within tight schedules and waiting periods will assist manufacturers to manufacturer in advance. F9 On-time deliveries assist in ensuring quick manufacturing processes.
F10
On-time agreements should be made with suppliers of fabrics to reduce the late arrival of fabrics. F11 Clothing material or fabric orders may arrive while the sales season has started.
F12
Ordering fabrics before the sales season starts, is crucial to avoid lost sales on clothing garments.
As indicated in Figure 4.8, the majority (over 57%) of the respondents were in agreement with eleven (11) of the twelve (12) statements relating to the effect of the late arrival of clothes, as listed below: Manufacturers should order fabrics from both local and international suppliers (76,6%). It is not possible to have control over the timely arrival of fabrics (51,7%). Fabrics and clothing material used in clothing manufacturing can be purchased from local suppliers (66%). Orders placed with overseas suppliers may arrive late for manufacturing (62,5%). Late arrival of clothing materials or fabrics should be considered when estimating future clothing needs (82,2%). The timely arrival of clothing material or fabrics may reduce the level of errors when estimations regarding future customer needs are implemented (75%). Operating within tight schedules and waiting periods will assist manufacturers in the manufacturing process (67,9%). On-time deliveries assist in ensuring quick manufacturing processes (84%). On-time agreements should be made with suppliers of fabrics to reduce the late arrival of fabrics (83,3%). Clothing material or fabric orders may arrive when the sales season has already started (64,3%). Ordering fabrics before the sales season starts is crucial to avoid lost sales on clothing garments (80,4%).
It is important to note that the clothing industry stakeholders were in strong disagreement with only one of the twelve (12) statements that were tested in Figure 4.10. The clothing industry stakeholders were neutral or in disagreement with the statement regarding fabrics and clothing material used in clothing manufacturing that can only be purchased overseas (19,7%).
Conclusion
The clothing industry has also experienced a decline in the number of clothing organisations, a decline in the manufacturing output, as well as fluctuating employment. This article explored factors affecting demand planning in the clothing industry in Gauteng, South Africa. An exploratory and descriptive research design, based on the convenience sampling technique, was conducted among clothing industry stakeholders in Gauteng. From a theoretical point of view, the article discusses some of the factors affecting demand planning in the clothing industry. The factors stem from the incorrect scheduling of clothing material, planning for fashion clothes, the incorrect use of the point of sale (POS) system, an influx of clothing imports, incorrect estimations of clothes, recession as an unfavourable economic condition, and the late arrival of clothing material. The overall findings indicated that the scheduling of the manufacturing of customer orders was perceived as the most important factor to consider when planning for clothing demand. From this finding, it was specified that more than half (above 50%) of the respondents perceived planning for fashion clothes, clothing imports, the recession and the effect of the late arrival of fabrics as factors that affect demand planning. The findings also highlight that half (above 50%) of the respondents perceived POS system usage and the estimation of customer clothing requirements as factors that have an effect on demand planning. Scheduling the manufacturing of customer orders was seen as the factor with the most profound influence on demand planning. The findings also specify that the POS system usage and the estimation of customer clothing requirements are factors with the least effect on demand planning.
This study is unique compared to previous studies which focus on individual factors affecting demand planning. The uniqueness of the study looks at different factors which affects demand planning in Gauteng clothing industry, in South Africa. From the research findings, it clear that product history and teamwork are crucial when scheduling customer clothing orders. The respondents indicated that planning for fashion clothes is a challenge due to their short life span as compared to basic clothes the respondents emphasised that clothing manufacturers should be proactive in reacting to market changes. The findings evidently indicates that correct sales information stored on the POS system assists manufacturers to plan for customers' needs. The research findings revealed that clothing manufacturers need to have access to the POS system of retail customers in order to plan for actual clothing demands. The findings also indicated that the influx of clothing imports into South Africa results in inaccurate planning in manufacturing processes. It is clear that during global recession, the purchase of non-essential items, such as clothing, decreases. Hence, the importance of small-batch manufacturing during a period of recession was emphasised greatly in the responses. The respondents agreed with the statements on the effect of the late arrival of clothes. The respondents agreed that on-time deliveries assist in ensuring quick manufacturing. It is suggested the clothing industry of Gauteng in South Africa consider these factors in their demand planning process for accuracy and proper planning to exist processes. It is evident that all these factors may be the reasons for the challenges faced by the clothing industry with regard to demand planning, or that they may contribute to it. It is imperative that clothing industry stakeholders take into considerations these factors when planning for their demand. Through the application of proper demand planning practices, customer needs can be fully met, thus improving the performance of the clothing industry. | 2019-01-02T21:27:39.724Z | 2016-10-30T00:00:00.000 | {
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7151908 | pes2o/s2orc | v3-fos-license | Named Entity Recognition for Arabic Social Media
,
Introduction
Named Entity Recognition (NER) is the task of tagging names with a predefined set of named entity types (e.g. Location, Person) in opendomain text (Nadeau and Sekine, 2007). NER has been shown to improve Information Retrieval performance (Thompson and Dozier, 1997) and Question Answering (QA) performance where (Ferrndez et al., 2007) shows that, on average, questions contain ≈ 85% Named Entities.
In the current world of ubiquitous social media presence, processing informal genre is becoming ever more crucial. One of the prevalent genre in social media in need for text mining is microblog data such as Twitter. Twitter data is characterized by being massive. Off the shelf NER systems trained on formal genre such as newswire fail to process such data, thereby current research in information extraction has been specifically targeting this genre (Ritter et al., 2011). This problem is quite significant in English and is ever more pronounced in lower resourced languages such as Arabic.
Arabic has gained more attention recently due to the increased availability of annotated datasets. Arabic NER systems, as other languages, are domain dependent and mainly trained on news corpora or other well structured data that uses the Modern Standard Arabic (MSA) variety of the language (Benajiba et al., 2007) and (Abdallah et al., 2012). Arabic, in general, poses additional challenges to Natural Language Processing (NLP) tasks, as opposed to other languages, due its rich morphology and highly inflected nature. Moreover, Arabic is also one of those languages that exists in a state of diglossia where multiple forms of the language exist in the same context, the standard formal form, MSA, used in formal settings (education, formal speeches, etc.) and the spoken vernaculars that differ significantly from MSA, known as Dialectal Arabic (DA) that are used pervasively in informal settings such as in social media. Since MSA and DA co-exist, we note that people very often code switch between the two varieties within the same utterance which is reflected in microblog data. Hence NLP systems targeting the Twitter genre needs to account for this phenomenon.
Compared to English NER, here are some example challenges posed for Arabic NER (Abdul-Hamid and Darwish, 2010): • Lack of capitalization: Capitalization in Latin languages is a strong indicator of Named Entity (NE). However, in Arabic, proper nouns are not capitalized, which renders the identification of NEs more complicated; • Nominal Confusability: Some words can be proper nouns, nouns, or adjectives. For instance, jamiyolap 1 which means 'beautiful' can be a proper noun or an adjective. Another example, jamAl, which means 'beauty', is a noun but can be a common noun or a proper name; • Agglutination: Since Arabic exhibits concatenative morphology, we note the pervasive presence of affixes agglutinating to proper nouns as prefixes and suffixes (Shaalan, 2014). For instance, determiners appear as prefixes Al as in (AlqAhrp, 'Cairo'), likewise with affixival prepositions such as l, 'for' (ldm$q 'for/to/from Damascus'), as well as prefixed conjunctions such as w, 'and', as in (wAlqds 'and Jerusalem'); • Absence of Short Vowels (Diacritic Markers): Written MSA, even in newswire, is underspecified for short vowels, aka undiacritized, which results in higher ambiguity that can only be resolved using contextual information (Benajiba et al., 2009). Examples of ambiguity are: mSr, may be miSor as in 'Egypt' or muSir as in 'insistent'; qTr may be the name of the country 'Qatar' if vowelized/diacritized as qaTar, qaTor for 'sugar syrup', quTor for 'diameter'.
In addition to the afore mentioned challenges, in general, for Arabic NER in general compared to Latin-based languages, DA NER faces additional issues: • Lack of annotated data for supervised DA NER; • Lack of standard orthographies or language academics (Habash et al., 2013): Unlike MSA, the same word in DA can be rewritten in so many forms, e.g. mAtEyT$, mtEyt$, mA tEyT$, 'do not cry', are all acceptable variants since there is no one standard; • Lack of comprehensive Gazetteers: this is a problem facing all NER systems for all languages addressing NER in social media text, 1 We use the Buckwalter encoding system to render Arabic.
For a reference listing please see http://www.qamus.org/transliteration.htm since by definition such media has a ubiquitous presence of highly productive names exemplified by the usage of nick names, hence the PERSON class in social media NER will always have a coverage problem; • Applying NLP tools designed for MSA to DA results in considerably lower performance, thus the need to build resources and tools that specifically target DA (Habash et al., 2012).
The majority of existing NER systems rely on the use of gazetteers to improve the system accuracy (Kazama and Torisawa, 2007), however, large external resources are correlated with higher performance cost. In this paper, we study the impact of word representation and embedding features on Arabic NER performance for Twitter and Dialectal Arabic, and demonstrate that our proposed features show comparable and superior results to other NER systems that use large gazetteers. Our contributions are as follows: • Show the impact of using word representations and embedding on NER performance; • Propose a set of features that does not include the use of external resources; • Produce comparable NER performance to other systems that use large gazetteers.
Related Work
Significant amount of work in the area of NER has taken place. In (Nadeau and Sekine, 2007), the authors survey the literature of NER and report on the different sets of used features such as contextual and morphological features. Although more research has been employed in the area of English NER, Arabic NER has been gaining more attention recently. Similar to other languages, several approaches have been used for Arabic NER: Rulebased methods, Statistical Learning methods, and a hybrid of both.
In (Shaalan and Raza, 2009), the authors present rule-based NER system for MSA that comprises gazetteers, local grammars in the form of regular expressions, and a filtering mechanism that mainly focuses on rejecting incorrect NEs based on a blacklist. Their system yields a performance of 87.7% F1 measure for the Person label (PER), 85.9% for Location (LOC), and 83.15% for Organization (ORG) when evaluated against corpora developed by the authors. (Elsebai et al., 2009) proposed a rule-based system targeting PER in MSA. It uses the Buckwalter Arabic Morphological Analyser (BAMA) and a set of keywords. The proposed system yields an F-score of 89% when tested on an in-house annotated dataset of 700 news articles extracted from Aljazeera television website.
Although rule based approaches proved successful to some extent, most recent NER research focuses on Statistical Learning techniques due to the shortcomings of rule based approaches in terms of coverage and robustness (Nadeau and Sekine, 2007). For example, (Benajiba et al., 2007) proposes an MSA NER system (ANERsys) based on n-grams and maximum entropy. The authors also introduce ANERCorp corpora and AN-ERGazet gazetteers. further modify ANERsys in terms of the underlying machinery to use Conditional Random Fields (CRF) sequence labeling as the statistical learning framework. ANERsys uses the following features: part of speech (POS) tags, Base Phrase Chunks (BPC), gazetteers, and nationality information. The latter feature is included based on the observation that PER occur after mentioning the nationality, in particular in newswire data. In , a different classifier is built for each NE type. The authors study the effect of features on each NE type, then the overall NER system is a combination of the different classifiers that target each NE class label independently. The set of features used is a combination of general features as listed in and Arabic-dependent (morphological) features. Their system's best performance is 83.5% for ACE 2003, 76.7% for ACE 2004, and 81.31% for ACE 2005, respectively. (Benajiba et al., 2010) presents an Arabic NER system that incorporates lexical, syntactic, and morphological features and augmenting the model with syntactic features derived from noisy data as projected from Arabic-English parallel corpora. The system Fscore performance is 81.73%, 75.67%, 58.11% on ACE2005 Broadcast News, Newswire, and Web blogs, respectively. The authors in (Abdul-Hamid and Darwish, 2010) suggest a number of features, some of which we incorporate in our current DA-NER system, namely, the head and trailing 2grams (L2), 3-grams (L3), and 4-grams (L4) characters in a word. (Abdul-Hamid and Darwish, 2010) produce near state-of-the-art results with the use of generic and language independent features that we use to generate baseline results (BL). (Shaalan and Oudah, 2014) presents a hybrid approach that targets MSA and produces state-ofthe-art results. However, we could not get access to the exact rules employed, we were not able to replicate their results. The rule-based component is identical to their previous proposed rulebased system in (Shaalan and Raza, 2009). The features used in their study are a combination of the rule-based rules in addition to morphological, capitalization, POS tag, word length, and dot (i.e. if a word has an adjacent dot) features. All the previous work mentioned above focused on MSA, albeit with variations in genres to the extent exemplified by the ACE data and author generated data. However, unlike the work mentioned above, (Darwish and Gao, 2014) proposed an NER system that specifically targets microblogs as a genre, as opposed to newswire data. Their proposed language-independent system relies on a set of features that is similar to (Abdul-Hamid and Darwish, 2010), with the use of a simple yet effective domain adaptation approach (Daumé et al., 2010) based on a two-pass semi supervised method. Their NER system on Twitter data yields an overall F-score=65.2% (76.7% for LOC, 55.6% for ORG, and 55.8% for PER). In our prior work, (Zirikly and Diab, 2014), we proposed a small set of annotated DA data and DA-NER system that yields an F-score=70.3%. We used n-gram, gazetteers and an extensive set of morphological features. In our current work, we explore the impact of using word embedding features and how can word representations and embedding replace the use of dictionaries and even generate better performance.
Approach
In this paper, we adopt a supervised machine learning approach. Supervised approaches have been shown to outperform unsupervised approaches for the NER task (Nadeau et al., 2006). We use Conditional Random Fields (CRF) sequence labeling as described in (Lafferty et al., 2001). Guided by previous work, for example demonstrates that CRF yields better results over other supervised machine learning techniques for the task of NER. One of the goals of our empirical investigation is to show the impact of using skip-gram word embedding and word representations on the NER performance, with the potential of these features substituting the use of extensive large gazetteers.
Baseline
For our baseline system (BL), we reimplemented the features proposed in (Abdul-Hamid and Darwish, 2010) that produced the best results: previous and next word, and leading and trailing +/-(2-4) character ngrams. These features are chosen as a preferred set for the baseline since they are directly applicable to DA, as none of the features rely on the availability of morphological or syntactic analyzers. This baseline is also adapted from (Darwish and Gao, 2014). We opted for this baseline as opposed to (Darwish and Gao, 2014)'s NER system since they use Wikipedia gazetteers (WikiGaz) for their NER system. 2 However, we report the results of applying their features using exact match against the gazetteers' entries in Section 4.3.
Our NER Features
In addition to the features employed in the baseline BL, we introduce the following additional features in our NER system: • Gazetteers (GAZ): Although our work mainly targets NER systems without the use of external sources, but we added GAZ features for comparison purposes. The gazetteer used is the union of: i) ANERGaz: proposed by , which contains 2183 LOC, 402 ORG, and 2308 PER; and ii) WikiGaz: large Wikipedia gazetteer (Darwish and Gao, 2014), which contains 50141 LOC, 17092 ORG, and 65557 PER. We followed this strategy for text matching against gazetteer entries: -Exact match (EM-GAZ): For more efficient search, we use Aho-Corasick Algorithm (Aho and Corasick, 1975) that has linear running time in terms of the input length plus the number of matching entries in a gazetteer. When a word sequence matches an entry in the gazetteer; -Partial match(PM-GAZ): This feature is created to handle the case of compound gazetteer entries. If the token is part of the compound name then this feature is set to true. For example, if the gazetteer entry is yAsr ErfAt 'Yasser Arafat' and the input text is yAsr BrkAt then PM-GAZ for the token yAsr will be set to true. This is particularly useful in persons names; -Levenshtein match (LVM-GAZ): We use Levenshtein distance (Levenshtein, 1966) to compare the similarity between the input and a gazetteer entry. This is based on the observation that social media data might contain non-standard spelling of words since it contains the DA variety.
• Morphological Features: The morphological features that we employ in our feature set are generated by MADAMIRA (Pasha et al., 2014). The set comprises the following: -Part of Speech (POS) tags: We use POS tags generated from MADAMIRA within a window of ±1 (POS-1, POS0, POS1); -Capitalization (CAPS): In order to circumvent the lack of capitalization in Arabic, we check the capitalization of the translated NE which could indicate that a word is an NE . This feature is dependent on the English gloss generated by MADAMIRA. This feature is set to true when the gloss starts with a capital letter; -Aspect (ASP), person (PERS), procl-itics0 (PROC0), proclitics1 (PROC1), proclitics2 (PROC2), proclitics3 (PROC3), enclitics0 (ENC0); detailed description for these features is provided in Table 1 Question proclitic: No proclitic, Not applicable, Interrogative particle Proclitic2 Conjunction proclitic: No proclitic, Not applicable, Conjunction f, Connective particle f, Response conditional f, Subordinating conjunction f, Conjunction w, Particle w, Subordinating conjunction w Proclitic1 Preposition proclitic: No proclitic, Not Applicable, Interrogative i$, Particle b, Preposition b, Progressive verb particle b, Preposition E, Preposition ElY, Preposition fy, Demonstrative hA, Future marker H, Preposition k, Emphatic particle l, Preposition l, Preposition l + preposition b, Emphatic l + future marker H, Response conditional l + future marker H, Jussive l, Preposition l, Preposition mn, Future marker s, Preposition t, Particle w, Preposition w, Vocative w, vocative yA Proclitic Article proclitic: No proclitic, Not Applicable, Demonstrative particle A, Determiner, Determiner Al + negative particle mA, Negative particle lA, Negative particle mA, Negative particle mA, Particle mA, relative pronoun mA Enclitics Pronominals: No enclitic, Not applicable, 1st person plural/singular, 2nd person dual/plural, 2nd person feminine plural/singular, 2nd person masculine plural/singular, 3rd person dual/plural, 3rd person feminine plural/singular, 3rd person masculine plural/singular, Vocative particle, Negative particle lA, Interrogative pronoun mA, Interrogative pronoun mA, Interrogative pronoun mn, Relative pronoun mn, m, mA, Subordinating conjunction m, mA. • Brown Clustering IDs (BC): Brown clustering (Brown et al., 1992) is a hierarchical clustering approach that maximizes the mutual information of word bigrams. Word representations, especially Brown Clustering, have been shown to improve the performance of NER system when added as a feature (Turian et al., 2010). In this work, we use Brown Clustering IDs (BC) of variable prefix lengths (4,5,6,7,10,13 and the full length of the cluster ID) as features resulting in the following set of features BC4, BC5, BC6, BC7, BC10, BC13, and BC, respectively. For example if AmrykA 'America' has the brown cluster ID BC=11110010 then BC7=1111001, whereas BC10 and BC13 are empty strings. This feature is based on the observation that semantically similar words will be grouped together in the same cluster and will have a common prefix; • Word2vec Cluster IDs : Word2vec is an algorithm for learning embeddings using a neural network model (Mikolov et al., 2013). Embeddings are represented by a set of latent variables, where each word is represented by a specific instantiation of these variables. In our system, we apply K-means clustering on the word vectors and use the clusters IDs as features.
Datasets
We use Microblogs and Dialectal weblogs datasets for our experiments: • Twitter dataset: We use the training and test data split proposed in (Darwish, 2013), where the training dataset contains 3,646 tweets which were randomly selected from tweets that were authored in the period of May 3-12, 2012. The tweets were scraped from Twitter using the query lang:ar. The testing data contains 1,423 tweets that were randomly selected from tweets authored between November 23, 2011 andNovember 27, 2011. This dataset has also been used in (Darwish and Gao, 2014) for testing. Both datasets are annotated using the Linguistics Data Consortium ACE tagging guidelines; • Dialectal Arabic dataset (DA-EGY): The annotated data was chosen from a set of web blogs that are manually identified by LDC as Egyptian dialect and contains nearly 40k tokens. The data was annotated by one native Arabic speaker annotator who followed the Linguistics Data Consortium guidelines for tagging. We use the same 80/20 train/test 5-fold cross validation split proposed in (Zirikly and Diab, 2014) Table 2 shows dataset statistics, namely number of tokens, and the named entity types: PER, LOC, and ORG.
Brown Clustering and word2vec Data In our work, we run brown clustering and word2vec three times based on the data genre: i) Newswire #Tokens #PER #LOC #ORG Twitter -Train 55k 788 713 449 Twitter-Test 26k 464 587 316 DA-EGY 24k 311 155 19 Parametric features values We use the following values for the parametric features: • CTX features: we set context window = ±1 for words; • LM-GAZ: Threshold of the number of deletion, insertion, or modification ≤ 2; • BC: Length of the prefixes of brown clusters ID is set to 4,5,6,7,10,13, and full length of cluster ID.
Data Normalization and Preprocessing
Arabic normalization has proven to improve retrieval results (Darwish et al., 2012). We apply the following normalizations on training, testing, BC and word2vec input data: i) Number normalizations: [0 − 9] → 8; ii) Hamza normalization: hamza numerous forms are used interchangeably depending on the role of a word in the sentence. For instance, the term "his sky" can be written smA&h, smA'h, or smA}h, where the hamza takes its form based on the term being subject, object, or idafa (construct state indicating possessive), respectively , >, <, &, }, |, {, ', Y → A; iii) Normalizing elongated words: We remove consecutive repeated letters that occur > 2.
Tools In this work, we use the following tools: 1. MADAMIRA (Pasha et al., 2014): For tokenization preprocessing and morphological features such as gender and POS tags; 2. CRFSuite implementation (Okazaki, 2007).
Evaluation Metrics
We choose precision (PREC), recall (REC), and harmonic F-measure (F1) metrics to evaluate the performance of our NER system over accuracy. This decision is based on the observation that the baseline accuracy is always high as the majority of the words in free text are not named entities.
Results & Discussion
Twitter Results: Table 3 illustrates results of our NER system performance. We use the weighted macro-average across the three NEs (PER, LOC, ORG) to calculate the overall performance.
Although we were not able to replicate (Darwish and Gao, 2014) results with WikiGaz (F1=55% vs. 51.62%), but our proposed features coupled with BC and word2vec surpass their performance yielding an F1=57.84% without the use of any external resources vs. 59.59% with the use of gazetteers. 4 Although word2vec and BC increase F1 ≈ 10% over BL, we note that BC impact (+6%) is more significant in comparison to word2vec with only 3% improvement. It is worth mentioning that this is aligned with (Turian et al., 2010) observations that Brown Clustering yields better English NER performance as opposed to word embedding. This is due to Brown Clustering's ability to induce rare words compared to word embedding. We also note that our intuition for using Levenshtein Matching approach, LVM-GAZ, against gazetteers' entries to overcome nonstandardization issue in DA shows 0.8% improvement over EX-GAZ and PAR-GAZ. We should note that LVM-GAZ very much depends on the percentage of present DA variety in the data. The results achieved are promising, especially in the area of social media since generating gazetteers that have high coverage is a challenging and expensive task.
When observing the MORPH feature set in more details, we notice that CAPS and POS yield the highest improvement over the baseline, especially in the PER class, this is mainly due to the correct assignment of the Proper Noun POS tag to this class confirming that POS tag is a strong indicator for NE.
We study the impact of applying BC and word2vec on different data genre. We take as an example BC, shown in Figure 1. We note that genre variations impose minimum impact on word representations, thus we can induce that word2vec and BC presents robust and domain-independent features. Figure 1: BC Data Genre and Performance correlation DA-EGY Results: We apply the feature sets that yields the best result with and without the use of gazetteers in Table 3 to our second evaluation dataset DA-EGY. The reported result is the average of 5-fold cross validation. As proposed in (Zirikly and Diab, 2014), we omit ORG class because there is less than 0.05% instances of ORG in the annotated data, which does not represent a fair training data to the system. Our system outperforms the state-of-the-art results by ≈ 7% with the use of gazetteers, and ≈ 2% without the use of gazetteers. As shown in Table 4, we notice that FS15, which uses word2vec features and excludes BC features and gazetteers, generate very comparable results (72.61%) to the best gazetteers-free performance achieved 72.68%.
Conclusion & Future Work
In this paper we study the impact of word representations and embedding on Arabic NER system for social media data. We show that our proposed gazetteers-free features surpass other NER systems that use large gazetteers. This is a significant advantage since gazetteers are expensive to generate, especially in the area of social media due to the low coverage of dictionaries. We show that our proposed system improves NER performance and outperforms state-of-the-art results for Dialectal Arabic. In future work, we would like to test the impact of cross-lingual word embedding and representation
Acknowledgment
This work was supported by the Defense Advanced Research Projects Agency (DARPA) Contract No. HR0011-12-C-0014, the BOLT program with subcontract from Raytheon BBN. We would like to thank three anonymous reviewers for their comprehensive feedback | 2015-08-11T20:29:18.000Z | 2015-06-01T00:00:00.000 | {
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252820541 | pes2o/s2orc | v3-fos-license | The synergic impact of lignin and Lactobacillus plantarum on DSS-induced colitis model via regulating CD44 and miR 199a alliance
Chronic or recurrent immune system activation and inflammation inside the gastrointestinal tract is characterized by inflammatory bowel disease (IBD). Due to the lack of safety and efficacy of traditional medications, the use of food supplements for IBD management is on the rise. Numerous studies reported that, certain food supplements have a variety of therapeutic benefits for IBD. In the present study, a mouse model of IBD was used to the anti-colitis effects of lignin supplementation with Lactobacillus plantarum (L. plantarum) on intestinal inflammation. The animal model was treated with dextran sodium sulphate (DSS), the illness index increased, and colon length and body weight declined, but these effects were reversed when lignin and L. plantarum treated groups. In addition, lignin and L. plantarum supplementation inhibited the DSS induced increase in levels of cytokines TNF-α (250 pg/mL), INF-γ (180 pg/mL), IL-1β (70 pg/mL) and TGF- β (72 pg/mL). Gene and protein expression study revealed that Lignin and L. plantarum supplementation restored the expression of E-cad and suppressed the expression of STAT3 in DSS induced colitis model. Lignin and L. plantarum supplementation also suppressed CD44 expression (1.2 fold) by up regulating the expression of miR199a (1 fold) over DSS induced colitis. Our study suggests that Lactobacillus, lignin, and their synergistic treatments have protective roles against inflammatory bowel disease through changes in inflammatory cytokines, and miR 199a expression in DSS-induced colitis.
Introduction
Inflammatory bowel disease (IBD) is an inflammatory irritation in the bowel of host system. It is caused by a multifactorial disease interaction between factors (Microbial, immunological and genetical) in nature, which is progressed to the development of bowel syndrome (Friedrich et al. 2019). IBD is the global autoimmune idiopathic issue of the intestine (Blumberg et al. 1999). In IBD there are two major conditions namely ulcerative colitis (UC) and Crohn's Disease (CD). Under Ulcerative colitis condition, the inflammated mucosal layer of the colon leads to chronic inflammation (Baumgart and Sandborn 2007). Whereas, under Crohn's disease condition the whole gastrointestinal tract was affected. Both the inflammatory bowel disease is a major bottleneck in the healthcare system in developed countries and currently increasing in developing countries (Baumgart and Sandborn 2007). There is a significantly higher risk of neurodegenerative diseases, gastrointestinal tumors and 1 3 233 Page 2 of 13 psychological issues in patients with IBD. These risks make it more important to diagnose the disease earlier and provide appropriate attention in the clinical treatment process (Feng et al. 2022). The intestinal environmental factors, which are complex to the immune response yet not clear in the etiology of IBD (Orel 2014). IBD diagnosis was very challenging in the early period over 60 years before, now both UC and CD are diagnosed with the technology of fiberoptic colonoscopy and ileocolonoscopy (Mulder et al. 2014). The IBD is currently treated with many agents such as 5-aminosalicylic acid, steroids, antibiotics, probiotics, immunosuppressant, etc. These agents are successful in improving acute and chronic inflammation, however they do not prevent/reverse intestinal fibro stenosis (Vetuschi et al. 2022). This drawback of the current treatment strategies has steered research towards more effective treatment methods.
Microflora of the human intestine constitute bacteria, fungi and viruses. Bacteria are dominating 96% of the intestine, and around 5000 species were found in the human bowel. An enormous amount microorganisms are obtained in the large intestine of the human bowel, with 1 × 10 12 microbial cells indexed in the intestinal bowel. The microbiota of intestinal flora could influence host health in both direct and indirect ways by its metabolites. The profile of flora may vary based on host life style, genetic factors and diet (Basso et al. 2019). The beneficial microbes, which positively regulate the host immune system are known as probiotics.
Probiotic is distinct live microorganisms which is helping to protect the host bowel system against inflammations (Borchers et al. 2009). Lactobacillus is a significant microbiota of the digestive system, it is involved in host health improvement and so considered as probiotic. Lactobacillus involved in immunoregulatory properties of the host, which stimulate the intestinal immune response to fight against the inflammation in the bowel system. Probiotic could be classified as a two groups, such as immunostimulating and antiinflammatory (Macho Fernandez et al. 2011). These probiotics secrete short chain fatty acids, protease, butyrate, and vitamins etc., Few reports revealed that, Short chain fatty acid (SCFA) inhibit the adherence of pathogenic microbes in the host gut. Gut epithelium injured in IBD was restore by butyrate, which supplies the essential nutrient to colonocytes and deed as an agent for inactivating intracellular transcriptional factors pathways to synthesis the cytokines factors (Kanauchi et al. 2005). In the binding sites of the mucus layer the pathogenic microbes were dispatched from luminal mucosal (Veerappan et al. 2012). The probiotic enhances the gut barrier and mucous layer to reduce the deleterious microbes and other antigen (Collado et al. 2007).
Probiotics regulates various mRNA transcripts of bowel inflammatory symptoms. The Mucin 3 (MUC3) gene were regulated by Lactobacillus strains, in goblet cells of gut mucosa and tend to increased mucus production (Mack 2003). Probiotic help to produce and secrete more cytokines against inflammation by stimulating the lymphocytes. The probiotic products and metabolites regulate inflammatory cytokines such as Tumor necrosis factor alpha (TNFα), Interleukin 1 beta (IL1β), Transforming growth factor beta (TGFβ), etc., (Caballero-Franco et al. 2007).
Lactobacillus plantarum is a potential probiotics, which improves the host's health and benefits against bowel inflammation. L.plantarum has many health-promoting effects and is hence used frequently in food and pharmaceutical industries. Some of the benefits of L. plantarum are the prevention of irritable bowel disease, IBD, cancer, coronary heart disease, etc., (Le and Yang 2018). L.plantarum which inhibits activation of the Nuclear Factor Kappa B (NF-κB) signaling pathway and reduction of gastrointestinal pro-inflammatory factors (Dubuquoy et al. 2002). Lactobacillus sp. are regulating the cytokines factors to reduce the inflammation in the Bowel diseases. L.plantarum which suppressed the pathogens and regulates the production of cytokines (TNF-α, (IL)-1β, IL-6, IL-10, IL-12), and Interferon gamma (IFN-γ) to modulate the balance between T Helper 1 (Th1) and T Helper 2 (Th2) of the T-cells (Ahrne and Johansson Hagslatt 2011). It also modulates the T-cell maturation. L.plantarum induced Treg cells suppress the effector T-cells and activate anti-inflammatory cytokines such as IL10 and TGF β. The suppression of effector T-cells also activates hyperplasia of the goblet cell population (Mayne and Williams 2013;Kim et al. 2020). Lactobacillus interfere with the progression of macrophages Dextran sodium sulphate (DSS) induced IBD mouse. The M1 macrophage reciprocally regulate the inflammatory cytokines in IBD patients as well as IBD mouse (Liew et al. 2010, Khalifa et al. 2022. Prebiotics are indigestible carbohydrates that improve the health of the host by encouraging the growth and activity of specific bacterial species in the colon (Gibson and Roberfroid 1995;Schrezenmeir and de Vrese 2001). After cellulose, lignin is the second most prevalent natural aromatic polymer in terrestrial ecosystems, accounting for about 30% of organic carbon stored in the biosphere (Ayyachamy et al. 2013). Based on their protective properties against lipid peroxidation caused by oxygen radicals, lignin from various sources have been labeled as antioxidants (Ugartondo et al. 2009). In previous studies, lignin fed chicken improved the beneficial bacteria in ileal content and reduced the Escherichia coli. Broiler chicken with 1% lignin fed significantly increasing populations of lactobacilli and bifidobacteria and lowering Escherichia coli loads after the challenge (Baurhoo et al. 2007;Wang et al. 2015). Although lignin's prebiotic effect is modest, the prebiotic nature of lignin fragments may aid in the eradication of intestinal pathogens in cattle products, protecting people from illness. More research is needed to determine the complete prebiotic value of lignin and lignin, as well 1 3 Page 3 of 13 233 as the best dosage for health benefits, animal welfare, and product safety (Ayyachamy et al. 2013). But no studies have looked into whether probiotic bacteria can use lignin as a prebiotic in an in vivo animal model to cure colitis.
In this study, the effect of Lactobacillus plantarum, lignin and their synergistic effects were investigated against DSS induced C57BL/6J mice. The attenuation of inflammatory markers in IBD mice was evaluated by disease index, body weight, and fecal haem. The microscopic evaluation was done by Hematoxylin and eosin (H&E) staining and inflammatory markers were quantified using Enzyme-linked immunosorbent assay (ELISA), quantitative Real-time polymerase chain (qRT-PCR) and Western blot.
Effect of lignin against Lactobacillus plantarum
The effect of lignin (Sigma Aldrich, India) against L. plantarum (University of Gothenburg, Sweden) was performed by a slightly modified method by Etchepare et al. (2016). The overnight culture of L. plantarum was streak in De Man Rogosa and Sharpe agar (MRS agar) media (Himedia, India) and added 10% Dimethyl sulphoxide (DMSO) as a control and different concentrations of lignin (1 Micrometer (µm), 5 μm, 10 μm, 50 and 100 μm) into culture plate and maintained at 37 °C under continuous stirring [150 Rotation per minute (rpm)/minute (min)] to a total of 4 h. The results were expressed as log Colony forming unit (CFU)/ millilitre (ml).
Animals
The experiment was conducted as per The committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines after obtaining approval from the Institute of Animal Ethical Committee (018/IAEC/ KIMS/2019) from Karpaga Vinayaga Institute of Medical Sciences, Chengalpattu, and Tamilnadu, India. Three weeks old male mice (C57BL/6J) were procured from the National Institute of Nutrition, Hyderabad, India and maintained at 25 °C (°C) with 55% (%) humidity and 12 h (Hrs) of cycle Light/Dark in the Polypropylene cage at Karpaga Vinayaga Institute of Medical Sciences. The pellet feed containing sufficient nutrition (Carbohyrates-74%, Protein-22% and Fat 4%) was procured from Agrotech LLP, Pune and used to feed the animals ad libitum. The animals were also provided ad libitum asses to normal drinking water. Treatment was carried out using oral canula.
Colitis induction with treatment
The mice were segregated into five groups and each group holding six mice were used for the experiment. Animals provided with Phosphate buffer solution (PBS) [100 µl (µl) kilogram (kg)/Body Weight (BW)] -Group I (control), animals provided with 2.5% (%) of dextran sodium sulphate (DSS) -Group II (Induced Colitis control), animals provided with 2.5% DSS and Lignin (5 mg kg/BW) for treatment -Group III, and animals provided with 2.5% DSS, L. plantarum strain (1 × 10 8 load/250 µl) and Lignin (5 mg kg/BW) for treatment -Group IV. At the end of the study, the experimental mice were euthanized and sacrificed using Carbon dioxide CO 2 inhalation at the end of the experiment. Immediately, the colon tissues were preserved in 10% buffered formalin for histopathology. While the tissues that were required for other protein related studies and stored at -70 °C immediately upon dissection.
Colitis severity assessment
The preserved colon tissues were washed with Phosphate buffer solution (PBS) and used for assessment of inflammation in the mucous tissue. Macroscopic assessed to identify the damage of tissue and given the scoring as follows: 0 (No inflammation), 1 (Local hyperaemia), 2 (ulceration without hyperaemia), 3 (ulceration and inflammation at one site only), 4 (ulceration and inflammation at more sites) and 5 (ulceration more than 2 cm).
Quantitative real-time RT-PCR (qRT-PCR)
Total Ribonucleic acid (RNA) was extracted from tissues using Tri Isolation Reagent (Thermo Scientific, MA, USA) according to the standard protocol. The extracted RNA was quantified using Nano Drop® 2000 spectrophotometer (Thermo Scientific) and 100 Nano gram (ng)/target was used for Complementary DNA (cDNA) preparation. The upstream forward primers and downstream reverse primers for Signal transducer and activator of transcription 3 (STAT3), Epithelial cadherin (E-cad), Cluster of Differentiation 44 (CD44), and Glyceraldehyde-3-phospate dehydrogenase (GAPDH) are shown in Table 1. Quantitative real-time RT-PCR (qRT-PCR) was performed with template cDNA using the SYBR master mix method. The expression of miRNA was analysed using stem loop RT-PCR assay using U6SnRNA (Table 1) as internal control as described earlier (Ibrahim et al. 2021). Messenger RNA (mRNA) and Micro-RNA (miRNA) expressions were analyzed using quantitative real-time PCR in VII 7 A (Applied Biosystems, Waltham, MA, USA). Results were expressed as relative gene expression using the 2(-Delta Delta C(T)) method (Livak and Schmittgen 2001).
Western blot analysis
Tissues were lysed in Radio immuno precipitation assay (RIPA) lysis buffer (Santa Cruz, Paso Robles, CA, USA) and a 1× protease inhibitor cocktail. The lysate was prepared and preserved at − 80 °C. Protein concentrations of cell lysate were estimated using a Bradford assay at 630 nm (nm). The equivalent of 50 µg (µg) of protein extract was separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (pore size: 0.45 μm, Bio-Rad, Hercules, CA, USA). Membranes were blocked using 5% non-fat dry milk in TBS buffer, followed by probing with the primary antibodies overnight at 4 °C, according to the manufacturer's protocol. The primary antibodies STAT3 (rabbit monoclonal antibody 1:1000) (Biorybt-CB4 0WY), E-cad (rabbit polyclonal antibody 1:1000) (Biorybt, CB4 0WY), and β-actin (rabbit polyclonal antibody 1:2000) (Cell Signaling Technology, Beverly, MA, USA, 4967 S) were incubated overnight at 4 °C, and then washed with Tris buffered saline with Tween 20 (TBST). Washed blots were incubated with horseradish peroxidase-conjugated primary specific secondary antibody at room temperature for 1 h. The blots were visualized by an enhanced chemiluminescence (ECL) system (Pierce, Life Technologies, Austin, TX, USA) and scanned using a LICOR detection system and expressed bands were analyzed using Image Quant software and quantified by densitometry using ImageJ software v1.8 (Ibrahim et al. 2021).
Statistical analysis
The data are presented as the mean ± SD of a representative experiment from three different trials that yielded similar outcomes. The statistical significance between the variables was determined using one-way Analysis of variance (ANOVA) and a post-doc test was performed between the mean differences of P ≤ 0.05 using SPSS 22.0 ver.
Effect of lignin against Lactobacillus plantarum
To our knowledge, this is the first study to indicate that dietary lignin and Lactobacillus plantarum supplementation, either alone or in combination, can reduce Dextran sodium sulphate (DSS)-induced colitis in mice. Lignin toxicity was evaluated against L.plantarum. Lignin concentration from 1 to 100 micromolar µM was tested against L.plantarum. Significant bacterial toxicity was observed above 5 µM concentration whereas below 5 µM growth of Lactobacillus was not inhibiting (Fig. 1). Lignin concentration 5 µM was not significantly toxic P > 0.05. DMSO was used as a control and it showed a log 2 × 10 7 microbial load was recorded. As shown in Fig. 1, the probiotic effect of lignin on lactic acid bacteria is not significant till 5 µM as test concentration.
To determine the impact of lignin mediated L. plantarum against DSS induced colitis
The disease severity of treated lignin with/without L. plantarum was evaluated using Disease index, body weight and colon length. These parameters were observed from both experimental groups (Fig. 2a, b). The Lactobacillus group had a significant recovery disease index (Fig. 2a), body weight (Fig. 2b) and colon length (Fig. 2c) than DSS induced group (P ≤ 0.05). Meanwhile lignin with L. plantarum treated groups had potent reduction of disease index and improved colon length and body weight (P ≤ 0.05). The individual tested group showed less significane than the combined tested groups. Further, the microscopic score and cytokines were evaluated. The disease index was reduced from 4.5 to 2.2 in the DSS group and Lignin with L. plantarum group prospectively. Interestingly, the colon length was observed in the DSS group 4.1 cm whereas the lignin with Lactobacillus group showed an increased colon length of 7.1 centimeter (cm). The body weight of the DSS induced group was reduced from 22.2 gram (gm) to 15 gm, whereas the Lignin with Lactobacillus group was 24 gm, which showed gaining in the body weight compared with the DSS group.
Lactobacillus plantarum retard DSS induced colitis in mice
The histology examination showed multiple erosive lesions and extensive inflammatory cellular infiltrations were observed in the colon tissue of DSS mice. The macrophages, lymphocytes, neutrophils and a few eosinophils were observed in the infiltrated parts of colon tissue (Fig. 3a, b). A significant reduction of infiltrating was observed in the lignin and Lactobacillus group (Fig. 3c, d). The corresponding histological severity score was potentially reduced in Lignin with L. plantarum mice (Fig. 3e). Even though the colon tissue of lignin DSS mice showed inflammatory lesions but the severity of inflammation was lesser than DSS induced mice (P ≤ 0.05) (Fig. 3f).
Lignin controls the inflammatory cytokines in colitis mice
The estimation of cytokines was performed in DSS induced colitis mice. After the14th day of DSS induction the distal colon was excised and homogenated for analyzing inflammatory markers. Lignin with and without the treatment of L. plantarum showed a significant decreasing of inflammatory cytokines revealed that the reduction of infiltrated goblet cells in the site of mucus layer of the colon. The inflammatory cytokines Tumor necrosis factor alpha (TNFα) and Interleukin 1 beta (IL1β) were reduced significantly by P ≤ 0.05 in Lignin with L. plantarum mice compared to DSS induced mice. The immuno-modulatory cytokines Interferon gamma (IFN-γ) and Transforming growth factor beta (TGFβ) were negatively augmented in Lignin with and without L. plantarum treated mice compared to DSS induced mice. Lignin with L. plantarum showed a potentially significant P ≤ 0.05 than Lignin alone group (Fig. 4a, d). The levels of cytokines (TNF-α, INF-γ, IL-1β and TGFβ) increased in DSS-treated mice. The levels of all cytokines studied significantly decreased in Lactobacillus, lignin, and synergistic groups.
Lignin and L. plantarum modulates STAT 3 and E. cad expression in DSS mice
The study evaluates the level of Signal transducer and activator of transcription 3 (STAT3) in lignin treated colitis mice. Lignin treated mice have estimated after the 14th day of DSS induction revealed down regulation of STAT-3 mRNA and protein expression in colitis mice (Fig. 5a, b). The Messenger RNA (mRNA) of STAT 3 was not significantly changed by Lignin associated with L. plantarum treatment P ≥ 0.05, whereas, the protein expression was significantly decreased (P ≤ 0.05) in the treatment. Followed by this treatment cell adhesion epithelial molecule Epithelial cadherin (E.cad) was significantly regulated in inflammatory bowel regions.
The expression of E-cad was down regulated in colitis mice the level of E-cad mRNA and protein expression was increased in lignin with and without L. plantarum treated mice. The expression of the E.cad gene in Lignin with/ without L.plantarum was potentially increased compared to Lignin alone treated mice. These results revealed that the transformation of mesenchymal tissues into normal epithelial cells by reversion of cell adhesion molecule E.cad (Fig. 5c, d).
Micro RNA 199 depletes stem cell modification in DSS induced mice
MicoRNA (miRNA) is single stranded short nucleotide that interferes with the mRNA post transcriptional regulation of the host. CD-44 is an intestinal stem cell marker which is increased in the DSS induced mice, but in lignin with/ without L. plantarum, the marker suppressed their expression due to an increase of E.cad expression in the epithelial cells (Fig. 6a, b and c). The miR-199 level was decreased in colitis mice, whereas Lignin and L. plantarum treatment showed significant P ≤ 0.05 miR-199 progression in colitis mice. These reports conclude that miR-199 is involved in the expression of stem cell marker CD-44 and showed reciprocal regulation in Lignin with/without L. plantarum treated
Discussion
The results explored the biocompatibility of Lignin against Lactobacillus plantarum and the result revealed that the lignin as a biologically compatible prebiotic to L. plantarum was not toxicity upto 5 µM. According to a recent study, less cytotoxicity of lignin makes them suitable candidates for nano system development for cancer medication delivery and therapeutic applications (Imlimthan et al. 2020). In support of this study, (Koh et al. 2013) reported that the prebiotic, tagatose significantly enhanced the growth of probiotic Lactobacillus. The result determined disease index and body weight was reduced and colon length was increased in the lignin with Lactobacillus group. The macroscopic and metabolic regulations were compared with the Dextran sodium sulphate (DSS) group.
Host-probiotic interactions improve gut immune homeostasis and regulate inflammatory markers (Hairul et al. 2014). The reduction in Interleukin (IL)-1, IL-6 and IL-8 levels in Tumor necrosis factor alpha (TNF-α)s-induced cells can be explained by the ability of BA to suppress anti-inflammatory mediators, as further confirmed in an in vivo model. Comparable findings were obtained in many in vitro studies. Lactobacillus acidophilus, Bacillus coagulans and Bacillus subtilis exhibited potential anti-inflammatory effects on colorectal adenocarcinoma cells (Caco2), dendritic and peripheral blood mononuclear (PBM) cells (Khalifa et al. 2022). These results confirm that gut laminal and epithelial cells of mucous play a major role in probiotic interactions via secreting inflammatory mediators such as chemoattractant proteins, interleukins and infiltration signals. Similarly, (Kangwan et al. 2022) reported that Lactobacillus pentosus, which was obtained from fermented tea leaves, improved colon abnormality. Treatment of probiotics significantly protected the colon from being inflamed by mitigating histological and clinical damage traits, enhancing the intestinal barrier integrity, and attenuating inflammation symptoms induced by DSS induction. (Hairul et al. 2011;Khalifa et al. 2022).
The crypt structure was abnormal in the DSS group due to crypt loss, epithelial cell death, and significant inflammatory cell infiltration. Whereas, DSS-induced colon shortening and acute inflammation were reduced by the Lactobacillus, lignin, and syerngistic therapies. A considerable decrease in colon length as a result of intestinal injury is a frequent hallmark of DSS-induced colitis (Rumi et al. 2004;Mizoguchi and Mizoguchi 2008). Both macroscopic and microscopic observations revealed a decrease in the severity of DSS induced colitis across the treatment groups. Prebiotic fibres that are soluble and non-viscous were reported to alleviate the symptoms of inflammatory bowel diseases (Hardy et al. 2013). In rats, symbiotic Lactobacillus and fructo oligosaccharide supplementation was found to reduce intestinal and systemic inflammation in Crohn's disease (Lindsay 2006;Delcenserie et al. 2008).
Rumen Lactobacillus regulates the immune response and acts as an anti-inflammatory strain and balance the cytokine secretion, whereas the other two active strains were particular inhibition on anti-inflammatory cytokines (Arokiyaraj et al. 2014). Few studies reported that, the Lactobacillus sp. and some rumen bacteria stimulate low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8), while activating high levels of Interferon gamma (IFN-γ) and IL-12 these two active cytokines to promote cellular immunity and enhance the clearance of pathogens. Probiotic strains might boost host immune status through activation of the particular and nonspecific immune pathways. This also can involve modulating of humoral, cellular, and nonspecific immunity (Hairul et al. 2014;Delcenserie et al. 2008).
Several cytokines generated by lactic acid bacteria serve critical roles in immunological modulation, and they are involved in colitis pathophysiology. These pro-inflammatory cytokines are important in building an immunogenic setting and in the anti-inflammatory response (Kim et al. 2015;Mantovani et al. 1992). An earlier study revealed Lactobacillus and prebiotic (tagatose) have inhibited DSS induced pro-inflammatory cytokines and prevented initiation of the inflammatory response (Son et al. 2019). In this, study the levels of cytokines (TNF-α, INF-γ, IL-1β and TGF-β) increased in DSS-treated mice. The levels of all cytokines studied significantly decreased in Lactobacillus, lignin, and synergistic groups. STAT3 is involved in a number of autoimmune diseases, including inflammatory bowel disease (IBD) (Sugimoto 2008).
In both Ulcerative colitis (UC) and Crohn's disease (CD), there was an increase in total STAT3 protein relative to noninflammatory control cells, and total STAT3 corresponded with greater activated pSTAT3 in tissue sections from both UC and CD (Mudter et al. 2005). Lower expression of E-cadherin on gut epithelial and immune cells resulted in more gut inflammation in DSS-induced colitis (Ooi et al. 2013). In this study the mRNA of STAT 3 was not significantly changed by Lignin associated with L.plantarum treatment, where the protein expression was significantly down regulated. In the E.cad mRNA and protein expression in the lignin with or without L.plantarum were increased potentially which was compared to Lignin alone treated group. CD44 has been identified as a potential therapeutic target for inflammatory disorders such as IBD, rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases (Wittig et al. 2002). The expression of CD44, was stronger in the UC-associated lesions of colon carcinoma (Mikami et al. 2000). It is well known that the expression of CD44 variant isoforms increases in inflammatory infiltrates in mouse and human colitis, and that blocking or deleting CD44 isoforms prevents the development of experimental colitis. Previous investigations have showed that established experimental colitis is cured by short-term administration of anti-CD44 antibody, which prevents T cell extravasation and recruitment to the intestinal mucosa (Farkas et al. 2005). CD44 was identified as a direct target of miR-199a and contributed to a significant role in carcinogenesis (Henry et al. 2010;Gao et al. 2015). As discussed above, aberrant expressions of CD44 induces the infiltration of immune cells into the lamina propria. It is well established that 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p. Also, the overexpression of miR-199a-3p significantly inhibited CD44 expression (Gao et al. 2015). Thus induction of miR-199 expression targets/reduces the expression of CD44, which subsequently attenuates the infiltration of immune cells and finally plays an important role in the in control of DSS induced colitis. In this study revealed that the E.cad expression increased in the epithelial cells. The lignin with L.plantarum group controls the inflammatory progression in the colitis mice through miR-199.
Conclusion
In conclusion, we used a Dextran sodium sulphate (DSS)induced colitis mouse model to evaluate the synergic effects of Lignin and Lactobacillus plantarum against inflammatory bowel disease (IBD). Our findings suggest that combining Lignin with L.plantarum improves intestinal damage recovery and suppresses the recurrence of experimental colitis, indicating that this new protective or preventative strategy for IBD could be used in the food sector. | 2022-10-12T13:53:15.592Z | 2022-10-12T00:00:00.000 | {
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14080705 | pes2o/s2orc | v3-fos-license | Polarization selective beam shaping using nanoscale dielectric metasurfaces
Metasurfaces consisting of ultrathin nanostructures are utilized to control the properties of light including its phase, amplitude and polarization. Hereby, we demonstrate the capability of such structures to perform arbitrary polarization selective beam shaping using dielectric nanoscale metasurfaces implemented in silicon. By illuminating the structure with right handed circular polarization we reconstruct a desired image. When switching the polarization into its orthogonal state, we obtain the reconstruction of a different image. This demonstration shows the potential of using dielectric metasurfaces for high efficiency beam shaping applications in general, and specifically for polarization coded beam shaping. ©2015 Optical Society of America OCIS codes: (230.5440) Polarization-selective devices; (160.3918) Metamaterials. References and links 1. M. Born and E. Wolf, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light, 7th ed. 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Tervo, “Polarization conversion in conical diffraction by metallic and dielectric subwavelength gratings,” Appl. Opt. 46(20), 4258–4265 (2007). 8. C. Gu and P. Yeh, “Form birefringence dispersion in periodic layered media,” Opt. Lett. 21(7), 504–506 (1996). 9. J. Tervo, V. Kettunen, M. Honkanen, and J. Turunen, “Design of space-variant diffractive polarization elements,” J. Opt. Soc. Am. A 20(2), 282–289 (2003). 10. U. Levy, C.-H. Tsai, L. Pang, and Y. Fainman, “Engineering space-variant inhomogeneous media for polarization control,” Opt. Lett. 29(15), 1718–1720 (2004). 11. U. Levy, H.-C. Kim, C.-H. Tsai, and Y. Fainman, “Near-infrared demonstration of computer-generated holograms implemented by using subwavelength gratings with space-variant orientation,” Opt. Lett. 30(16), 2089–2091 (2005). 12. T. Kaempfe, P. Sixt, D. Renaud, A. Lagrange, F. Perrin, and O. Parriaux, “Linear to radial/azimuthal polarization converter in transmission using form birefringence in a segmented silicon grating manufactured by high productivity microelectronic technologies,” in (2014), Vol. 9130, p. 91300W–91300W–7. 13. Z. Bomzon, G. Biener, V. Kleiner, and E. Hasman, “Radially and azimuthally polarized beams generated by space-variant dielectric subwavelength gratings,” Opt. Lett. 27(5), 285–287 (2002). 14. G. M. Lerman and U. Levy, “Generation of a radially polarized light beam using space-variant subwavelength gratings at 1064 nm,” Opt. Lett. 33(23), 2782–2784 (2008). 15. W. S. Mohammed, A. Mehta, M. Pitchumani, and E. G. Johnson, “Selective excitation of the TE01 mode in hollow-glass waveguide using a subwavelength grating,” Photonics Technol. Lett. IEEE 17(7), 1441–1443 (2005). 16. N. Yu and F. Capasso, “Flat optics with designer metasurfaces,” Nat. Mater. 13(2), 139–150 (2014). #243526 Received 24 Jun 2015; revised 30 Jul 2015; accepted 16 Aug 2015; published 19 Aug 2015 © 2015 OSA 24 Aug 2015 | Vol. 23, No. 17 | DOI:10.1364/OE.23.022611 | OPTICS EXPRESS 22611 17. A. V. Kildishev, A. Boltasseva, and V. M. Shalaev, “Planar Photonics with Metasurfaces,” Science 339(6125), 1232009 (2013). 18. A. Pors, M. G. Nielsen, R. L. Eriksen, and S. I. Bozhevolnyi, “Broadband Focusing Flat Mirrors Based on Plasmonic Gradient Metasurfaces,” Nano Lett. 13(2), 829–834 (2013). 19. M. Decker, I. Staude, M. Falkner, J. Dominguez, D. N. Neshev, I. Brener, T. Pertsch, and Y. S. Kivshar, “HighEfficiency Dielectric Huygens’ Surfaces,” Adv. Opt. Mater. 3(6), 813–820 (2015). 20. P. Moitra, B. A. Slovick, Z. G. Yu, S. Krishnamurthy, and J. Valentine, “Experimental demonstration of a broadband all-dielectric metamaterial perfect reflector,” Appl. Phys. Lett. 104(17), 171102 (2014). 21. D. Lin, P. Fan, E. Hasman, and M. L. Brongersma, “Dielectric gradient metasurface optical elements,” Science 345(6194), 298–302 (2014). 22. Y. F. Yu, A. Y. Zhu, R. Paniagua-Domínguez, Y. H. Fu, B. Luk’yanchuk, and A. I. Kuznetsov, “Hightransmission dielectric metasurface with 2π phase control at visible wavelengths,” Laser Photonics Rev. 9(4), 412–418 (2015). 23. A. Arbabi, Y. Horie, M. Bagheri, and A. Faraon, “Complete control of polarization and phase of light with high efficiency and sub-wavelength spatial resolution,” arXiv:1411.1494v1 [physics.optics] (2014). 24. A. Arbabi, Y. Horie, A. J. Ball, M. Bagheri, and A. Faraon, “Subwavelength-thick lenses with high numerical apertures and large efficiency based on high-contrast transmitarrays,” Nat. Commun. 6, 7069 (2015). 25. R. Paniagua-Dominguez, Y. F. Yu, A. E. Miroschnichenko, L. A. Krivitsky, Y. H. Fu, V. Valuckas, L. Gonzaga, Y. T. Toh, A. Y. S. Kay, B. Luk’yanchuk, and A. I. Kuznetsov, “Generalized Brewster-Kerker effect in dielectric metasurfaces,” 12 (2015). 26. S. Liu, M. B. Sinclair, T. S. Mahony, Y. C. Jun, S. Campione, J. Ginn, D. A. Bender, J. R. Wendt, J. F. Ihlefeld, P. G. Clem, J. B. Wright, and I. Brener, “Optical magnetic mirrors without metals,” Optica 1(4), 250 (2014). 27. K. E. Chong, I. Staude, A. James, J. Dominguez, S. Liu, S. Campione, G. S. Subramania, T. S. Luk, M. Decker, D. N. Neshev, I. Brener, and Y. S. Kivshar, “Polarization-independent silicon metadevices for efficient optical wavefront control,” Nano Lett. 15(8), 5369–5374 (2015). 28. Y. H. Fu, A. I. Kuznetsov, A. E. Miroshnichenko, Y. F. Yu, and B. Luk’yanchuk, “Directional visible light scattering by silicon nanoparticles,” Nat. Commun. 4, 1527 (2013). 29. Y. Yang, W. Wang, P. Moitra, I. I. Kravchenko, D. P. Briggs, and J. Valentine, “Dielectric Meta-Reflectarray for Broadband Linear Polarization Conversion and Optical Vortex Generation,” Nano Lett. 14(3), 1394–1399 (2014). 30. N. Yu, P. Genevet, M. A. Kats, F. Aieta, J.-P. Tetienne, F. Capasso, and Z. Gaburro, “Light Propagation with Phase Discontinuities: Generalized Laws of Reflection and Refraction,” Science 334(6054), 333–337 (2011). 31. W. T. Chen, K.-Y. Yang, C.-M. Wang, Y.-W. Huang, G. Sun, I.-D. Chiang, C. Y. Liao, W.-L. Hsu, H. T. Lin, S. Sun, L. Zhou, A. Q. Liu, and D. P. Tsai, “High-Efficiency Broadband Meta-Hologram with PolarizationControlled Dual Images,” Nano Lett. 14(1), 225–230 (2014). 32. C. Pfeiffer, C. Zhang, V. Ray, L. J. Guo, and A. Grbic, “High performance bianisotropic metasurfaces: asymmetric transmission of light,” Phys. Rev. Lett. 113(2), 023902 (2014). 33. E. Hasman, V. Kleiner, G. Biener, and A. Niv, “Polarization dependent focusing lens by use of quantized Pancharatnam–Berry phase diffractive optics,” Appl. Phys. Lett. 82(3), 328–330 (2003). 34. I. Moreno, J. A. Davis, T. M. Hernandez, D. M. Cottrell, and D. Sand, “Complete polarization control of light from a liquid crystal spatial light modulator,” Opt. Express 20(1), 364–376 (2012). 35. R.-C. Tyan, P. C. Sun, and Y. Fainman, “Polarizing beam splitters constructed of form-birefringent multilayer gratings,” In 2689, 82–89 (1996). 36. B. Walther, C. Helgert, C. Rockstuhl, F. Setzpfandt, F. Eilenberger, E.-B. Kley, F. Lederer, A. Tünnermann, and T. Pertsch, “Spatial and spectral light shaping with metamaterials,” Adv. Mater. 24(47), 6300–6304 (2012). 37. R. Gerchberg and W. Saxton, “A practical algorithm for the determination of phase from image and diffraction plane pictures,” Optik 35, 237–250 (1971).
Introduction
Controlling the refractive index of materials at will is a prime goal of photonics research.It is well known that the refractive index can be manipulated by structuring the material at the subwavelength scale [1], using the concept of effective index.Over the years, this capability had led to a large number of demonstrations in which artificial dielectric materials has been created [2][3][4][5][6][7][8][9][10][11].In particular, such structures exhibit strong birefringence properties, also known as "form birefringence", which can be an order of magnitude higher than the birefringence of natural materials.Using this property, numerous demonstrations of polarization transformations could be achieved, including for example the generation of radially and azimuthally polarized light [12][13][14][15].Following this plethora of demonstrations, recent years brought into the forefront the concept of metallic metasurface, in which thin metallic nanostructures have been used for the purpose of controlling the properties of light [16][17][18] polarization of the incident beam, yet with limited energetic efficiency due to the ohmic loss in the metal.The latter claim is particularly true for transmission type devices.This limitation has tailored the research focus back towards dielectric metasurfaces, with several recent important demonstrations, e.g. the use of thin silicon nanoantennas for phase and polarization control [19][20][21][22][23][24][25][26][27] as well as directional light scattering and reflection [28,29].
Hereby, we demonstrate the capability of a dielectric metasurface to perform arbitrary polarization selective beam shaping.While polarization selectivity in such structures has been observed, e.g. for the purpose of polarization and wavelength dependent focusing and splitting [30][31][32][33][34][35][36], achieving an arbitrary polarization selective beam shaping with a single dielectric metasurface is yet to be demonstrated.Specifically, we show here for the first time that by modifying the polarization of an incident beam of light one can switch between two arbitrarily chosen images.It should be noticed that polarization selective hologram was recently demonstrated in metallic metasurfaces in reflection configuration [32].
Theoretical background
We begin by defining the problem in hand.Given a single metasurface, we would like to illuminate it by a right handed circularly polarized light and achieve a reconstruction of a desired image at a specific distance away of the metasurface.Furthermore, by switching the polarization to its left handed orthogonal state, the same metasurface should provide a reconstruction of a different image per our choice, which is located at the same image plane.This is shown schematically in Fig. 1.We now turn into selecting our metasurface and study its properties.Our metasurface consists of a pixilated structure.In each pixel, a subwavelength periodic structure is defined and etched into the silicon substrate.A schematic representation of this structure is given in Fig. 2. Fig. 2. Schematic representation showing a top view of our dielectric metasurface.In each pixel a nanoscale subwavelength 1-D grating is defined.The orientation of this structure is kept as a free parameter and is used to control the phase of the light emanating from our metasurface.
We consider a plane wave transmitted through the metasurface located at z = 0, with x and y being the transverse coordinates.Neglecting Fresnel reflections, the transmitted field is given by, are the rotation and the Jones matrix, respectively, and in V represents the Jones vector of the incident field.The matrix G describes the birefringence of the subwavelength grating in each pixel.The obtained phase retardation, φ(x,y) is determined by the geometry of the subwavelength grating (duty cycle and grating depth), and by the refractive index of the substrate material.θ(x,y) represents the orientation angle of the subwavelength grating at each pixel with respect to the x axis (see Fig. 2).The validity of Eq. ( 1) relies on the thin element approximation, which holds as long as the pixel size is large and the grating depth is not too large compared to the wavelength.Assuming constant retardation for each of the pixels (i.e. the grating is identical at each pixel, with the result of space invariant values of φ and G), the rotation angle ( ) , x y θ is the only free parameter.For right and left hand circularly polarized (RCP and LCP respectively) incident light with the corresponding Jones vectors V inR = [1, i] T and V inL = [1, -i] T , we can now express Eq. ( 1) as [6,10,11], respectively.
The cosine term can be eliminated by designing the grating parameters such that a π phase retardation (φ = π) is achieved.For such a case, the metasurface provides pure phase modulation, with the obtained phase being twice the rotation angle.Therefore, the desired phase modulation can be achieved simply by varying the orientation of the periodic structure in each pixel, such that a continuous phase modulation is achieved using a binary grating.This approach eliminates the need for complicated multiple step or gray scale fabrication procedures, significantly reducing the fabrication complexity and cost.We used pixel size of 8 micron to be larger then the wavelength of optical fields to allow validity of thin element approximation [37].The pixel size can be further decreased down to about 3 microns [11] while preserving the good performance of the hologram.The 8 micron pixel size was chosen as a convenient number which is comparable to commercial available liquid crystal on silicon spatial light modulators.Further reduction of the pixel size is possible, nut requires a slightly different mechanism, in which the scattering efficiency from a single rod is enhanced by operating close to resonance.
Design and simulations
Our device was designed based on a modified version of the Gerchberg Saxton (G-S) algorithm [37].Specifically, we take advantage of the phase degree of freedom, namely, we are only interested in the amplitude of the reconstructed images, and thus their phases can be used as free parameters.
Our goal is to achieve two independent images in the Fresnel domain, at a distance Z0 away of the metasurface plane.If illuminated by RCP the complex transmission function of the metasurface is given by H(x,y), while for LCP illumination, the complex transmission function of the metasurface is given by -H*(x,y).In the output plane, the images are given by the Fresnel transform, i.e.
Can the two distributions ( ) represent two non-identical intensity functions?Indeed, this is possible.We now release all restrictions on the displayed phase, and only require that the amplitude of those distributions should match the desired to images.
We now need to find the function H(x,y) which can generate the desired distributions I 1 and I 2 .Such a function can be found by using the G-S algorithm, bearing in mind that the amplitude distribution of f 1 and f 2 are the constraints i.e.
Fabrication and measurements
After calculating the value of H(x,y) we can now turn into fabricating the actual device which will produce the desired phase distribution.First, we find the desired rotation angle for each pixel which is simply half of the phase of H(x,y).Once the orientation of the grating is know, we have all the needed parameters for the device fabrication.Our metasurface was fabricated on a double sided polished 2-inch p-type silicon wafer.First, an etching mask was defined in the resist (ZEP 520A) by Electron Beam Lithography (Raith E_Line 150).Next the pattern was transferred to the silicon substrate by Reactive Ion Etching (Corial 200RL) of silicon (depth ~500nm to provide π retardation).Finally the resist was stripped and the fabricated element was inspected in Scanning Electron Microscope (SEM), and a top view of the final structure is shown in a Fig. 3.The metasurface consist of 128X128 pixels, 8 micron each with sub wavelength grating ( 400nm Λ = , duty cycle 50%) embedded into each pixel.To characterized the polarization selective dielectric metasurface we illuminated the device at a wavelength of 1550 nanometer using a single lens imaging system (F = 150mm) with a magnification of 4. A fiber coupled laser (Thorlabs S3FC1550) was connected to the laser collimator followed by a quarter wave plate that was mounted on a manual rotation stage for converting the incident linearly polarized beam into left and right circularly polarized beam.A near-infrared InGaAs 2-D focal plane array (Sensors Unlimited) was placed at some distance away of the back focal plane of the optical system in order to capture the images in the Fresnel domain.The schematic diagram of measurement system is shown in Fig. 4. The obtained experimental results for RCP and LCP incident beams are shown in Fig. 5 a,b respectively.The corresponding computer simulation results with the same color scheme as the obtained results are presented in Fig. 5 c,d respectively.As an example, the two set of letter "POL" and "SEL" were selected, to represent the polarization selective property of our dielectric metasurface.The calculated purity of the reconstructed image in the simulation, defined as a ratio between the all optical power of the image and the integrated optical power of the reconstructed object, was found to be 17% and 20% for the RCP and LCP incident beam respectively.In our measurements we found the purity of the conversation for LCP and RCP to be ~13% and ~11% respectively.This can be further improved by using a more elaborated optimization process.The efficiency (the ratio between an optical power in the image plane and the incident power before the element) was measured to be 57%.Most of the power loss (~30%) is due to Fresnel reflection in the interface between the air and the silicon.This can be easily overcome by using an additional antireflective coating layer of silicon nitride on the back side of the fabricated element.
Summary
In conclusion we demonstrate the design, fabrication and experimental characterization of a dielectric metasurface acting as a polarization selective computer generated hologram, and operating at near infrared wavelength of 1550 nm.The device is based on allocating a sub wavelength grating to each pixel.By controlling the orientation of this grating one can achieve desired phase modulation.Taking advantage of the conjugate phase modulation obtained by illuminating the dielectric metasurface with LCP and RCP, we were successful in demonstrating of two independent images, for the two orthogonal polarization states.The reconstruction is obtained in the Fresnel domain.The energy in the reconstructed letters with respect to the incident power is given by the multiplication of the efficiency and the purity, i.e. it is in the range of 6%.Further improvement can be achieved by the use of AR coating and advanced optimization.The quality of the reconstructed images can be further improved by using additional degrees of freedom, e.g.grating depth and/or duty cycle.The demonstrated concept paves the way for achieving arbitrary polarization selective beam shaping using a single dielectric metasurface.
the phase distributions are free parameters with no restrictions.Applying the G-S algorithm with these constraints, we can find the function H(x,y) that would reconstruct the two desired images with minimal error.
Fig. 3 .
Fig. 3. Left -Scanning Electron Microscope micrographs showing a top view of fabricated device.Right -Magnified image.Scale bar is 2 micron in both figures.
Fig. 5 .
Fig. 5. Experimental result, showing the obtained images obtained by illuminating our dielectric metasurface with a) left and b) right circularly polarized light.The corresponding simulated results are shown in Fig. 5c and 5d respectively. | 2017-04-26T02:27:25.211Z | 2015-08-24T00:00:00.000 | {
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238681849 | pes2o/s2orc | v3-fos-license | An Overview of the Practices and Management Methods for Enhancing Seed Production in Conifer Plantations for Commercial Use
: Flowering, the beginning of the reproductive growth, is a significant stage in the growth and development of plants. Conifers are economically and ecologically important, characterized by straight trunks and a good wood quality and, thus, conifer plantations are widely distributed around the world. In addition, conifer species have a good tolerance to biotic and abiotic stress, and a stronger survival ability. Seeds of some conifer species, such as Pinus koraiensis , are rich in vitamins, amino acids, mineral elements and other nutrients, which are used for food and medicine. Although conifers are the largest (giant sequoia) and oldest living plants (bristlecone pine), their growth cycle is relatively long, and the seed yield is unstable. In the present work, we reviewed selected literature and provide a comprehensive overview on the most influential factors and on the methods and techniques that can be adopted in order to improve flowering and seed production in conifers species. The review revealed that flowering and seed yields in conifers are affected by a variety of factors, such as pollen, temperature, light, water availability, nutrients, etc., and a number of management techniques, including topping off, pruning, hormone treatment, supplementary pollination, etc. has been developed for improving cone yields. Furthermore, several flowering-related genes ( FT , Flowering locus T and MADS-box, MCMI , AGAMOUS , DEFICIENCES and SRF ) that play a crucial role in flowering in coniferous trees were identified. The results of this study can be useful for forest managers and for enhancing seed yields in conifer plantations for commercial use.
Introduction
Conifer species are typically tall perennial and evergreen trees or shrubs. They are the largest and most important species of gymnosperms with high economic and ecological values, which have a potential life span of 1000 years under natural growth conditions [1]. Conifers include about 613 species, mainly in the Pinaceae, Taxodiaceae and Cupressaceae families that are widely distributed over the world [2,3]. They appeared on earth's surface about three hundred million years ago [4]. The number of species of angiosperms is estimated at about 300,000, conifers dominated the forests of the Jurassic until the rise Figure 1. Flowering and seed yields in conifers are affected by a variety of factors (pollen, temperature, light, water availability, nutrients, etc.) and that for improving cone yields a number of management techniques have been tested, including topping off, pruning, fertilization, hormone regulation, supplementary pollination, etc. Panel (a) represents the 5-10-year-old tree of conifer species (P. koraiensis for example), panel (b) is topping off, panel (c) is pruning, panel (d) shows ideal tree shape, panel (e) shows fertilization and watering, panel (f) depicts hormone application, panels (g) and (h) are female and male cones, respectively, panel (i) shows pollen storage, panel (j) depicts supplementary pollination and panel (k) shows improved cone yield.
Pollen
Pollen, the material basis for transmitting genetic information, is one of the important factors for seed formation [20]. Under natural conditions, most conifer species are pollinated by wind (anemophilous pollination). Pollen can travel hundreds of miles and at thousands of feet in altitude while maintaining some viability [21,22]. A typical young reproductive P. taeda can shed about one kg of pollen in a two-week period. Each year, about one billion seedlings of P. taeda are planted in the Southeastern US, with a potential Figure 1. Flowering and seed yields in conifers are affected by a variety of factors (pollen, temperature, light, water availability, nutrients, etc.) and that for improving cone yields a number of management techniques have been tested, including topping off, pruning, fertilization, hormone regulation, supplementary pollination, etc. Panel (a) represents the 5-10-year-old tree of conifer species (P. koraiensis for example), panel (b) is topping off, panel (c) is pruning, panel (d) shows ideal tree shape, panel (e) shows fertilization and watering, panel (f) depicts hormone application, panels (g) and (h) are female and male cones, respectively, panel (i) shows pollen storage, panel (j) depicts supplementary pollination and panel (k) shows improved cone yield.
Pollen
Pollen, the material basis for transmitting genetic information, is one of the important factors for seed formation [20]. Under natural conditions, most conifer species are pollinated by wind (anemophilous pollination). Pollen can travel hundreds of miles and at thousands of feet in altitude while maintaining some viability [21,22]. A typical young reproductive P. taeda can shed about one kg of pollen in a two-week period. Each year, about one billion seedlings of P. taeda are planted in the Southeastern US, with a potential yield of ten million tons of pollen over a short rotation from this species alone. Pollen dispersal not only promotes gene flow between different populations, it also effectively increases species diversity and richness [23]. In conifer species, pollen viability and quantity are crucial for the success of pollination. Low pollen viability, high abortion rate and unsynchronized flowering are common in conifers and thereby reduce the rate of successful zygotic fertilization [24]. Environmental conditions have a great impact on the flowering period, and pollen viability is generally reduced or lost during abnormal weather conditions [25,26]. In addition, pollen development and maturation are easily disturbed by pollutants due to industrialization [27]. Combined with the above factors, the probability of normal pollination of female cones is low in conifers, which has a direct effect on the formation and development of seed. The low viability of pollen is to some extent compensated by extraordinary pollen abundance. The synchronous release of pine pollen, for example, creates visible yellow pollen clouds, which blanket the land as a fine layer of snow. Pine pollen has been collected and distributed as a food and as a medicine for 5000 years (Tang Materia Medica 657-659 C.E.).
Temperature and Light
Low temperature affects germination by regulating the vernalization of seeds [28]. Vernalization is a process that depends on a chilling requirement to produce flowering and a good fruit crop. For example, peaches need a few hundred hours below 7 • C to satisfy the chilling requirement to break dormancy, promote flowering and have successful fruit production (https://www.greenwoodnursery.com/peach-tree-chilling-requirements accessed on 31 March 2021). The overall germination rate and the time needed for germination of seeds varies significantly under different temperature conditions [29][30][31]. A suitable temperature is also a precondition for flower bud formation and an important factor affecting pollen longevity and viability [32]. In addition, the number of male cones is generally more than female cones in conifers, which is not an ideal situation. Many experimental results may be caused by the different mechanisms of male and female flower buds in response to temperature, as temperature indirectly regulates the sex expression of conifer species by changing the hormone balance in flower buds [33,34]. Conifer seeds typically can be stored in the cold, e.g., at around zero degrees + or −2 • C for several months, then planted to break dormancy. Temperature signals can also regulate the activity of various enzymes and affect the metabolism in various biochemical reactions of conifer species; thus, becoming a vital participant in photosynthesis and respiration [35][36][37].
Light is also a crucial environmental signal and the main energy source for the plant's photosynthesis and respiration [38]. There is a high canopy density with many lateral and dead branches that affect the supply of normal light in conifers. Light intensity, spectral composition and photoperiod are important factors for conifer species growth and development, and they influence numerous physiological and biochemical reactions that cause changes in their morphology and reproductive characteristics [39]. In addition, different tree species have different demands and reaction mechanisms for light, which are affected by external environmental factors as well as their biological characters [40,41]. With the increase in forest age and the crown canopy density, the available light under the canopy will decrease, and flowering and reproduction will inevitably be affected. Therefore, an appropriate planting density, pruning and topping off can ameliorate the insufficient light of conifer species [42].
Water and Nutrient Fertilization
Water and nutrient fertilization are equally indispensable for plant seed formation and development [43]. Plant roots absorb nutrients from the soil, which is rich in minerals and organic matter to feed seed formation. Nutrient fertilization typically provides nitrogen, phosphate and potassium. Nitrogen deficiency often limits growth due to the need for substantial amounts being needed for biosynthesis of proteins and nucleic acids, while phosphorous is needed for energy metabolism and nucleic acid biosynthesis. Potassium is needed for salt balance, transport of water and nutrients. Fertilization maintains the stability of mineral circulation in the soil, which ensures the support capacity of the soil for plants and promotes the biosynthesis of proteins, amino acids and vitamins [44,45]. Plant growth and development are the result of the interaction of water and fertilizer. Water provides a good moist environment for plant growth, which determines the activity of roots and microbes, and contributes to the construction of a good root system [46]. In the Southeastern US, with extensive plantations of southern pines, fertilization is a common practice. In this region, chronic deficiencies are found for both nitrogen and phosphorus [47]. The internal rate of return from mid-rotation fertilization of nitrogen and phosphorus was calculated to be 16%. Because most conifer species grow in mountainous areas with poor soil and harsh environmental conditions, the nutrients are typically limited in the soil [48]. Water and fertilization not only can effectively improve the soil environment, but can regulate the nutrient supply and growth, photosynthesis and other metabolic processes [49,50].
Molecular Mechanisms
The molecular mechanisms of flowering and seed production are complex, and the related genes can directly or indirectly interfere with the sex differentiation of flower, time of anthesis and seed development, resulting in the seed formation differences among individuals or species. Flower-related genes, such as FT (Flowering locus T) and MADSbox (MCMI, AGAMOUS, DEFICIENCES and SRF box), have been identified to play a crucial role in flowering in conifer trees.
In the study of P. massoniana, Chen [51,52] cloned PmFT1 and PmEMF2 genes by RT-PCR and RACE technology, and found that the two genes were highly expressed during the development of male and female cones, respectively, suggesting that they were involved in the development of flowers and seed formation. The CO gene is an important member of the regulation of sunshine length between the circadian clock and flowering time genes, which can combine light signals with circadian clock signals to regularly activate the expression of the FT gene; thus, inducing flowering. In Ginkgo biloba, the study of the effects of photoperiod on the GbCO gene transcription and seedling growth showed that GbCO activates FT transcription to control flowering [53,54]. The plant LEAFY gene encodes a class of plant-specific transcription factors, which play an important role in the transition from vegetative to reproductive growth of flowering plants. The LEAFY and UFO genes have a similar function in conifers; the LEAFY gene interacts with the UFO gene by cloning and yeast two-hybrid techniques, and it is speculated that the UFO gene in Metasequoia glyptostroboides may act as a transcriptional cofactor to regulate the LEAFY gene activity and, thus, participate in the regulation of the flower meristem development [55]. MADS-box genes, a class of important transcriptional regulatory factors in eukaryotes (animals, plants and fungi), play an important role in growth and development regulation and signal transduction [56,57]. There are studies that show that three MADS-box genes (PrMADS1, PrMADS2 and PrMADS3), LEAFY/FLORICAULA and NEEDLY (NLY) were highly participated in the early stages of initiation and differentiation of P. radiata male and female cone buds, as well as vegetative buds [58]. In another study, a transcriptome data analysis showed that the enhanced transcriptional activity of MADS-box transcription factors was also closely related to the formation of early cones, in particular the process of sex reversal [59,60]. Thus, it can be also speculated that MADS-box transcription factors are a key gene family in the molecular mechanism of conifer flowering and seed production. The research on the molecular mechanism of flowering and seed production of conifers is still scarce. Therefore, it is still the mainstream direction of future research. Despite a long growth cycle and large genome compared with the broad-leaved trees, conifer species were developed and used with some traditional tree breeding methods.
Technical Measures of Breeding and Management
In forest tree breeding, selecting parents with good characters is the basis of tree improvement. Efficient management technology is the guarantee of a high quality and yield. Conventional methods, including artificial pollination, hormone treatment, pruning, water and fertilization coupling, can promote the flowering and reproduction of conifer species. One technical measure that greatly improves the efficient management of a genetic improvement program is genetic fingerprinting, which provides a high level of quality control on genetic selection [61]. In addition to providing an accurate inventory, DNA markers can estimate genetic diversity, define genetic load, estimate the degree of inbreeding, identify quantitative trait loci (QTLs) and using high throughput genomic markers allow for a highly efficient genomic selection where many genomic markers associated with favorable traits can be selected simultaneously [62].
Supplementary Pollination
Plant inbreeding refers to the combination of male and female gametophytes on the same plant, or the mating between individuals with the same genotype [63]. Because of the high genetic diversity and load, close genetic relationships can lead to poor selection. Inbred lines are rare in conifer breeding programs [64,65]. The genetic characters of inbred offspring are mediocre, including growth, cone and wood traits [66]. Most conifer species are monoecious with a high possibility of inbreeding, which causes a high abortion rate of flowers and low cone yields [67]. Therefore, supplemental pollination avoids inbreeding and increases the efficiency of crosses. This method can be applied to open pollination or to controlled crosses to effectively make up for low natural pollination. The pollen with desired genes is scattered on the stigma of female flowers to create more fertilization events [66]. Understanding the details of flowering is needed to improve the sustainability and effectiveness of pollination. In the study of Douglas fir, (Pseudotsuga menziesii), the rate of external pollination and the inbreeding rates were 10% to 28% and 12% to 17%, respectively [68]. In addition to high inbreeding, pollen contamination from unwanted genotypes is a serious detriment to defined breeding. In Scots pine (P. sylvestris), a hand pollinator was used on female cones at the end of May. The amount of pollen per individual plant was 0.06~0.08 mL, and the success rate was 66-84%; whereas the success rate was 10-23% when the pollination amount per plant was 0.03-0.05 mL that was applied by a long aluminum pole duster [69]. The highest success rate of pollination was 69% using pressurized backpack sprayers to pollinate the female flowers. Therefore, methods and amounts are also critical factors affecting the success of pollination.
Supplementary pollination can introduce elite pollen into female cones to broaden the genetic base, reduce pollen contamination, increase cone yield and maximize genetic gain [70]. Understanding the flowering habits of each individual and selecting elite parents are crucial for the efficient improvement of conifer species. It is essential to select the best plants with good characters, fast growth, a high yield and allowing a robust pollen collection. Furthermore, weather condition is a key factor affecting the success rate of pollination [71]. As Additionally, the well-timed isolation of cones (method and bags) of selected mother trees has a crucial importance in the process of artificial pollination.
Topping Off
Topping off (cutting off the apical meristem) inhibits apical dominance, controls shape and height, improves light conditions and increases seed yields of trees [72]. When topping off was carried out on P. sylvestris for seven consecutive years, the cone yields for each individual tree increased by 95.5% and quality by 17.2% [73]. In Pinus tabulaeformis, the upper two-wheel branches were topped off, and the results showed an average growth rate for female cones and male cones increased by 372% and 238% [74]. Sun [75] carried out topping off on P. koraiensis for three years, and the best result for topping off was to cut the first wheel branch, and then the numbers of female and male cones increased by 2-4-fold. Similar results were obtained in Chen et al. [76] and Tan et al. [77] studies on Pinus massoniana.
Topping off can reduce the tree height of conifers, which is more convenient for cone collection and improves cone quality. The topping off treatment should be carried out in late autumn or early spring because conifer species grow slowly and have low physiological activity during those periods [74,75]. The target trees should be 10~20 m tall and have vigorous growth. For such trees, it is advisable to cut off a fifth to quarter of the tree height.
For some conifer species with a height of 20~30 m, a quarter to third of the overall height can be topped off. For particularly tall trees, the topping off height should not exceed one half of the tree height to avoid inhibition of further growth. After a topping off treatment, some new branches of conifer species will regenerate at the top in the second year, which will serve as cone setting branches, thereby increasing cone yields [74].
Adult conifer species with big crowns hardly form ideal tree shapes under natural conditions. Thus, some measures such as topping off and branch pruning should be used to control the tree shape and improve cone yields [78] (Figure 2). Many new branches are induced from new lateral meristems after topping off in the first year, then the trees create a new leader and regain apical dominance. Therefore, the topping off treatment should be implemented on lateral branches in consecutive years. The final ideal tree shape is umbrella-like and has a large canopy, low tree height, many lateral branches and new shoots [76]. When conifer species reach the mature seed production age, the number of flowers and fertile seeds increases significantly. cut the first wheel branch, and then the numbers of female and male cones increased by 2-4-fold. Similar results were obtained in Chen et al. [76] and Tan et al. [77] studies on Pinus massoniana.
Topping off can reduce the tree height of conifers, which is more convenient for cone collection and improves cone quality. The topping off treatment should be carried out in late autumn or early spring because conifer species grow slowly and have low physiological activity during those periods [74,75]. The target trees should be 10~20 m tall and have vigorous growth. For such trees, it is advisable to cut off a fifth to quarter of the tree height. For some conifer species with a height of 20~30 m, a quarter to third of the overall height can be topped off. For particularly tall trees, the topping off height should not exceed one half of the tree height to avoid inhibition of further growth. After a topping off treatment, some new branches of conifer species will regenerate at the top in the second year, which will serve as cone setting branches, thereby increasing cone yields [74].
Adult conifer species with big crowns hardly form ideal tree shapes under natural conditions. Thus, some measures such as topping off and branch pruning should be used to control the tree shape and improve cone yields [78] (Figure 2). Many new branches are induced from new lateral meristems after topping off in the first year, then the trees create a new leader and regain apical dominance. Therefore, the topping off treatment should be implemented on lateral branches in consecutive years. The final ideal tree shape is umbrella-like and has a large canopy, low tree height, many lateral branches and new shoots [76]. When conifer species reach the mature seed production age, the number of flowers and fertile seeds increases significantly.
Thinning and Pruning
Thinning, a primary forestry tree management method, is the main treatment to reduce stand density. Its main function for trees is to provide light and to improve nutrient utilization efficiency [79][80][81], as well as carbon uptake and drought resistance [82]. Thinning allows faster growth rates, reduces root competition and combined with pruning results in longer rotation times and great improvements in wood quality. Thinning treatment of P. koraiensis, retaining 300 trees per hectare, with 75% intensity, increased the cone yield of each individual tree by 8~10-fold. When 750 trees per hectare were reduced by 37.5% thinning intensity, the total yield of cones increased three-fold compared to an un-thinned stand [83]. In Pinus pinea (stone pine), when 350 trees were retained per hectare, the cone yield of each individual tree increased by 1.48 times, while a high thinning intensity reduced the cone yield [84]. Appropriate timing and reasonable intensity are the basic requirements of thinning. Based on the evaluation of different traits such as growth, wood and cone characters, some individuals with poor performance were selected as the thinning targets [85,86]. Based on aforementioned research, the optimal thinning time was before the stage of tree sap flow or after cone shedding. For different site conditions and species characteristics, optimal thinning intensity may be different, and a poor choice of thinning methods may result in the reduction in yield [82]. In summary, conifer forests with a high density should maintain 500~700 trees per hectare after thinning in the first year, and thinning intensity should maintain 300~400 trees per hectare after 2~3 years.
Pruning is another effective measure to improve the light transmittance of trees in plantations. It refers to the removal of parts or redundant lateral branches, which can improve tree shape and increase the effect of ventilation and light transmittance [87]. Pruning may have dramatic effects on wood quality by reducing knots and promoting the growth of long straight stems. The content of trace elements in needles changes after pruning, which can improve the biomass and productivity of trees [88][89][90]. In P. sylvestris and P. koraiensis, the yields and quality of individual trees were significantly improved after pruning [91,92]. In another study, 3-4 main branches were kept in each round of the P. sylvestris crown while the remaining branches were cut off results in an increase in the number of cones of each individual tree by 14.3% [93]. Therefore, reasonable pruning plays a key role in the growth and development of conifers [94,95]. The stage of tree sap flow is the optimal time for pruning because the ability of trees to recover is strong and wound healing is fast.
Girdling and Cutting Roots
Girdling is a method involving wounding of the phloem of the trunk or main branches to regulate the distribution of nutrients in the trees so as to promote flowering and reproduction. Studies on girdling in conifers are focused on its impacts on cell activity [96], wood characteristics [97,98] and photosynthesis and respiration [99], but only a few investigate its effect on flowering and reproduction dynamics. In a study on Japanese larch (Larix leptolepsis), girdling carried out at 1.2 m above the ground in April resulted in a significantly higher average cone yield than the control group [100].
Girdling should be carried out on trees growing in good soil conditions, with vigorous growth and no infestation/infection by pests or pathogens. The girdling time should be one month before bud differentiation. The specific method would be to girdle the bark of the trunk or main branches with a knife around the trunk with a width of 1~2 cm below the DBH (100~120 cm from the ground) and cut the bark to the xylem in depth. If the tree is very large, the girdling width can be enlarged. After 3~5 years, the number of flowers and developing seeds will improve. Girdling does damage to the tree itself, which may lead to a weakened resistance to pests and pathogens, and nutrients should be added in time to accelerate conifer growth after girdling.
Conifer species absorb nutrients from the soil by their roots. Root cutting is the process of removing excess virtual roots from the ground and limiting root growth. The study conducted on winter wheat (Triticum aestivum) has shown that the water consumption and water use efficiency were low after root cutting [101,102]. It also affects the photosynthesis, translocation and stress resistant of plants [103]. However, root cutting is rarely conducted in conifer species. In a study of P. asperata, root cutting was carried out in three seed orchards of different ages in May at about 2 m from the trunk on both sides of each tree with sharp steel plates to a soil depth of 40 cm. There was no significant difference in the number of male cones, but the number of female cones was doubled [104]. In P. koraiensis, a circular groove was opened at a distance of 1.7 m from the trunk to cut off roots in April, and the number of female and male flowers increased two to three-fold and one to two-fold, respectively [75]. Summing up the above, the treatment time of conifer species generally chosen before the stage of tree sap flow and an annular groove is dug around the tree 1.5~2 m away from the trunk of the tree.
Water and Fertilization Management
The number of flowers is related to the nutrient supply of the plant and the external soil environment, and some plants growing in low fertility soil produce few flowers because of nutrient deficiencies. Thus, it will be necessary to apply nutrient fertilizers and to maintain disease resistance [105,106]. Suitable water and fertilizer treatments differ according to soil conditions and biological characteristics that can effectively shorten the breeding cycle [107]. Good water and fertilizer management can not only increase production and maintain nutrient supply, but also improves photosynthesis and respiration [108,109].
The main inorganic nutrients for conifer cultivation include nitrogen (N), phosphorus (P), potassium (K), calcium (C), iron (Fe), zinc (Zn), boron (B), copper (Cu) and magnesium (Mg). The corresponding fertilization and efficacy of each element are shown in Table 1. Generally, N, P and K fertilizers are used in the management of conifer species, which play significant roles in cone development. According to different environmental conditions, the combination of many nutrient elements is needed to maintain the normal growth of plants [110]. Fertilization timing and dose are crucial for vegetative and reproductive growth of trees. In studies of conifer species, fertilizers are applied before flower bud differentiation in the spring, which can shorten flowering time of P. taeda clones [50]. In a study of the application of N, P, K and other fertilizers on Chinese fir (Cunninghamia lanceolata), the compound fertilizer (0 g N, 400 g P, 50 g K, 50 g Mg and 50 g B) increased the average number of cones in each individual plant by 0.54-fold [111]. In a study of 1.5 generations of P. massoniana, the application of N, P, K and micronutrient fertilizer resulted in an increase in the number of cones by 2.76-fold when 150 g N, 90 g P and 60 g micronutrients were applied to each tree. The ratio of male and female cones was more suitable for the development of seed orchards after applying 120 g N, 72 g K and 60 g of micronutrients. When the treatment involved 150 g N, 26 g P and 60 g of micronutrient being applied to each individual tree, the number of cones increased by more than threefold [112]. In a study conducted on Fujian cypress (F. hodginisi), the application of 0.3 kg N, 0.4 kg P and 0.3 kg K on each individual tree increased the number of cones from each individual tree by 200% and the number of male and female cones by 129% and 338%, respectively, demonstrating that fertilization could improve the ratio of male and female cones of F. hodginsii and promote the flowering and reproduction of the trees [113]. Based on the results above, we summarized a set of water and fertilization management measures for conifer species. In the stage of tree sap flow, fertilizer treatments are implemented when the soil thaws. Compound fertilizer in a ratio of N:P:K = 10:2:1 should be applied in a circular ditch 20~30 cm depth at a distance of 1.5 m away from trees. For conifer tree species with heights less than 5 m, 0~1 kg of fertilizer should be applied to each tree. The optimal dose for trees 6 to 15 m high is 1~2 kg, while the suitable dose for trees more than 15 m high is 2~5 kg. In terms of nutrients, N and P are the most important nutrients affecting formation of female cones. Besides fertilization, water is a key factor to promote the plant growth and reproduction process. In the areas of high altitude, where the soil is not suitable for storing water, irrigation is indispensable [121,122]. Dobbertin et al. [46] studied the effect of irrigation in a P. sylvestris forest in Switzerland by applying water quantities that doubled the long-term annual precipitation of the site. The results showed irrigation treatment increased the growth of foliage, stems and shoots, which provided basic conditions for future seed production [46]. Therefore, the conifer species planted in drought-affected areas need timely watering. However, there is a lack of reports on the related mechanisms of action between water and seed production, which will be a crucial research direction in the future. Reasonable irrigation measures and fertilization strategies provide significant improvements in flowering and reproduction of conifers.
Phytohormone Treatments
Plant hormones are an important regulator of embryogenesis, organ development and flowering [123,124]; they can enhance stress resistance and increase yield and planting efficiency [125,126]. Phytohormones have a wide range of effects and several phytohormones affect seed formation. Auxin, (primarily indole acetic acid, IAA), gibberellins (GA), cytokinin (CTK), abscisic acid (ABA) and ethylene (ETH) are widely used in agricultural production and forest tree physiological investigations [127]. IAA, GA and CTK have the most direct activity on flowering, while ABA and ETH affect reproduction and seed production [128]. IAA is the most abundant of the auxins found in conifers, which is produced in the apical bud, then is transported down the stem and inhibits lateral bud formation. The suppression of lateral branching results in the apical dominance of the main central stem giving rise to tall straight trees able to outcompete branching trees for light and to minimize the damage to lateral branches from wind or snow. Cytokinin, known to stimulate shoot development and lateral branching are a class of molecules derived from adenine. They are synthesized in the roots and move through the xylem to leaves. In conifers, the growth and morphology of the stem and crown depend on the ratio of auxin and cytokinin.
Gibberellins (GAs) are a group of more than a hundred tetracyclic diterpenoid carboxylic acids affecting organ growth, seed dormancy, germination, flowering and senescence [129]. GAs are composed of 19 or 20 carbon atoms, and the C-19 GAs are the most active. The mutation of enzymes in the GA biosynthetic pathway are dwarfs due to the inhibition of internode growth. GAs have a key role in seed dormancy and germination in addition to metabolic effects on biosynthesis of phospholipids, nucleic acids and proteins. GAs are synthesized in the plastids and when breaking dormancy, they trigger de novo synthesis of hydrolytic enzymes in the seeds. Cytokinin and gibberellins stimulate flowering in conifers [130,131]. Ethylene is intriguing because it affects both the growth and senescence, often with contradictory effects [131]. The complexity of its effects may reside in its interaction with auxins, cytokinin, gibberellins and abscisic acid. In general, ethylene inhibits flowering and senescence, for example, in Arabidopsis and rice, but stimulates flowering in Bromeliads. In conifers, ethylene stimulates a response to wounding, to systemically acquired resistance and promotes the formation of traumatic resin ducts. Ethylene affects the branch angle (hyponasty) and, therefore, has some influence on the apical control of branching [132]. Auxin induces an ethylene response, confounding the interpretation of results because the exogenous application of auxin may induce an ethylene response.
The abundances of GA, IAA and CTK are high during bud differentiation of oriental arborvitae (P. orientalis), suggesting that these hormones have a significant influence on the internal mechanism of bud formation [133]. IAA and CTK are also involved in cellular proliferation and differentiation, organ formation and stem development. GA is the main regulator of the flowering pathway, and it mainly regulates growth, flower development and seed formation [134,135]. Previous studies on G. biloba and P. asperata found that the changes of hormone composition at different development stages would determine whether it would be a major or minor seed year. A high content of ZR (zeatin riboside, a cytokinin), IAA and GA are conducive to a high seed yield [136]. An imbalance of the male and female differentiation limits a high seed yield in conifer species, but the application of hormones can effectively restore the balance [137]. Table 2 summarizes studies on the application of hormones to promote flowering and reproduction in several conifer species. Among the different hormones tested, GA 4/7 was the most widely used to induce blooming in conifers [138]. In Chinese hemlock (T. chinensis), the cone yield was increased 2-5-fold after extensive spraying with a GA 4/7 solution [145]. The injection of GA 4/7 for two consecutive years from May to June resulted in an increase in the number of female flowers of Scots pine (P. sylvestris) by 63~120% and 168~282% in the first and second years, respectively [141]. In western larch (Larix occidentalis), doses were graded according to the DBH so that 60 mg was injected for every 5 cm of DBH. Additionally, it was found that cone yield was increased up to seven-fold when the injection concentration was 120 mg/mL GA 4/7 [150]. In Zhao's 2007 study [142], 6-BA (6-benzylaminopurine, a cytokinin), CCC (chlormequat chloride, a GA synthesis inhibitor) and GA 4/7 were injected into plant stems, and better flowering was observed with GA 4/7 in Chinese pine (P. tabulaeformis). The number of female and male cones was the highest at 500 mg/L and 1000 mg/L, respectively. After a stem injection of 20 mg GA 4/7 in Chinese spruce (P. asperata) in June, the yield of cones for each individual increased 12-fold [147]. In another study, the number of P. asperata cones also increased with a hormone dose [151]. In a study of Fujian cypress (Fokienia hodginsii), branches were sprayed with GA 3 , NAA (naphthaleneacetic acid, a synthetic auxin) and 2,4-D (a synthetic auxin used as an herbicide against dicots) during the flowering period. NAA had the greatest influence on the yield of male and female flowers and cones. At 200 mg/L, the increase in female flowers reached 111%, and the increase in cones was 9.27% higher than the control group [113]. In May, the number of male and female cones of Korean pine (P. koraiensis) was increased significantly by stem injection with GA 3 , GA 4 and GA 7 . Up to a 50 mg dose, the number of male and female cones increased with the increase in concentration [139]. Many hormones interact to regulate the various stages of flowering and seed development and germination, so hormone interactions have profound significance in promoting seed production of conifer species [143]. Receptors for endogenous hormones' action are different, which involve bud differentiation and sexual reversal by a unique signal transmission mechanism [152,153]. The balance of hormones shortened the time needed for flowering and seed formation. Under natural conditions, conifer species not only have a long breeding cycle, but also an uneven male and female gametic ratio. As the most important endogenous signaling mechanisms, phytohormones play crucial roles in the processes of growth, development, reproduction, metabolism and adaptation of conifer species [154,155].
Previous results lead to some suggestions for hormone treatments to promote flowering and seed development in conifers: (1) stem injection and spraying are the best methods for hormone treatment; (2) DBH is a useful reference for hormone dosage because the DBH growth of conifer species is directly related to the age of the tree. When DBH is below 15 cm, 30~45 mg of hormone should be injected, the concentration should be 200~350 mg/L and spraying should continue until the branches are moist. If the DBH is 15~30 cm, hormone injection should be 45~60 mg per plant, and solution concentration should be set at 350~500 mg/L. Similarly, for every 15 cm increase in DBH, stem injection amounts should increase by 15 mg and the concentration increased to 150 mg/L. (3) Hormones should be applied before flower bud differentiation in winter and (4) GA is preferred for the promotion of flowering and seed yield. To obtain synergy in the development of male and female cones in annual cone producing conifer species, such as Tsuga chinensis, the application of a mixture of GA 4/7 and GA 3 with equal doses one month before flower bud differentiation gives the best results. The method is also suitable for some conifers with two-year growth cycles, such as P. koraiensis, P. taeda and Pinus thunbergii. Before snow comes, GA 3 should be injected to preserve pollen viability, to successfully achieve fertilization in the following year. In addition, NAA solutions can be sprayed during flowering to ensure a favorable cone-setting. CCC can be sprayed on cones twice within 7 days to ensure quality.
Conclusions and Outlook
The yield and quality of conifer cones and seeds have become a greater focus of research over the years. Many experiments about flowering and reproduction in conifers have been carried out and a substantial amount of information obtained. Based on the synthesis of available information, the present work revealed that flowering and seed production in conifers are affected by a variety of biotic and abiotic factors, That can seriously impact cone yields in plantations for commercial use. On the other hand, there are useful tools and management techniques that can be adopted in order to sustain or improve cone yields, including topping off, branch pruning, girdling, hormone application, fertilization, irrigation and supplementary pollination. Furthermore, several flowering-related genes (FT, Flowering locus T and MADS-box, MCMI, AGAMOUS, DEFICIENCES and SRF) that play a crucial role in flowering in conifer trees were identified. In recent years, genome selection (GS) and genetic engineering have been added to traditional approaches to conifer species breeding research. Even though conifers have very large genomes, molecular technology is being applied to the genetic improvement of economic traits for conifer species, especially in improving flowering and reproduction traits. Despite research advances, there is an urgent need to produce species-specific technical management guidelines for conifers, aiming to sustain high cone yields. This work can be considered as a first attempt of a framework to reach the goal; however, more extended and systematic research is needed. Data Availability Statement: Data for this study can be made available with reasonable request to the author.
Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial of financial relationships that could be construed as a potential conflict of interest. | 2021-09-27T18:30:51.929Z | 2021-08-18T00:00:00.000 | {
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269465312 | pes2o/s2orc | v3-fos-license | How useful are indirect radiographic measurements of hip instability in borderline hip dysplasia? An MRI-based comparison to a healthy cohort
Purpose Symptomatic hips with borderline hip dysplasia (BHD) morphology pose a challenge in differentiating stable from unstable hips. The current study aims to compare indirect radiographic signs of instability in a symptomatic BHD population to those in a healthy cohort. Methods The study group consisted of patients with a lateral centre–edge angle (LCEA) with values 18° ≤ LCEA < 25° who underwent corrective periacetabular osteotomy (PAO) and reported an improvement in patient-reported outcome measures (PROMs). The comparison group consisted of a healthy cohort of athletes who did not complain of any hip-related symptoms and who had normal values of their hip morphological parameters (LCEA, acetabular index (AI°), alpha angle (α°), femoral version, acetabular version). Indirect signs of instability consisting of the femoro-epiphyseal acetabular roof index (FEAR), iliocapsularis-to-rectus-femoris (IC/RF) ratio and labral dimensions (height-to-length ratio) were assessed in both groups. Partial Pearson correlation, logistic multiple regression analysis and Receiver-Operating Characteristic (ROC) curve analysis were performed to determine correlations, as well as the sensitivity and specificity of these signs to differentiate between healthy hips and BHD. Results On binary logistic multiple regression analysis, the FEAR Index was the only independent predictor to differentiate between BHD and healthy hips (p < 0.001). The IC/RF ratio did not achieve significance. The calculated area under the curve (AUC) was 0.93 (0.87 – 0.99, CI 95%, p < 0.001) for the FEAR Index and 0.81 (0.70 – 0.92, CI 95%, p < 0.001) for the height-length ratio. Using the predefined cut-off values (dysplastic—FEAR Index ≥ 5° or labral height-to-length ratio ≤ 0.5), 27% sensitivity/100% specificity and 20% sensitivity/ 100% specificity, were achieved. ROC analysis provided the following new thresholds: FEAR Index ≥ -5° (73% sensitivity/97% specificity); labral height-to-length ratio ≤ 0.8 (70% sensitivity, 79% specificity). Conclusion In our cohort, the FEAR index was an independent parameter that could differentiate between borderline dysplastic and asymptomatic hips. The previously published values for both the FEAR index and labral hypertrophy ratio had a poor sensitivity in differentiating symptomatic unstable BHD from healthy hips. The cut-off values of ≥ -5° (FEAR index) and ≤ 0.8 (labral height-to-length ratio) provided acceptable sensitivity and specificity when comparing to morphological healthy hips.
Introduction
Borderline hip dysplasia (BHD) represents a challenging and controversial topic in adult hip-preservation surgery [1][2][3][4][5].Not every patient with a borderline hip dysplasia may show clinical symptoms and it is unclear which hips and how quickly would develop an early osteoarthritis [6,7].The management and diagnostic work-up of a Level of Evidence: III.Investigation performed at the Balgrist University Hospital, Zurich, Switzerland.
Extended author information available on the last page of the article hip with borderline dysplasia is complex, as it usually encompasses other concomitant pathologies of hip, such as: femoroacetabular impingement, labral tears, versional abnormalities and extra-articular impingement syndromes [8][9][10][11].Arthroplasty is ultimately also impacted with difficulties in using traditional landmarks and techniques for osteoarthritis (OA) secondary to hip dysplasia [12,13].
Various efforts have been made to select the correct treatment strategy for each respective patient to avoid postoperative residual hip related symptoms [12][13][14].One of these efforts has been the evaluation of hip stability [15].There is therefore a lack of a systematic approach that would clarify which morphological pathology to address.
Different indirect parameters to evaluate hip stability have been established, such as the Femoro-Epiphyseal Acetabular Roof (FEAR) index [16], iliocapsularis-torectus-femoris (IC/RF) ratio [17], and labral hypertrophy [18].Previous studies focused on single indirect instability parameters, such as the FEAR index, and in cohorts that included not only BHD patients but also patients with severely dysplastic hips (i.e., lateral centre-edge angle (LCEA) < 18°) [16,19].Furthermore, both of these studies lacked a healthy control group and instead used patients consulting the trauma unit [16] or suffering from femoroacetabular impingement (FAI) [19].Consequently, there is a lack of validation of these indirect instability parameters against healthy individuals without any hip related symptoms.
Distinguishing unstable from stable hips with BHD facilitates the surgical decision-making, as both periacetabular osteotomy (PAO) and hip arthroscopy (HA) are considered for surgical treatment of this patient subgroup [20][21][22] with promising mid-term outcomes.While patients with BHD and without signs of hip instability can probably be considered for a less invasive HA, patients with BHD and signs of instability presumably should be considered for a more invasive PAO [16,19].
Physicians treating these patients are most interested in linking radiographic parameters to the likelihood of response to treatment.Contrasting BHD with symptoms to asymptomatic individuals with normal radiographs may provide valuable information in the decisionmaking.Therefore, the aim of this study was to evaluate three indirect hip instability measures using magnetic resonance imaging (MRI) in a cohort of BHD patients who had undergone PAO with correction of deformity and postoperative improved patient-reported outcome measures (PROMs), indicating PAO as the correct treatment strategy of a symptomatic unstable hip, and to compare these measurements to healthy individuals without any hiprelated morphological abnormalities.
Symptomatic borderline hip dysplasia cohort
Patients were selected from a consecutive retrospective cohort comprising patients with BHD (Wiberg's lateral centre-edge angle (LCEA°) with values of 18° ≤ LCEA < 25°) who underwent PAO for symptomatic hip instability between January 2009 and January 2016 and had correction of their deformity (LCEA°), as well as improvements in their PROMs (the modified Harris Hip Score (mHHS) [23], the subjective hip value (SHV), the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index [24]) at a minimum five year follow-up.Clinical instability was defined as primary symptoms of static hip pain in extension with worsening symptoms after jogging, longer periods of walking or standing or lifting /carrying heavy objects.This in the context of no clear signs of FAI-related symptoms.Hips receiving concomitant procedures, including femoral osteotomies, were excluded.
The healthy cohort
The control group consisted of data from healthy football players originally collected for a different study purpose and who had no hip or other groin complaints.All subjects in the control group participated in Swiss competitive amateur football.Exclusion criteria were any prior surgery to the hip, knee or ankle and hip, knee or ankle pain.All members of the control group underwent a native MRI scan of both hips.Examinations were performed using a 3 T MR scanner (MAGNETOM Prisma, Siemens Healthcare, Erlangen, Germany) and a dedicated hip coil.
Radiographic assessment
The radiological evaluation was performed by a fellowshiptrained board-certified musculoskeletal radiologist (with 8 years of experience) blinded to all clinical data.Using anteroposterior (AP) and lateral axial radiographs of the hip joint, the following radiographic parameters were evaluated: Wiberg's lateral centre-edge angle (LCE°) [28], CCD angle [29], acetabular index (AI°) [30], alpha angle (α°) [31], and the presence or absence of acetabular retroversion (crossover sign, sciatic spine sign and posterior wall sign) [30].Measurement of the femoral torsion was performed using a validated previously described method by Sutter [32,33].Acetabular retroversion was determined by the concomitant presence of crossover, posterior wall and sciatic spine signs on conventional radiographs in the BHD group, which provided a specificity of 94%, as described by Lerch et al. [34] In the healthy cohort, acetabular retroversion was measured as described by Anda [35] on pelvic axial slices where the baseline was drawn between the femoral heads of both hips.A normal value was considered between 12° and 20° [36].
Statistical analysis
Data distribution was determined using the Shapiro Wilk test.Normally distributed data were tested with the paired t test.Nonnormally distributed data were tested with the Wilcoxon signed rank test (paired) or Mann-Whitney U test (nonpaired data).The chi-squared test was performed for comparison of proportions.
A binary model was constructed due to the dichotomous nature (yes/no) of the dependent variables used (presence or absence of radiographic signs) to assess the relationship between the association of morphological parameters with the group of interest.
A binary multiple regression analysis was utilized to look for independent predictors of instability (FEAR Index, IC/ RF circumference ratio, IC/RF cross-sectional ratio, labral height/length ratio).An a-priori power analysis for a multiple regression revealed a minimum required sample size with 63 for an anticipated effect size (f 2 ) = 0.45 (medium effect size) and a desired statistical power of 0.8 (total number of predictors-4).The multiple regression analysis allowed the inclusion of all instability signs in the regression equation for the independent calculation of each predictor.
Partial Pearson correlation analysis (after controlling for the femoral version and CCD values) was performed to exclude possible confounders for the detection of indirect radiographic signs of instability.
The Receiver-Operator Characteristic (ROC) curve was used to identify the sensitivity and specificity of previously reported cut-off values for the FEAR index (> 3° and > 5°) [19] and the height-to-labrum ratio (< 0.5) [39], as well as to determine the best cut-off values for IC/RF ratios for the differentiation between healthy and BHD study participants.
All statistical tests were 2-sided, and a p value of < 0.05 was considered statistically significant.Analysis was performed with SPSS (version 23.0;IBM SPSS Statistics).
Demographics
A total of 30 hips of 30 patients (24 female, 6 male) with BHD were included.This consisted of 20 right and ten left hips.The mean age was 25.2 ± 6.3 years (range 16 to 37 years).The mean body mass index (BMI) was 23.1 ± 3.0 kg/m 2 (range 18.1 to 29.3 kg/m 2 ).An overview of the PROMs of the BHD patients is given in Table 1.A significant improvement was observed in all participants in the study group who underwent PAO.Improvements were observed for all measures of PROMs, including: mHHS, WOMAC and SHV.Regarding the healthy control group, 33 hips of 21 controls (8 female, 13 male) were included.This consisted of 18 right and 15 left hips.The means age of the control group was 22.0 ± 3.2 years (range 17 to 28 years).The mean BMI of the control group was 23.0 ± 1.6 kg/m 2 (range 20.9 to 27.1 kg/m 2 ).There were no significant differences between groups in terms of age (p = 0.09, Mann-Whitney U Test) or BMI (p = 0.96, Mann-Whitney U Test).
Radiographic parameters
An overview of the measured radiographic parameters, including indirect instability parameters, is given in Table 2.All hips included in the healthy cohort had normal values for the alpha angle, LCEA, AC index, femoral version and acetabular version.
Indirect radiographic instability signs
The binary logistic multiple regression analysis revealed the FEAR Index to be an independent predictor for instability and was able to differentiate between BHD and healthy hips (p < 0.001).The height-to-length ratio showed a nearly significant statistical value (p = 0.091) and may have achieved significance in a larger sample size.The IC/RF circumference or cross-sectional ratios were not independent predictors (p = 0.943 and p = 0.829, respectively).After controlling for CCD angle, femoral version and acetabular version with partial Pearson correlation analysis, an increased FEAR Index still showed a significant correlation with the BHD group as opposed to healthy hips (r = 0.671, p < 0.001).
A ROC curve analysis (confidence interval (CI), 95%) was performed for both the FEAR Index and for the heightlength ratio of the labrum to determine the utility (sensitivity and specificity) of these indirect signs in differentiating between BHD and healthy hips.The area under the curve (AUC) was 0.93 (0.87 -0.99, CI 95%, p < 0.001) for the FEAR Index (Fig. 1) and 0.813 (0.703 -0.923, CI 95%, p < 0.001) for the height-length ratio (Fig. 2).The IC/RF circumference ratio and IC/RF cross-sectional area ratio (Fig. 3) did not achieve a significant result in the ROC analysis (p = 0.137 and p = 0.165, respectively).We therefore chose to not define any cut-off values for the further calculation of the sensitivity or specificity values.
When using the already predetermined values for the FEAR Index of ≥ 3° [19] or ≥ 5° according to the initial publication from Beck [16], we obtained 35% sensitivity and 100% specificity and 27% sensitivity and 100% specificity, respectively, when identifying hips with symptomatic BHD and differentiating these hips from healthy controls (Fig. 1).On the other hand, the previously published cut-off value for the height-to-length ratio of the labrum (hypertrophic ≤ 0.5) [39] yielded a sensitivity of 20% and a specificity of 100% in identifying BHD hips in our cohort (Fig. 2).
Discussion
The most important finding of this study is that the FEAR index and the assessment of labral hypertrophy as indirect hip instability parameters could reliably and independently identify unstable symptomatic BHD hips compared to healthy controls, whereas the IC/RF circumference and cross-sectional ratio showed no statistically significant difference between the two groups and no utility.In the original publication of the FEAR index [16], the cut-off value for hip instability was defined to be ≥ 5°, resulting in a sensitivity of 78% and a specificity of 80% for the FEAR index.In their study, the authors compared 39 patients who received PAO or femoroacetabular impingement procedures to 20 134.9 ± 3.7 (range 125 to 142) 132.0 ± 4.8 (range 123 to 145) n.s FEAR Index (°) -0.1 ± 6.5 (range -13 to 10) -13.9 ± 6.3 (range -29 to -4) < 0.001 Labral Height-to-Length Ratio 0.7 ± 0.2 (range 0.4 to 1.2) 0.9 ± 0.1 (range 0.6 to 1.1) < 0.001 Labral Hypertrophy (n) 13 0 < 0.001 IC/RF Circumference Ratio 1.0 ± 0.2 (range 0.6 to 1.7) 0.9 ± 0.2 (range 0.7 to 1.3) n.s IC/RF Cross-sectional Ratio 1.0 ± 0.3 (range 0.3 to 2.0) 0.9 ± 0.3 (range 0.4 to 1.6) n.s patients consulting their trauma unit.The FAI and trauma cohorts had normal acetabular coverage with LCEA > 25°.However, in addition to patients with BHD, the PAO cohort also included patients with severely dysplastic hips (i.e., LCEA < 18°), without differentiating severe dysplasia from BHD.Furthermore, the control group was formed by recruiting trauma patients, wherefore there was no guarantee for lack of symptoms.A more recent study by Meyer et al. [19] evaluated the FEAR index in patients who have undergone hip arthroscopy for FAI or PAO for developmental dysplasia of the hip (DDH).As the range of LCEA was not given, but instead standard deviations, we could not evaluate how many of the DDH patients had radiographic BHD and how many severe dysplasia.Postoperatively, they calculated the sensitivity and specificity of the FEAR index to detect the actual surgical procedure performed in their patients.Using a threshold value of 3°, the FEAR index showed a sensitivity and specificity of 80% and 81%, respectively, for correctly predicting the performed procedure.However, an evaluation of whether the correct surgical procedure was actually chosen is lacking, as well as a comparison to healthy controls.
Our own analysis identified a new threshold for the FEAR index that achieved better utility: FEAR Index ≥ -5° with 73% sensitivity/97% specificity.It may well be that some hips with FEAR values below the previous published cut-offs (< 5° or < 3°), can still present borderline dysplastic features and develop symptoms at a specific point of time in life.The improved sensitivity of this new threshold might suggest that a larger variation of hips can still be suspected for symptoms due to borderline dysplasia.
The strength of our study is represented by the validation of dysplastic symptoms.All patients in the study group underwent PAO in isolation without concomitant procedures and had an improvement in postoperative PROMs.Using a threshold value of ≥ 3° or ≥ 5°, the specificity and sensitivity of the FEAR index in our study were 100% and 35% and 100% and 27%, respectively, when identifying hips with BHD and differentiating these hips from healthy controls.
Regarding labral hypertrophy, a height-to-length ratio of < 0.5 is described as another hip instability parameter.Nwachukwu et al. [41] showed a strong correlation between labral hypertrophy and BHD in a cohort of patients who underwent hip arthroscopy for FAI.However, calculation of sensitivity and specificity was not performed in their study.Using the established threshold value of < 0.5, the labral heightto-length ratio showed a sensitivity of 20% and a specificity of 100% in identifying BHD hips in our cohort.In contrast, the ROC analysis performed in this study offered a threshold value of ≤ 0.8 that provided a 70% sensitivity and 79% specificity, which could be an alternative worth considering.The IC/RF ratio is described as a valuable secondary sign to identify the predominant pathology in patients with BHD and concomitant cam-type deformity [17].However, using our data, the IC/RF circumference ratio and the IC/RF crosssectional area ratio were not independent predictors for symptomatic BHD and the ROC analysis did not achieve statistical Fig. 2 ROC Curve Analysis for the Height-to-Length Labral Ratio Fig. 3 ROC Curve Analysis for the IC/RF circumference and cross-sectional ratios significance with a poor AUC; therefore, we decided not to define any threshold values for these parameters using our data.
This study has some limitations.Acetabular retroversion in the BHD group could not be measured due to the lack of whole pelvic computed tomography or magnetic resonance scans.Hence, the presence of crossover, posterior wall and sciatic spine signs on conventional pelvic radiographs were used to identify patients with acetabular retroversion.Lerch et al. [34] showed a specificity of 94% in identifying acetabular retroversion by the presence of these three indirect conventional radiographic signs.We had four patients that we needed to include in the healthy cohort that had a femoral version between 25° and 29°, which some authors may consider pathologic.Some included patients had a coxa valga with CCD angle exceeding 140°, which could represent a pathologic finding.We chose to keep these hips in order to achieve the minimal sample size for adequate statistical power.Although interrater and intrarater reliability for radiographic parameters were not evaluated in our study, previous reliable reproducibility of these measurements has been already proven in other studies [19,42].
Conclusion
In our cohort, the FEAR index was an independent parameter that could differentiate between borderline dysplastic and asymptomatic hips.The previously published values for both the FEAR index and labral hypertrophy ratio had a poor sensitivity in differentiating symptomatic unstable BHD from healthy hips.The cut-off values of ≥ -5° (FEAR index) and ≤ 0.8 (labral height-to-length ratio) provided acceptable sensitivity and specificity when comparing to morphological healthy hips.
Acknowledgements None to declare.
Author contribution statement All authors have made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted, (4) being 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.
Fig. 1
Fig. 1 ROC Curve Analysis for the FEAR Index
Table 1
Patient-reported Outcomes Abbreviations: mHHS modified Harris Hip Score, SHV Subjective hip value, WOMAC Western Ontario and McMaster Universities, Osteoarthritis Index
Table 2
Radiographic ParametersAbbreviations: LCEA Lateral center-edge angle, AC-Index Acetabular index, CCD-angle Caput-collumdiaphyseal angle, FEAR Femoro-Epiphyseal Acetabular Roof Index; IC/RF iliocapsularis-to-rectus-femoris ratio; n.s not significant Data distribution was determined using the Shapiro Wilk test.Normal-distributed data were tested with the paired t test.Non-normally distributed data were tested with the Wilcoxon signed rank test (paired) or Mann-Whitney U test (nonpaired data).Chi-squared test was performed for comparison of proportions | 2024-05-01T15:30:18.691Z | 2024-04-29T00:00:00.000 | {
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19187356 | pes2o/s2orc | v3-fos-license | Disseminated Pasteurella multocida infection: Cellulitis, osteomyelitis, and myositis
A 67-year-old man with poorly controlled type II diabetes mellitus was evaluated for right lower extremity erythema and swelling and left-sided lower back pain. He was found to have Pasteurella multocida bacteremia; magnetic resonance imaging showed osteomyelitis of the lumbar spine with myositis in the adjacent left paraspinal muscles. He was initially treated with intravenous antibiotics and was later transitioned to oral amoxicillin. He recovered completely with six weeks of antimicrobial therapy.
Introduction
Pasteurella multocida should be considered as a possible cause of skin and soft tissue infection in patients with animal exposure, particularly to cats and dogs. These infections most commonly occur after bites or scratches, although they can also result from licks when the skin is not intact [1]. Symptoms often develop within 24-48 hours of the initial exposure [1]. Complications such as soft tissue abscesses, osteomyelitis, septic arthritis, and necrotizing fasciitis can occur [1], and are thought to arise predominantly from local extension of the infection [1][2][3][4]. This report describes a case of invasive disease due to P. multocida characterized by cellulitis complicated by vertebral osteomyelitis and paraspinal myositis at a distant anatomical site; these complications are thought to have occurred as a result of hematogenous spread of the infection. This constellation of clinical findings has not previously been described in the English-language literature.
Case presentation
A 67-year-old man with uncontrolled type II diabetes mellitus (hemoglobin A1c of 13.9 11 months prior to presentation), diabetic peripheral neuropathy, chronic foot wounds, and prior left below-the-knee and right fifth-digit ray amputations was evaluated for three days of right lower extremity erythema, pain, and swelling. The patient also described new, severe pain in the left lumbar paraspinal muscles during this time period. On the day prior to presentation, he developed subjective fever and chills. He reported exposure to a pet cat at home but denied any known scratches or bites. At presentation, the patient was afebrile with a blood pressure of 85/55 mmHg, heart rate of 120 beats per minute, and oxygen saturation of 97% while breathing ambient air. The right lower extremity was warm to touch with erythema and pitting edema extending from the ankle to the knee; there was full range of motion in the ankle and knee joints. A blackened callus was present at the right fifth-digit ray amputation site, where the wound had previously been left to heal via secondary intention under a wound vacuum; it was without drainage or purulence at the time of presentation. His left-sided amputation stump appeared clean without erythema or swelling. The patient also had pain to palpation of the left paraspinal muscles in the L1-L5 region; there were no overlying skin changes in this area. Laboratory findings were notable for a white-blood-cell count of 5200 cells/microliter with 96% neutrophils and a lactic acid of 3.7 mmol/L (normal range: 0.5-2.2 mmol/L). The patient's hypotension and tachycardia improved with intravenous fluid resuscitation. He was given empiric vancomycin and ceftriaxone and admitted to the hospital. Admission blood cultures grew pan-sensitive P. multocida in two aerobic bottles and two anaerobic bottles. Contrast-enhanced magnetic resonance imaging of the lumbar spine showed osteomyelitis of the L4-L5 pedicles ( Fig. 1) with associated myositis of the L1-L5 paraspinal musculature (Fig. 2). The patient improved with three days of ceftriaxone. He was then transitioned to oral amoxicillin, of which he completed a six-week course. All symptoms had resolved at the end of antimicrobial therapy.
Discussion
We report a case of an older man with invasive P. multocida infection manifesting as cellulitis and bacteremia complicated by vertebral osteomyelitis and paraspinal myositis at a distant anatomical site. Although the patient denied any bites from his cat, he was suspected to have acquired the infection from his chronic wounds coming into contact with the cat's saliva. Osteomyelitis and myositis were thought to have occurred due to hematogenous spread of the infection from the right lower extremity.
Our case is also unique in that, at the time of initial presentation, the patient had already developed complications of bacteremia. Osteomyelitis more commonly presents weeks to months after signs and symptoms of the initial infection [1].
Regarding treatment, oral amoxicillin is thought to be effective against P. multocida if the isolate does not produce beta-lactamase and appropriate drug levels can be attained in the target tissues [18]. In this case, the patient's isolate was sensitive to amoxicillin, which was dosed at 500 mg every eight hours to achieve sufficient blood levels. In addition, oral antibiotic therapy can be used to treat vertebral osteomyelitis in the appropriate clinical situation [19]. The chosen regimen treated the patient effectively.
Our patient's risk factors for P. multocida cellulitis and bacteremia were exposure to a cat, poorly controlled diabetes, and chronic wounds.
In the presence of bacteremia, the patient's musculoskeletal complaints were highly concerning for a focus of metastatic infection. Practitioners should consider P. multocida infection in the appropriate clinical context, and should maintain a high index of suspicion for invasive disease.
Consent
Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. | 2018-04-03T02:21:03.430Z | 2017-09-15T00:00:00.000 | {
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2991298 | pes2o/s2orc | v3-fos-license | The effect of morphology on the superconductor-insulator transition in 1-D nanowires
We study the effect of morphology on the low temperature behavior of superconducting nanowires of length $\approx$100 nm. A well-defined superconductor-insulator transition (SIT) is observed only in homogenous wires, in which case the transition occurs when the normal resistance is close to $h/4e^2$. Inhomogeneous wires, on the other hand, exhibit a mixed behavior, such that signatures of the superconducting and insulating regimes can be observed in the same sample. The resistance versus temperature curves of inhomogeneous wires show multiple steps, each corresponding to a weak link constriction (WLC) present in the wire. Similarly, each WLC generates a differential resistance peak when the bias current reaches the critical current of the WLC. Due to the presence of WLCs an inhomogeneous wire splits into a sequence of weakly interacting segments where each segment can act as a superconductor or as an insulator. Thus the entire wire then shows a mixed behavior.
Evidence for a superconductor-insulator quantum phase transition (SIT) in one-dimensional (1D) wires has been found in a number of experiments. 1,2 Yet, other studies have demonstrated a crossover, as opposed to an SIT, in thin wires where superconductivity disappears gradually, as diameter is reduced, presumably due to an increasing number of quantum phase slips (QPS). 3,4,5 Thus the existence and possible origins of superconductor-insulator transitions in 1D remain important open problems. In particular it is not known how the SIT depends on the morphology of nanowires.
In two-dimensional system, for example, the crucial role of morphology (i.e. granularity) on the SIT is well known. 6,7,8 For uniform films, as the film thickness is reduced, a reduction of the critical temperature is observed while the superconducting transition remains sharp. The SIT occurs when the square resistance of the film reaches a critical value close to the quantum resistance R Q =h/4e 2 =6.5 kΩ. 9 In nonhomogeneous (granular) films, on the other hand, a reduction of the film thickness results in a crossover between superconducting and insulating regimes, with very broad resistive transitions in the thinnest superconducting samples. 6,10,11 Our goal here is to determine the morphological requirements for 1D nanowires under which an SIT can occur.
In this Communication, we present a comparative study of homogeneous and inhomogeneous nanotubetemplated wires and find a qualitatively different behavior at low temperatures. Homogeneous samples (of length ≈100 nm) show an SIT which occurs when the wire's normal resistance is close to R Q , confirming previous results where different nanotubes were utilized as substrates. 2 Inhomogeneous wires, on the other hand, exhibit a mixed behavior displaying properties of superconductors and insulators at once. Such samples frequently show multiple steps in the resistive transitions but no resistive tails typical of QPS. 3,4,5 We propose a model which regards the inhomogeneous wire as a sequence of weak link constrictions (WLC) connected in series. Each WLC has a certain dissipative size and corresponding normal resistance, depending on which the WLC can be either superconducting or insulating. Homogeneous (and short enough) wires do not show such mixed behavior because each wire acts as a single WLC.
The wires were fabricated using a molecular templating technique, 2 in which a single-walled carbon nanotube was suspended over a 100 nm wide trench etched into a multilayered Si/SiO 2 /SiN substrate. Unlike previous studies, 2,4 here we use fluorinated single-wall nanotubes (FSWNT), which are known to be insulating. 12 The substrate with suspended nanotubes was then sputtercoated 13 in one of two ways: (i) with amorphous 14 Mo 0.79 Ge 0.21 or (ii) with a slightly thinner amorphous Mo 0.79 Ge 0.21 film followed (in the same vacuum cycle) by a 2 nm Si film. Contact pads were defined using photolithography, followed by (i) wet etching in H 2 O 2 for MoGe or (ii) reactive ion etching followed by wet H 2 O 2 etching for Si-coated samples. While both processes produce samples with the same geometry ( Fig. 1a) and similar dimensions (Table I), these two fabrication methods result in different wire morphologies as revealed by TEM imaging and confirmed by transport measurements (see below). The first process results in homogeneous nanowires (Fig. 1b), which we will refer to as "bare" wires. The second method yields inhomogeneous wires, which will be called "Si-coated" wires ( Fig. 1c). Although these two fabrications methods give qualitatively different wires, the exact mechanism of this is not clear. The most probable explanation is that Si introduces some surface tension that leads to the creation of grains. On the other hand, if exposed to air, the outer layer of MoGe wires oxidizes unless coated by a protective film, in our case Si. To ensure that the conducting cores of bare wires have similar dimensions as the cores of the Si-coated wires, the bare wires must be made thicker to compensate for the expected surface oxidation. Since thicker wires are always considerably more homogeneous than thin ones, the bare wires exhibit a much higher degree of homogeneity.
The oxidized layer thickness (in bare wires) can be es- timated as follows (Fig. 2). For each sample, the wire length, L, divided by the normal state resistance, R N , is plotted versus SEM measured width, w in Fig. 2a. The bare wire data (circles) can be well approximated by a linear fit (dashed line), providing evidence for their homogeneous structure. The fitting parameters give an oxidized layer thickness ≈2.3 nm (half of the fit's x-axis intercept value) and the resistivity for MoGe ρ≈180 µΩ-cm (obtained from the fit's slope and the wire's average thickness, d ave ≈4.7 nm, estimated under the assumption that the top 2.3 nm of the sputtered MoGe is oxidized), in agreement with published values. 2,14 No reasonable linear fit could be obtained for the Si-coated wires (crosses), further indicating their inhomogeneous structure. In Fig. 2b we compare the T c 's of bare MoGe films and Si-coated MoGe films (circles and crosses, respectively) plotted versus their thicknesses. By shifting the data for bare films by 2.7 nm to the left the two families of data points overlap (Fig. 2b). It is therefore concluded that the oxidized layer thickness is 2.7 nm, similar to the above estimate.
Voltage vs. current measurements, V (I), were performed by current biasing the sample through a large (∼1 MΩ) resistor. Zero-bias resistance, R(T ), was obtained from the slope of the linear part of the V (I) curves as temperature was varied. Similar to Ref. 16, transport measurements were performed in 4 He or 3 He cryostats equipped with rf-filtered leads. The differential resistance vs. bias current, dV (I)/dI, was measured using an AC excitation on top of a DC current offset generated by a low-distortion function generator (SRS-DS360), again in series with a (∼1 MΩ) resistor.
Zero-bias resistance vs. temperature measurements are compared for homogeneous (Figs. 3a,b) and inhomogeneous (Figs. 3c,d) nanowires. First note that the rightmost superconducting transition observed on all samples is due to the electrodes (i.e. thin MoGe films connected to the ends of the wire). Below the temperature of this transition (T c,f ilm ) only the wire contributes to the measured resistance. Therefore, in what follows we will only be interested in temperatures T <T c,f ilm . We first discuss the family of R(T ) curves for bare wire samples, presented in Fig. 3a, which show a clear dichotomy. In the log-linear representation, samples A1-A8 all have negative curvature (d 2 log(R)/dT 2 <0) with more than half of these samples reaching immeasurably low resistances. Therefore, we refer to these samples as superconducting. Although samples A6, A7, and A8 do not reach such low resistances, their negative curvature as well as their decreasing resistance with decreasing temperature (Fig. 3b) lead us to believe they too are superconducting. Due to their smaller widths we assume that their critical temperatures are suppressed 15 such that they do not go through their full superconducting transition within the temperature range studied. Samples A9-A12 show only the transition due to the film electrodes. Their curvature is always positive (for T <T c,f ilm ) with increasing resistances as temperature is decreased. Consequently we consider these samples to be insulating. The insulating regime can be either due to a suppression of T c to zero 15 or due to a proliferation of QPS 17 (or both). Observation of these two regimes indicates that a superconductorinsulator transition occurs in this family of nanowires, confirming previous results. 2 As illustrated in Fig. 3b, the transition from superconducting to insulating behavior occurs when the wire's R N ≈R Q . Therefore it can be suggested that the R N of the wire is the parameter that controls the SIT. However, since wire length was not varied over a wide range we cannot exclude the possibility that the cross-sectional area of the wire is the true control parameter.
Measurements presented in Fig. 3 demonstrate that bare and Si-coated wires are qualitatively different. In the Si-coated wires the R(T ) curves are less predictable, show multiple steps or "humps" (Figs. 3c,d), and in some cases exhibit a mixed behavior that does not always allow them to be clearly identified as either superconducting or insulating. For example, sample B4 shows three steps on the R(T ) curve and a tail at the lowest temper- ature, which could be the beginning of the fourth transition. The first step, as always, reflects the superconducting transition in the electrodes, but the other steps correspond to some weak links in the wire itself. Some Sicoated wires exhibit both positive and negative curvature at different temperatures. Such mixed behavior is seen in samples B5 and B6, with their resistance initially dropping as in superconducting wires (i.e. with a negative curvature) but then starting to increase as in insulating wires. The conclusion is that no clear SIT at R N ≈R Q can be observed in the family of non-homogeneous wires.
A clear difference between bare and Si-coated wires is also found in the dV (I)/dI measurements (Fig. 4). Bare MoGe wires (left column) again show one of only two distinct types of behavior. The superconducting wires exhibit a large minimum centered around zero-bias (e.g. A4 and A5) and peaks at the bias current which is equal to the critical current of the nanowire. The insulating samples show a single zero-bias resistance peak (e.g. A11 and A12). This is in agreement with previous experiments. 2 The Si-coated MoGe wires, on the other hand, frequently show various combinations of such types of behavior. For example, samples B5 has a clear resistance depression in the range ±150 nA with the critical current peaks at the limits of this depression, and, in addition to this, a narrow zero-bias resistance peak. Such mixed behavior indicates that some parts of the wire are superconducting while some other parts act like insulating wires. Sample B6 also shows similar results. This fact again confirms that these wires are not homogeneous.
The properties of inhomogeneous wires can be understood by assuming that they contain a sequence of independent weak link constrictions, each surrounded by a dissipative region, the size of which determines the normal resistance of the WLC. Since the free energy barriers for phase slips are different in each such WLC and the constrictions are connected in series it is clear that R(T ) curves should show crossings 5 and multiple steps (such as for sample B4 in Fig. 3c). Moreover some links can be insulating if their normal resistance is large enough, in the same sense as homogeneous insulating wires. For example sample B5 shows a mixed behavior that is explained by assuming it contains two WLC -one superconducting and one insulating. The R(T ) curve then should show one superconducting hump and a tail with increasing resistance at lower temperatures, typical of insulating wires. Likewise, a single superconducting feature and an insulating zero-bias peak should be observed in the dV (I)/dI curve. These characteristics are indeed observed (Fig. 3d and Fig. 4). In some wires all WLC can be superconducting, as for example in sample B1 (and possibly B2, B3 and B4). In the case of B1, two humps in the R(T ) curve (at T ≈5 K and T ≈2 K) and two superconducting peaks on dV (I)/dI, corresponding to two critical currents (at I≈15 nA and I≈60 nA), are observed, indicating that two superconducting WLCs are present. Finally, since each insulating WLC produces a single dV (I)/dI peak located at zero current, the wires with a few insulating WLCs should nevertheless exhibit only one peak at zero bias, as could be the case for sample B7 in Fig. 4.
All measured homogeneous samples act as superconductors if they satisfy R N <R Q and as insulators otherwise (Fig. 3b). Inhomogeneous wires frequently violate this condition, as for example sample B2 (Fig. 3d). This can be explained by the WLC model and by assuming that each WLC can be either insulating or superconducting depending on its own normal resistance. The sample B2 has shown two distinguishable peaks on its dV (I)/dI curve (not shown), corresponding to two superconducting WLCs with slightly different critical currents. Since this sample's normal resistance is 8.30 kΩ and since two similar WLCs are detected in this wire, it can be estimated that the normal resistance of each WLC is ≈4.15 kΩ. Thus each WLC is superconducting and hence the entire wire is superconducting as well.
Since each WLC is manifest by a jumpwise increase of the sample resistance at the WLC's critical current, it is reasonable to associate WLCs with phase slip centers 18,19 (which are usually positioned at weak spots along the wire). The normal resistance of a WLC is then the resistance of the dissipative region or the region which is populated by quasiparticles generated by the phase slip center. This dimension is the quasiparticle diffusion length, Λ Q ≈(Dτ E ) 1/2 ≈100 nm, where D≈10 −4 m 2 s is the diffusion constant and τ E ≈10 −10 s is the inelastic scattering time. 18,19,20 If constrictions are present in the wire, Λ Q is further reduced and equals the distance between the constrictions. Our short homogeneous wires are not longer than Λ Q , so each wire acts as a single WLC. These wires therefore show a clear SIT. Inhomogeneous wires have more than one WLC (i.e. will develop more than one phase slip center under strong current bias) and consequently show a mixed behavior.
In summary, we have found that the morphology of superconducting nanowires has a strong effect on the observed superconductor-insulator transition. A clear SIT is only found in homogeneous wires whereas inhomogeneous samples show a smeared transition and mixed behavior. In the later case, the results are understood by the assumption that inhomogeneous wires are composed of weakly coupled sections connected in series. Each section exhibits either superconducting or insulating behavior while the entire wire shows a mixed behavior. This work is supported by NSF CAREER Grant No. DMR 01-34770 and by the Alfred P. Sloan Foundation. Some fabrication was performed at CMM-UIUC supported in part by DOE grant DEFG02-96-ER45439. | 2016-03-22T00:56:01.885Z | 2004-01-19T00:00:00.000 | {
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257162525 | pes2o/s2orc | v3-fos-license | Effects of climatic factors on the sedimentation trends of Tarbela Reservoir, Pakistan
The study explores the factors affecting the sedimentation trends in the surrounding catchment area of the Tarbela Dam for the period 1980–2017, specifically rain and temperature. Furthermore, this study also investigates the seasonal effects of sediment loads coming into the reservoir from 2007 to 2012. The statistical techniques were applied for the estimation of the effects of climate (rainfall and temperature) on the sedimentation and the inflow of Tarbela Dam and its surrounding catchment area. The results revealed that sediment deposition pattern points toward the greater bed elevation at 7–12 range lines that lie near the Main Embankment Dam, also that Barandu River carried more bulk of inflow to the Indus, as compared to Siran River and thus contributed a greater amount of sediments to the reservoir. Summer monsoon season also contributed to the greatest bulk of sediments to the Tarbela Dam and its surrounding catchment area. The regression confirmed that for one degree increase in temperature there will be a 7 Million Acre Feet (MAF) increase; for a 1 mm (mm) increase in rainfall there will be a 1 MAF decrease and for a 1 Cusec increase in the inflow coming into the reservoir, there be a 4 MAF increase in sedimentation of the reservoir. Thus, the contribution of the temperature in sediment generation is found to be greater than rainfall. The study recommended that for the proper functioning of Tarbela Dam, the shift in the climate of the Upper Indus Basin (UIB) might be regularly monitored as rising temperatures in UIB would result in greater glacial melt and hence greater sediments to settle down into the reservoirs downstream.
Introduction
Reservoirs around the world are facing the challenges of sedimentation. For the year 1986, it was estimated that there is an economic loss of US$ 6 billion each year due to the loss of 1% of the world's gross reservoir storage [1]. The process of sedimentation begins right from the movement of runoff, either generated from rainfall, wind or snowmelt. The reservoirs serve as an efficient sediment trap because the velocity of water is very low here. Sediment inflow has a strong impact on the reservoir's storage capacity. Most reservoirs across the globe suffer from the menace of sedimentation because an increase in the flow depth and consequently a decrease in the flow velocity cause a reduction in the capacity of the reservoir's sediment transport, which in turn leads to settling of the bulk of incoming sediments. Bed elevation of reservoir rises, and aggravation is caused as the sediments get deposited across the entire reservoir. These sediments are deposited in a deltaic formation, with coarser sediments in the headwater area of the reservoir, while the finer sediments can get transported to the dam by the density currents [2].
Seasonal variations in the catchment area have great impacts on the amounts of sediment generation. [3] mention that seasonal shifts in rainfall patterns cause changes in the physical, biological and chemical characteristics of the hydrologic systems. Similarly, the gradual rising temperatures and prolonged summers in the catchment area can lead to increased glacial melting that in turn lead to greater sediment generation. According to [4], sedimentation is a parameter to define the capacity of the reservoirs, and sedimentation yield controls the amount of sedimentation reaching a reservoir. [5] indicates the sediment yield as "the sediment discharge through a river outlet per unit catchment area per unit time." Sediment yield in turn is dependent upon the rate of soil erosion in the catchment area.
The sediment load increase can cause a reduction in the water quality of a river as it carries pollutants, leads to reduction in light penetration, a reduction in the river depth and in extreme cases clogging of the gills of the fish [6,7]. [7] states that the quality of the surface water deteriorates due to increase in sediment loads that the rivers carry, and thus, it is essential to control the rate of soil erosion by understanding the processes involved in the transportation of suspended sediments.
The amount of sediment generation is also dependent upon factors such as land use practices, removal of vegetal cover, which causes a reduction in infiltration and leads to increased runoff [8 and 9], whereas the uncertain pattern of sedimentation of reservoirs is attributed to both inflow, sediment flux, particle size, geometry and structure of reservoir and their operational rules [10,31]. The varying loads of sediments are dependent on the boundary condition.
The river Indus carries huge loads of sediments and has one of the highest rates of sediment transport reported in the literature [11]. This is because it passes through Himalayas and Karakoram, both geomorphically extremely active regions [12]. These are young fold mountains that are subject to rapid degradation. These are prone to tectonic instability, have high relief and receive most of the runoff from the glaciers [13]. According to [14], the average annual inflow to Tarbela is 81,000 million cubic meter (Mm 3) , while the annual sediment inflows vary between 100 and 300 metric tons (MT) for 1967-1996. [15] are of the view that the sediments from the catchment area reach Tarbela reservoir where due to insufficient velocity favorable conditions for particle settling are generated, due to which reservoir's important storage capacity is lost. Eighty percentage of the total sediment load gets deposited in the Tarbela reservoir during July-August [15]. [16] states that 97% of the sediments are carried during the large flows in summer season between May and September, and a peak is witnessed because of the snowmelt in July and August. [17] state that snowmelt with 35-44% inflow input, contributes more inflow to the Indus River than glacial melt, with a percentage of 25-36%. The authors further affirm that just upstream Tarbela Reservoir, Indus River carries annual flow, which is 70% composed of melt waters. Among this 70%, 44% is obtained through snowmelt, while 26% through glacial melt. The contribution to river discharge through glacial melt is decreasing rapidly due to loss of glacial masses at higher elevations. These losses can be attributed to changes in mean annual temperature of UIB.
Snow and glacial melt contribute 90% of the inflow coming into the Tarbela reservoir. These glaciers crush the rocks and provide the bulk of the sediments that get settled in the reservoir [18]. [19] enlist the factors that have an impact on the trap efficiency: fall velocity of various sediment particles, flow rate of these sediments and their velocity through the reservoir. [20] explain that UIB has three regions based on runoff sources, Region 1 is temperature controlled and consists of glacierized sub basins, Region 3 gets its runoff from monsoon and western disturbances while the Region 2 is a combination of the above two sources of runoff. Based on the difference in sources of runoff, sediment generation rate and time also differ. Sediment yield also increases with the increase in the drainage area, for, e.g., "from 355 t km −2 yr −1 at Kharmong to 1197 t km −2 yr −1 at Besham Qila": (p 382), and the main reason is the contribution of runoff by many small and steep catchments.
Tarbela's trap efficiency is very high because its shape leads to the deposition of almost entire incoming sediment. According to estimates, since 1974 there has been a 30% decrease in the live storage of the reservoir till 2009. This drastic decrease in live storage means a decrease in the supply of irrigation water and less production of hydroelectricity [16]. The trapping efficiency of Tarbela is among the highest in the world's reservoirs. Thus, it is necessary to monitor the factors that affect sedimentation. [20] identify that between Besham Qilla and Tarbela the three most important tributaries that bring the bulk of sediments into the dam are Gorband, Barandu and Siran. Their research provides the specific sediment yield of these rivers at different gauging stations, which is very helpful for detailed analysis.
Tarbela has been filled to its maximum elevation, i.e., 472 m in almost all years. Usually, the reservoir reaches its maximum level by August 20 and remains full till mid-September. During this period, the reservoir is well above the delta and a deposit of new sediments is formed on top of the existing delta surface. Sedimentation is a cyclic process. The reservoir starts providing water for irrigation after September, and the reservoir reaches its minimum level in April or early May, and it is in late May or early June when the snowmelt starts, and high flows start again [18].
Since its impoundment in 1974, the reservoir has lost 20% of its gross capacity and nearly 40% of the original dead storage. The live storage is being lost and sediments threaten to block the intake tunnels dealing with irrigation and power generation [12]. Although many studies have been conducted on sedimentation trends of Tarbela reservoir, however, the relationship between the key variables affecting sedimentation and the study of sediment load input season-wise have been a neglected area in the literature. The objective of this study is to explore the effects of rainfall and temperature on the sedimentation and the inflow of Tarbela Dam and its surrounding catchment area for the period of 1980-2017. Furthermore, this study also investigates the seasonal effects of sediment loads coming into the reservoir from 2007-2012.
Characteristics of Tarbela Dam, Pakistan
The Tarbela Dam is basically named after the town of Tarbela which is located in Haripur and Swabi district. Its coordinates are 34°05′23″ N-72°41′55″ E (Fig. 1). It is the largest natural dam in the world which was constructed over Indus River and completed in 1976. It is used to produce hydroelectric power, for irrigation purposes and to flood control. The average daily temperature varies from summer to winter. In the summer, it goes up to 40 °C in June and lowest in winter at 3 °C. In monsoon season, relative humidity also plays its role and exceeds up to 50%.
Data analysis
Secondary data of the variables of bed elevation, inflow of sediments, main reservoir sedimentation, daily inflow of water in the reservoir and deposition/erosion at longitudinal profile points were collected from the office of the Tarbela Dam Project, Haripur, for a period of thirty years, i.e., 1980-2010. In order to have an estimate of the amount of sediments being contributed by the two tributaries of Indus, Siran and Barandu rivers that lie just north of Tarbela reservoir, the annual discharge datasets were obtained for the years 1980-2010, for the Daggar and Phulra gauging stations from WAPDA regional office. Statistical analysis, including comparative line and bar graphs, multiple linear regression, ANOVA and correlation analysis between sedimentation and inflow variables were performed in SPSS 16.0.
The rate of sedimentation in Tarbela reservoir is dependent upon inflow coming into the reservoir, temperature and rainfall in the catchment area north of Tarbela. The relationship between dependent variable sedimentation with the independent variables of inflow, rainfall and temperature was explored through multiple linear regression model in SPSS. Multiple linear regression (MLR) has also been used in various other international studies focused on sedimentation and bed load analysis. [21 and 32] applied MLR to prove linear relationship between variables, in this case river discharge and suspended sediment load of River Rio Chama, USA. [22] used MLR in their study for bed load analysis of Malaysian rivers.
Deposition/erosion range line-wise
The analysis of the bed elevation data concludes that the bed elevation had drastically increased since Tarbela's inception along all the range lines (Fig. 2). The location of the range lines of the Tarbela Dam reservoir is shown in Fig. 3.
During the period of 2011-2012, the elevation of underwater delta was found to be maximum between range lines 7 to 12, as has been shown in Fig. 4, meaning that deposition was more near the (Main Embankment dam) MED. As we move away from MED the deposition decreases and even reaches a point of erosion between rangeline 13 ¼-13 ¾, 17 and 21-27.
Factors affecting the sedimentation at Tarbela Reservoir
Scarce vegetation along with the steepness of the valley walls contributes to the higher sediment generation rate in the catchment. The bulk of inflow determined the amount of sediments that will be carried with it. The amount of inflow coming into the reservoir is highly dependent on the seasonal shift. Sedimentation is greatly influenced by factors like slope, amount of inflow, season, geology of the catchment area, etc. However, in the current study only the impact of tributaries, temperature and rainfall in the catchment area, i.e., Upper Indus Basin (UIB), the seasonal bulk of sediments and inflow coming into the reservoir has been analyzed.
River Siran and Barandu
River Siran and Barandu, gauged at Daggar and Phulra gauging stations (Fig. 5), just south of Besham Qilla gauging station, are the tributaries of Indus north of Tarbela reservoir. Thus, they also contribute to the addition of the sediment load of Tarbela reservoir. Among the two tributaries, (Fig. 6) the annual average discharge of river Barandu is higher than river Siran. Figure 6 depicts the great fluctuations in the discharge of river Barandu, which is an important tributary of Indus in terms of sediment contribution, since it lies near Tarbela reservoir.
The annual average discharge at Phulra was highest in 2010, when Pakistan experienced its worst flood, and lowest in the year 2001, the year in which Pakistan was facing drought conditions. On the other hand, Siran River contributes less water and sediments to the Indus and the graph presents little fluctuations in the discharge of Siran during 1993-2012. [3] also concluded similar findings of higher suspended loads of sediments in Chalok River of Malaysia, during the wet periods. [23] supports the fact that the three months of July-Sep are most influential in deciding the amount of sedimentation that will get trapped in the reservoir each year, by mentioning that 80% of the sediment of Tarbela gets trapped in the reservoir during these three months. [16] is also of the view that 97% sediments are carried during heavy flows witnessed between May and September, while another peak is witnessed due to snowmelt in July and August. In the current study, Nov to Feb are classified as winter season, March till June is classified as summer and July-Oct as the summer monsoon season. Figure 9 indicates the strong positive correlation between the two variables. As the inflow in the dam increases, so does the amount of incoming sediments. This relation is especially evident in the years 1984,1990,1994,1996,1999,2006,2010,2014 and 2016 (Fig. 10).
Relationship between sedimentation and inflow in Tarbela
The MLR model was applied after all the five assumptions of the model were met. Next the overall significance of the model was tested through ANOVA. The p value was < 0.001 proving that the model is significant.
The adjusted R square value in the model summary is: 0.439. Almost 43% of variability in sedimentation is accounted by the temperature and rainfall in the upper Indus basin and the amount of inflow coming into the Tarbela reservoir. Based on the β coefficients (one goes with each predictor), (based on the model summary) the equation for the regression line is presented in Eq. 1.
Independent variable coefficient is statistically nonsignificant except for inflow as model predicts that: For every 1 • increase in temperature causes 0.007 MAF increase in sedimentation. Similarly, with every 1 mm increase in rainfall, there will be a decrease in sedimentation of 0.001 MAF. However, inflow increase of 1 Cusec coming into the reservoir causes 0.004 MAF increase in sedimentation. The significance values in the coefficient table also proves inflow as a variable to have higher significant p value of 0.000. Thus, the regression model which was applied using sedimentation as dependent variable, and temperature, rainfall and inflow as independent variables, presents meaningful relationships between the dependent variable with the independent variables. It can be concluded that among the three independent variables, inflow is the one Thus, we can say that while monitoring sedimentation trends of Tarbela reservoir, a seasonal link with bulk of sediment, i.e., maximum sediments get settled in the reservoir during monsoon season, was identified. The second finding explores a strong correlation between inflow and sedimentation. Therefore, in the monsoon season when the flow is maximum due to rains and snowmelt, the incoming sediment rate is also high. The third finding was that among the three independent variables of inflow, temperature and rain affecting the dependent variable of sedimentation, 1 Cusec rise in inflow results in a 0.004 MAF increase in sedimentation with a high significant p value of 0.000, while 1 o rise in temperature will lead to 0.007 MAF increase in sedimentation but with lesser significance of The sedimentation rate of Tarbela reservoir was proved to be dependent on seasons with maximum being recorded in the monsoon. A very strong positive correlation of sediment with inflow was proved with the help of Pearson correlation with a p value < 0.001. [24] also concluded in his study that summer monsoon is the season when runoff in Indus is at its peak. The reason of course is the high snowmelt runoff accompanied by monsoon rains; this situation leads to peak floods. Thus, if the runoff in Indus will be maximized in the summer monsoon, so would be the inflow and sediments coming in Tarbela reservoir, as proven in this study. It can be suggested that monsoon season sediment generation needs to be monitored on regular basis and yearly monsoon sediment generation trends must be established for efficient management of sedimentation in this reservoir.
The annual average sediments deposited in the reservoir for the period of 1980-2012 was 0.100 MAF. The MLR model proved that among the three independent variables of temperature, rainfall and inflow, inflow as a variable had the strongest impact on the sedimentation of Tarbela reservoir, so much so that 1 Cusec rise in inflow coming into the reservoir can cause 0.004 MAF increase in sedimentation. Rainfall, as a variable, has negative impact on sedimentation as River Indus is more dependent on snow and glacial melt for its runoff, than on rains. Therefore, future studies are recommended to be focused on the glacial retreat in the UIB region and proposed models to forecast resultant sediment generation rate.
Conclusion and recommendations
In the current study, the statistical techniques were applied for the estimation of the effects of climate (rainfall and temperature) on the sedimentation and the inflow of Tarbela Dam and its surrounding catchment area for the period of 1980-2017. The results revealed that summer monsoon season contributed to the greatest bulk of the sediments to Tarbela for the .619 acre feet of sediments were found to be deposited in the reservoir. [15] mentioned a similar inflowing sediment bulk for Tarbela reservoir, where 80% of its sediment load were received during July and August. [3] also reported similar increased sediment inflow for Chalok River, Malaysia, during the wet season. This study has examined the relationship between sedimentation of Tarbela reservoir with rainfall, temperature and inflow, for the period of 1980-2017 and MLR proved inflow in the reservoir as a variable, have most a strong relationship with sediment generation. The increase of sedimentation due to increase of 1 Cusec rise in mean annual inflow coming into the reservoir, boost toward the harmful results, since the flow of the Indus and its tributaries is high in summer monsoon, thus the sediment load gets transported downstream and most of it ends up settling in Tarbela reservoir due to its high trap efficiency. Resultantly, the loss of the operational capacity of the dam has an installed capacity of 3478 MW and has the potential to be enhanced to 6300 MW in near future [25].
In order to keep the Tarbela Dam functioning for a long time, it is recommended that measures must be taken to monitor the changing climatic trends that are leading to fluctuations in temperature and precipitation means of UIB, which would result in changes of runoff, magnitude and the amount of sediment generation. It is recommended that sediment budget of UIB may be formulated by focusing on the relation between the source region and the delivery process. For future studies in the region, it is recommended that gauging network may be made extensive and more reliable. Proper watershed management of Indus is recommended as inflow of sediments in a reservoir might be controlled to great extent by carefully planned watershed management. | 2023-02-25T14:11:33.016Z | 2021-01-01T00:00:00.000 | {
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